Archive for the ‘Water Transporters’ Category

Studies of Respiration Lead to Acetyl CoA

Curator: Larry H. Bernstein, MD, FCAP

In this series of discussions it has become clear that the studies of carbohydrate metabolism were highlighted by Meyerhof’s work on the glycolytic pathway, and the further elucidation of a tie between Warburg’s studies of impaired respiration for malignant aerobic cells relying on glycolysis, comparanle to Pasteur’s observations 60 years earlier by for yeast.   The mitochondrion was unknown at the time, and it took many years to discover the key role played by oxidative phosphorylation and Fritz Lipmann’s discovery of “acetyl coenzyme A, and the later explanation of electron transport.  This was crucial to understanding cellular energetics, which explains the high energy of fatty acid catabolism from stored adipose tissue.  I shall here embark on a journey to trace these important connected developments.

  1. Signaling and signaling pathways
  2. Signaling transduction tutorial.
  3. Carbohydrate metabolism

3.1  Selected References to Signaling and Metabolic Pathways in Leaders in Pharmaceutical Intelligence

  1. Lipid metabolism

4.1  Studies of respiration lead to Acetyl CoA

  1. Protein synthesis and degradation
  2. Subcellular structure
  3. Impairments in pathological states: endocrine disorders; stress hypermetabolism; cancer.


In some reactions, the purpose of phosphorylation is to “activate” or “volatize” a molecule, increasing its energy so it is able to participate in a subsequent reaction with a negative free-energy change. All kinases require a divalent metal ion such as Mg2+ or Mn2+ to be present, which stabilizes the high-energy bonds of the donor molecule (usually ATP or ATP derivative) and allows phosphorylation to occur.This is a major focus of this discussion.

In other reactions, phosphorylation of a protein substrate can inhibit its activity (as when AKT phosphorylates the enzyme GSK-3). When src is phosphorylated on a particular tyrosine, it folds on itself, and thus masks its own kinase domain, and is thus turned “off”. In still other reactions, phosphorylation of a protein causes it to be bound to other proteins which have “recognition domains” for a phosphorylated tyrosine, serine, or threonine motif. In the late 1990s it was recognized that phosphorylation of some proteins causes them to be degraded by the ATP-dependent ubiquitin/proteasome pathway. This is all that needs to be said at this time about proteins.


Oxidative Phosphorylation

ATP is the molecule that supplies energy to metabolism. Almost all aerobic organisms carry out oxidative phosphorylation. This pathway is probably so pervasive because it is a highly efficient way of releasing energy, compared to alternative fermentation processes such as anaerobic glycolysis.

During oxidative phosphorylation, electrons are transferred from electron donors to electron acceptors such as oxygen, in redox reactions. These redox reactions release energy, which is used to form ATP. In eukaryotes, these redox reactions are carried out by a series of protein complexes within the cell’s intermembrane wall mitochondria, whereas, in prokaryotes, these proteins are located in the cells’ intermembrane space.

O t to  W a r b u r g
Nobel Lecture, December 10, 1931

The oxygen-transferring ferment of respiration

The effects of iron are very great, and it follows that oxidation and reduction of the ferment iron must occur extremely rapidly. In fact, almost every molecule of oxygen that comes into contact with an atom of ferment iron reacts with it.  Complex-bound bivalent iron in compounds reacts, in vitro as well as in the cell, with molecular oxygen. tt is not yet possible to reduce in vitro trivalent iron with the cell fuel: it is always necessary to add a substance of unknown composition, a ferment, that activates the combustible material for the attack of the iron. It must, therefore, be concluded that activation of the combustible substance in the breathing cell precedes the attack of the ferment iron; this corresponds with “hydrogen activation” as postulated in the theory of Wieland and Thunberg. According to the results of a joint research with W. Christian, this is a cleavage comparable with those known as fermentation.

It is possible that the interplay of splitting ferment and oxygen-transferring ferment does not fully explain the mechanism of cellular respiration; that the iron that reacts with the molecular oxygen does not directly oxidize the activated combustible substances, but that it exerts its effects indirectly through still other iron compounds – the three non-auto-oxidizable cell hemes of MacMunn, which occur in living cells according to the spectroscopic observations of MacMunn and Keilin, and which are reduced in the cell under exclusion of oxygen. It is still not possible to answer the question whether the MacMunn hemes form part of the normal respiratory cycle, i.e., whether respiration is not a simple iron catalysis but a four-fold one. The available spectroscopic observations are also consistent with the view that the MacMunn hemes in the cell are only reduced when the concentration of activated combustible substance is physiologically above normal. This will suffice to indicate that oxygen transfer by the iron of the oxygen transferring ferment is not the whole story of respiration. Respiration requires not only oxygen-transferring ferment and combustible substance, but oxygen-transferring ferment and the living cell.

Inhibition of cellular respiration by prussic acid was discovered some 50 years ago by Claude Bernard, and has interested both chemists and biologists ever since. It takes place as the result of a reaction between the prussic acid and the oxygen-transferring ferment iron, that is, with the ferment iron in trivalent form. [In the prussic acid reaction] the oxidizing OH-group of the trivalent ferment-iron is replaced by the non-oxidizing CN-group, thus bringing transfer of oxygen to a standstill. Prussic acid inhibits reduction of the ferment iron. Inhibition of respiration by carbon monoxide was discovered only a few years ago. [Given] the initial reaction in respiration, then, in the presence of carbon monoxide, the competing reaction will also occur and, varying with the pressures of the carbon monoxide and of the oxygen, more or less of the ferment iron will be removed from the catalytic process on account of fixation of carbon monoxide to the ferment iron. Unlike prussic acid, therefore, carbon monoxide affects the bivalent iron of the ferment. Carbon monoxide inhibits oxidation of the ferment iron.

Thus inhibition of respiration by carbon monoxide, unlike that by prussic acid, depends upon the partial pressure of oxygen. The toxic action of prussic acid in the human subject is based on its inhibitory action on cellular respiration. The toxic effect of carbon monoxide on man has nothing to do with inhibition of cellular respiration by carbon monoxide but is based on the reaction of carbon monoxide with blood iron. For, the effect of carbon monoxide on blood iron occurs at pressures of carbon monoxide far from the level at which cellular respiration would be inhibited.

If carbon monoxide is added to the oxygen in which living cells breathe, respiration ceases, as has already been mentioned, but if exposure to ultraviolet or visible light is administered, respiration recurs. By alternate illumination and darkness it is possible to cause respiration and cessation of respiration in living, breathing cells in mixtures of carbon monoxide and oxygen. In the dark, the iron of the oxygen-transferring ferment becomes bound to carbon monoxide, whereas in the light the carbon monoxide is split off from the iron which is, thus, liberated for oxygen transfer. This fact was discovered in 1926 in collaboration with Fritz Kubowitz. Photochemical dissociation of iron carbonyl compounds was discovered in 1891 by Mond and Langer, by exposing iron pentacarbonyl. This reaction is specific for carbonyl compounds of iron, most of which appear to dissociate in the presence of light, e.g., carbon-monoxide hemoglobin (John Haldane, 1897) carbon-monoxide hemochromogen (Anson and Mirsky, 1925), carbon-monoxide pyridine hemochromogen (H. A. Krebs, 1928), and carbon-monoxide ferrocysteine (W. Cremer, 1929).

When the photochemical dissociation of iron carbonyl compounds is measured quantitatively (we followed hereby Emil Warburg’s photochemical experiments), by using monochromatic light and comparing the amount of light energy absorbed with the amount of carbon monoxide set free, it is found that Einstein’s law of photochemical equivalence is very exactly fulfilled. The number of FeCO-groups set free is equal to the number of light quanta absorbed, and this is independent of the wavelength employed.

Photochemical dissociation of iron carbonyl compounds can be used to determine the absorption spectrum of a catalytic oxygen-transferring iron compound. One combines the catalyst in the dark with carbon monoxide, and so abolishes the oxygen-transferring power of the iron. If then this is exposed to monochromatic light of various wavelengths and of measured quantum intensity, and the effect of light W measured the increase in the rate of catalysis – it is found that the effects of the light are proportional to the quanta absorbed. The arrangement becomes very simple if the catalyst is present, as is usually the case, in infinitesimally low concentration in the exposed system. Then the thickness of the layers related to the amount of absorption of light can be considered to be infinitely thin, the number of quanta absorbed is proportional to the number of quanta supplied by irradiation.

In collaboration with Erwin Negelein, this principle was employed to measure the relative absorption spectrum of the oxygen-transferring respiratory ferment. The respiration of living cells was inhibited by carbon monoxide which was mixed with the oxygen. We then irradiated with monochromatic light of various wavelengths and of measured quantum intensity, and [measured] the increase of respiration together with the relative absorption spectrum. Only practically colorless cells are suitable for this type of experiment, [which requires] a layer infinitely thin with regard to light absorption.

Imagine living cells whose respiration is inhibited by carbon monoxide. If these are irradiated, respiration does not increase suddenly from the dark to the light-value, but there is a definite, although very short, interval until the combination of carbon monoxide with the ferment is broken down by the light. Even without calculation, it is obvious that the rate of increase in the effect of light must be related to the depth of colour of the ferment. If the ferment absorbs strongly, the -monoxide compound will be rapidly broken down, and vice versa.

The time of increase of the action of light can be measured. The time taken for a given intensity of light to cause dissociation of approximately half the carbon-monoxide compound of the ferment can be measured and, from this time, and from the effective intensity of light, the absolute absorption coefficient of the ferment for every wavelength can be calculated. The absorption capacity of the ferment, measured in accordance with this principle, was found to be of the same order as the power of light absorption of our strongest pigments. If one imagines a ferment solution of molar concentration, a layer of 2 x 10-6 cm thickness would weaken the blue mercury line 436 µµ up by half. The fact that the ferment in spite of this cannot be seen in the cells is due to its low concentration.

Monochromators and color filters were used to isolate the lines from these sources of light. If the absorption coefficient is entered as a function of the wavelength, the absorption spectrum of the carbon-monoxide compound of the ferment is obtained. The principal absorption-band or y-band lies in the blue.
This is the spectrum of a heme compound, according to the position of the bands, the intensity state of the bands, and the absolute magnitude of the absorption coefficients.

It appeared essential to have a control to ascertain whether heme as an oxidation catalyst of carbon monoxide and prussic acid really behaves like the ferment. If cysteine is dissolved in water containing pyridine, and a trace of heme is added, and this is shaken with air, the cysteine is catalytically oxidized by the oxygen-transferring power of the heme. According to Krebs, the catalysis is inhibited by carbon monoxide in the dark, but the inhibition ceases when the mixture is illuminated. Prussic acid too acts on this model on cellular respiration, inasmuch as it combines with the trivalent heme and inhibits its reduction. Just as in life, inhibition by carbon monoxide is dependent on the oxygen pressure, while inhibition by prussic acid is independent of the oxygen pressure.

In conjunction with Negelein, this model was also used to test the ferment experiments quantitatively. Heme catalysis in the model was inhibited by carbon monoxide in the dark. Then monochromatic light of known quantum intensity was used to irradiate it, and the absorption spectrum of the catalyst calculated from the effect of the light which was known from direct measurements on the pure substance. The calculation gave the absorption spectrum of the heme that had been added as a catalyst, and so the method was verified as a technique for the determination of the ferment spectrum, both the calculation and the measurement method.

The positions of the principal band and a-band of the ferment are:

Principal band            α-band

433 µµ                    590 µµ

These will be referred to as the “ferment bands” because the ferment was the first for which they were determined. Hemes are the complex iron compounds of the porphyrins, in which two valencies of the iron are bound to nitrogen. The porphyrins, of which Hans Fischer determined the chemical structure, are tetrapyrrole compounds in which the four pyrrole nuclei are held together by four interposed methane groups in the cr-position. Green, red, and mixed shades of hemes are known. If magnesium is replaced by iron in chlorophyll, green hemes are obtained. Their color is due to a strong band in the red which is already recognized in chlorophyll. The ferment does not absorb in the red and cannot, therefore, be a green heme. Red hemes are the usual hemes in blood pigment and in its related substances, such as mesoheme and deuteroheme. Coproheme is also a red heme which is an iron compound of the coproporphyrin that H. Fischer recognized in the body. Other red hemes are 20 µµ further from the red than the ferment bands. It follows that the ferment is not a red heme.

The pheoporphyrins are closely related to blood pigment but, as H. Fischer showed, pheoporphyrin a is simply mesoporphyrin in which the one propionic acid has been oxidized so that ring closure with the porphyrin nucleus is made possible. Pheoporphyrin a is a reduction product of chlorophyll a or an oxidation product of blood pigment, and connects together, in an amazingly simple manner, the principal pigments of the organic world the blood pigment and the leaf pigment.

Chlorophyll b has, in general, bands of longer wavelength than chlorophyll a, and for this reason,

  1. Christian and I applied Fischer’s reduction method to it. In this way we obtained pheoheme b, which, when linked with protein, corresponds with the ferment in respect to the position of the principal band. The principal band of the carbon-monoxide compound of pheohemoglobin b is 435 µµ.
  2. However, while the principal band of pheohemoglobin corresponds with the ferment bands within the permitted limits, the α-band shifts so far beyond them because it lies too near the red. It is, nevertheless, interesting that
  3. when ‘chlorophyll b is reduced, one obtains a pheoporphyrin of which the heme of all the pheohemes that have been demonstrated up to the present time is the most like the ferment.


Still nearer the ferment in its spectrum, is a heme occurring in Nature. This is

  • spirographis heme, which has been isolated from chlorocruorin, the blood pigment of the bristle-worm Spirographis,

in collaboration with Negelein and Haas, the bands of spirographis heme, coupled to globin, are :

  • carbon-monoxyhemoglobin of spirographis:  principle band, 434 µµ; α-band, 594 µµ.

Spirographis heme differs from the red hemes by the surplus or ketone oxygen-atom, and is classified as pheoheme. Like Fischer’s pheohemes, spirographis heme is intermediate between chlorophyll and blood pigment in respect of

  • the degree of oxidation of the side-chains.

The two hemes with a spectrum most like that of the ferment – pheoheme b and spirographis heme – possess a remarkable property. If they are dissolved in dilute sodium-hydroxide solution, in the form of ferrous compounds,

  • the absorption bands slowly wander towards the blue, near the bands of blood heme. In this way,
  • mixed-color hemes have been converted into red hemes.

On acidification, the change reverts: the <<blood bands>> disappear and

  • the ferment bands appear.

This experiment shows that

  1. oxidation of the side-chains does not suffice to give rise to the ferment bands, but
  2. some process of the type of anhydride formation must also occur.

The unique intermediate status of the ferment-like hemes demonstrated by these simple experiments suggests

  1. the suspicion that blood pigment and leaf pigments have both arisen from the ferment –
  2. blood pigments by reduction, and leaf pigment by oxidation.
  • For evidently, the ferment existed earlier than hemoglobin and chlorophyll.

The investigations on the oxygen-transferring ferment have been supported from the start by the Notgemeinschaft der deutschen Wissenschaft and the Rockefeller Foundation, without whose help they could not have been carried out. I have to thank both organizations here.

A L B E R T S Z E N T- GY Ö R G Y I      Nobel Lecture, December 11, 1937

Oxidation, energy transfer, and vitamins

A living cell requires energy not only for all its functions, but also

  • for the maintenance of its structure.
  • The source of this energy is the sun’s radiation.

Energy from the sun’s rays is trapped by green plants, and

  • converted into a bound form, invested in a chemical reaction.

When sunlight falls on green-plants, they liberate oxygen from carbon dioxide, and

  1. store up carbon, bound to the elements of water, as carbohydrate.

The radiant energy is now locked up in this carbohydrate molecule. This molecule is our food. When energy is required,

  • the carbohydrate is again combined with oxygen to form carbon dioxide, oxidized, and energy released.

Investigations during the last few decades have brought hydrogen instead of carbon, and instead of CO2 water, the mother of all life, into the foreground. It is becoming increasingly probable that

  1. radiant energy is used primarily to break water down into its elements,
  2. while CO2, serves only to fix the elusive hydrogen thus released.

While this concept of energy fixation was still being developed, the importance of hydrogen in the reversal of this process, whereby energy is liberated by oxidation, had already been confirmed by H. Wieland’s experiments.

Our body really only knowns one fuel, hydrogen. The foodstuff, carbohydrate, is essentially a packet of hydrogen, a hydrogen supplier, a hydrogen donor, and the main event during its combustion is

  • the splitting off of hydrogen.

So the combustion of hydrogen is

  • the real energy-supplying reaction;

To the elucidation of reaction (6), which seems so simple, I have devoted all my energy for the last fifteen years.

When I first ventured into this territory, the foundations had already been laid by the two pioneers H. Wieland and
O. Warburg, and Wieland’s teaching had been applied by Th. Thunberg to the realm of animal physiology.Wieland and Thunberg showed, with regard to foodstuffs, how

  1. the first step in oxidation is the “activation” of hydrogen, whereby
  2. the bonds linking it to the food molecule are loosened, and
  3. hydrogen prepared for splitting off.

But at the same time oxygen is also, as Warburg showed,

  • activated for the reaction by an enzyme.
  • the hydrogen-activating enzymes are called dehydrases or dehydrogenases.

Warburg called his oxygen-activating catalyst, “respiratory enzyme”.These concepts of Wieland and Warburg were apparently contradictory, and

  1. my first task was to show that the two processes are complementary to one another, and that
  2. in muscle cells activated oxygen oxidizes activated hydrogen.

This picture was enriched by the English worker D. Keilin, who showed that

  • activated oxygen does not oxidize activated hydrogen directly, but
  • that a dye, cytochrome, is interposed between them.

In keeping with this function, the “respiratory enzyme” is now also called “cytochrome oxidase”.

About ten years ago, when I tried to construct this system of respiration artificially and added together the respiratory enzyme with cytochrome and some foodstuff together with its dehydrogenase, I could justifiably expect that this system would use up oxygen and oxidize the food. But the system remained inactive. I found that

  • the dehydrogenation of certain donors is linked to the presence of a co-enzyme.

Analysis of this co-enzyme showed it to be a nucleotide, identical with v. Euler’s co-zymase, which H. v. Euler and R. Nilsson had already shown to accelerate the process of dehydration. As a result of Warburg’s investigations,this co-dehydrogenase has recently come very much into the foreground. Warburg showed that

  • it contains a pyridine base, and that it accepts hydrogen directly
    [pyridine nucleotide, triphosphopyridine nucleotide, TPN]

from food when the latter is dehydrogenated. It is therefore, the primary H-acceptor.

While working on the isolation of the co-enzyme with Banga, I found a remarkable dye, which showed clearly by its reversible oxidation that it, too, played a part in the respiration. We called this new dye cytoflav. Later Warburg showed that

  • this substance exercised its function in combination with a protein.

He called this protein complex of the dye, “yellow enzyme”. R. Kuhn, to whom we owe the structural analysis of the dye, called the dye lactoflavin and, with Györgyi and Wagner-Jauregg, showed it to be identical with vitamin B,.But the respiratory system stayed inactive even

  • after the addition of both these new components, codehydrogenase and yellow enzyme.

The C4-dicarboxylic acids and their activators which Thunberg discovered are

  • interposed between cytochrome and the activation of hydrogen as intermediate hydrogen-carriers.

In the case of carbohydrate, hydrogen from the food is first taken up by oxaloacetic acid, which

  • is reacted with the cytoplasmic malic dehydrogenase (and pyridine nucleotide –
    reduced DPN[H])
    , and thereby activated.

By taking up two hydrogen atoms, oxaloacetic acid is changed into malic acid.

  • OAA + NADH – (MDH) – malate + NAD+ + H+

This malic acid now passes on the H-atoms, and thus reverts to oxaloacetic acid,

  • which can again take up new H-atoms.

Malate + NAD+ + H+ — MDH – OAA + NADH

The H-atoms released by malic acid are taken up by fumaric acid, which is similarly

  • activated by the so-called succinic dehydrogenase.

The uptake of two H-atoms

  • converts the fumarate to succinate, to succinic acid.

The two H-atoms of succinic acid are then

  • oxidized away by the cytochrome.

Finally the cytochrome is oxidized by the respiratory enzyme, and

  • the respiratory enzyme by oxygen.

The function of the C4-dicarboxylic acids is not to be pictured as consisting of a certain amount of C4-dicarboxylic acid in the cell which is alternately oxidized and reduced. Fig. 2 corresponds more to the real situation. The protoplasmic surface, which is represented by the semi-circle, has single molecules of oxaloacetate and fumarate attached to it as prosthetic groups. These fused, activated dicarboxylic molecules then temporarily bind the hydrogen from the food. The co-dehydrogenases and the yellow enzymes also take part in this system. I have attempted to add them in at the right place.

This diagram, which will probably still undergo many more modifications, states that the “foodstuff” – H-donor – starts by

  1. passing its hydrogen, which has been activated by dehydrase, to the co-dehydrogenase.
  2. The coenzyme passes it to the oxaloacetic acid*.
  3. The malic acid then passes it on again to a co-enzyme,
  4. which passes the hydrogen to the yellow enzyme.
  5. The yellow enzyme passes the hydrogen to the fumarate.
  6. The succinate so produced is then oxidized by cytochrome,
  7. the cytochrome by respiratory enzyme,
  8. the respiratory enzyme by oxygen.

So the reaction 2H + O – H2O, which seems such a simple one,

  • breaks down into a long series of separate reactions.

With each new step, with each transfer between substances,

  • the hydrogen loses some of its energy,
  • finally combining with oxygen in its lowest-energy compound.

So each hydrogen atom is gradually oxidized in a long series of reactions, and

  • its energy released in stages.

This oxidation of hydrogen in stages seems to be one of the basic principles of biological oxidation. The reason for it is probably mainly that

  • the cell would not be able to harness and transfer to other processes
  • the large amount of energy which would be released by direct oxidation.

The cell needs small change if it is to be able to

  • pay for its functions without losing too much in the process.

So it oxidizes the H-atom by stages, converting the large banknote into small change.

About half of all plants – contain a polyphenol, generally a pyrocatechol derivative, together with an enzyme, polyphenoloxidase, which oxidizes polyphenol with the help of oxygen. The current interpretation of the mode of action of this oxidase was a confused one. I succeeded in showing that the situation was simply this, that

the oxidase oxidizes the polyphenol to quinone with oxygen.

  • In the intact plant the quinone is reduced back again
  • with hydrogen made available from the foodstuff.

Phenol therefore acts as a hydrogen-carrier between oxygen and the H-donor, and we are here again faced with a probably still imperfectly understood system for

  • the stepwise combustion of hydrogen.


Vitamin C

If benzidine is added to a peroxide in the presence of peroxidase, a deep-blue color appears immediately, which is caused by the oxidation of the benzidine. This reaction does not occur without peroxidase. I simply used some juice which had been squeezed from these plants instead of a purified peroxidase, and added benzidine and peroxide, and the blue pigment appeared, after a small delay of about a second. Analysis of this delay showed that it was due to the presence of a powerful reducing substance, which reduced the oxidized benzidine again, until it had itself been used up. Thanks to the invitation from F. G. Hopkins and the help of the Rockefeller Foundation, I was able ten years ago to transfer my workshop to Cambridge, where for the first time I was able to pay more serious attention to chemistry. Soon I succeeded in isolating the substance in question from adrenals and various plants, and in showing that it corresponded to the formula C6H8O6 and was related to the carbohydrates. This last circumstance induced me to apply to Prof. W. N. Haworth, who immediately recognized the chemical interest of the substance and asked me for a larger quantity to permit analysis of its structure.

The Mayo Foundation and Prof. Kendall came to my help on a large scale, and made it possible for me to work, regardless of expense, on the material from large American slaughter-houses. The result of a year’s

work-was 25 g of a crystalline substance, which was given the name “hexuronic acid”. I shared this amount of the substance with Prof. Haworth. He undertook to investigate the exact structural formula of the substance. I used the other half of my preparation to gain a deeper understanding of the substance’s function. The substance could not replace the adrenals, but caused the disappearance of pigmentation in patients with Addison’s disease.

In 1930 I settled down in my own country at the University of Szeged. I also received a first-rate young American collaborator, J. L. Svirbely, who had experience in vitamin research, but besides this experience brought only the conviction that my hexuronic acid was not identical with vitamin C. In the autumn of 1931 our first experiments were completed, and showed unmistakably that hexuronic acid was power- fully anti-scorbutic, and that the anti-scorbutic acitvity of plant juices corresponded to their hexuronic acid content. We did not publish our results till the following year after repeating our experiments. At this time Tillmans was already directing attention to the connection between the reducing strength and the vitamin activity of plant juices. At the same time King and Waugh also reported crystals obtained from lemon juice, which were active anti-scorbutically and resembled our hexuronic acid.

My town, Szeged, is the centre of the Hungarian paprika industry. Since this fruit travels badly, I had not had the chance of trying it earlier. The sight of this healthy fruit inspired me one evening with a last hope, and that same night investigation revealed that this fruit represented an unbelievably rich source of hexuronic acid, which, with Haworth, I re-baptized ascorbic acid. I also had the privilege of providing my two prize-winning colleagues P. Karrer and W. N. Haworth with abundant material, and making its structural analysis possible for them. I myself produced with Varga the mono-acetone derivative of ascorbic acid, which forms magnificent crystals; from which, after repeated dissolving and recrystallization, ascorbic acid can be separated again with undiminished activity. This was the first proof that ascorbic acid was identical with vitamin C.

Returning to the processes of oxidation, I now tried to analyse further the system of respiration in plants, in which ascorbic acid and peroxidase played an important part. I had already found in Rochester that the peroxidase plants contain an enzyme which reversibly oxidizes ascorbic acid with two valencies in the presence of oxygen. Further analysis showed that here again a system of respiration was in question, in which hydrogen was oxidized by stages. I would like, in the interests of brevity, to summarize the end result of these experiments, which I carried out with St. Huszák. Ascorbic acid oxidase oxidizes the acid with oxygen to reversible dehydroascorbic acid, whereby the oxygen unites with the two labile H-atoms from the acid to form hydrogen peroxide. This peroxide reacts with peroxidase and oxidizes a second molecule of ascorbic acid. Both these molecules of dehydro-ascorbic acid again take up hydrogen from the foodstuff, possibly by means of SH-groups. But peroxidase does not oxidize ascorbic acid directly. Another substance is interposed between the two, which belongs to the large group of yellow, water-soluble phenol-benzol-r-pyran plant dyes (flavone, flavonol, flavanone). Here the peroxidase oxidizes the phenol group to the quinone, which then oxidizes the ascorbic acid directly, taking up both its H-atoms.

At the time that I had just detected the rich vitamin content of the paprika, I was asked by a colleague of mine for pure vitamin C. This colleague himself suffered from a serious haemorrhagic diathesis. Since I still did not have enough of this crystalline substance at my disposal then, I sent him paprikas. My colleague was cured. But later we tried in vain to obtain the same therapeutic effect with pure vitamin C. Guided by my earlier studies into the peroxidase system, I investigated with my friend St. Rusznyák and his collaborators Armentano and Bentsáth the effect of the other link in the chain, the flavones. Certain members of this group of substances, the flavanone hesperidin (Fig. 5) and the formerly unknown eriodictyolglycoside, a mixture of which we had isolated from lemons and named citrin, now had the same therapeutic effect as paprika itself.

H U G O T H E O R E L L          Nobel Lecture, December 12, 1955

The nature and mode of action of oxidation enzymes


Practically all chemical reactions in living nature are started and directed in their course by enzymes. This being the case, Man has of course since time immemorial seen examples of what we now call enzymatic reactions, e.g. fermentation and decay. It would thus be possible to trace the history of enzymes back to the ancient Greeks, or still further for that matter. But it would be rather pointless, since to observe a phenomenon is not the same thing as to explain it. It is more correct to say that our knowledge of enzymes is essentially a product of twentieth-century research.

Jöns Jacob Berzelius, wrote in his yearbook in 1835: “…The catalytic force appears actually to consist thought herein that through their mere presence, and not through their affinity, bodies are able to arouse affinities which at this temperature are slumbering…”  Enzymes are the catalyzers of the biological world, and Berzelius’ description of catalytic force is surprisingly far-sighted…  if one could once understand the mechanism it would doubtless prove that the forces of ordinary chemistry would suffice to explain also these as yet mysterious reactions.

The year 1926 was a memorable one. The German chemist Richard Willstitter gave a lecture then in Deutsche Chemische Gesellschaft, in which he summarized the experiences gained in his attempts over many years to produce pure enzymes. Willstätter drew the conclusion that the enzymes did not belong to any known class of chemical substances, and that the effects of the enzymes derived from a new natural force, thus taking the view that 90 years earlier Berzelius thought to be improbable. That same year, through an irony of fate, the American researcher J. B. Sumner published a work in which he claimed to have crystallized in pure form an enzyme, urease. In the ensuing years J. H. Northrop and his collaborators crystallized out a further three enzyme preparations, pepsin, trypsin, and chymotrypsin, like urease, hydrolytic enzymes that split linkages by introducing water. If these discoveries had been undisputed from the outset it would probably not have been 20 years before Sumner, together with Northrop and Stanley, received a Nobel Prize.

When in 1933 I went on a Rockefeller fellowship to Otto Warburg’s institute in Berlin, Warburg and Christian had in the previous year produced a yellow-coloured preparation of an oxidation enzyme from yeast. The yellow colour was of particular interest: it faded away on reduction and returned on oxidation with e.g. oxygen, so that it was evident that the yellow pigment had to do with the actual enzymatic process of oxido-reduction. It was possible to free the yellow pigment from the high-molecular carrier substance, whose nature was still unknown, for example by treatment with acid methyl alcohol, whereupon the enzyme effect disappeared. Through simultaneous works by Warburg in Berlin, Kuhn in Heidelberg and Karrer in Zurich the constitution of the yellow pigment (lactoflavin, later riboflavin or vitamin B,) was determined. It was here for the first time possible to localize the enzymatic effect to a definite atomic constellation: hydrogen freed from the substrate (hexose monophosphate) is, with the aid of a special enzyme system (TPN-Zwischenferment) whose nature was elucidated somewhat later, placed on the nitrogen atoms of the flavin (1) and (10), giving rise to the colourless leucoflavin. This is reoxidized by oxygen, hydrogen peroxide being formed, and may afterwards be reduced again, and so forth. This cyclic process then continues until the entire amount of substrate has been deprived of two hydrogen atoms and been transformed into phosphogluconic acid; and a corresponding amount of hydrogen peroxide has been formed. At the end of the process the yellow enzyme is still there in unchanged form, and has thus apparently, as Berzelius expressed himself, aroused a chemical affinity through its mere presence.

The polysaccharides, which constituted 80-90% of the entire weight, were completely removed, together with some inactive colourless proteins. After fractionated precipitations with ammonium sulphate I produced a crystalline preparation which on ultracentrifuging and electrophoresis appeared homogeneous. The enzyme was a protein with the molecular weight 75,000 and strongly yellow-colored by the flavin part. The result of the Flavin analysis was 1 mol flavin per 1 mol protein. With dialysis against diluted hydrochloric acid at low temperature the yellow pigment was separated from the protein, which then became colorless. In the enzyme test the flavin part and the protein separately were inactive, but if the flavin part and the protein were mixed at approximately neutral reaction the enzyme effect returned, and the original effect came back when one mixed them in the molecular proportions 1 : 1. That in this connection a combination between the pigment and the protein came about was obvious, moreover, for other reasons: the green-yellow colour of the flavin part changed to pure yellow,and its strong. yellow fluorescence disappeared with linking to the protein.

In my electrophoretic experiments lactoflavin behaved as a neutral body, while the pigment part separated from the yellow enzyme moved rapidly towards the anode and was thus an acid. An analysis for phosphorus showed 1 P per mol flavin, and when after a time (1934) I succeeded in isolating the natural pigment component this proved to be a lactoflavin phosphoric acid ester, thus a kind of nucleotide, and it was obvious that the phosphoric acid served to link the pigment part to the protein. I will now show some simple experiments with the yellow enzyme, its colored part, which we now generally refer to as FMN (flavin mononucleotide), and the colorless enzyme protein.

  • The ferment-solution is pure yellow, the FMN-solution green-yellow,owing to the 1st that the light-absorption band in the blue of the free FMN is displaced somewhat in the long-wave direction on being linked with the protein component. A reducing agent (Na2S2O4) is now added to the one cuvette, it is indifferent which. The colour disappears in consequence of the formation of leucoflavin. Oxygen-gas is bubbled through the solution: the colour comes back as soon as the excess of reducing agent has been consumed. The experiment demonstrates the reaction cycle of the yellow enzyme: reduction through hydrogen from the substrate side, reoxidation with oxygen-gas.
  • A flask containing FMN-solution so diluted that its yellow color is not descernible to the eye is placed on a lamp giving long-wave ultraviolet light. The solution gives a strong, yellow fluorescence which disappears on reduction and returns on bubbling with oxygen-gas.
  • Two flasks are placed on the fluorescence lamp. The one contains a diluted solution of the free protein in phosphate buffer (pH 7), the other phosphate buffer alone. An equal amount of FMN-solution is dripped into each flask. In the flask with protein the fluorescence is at once extinguished,

but in the flask with buffer-solution alone it remains. The experiment demonstrates the resynthesis of  yellow enzyme, and since the fluorescence is extinguished by the protein, one may draw the conclusion that some group in the protein is in this connection linked to the imino-group NH(3) of the flavin, which according to Kuhn must be free for the fluorescence to appear.

The significance of these investigations on the yellow enzyme may be summarized

as follows.

  1. The reversible splitting of the yellow enzyme to apo-enzyme + coenzyme in the simple molecular relation 1 : 1 proved that we had here to do with a pure enzyme; the experiments would have been incomprehensible if the enzyme itself had been only an impurity.
  2. This enzyme was thus demonstrably a protein. In the sequel all the enzymes which have been isolated have proved to be proteins.
  3. The first coenzyme, FMN, was isolated and found to be a vitamin phosphoric acid ester. This has since proved to be something occurring widely in nature: the vitamins nicotinic acid amide, thiamine and pyridoxine form in an analogous way nucleotide-like coenzymes, which like the nucleic acids

themselves combine reversibly with proteins.

During the past 20 years a large number of flavoproteins with various enzyme effects have been produced. Instead of FMN many of them contain a dinucleotide, FAD, which consists of FMN + adenylic acid.

We constructed a very sensitive apparatus to record changes in the intensity of the fluorescence, and were thus able to follow the rapidity with which the fluorescence diminishes when FMN and protein are combined, or increases when they are split. Under suitable conditions the speed of combination is very high. Thanks to the great sensitivity of the fluorescent method my Norwegian collaborator Agnar Nygaard and I were able to make accurate determinations of the speed-constant simply by working in extremely diluted solutions, where the speed of combination is low because an FMN molecule so seldom happens to collide with a protein-molecule. We then varied the degree of acidity, ionic milieu and temperature, and we treated the protein with a large number of different reagents which affect in a known way different groups in proteins. In this way we succeeded with a rather high degree of certainty in ascertaining that phosphoric acid in FMN is linked to primary amino-groups in the protein, and the imino-group (3) in FMN to the phenolic hydroxyl group in a tyrosine residue, whereby the fluorescence is extinguished.

We still do not quite understand how through its linkage to the coenzyme the enzyme-protein “activates” the latter to a rapid absorption and giving off of hydrogen. But something we do know. The so-called oxido-reduction potential of the enzyme is in any case of great importance, and it is determined by a simple relation to the dissociation constants for the oxidized and for the reduced coenzyme-enzyme complex. The dissociation constants are in their turn functions of the velocity constants for the combination between coenzyme and enzyme and for the reverse process, and these velocity constants we have been able to determine both in the yellow ferment and in a number of enzyme systems. Without going into any details I may mention that the linkage of coenzyme to enzyme was found to have surprisingly big effects upon the potential of the former.

Alcohol dehydrogenase


Alcohol dehydrogenases occur in both the animal and the vegetable kingdoms, e.g. in liver, in yeast, and in peas. They are colourless proteins which together with DPN may either oxidize alcohol to aldehyde, as occurs chiefly in the liver, or conversely reduce aldehyde to alcohol, as occurs in yeast.

The yeast enzyme was crystallized by Negelein & Wulf (1936) in Warburg’s institute, the liver enzyme (from horse liver) by Bonnichsen & Wassén at our institute in Stockholm in 1948. These two enzymes have come to play a certain general rôle in biochemistry on account of the fact that it has been possible to investigate their kinetics more accurately than is the case with other enzyme systems. The liver enzyme especially, we have on repeated occasions studied with particular thoroughness, since especially favourable experimental conditions here presented themselves. For all reactions with DPN-system it is possible to follow the reaction DPN+ + 2H =+ DPNH + H+ spectrophotometrically, since DPNH has an absorption-band in the more long-wave ultraviolet region, at 340 rnp, and thousands of such experiments have been performed all over the world. A couple of years ago, moreover, we began to apply our fluorescence method, which is based on the fact that DPNH but not DPN fluoresces, even if considerably more weakly than the flavins. Asregards the liver enzyme there is a further effect, which proved extremely useful for certain spectrophotometrical determinations of reaction speeds; together with Bonnichsen I found in 1950 that the 340 rnp band of the reduced coenzyme was displaced, on combination with liver alcohol dehydrogenase, to 325 rnp, and together with Britton Chance we were thus able with the help of his extremely refined rapid spectrophotometric methods to determine the velocity constant for this very rapid reaction. This reaction belongs to the 3 bost problem involving the enzyme, the coenzyme, and the substrate, and both the coenzyme and the substrate occur in both oxidized and reduced forms.

It is a curious whim of nature that the same coenzyme which in the yeast makes alcohol by attaching hydrogen to aldehyde also occurs in the liver to remove from alcohol the same hydrogen, so that the alcohol becomes aldehyde again, which is then oxidized further
Heme proteins

In 1936 we had obtained cytochrome approximately 80% pure, and in 1939 close to 100%.It is a beautiful red, iron-porphyrin-containing protein which functions as a link in the chain of the cell-respiration enzymes, the iron atom now taking up and now giving off an electron, and the iron thus alternating valency between the 3-valent ferri and the 2-valent ferro stages. It is a very pleasant substance to work with, not merely because it is lovely to look at, but also because it is uncommonly stable and durable. From 100 kg horse heart one can produce 3-4 grams of pure cytochrome c. The molecule weighs about 12,000 and contains one mol iron porphyrin per-mol.

Exp. 4. Two cuvettes each contain a solution of ferricytochrome c. The colour is blood-red. To the one are added some grains of sodium hydrosulphite: the color is changed to violet-red (ferrocytochrome). Oxygen is now bubbled through the ferrocytochrome-solution: no visible change occurs. The ferrocyto-chrome can thus not be oxidized by oxygen. A small amount of cytochrome oxidase is now added: the ferricytochrome color returns.

From this experiment we can draw the conclusion that reduced cytochrome c cannot react with molecular oxygen. In a chain of oxidation enzymes it will thus not be able to be next to the oxygen. The incapacity of cytochrome to react with oxygen was a striking fact that required an explanation. Another peculiarity was the extremely firm linkage between the red heme pigment and the protein part; in contradistinction to the majority of other heme protides, the pigment cannot be split off by the addition of acetone acidified with hydrochloric acid. Further, there was a displacement of the light-absorption bands which indicated that the two unsaturated vinyl groups occurring in ordinary protohemin were saturated in the hematin of

the cytochrome. In 1938 we succeeded in showing that the porphyrin part of the cytochrome was linked to the protein by means of two sulphur bridges from cysteine residues in the protein of the porphyrin in such a way that the vinyl groups were saturated and were converted to α-thioether groups. The firmness of the linkage and the displacement of the spectral bands were herewith explained. This was the first time that it had been possible to show the nature of chemical linkages between a “prosthetic” group (in this case iron porphyrin) and the protein part in an enzyme.

The light-absorption bands of the cytochrome showed that it is a so-called hemochromogen, which means that two as a rule nitrogen-containing groups are linked to the iron, in addition to the four pyrrol-nitrogen atoms in the porphyrin. From magnetic measurements that I made at Linus Pauling’s institute in Pasadena and from amino-acid analyses, titration curves and spectrophotometry together with Å. Åkeson it emerged (1941) that the nitrogen-containing, hemochromogen-forming groups in cytochrome c were histidine residues, or to be more specific, their imidazole groups.   Recently we have got a bit farther. Tuppy & Bodo in Vienna began last year with Sanger’s method to elucidate the amino-acid sequence in the hemin-containing peptide fragment that one obtains with the proteolytic breaking down of cytochrome c, and succeeded in determining the sequence of the amino acids nearest the heme. The experiments were continued and supplemented by Tuppy, Paléus & Ehrenberg at our institute in Stockholm with the following result:

The peptide chain 1-12 (“Val”) = the amino acid valine, “Glu” = glutamine,”Lys” = lysine, and so forth) is by means of two cysteine-S-bridges and a linkage histidine-Fe linked to the heme. When in 1954 Linus Pauling delivered his Nobel Lecture in Stockholm he showed a new kind of models for the study of the steric configuration of peptide chains, which as we know may form helices or “pleated sheets” of various kinds. It struck me then that it would be extremely interesting to study the question as to which of these possibilities might be compatible with the sulphur bridges to the hemin part and with the linkage of nitrogen containing groups to the iron. Pauling was kind enough to make me a present of his peptide-model pieces, which I shall show presently. This is thus the second time they figure in a Lecture.

Anders Ehrenberg and I now made a hemin model on the same scale as the peptide pieces and constructed models of hemin peptides with every conceivable variant of hydrogen bonding. It proved that many variants could be definitely excluded on steric grounds, and others were improbable for other reasons. Of the original, at least 20 alternatives, finally only one remained – a left-twisting a-helix with the cysteine residue no. 4 linked to the porphyrin side-chain in 4-position, and cysteine no. 7 to the side-chain in 2-position. The imidazole residue fitted exactly to linkage with the iron atom. The peptide spiral becomes parallel with the plane of the heme disc.

Through calculations on the basis of the known partial specific volume of the cytochrome we now consider it extremely probable that the heme plate in cytochrome c is surrounded by peptide spirals on all sides in such a way that the heme iron is entirely screened off from contact with oxygen; here is the explanation of our experiment in which we were unable to oxidize reduced cytochrome c with oxygen-gas. The oxygen simply cannot get at the iron atom. There is, on the other hand, a possibility for electrons to pass in and out in the iron atom via the imidazole groups.  It strikes us as interesting that even at this stage the special mode of reacting of the cytochrome is beginning to be understood from what we know of its chemical constitution.

F r i t z  L i p m a n n           Nobel Lecture, December 11, 1953

Development of the acetylation problem: a personal account


In my development, the recognition of facts and the rationalization of these facts into a unified picture, have interplayed continuously. After my apprenticeship with Otto Meyerhof, a first interest on my own became the phenomenon we call the Pasteur effect, this peculiar depression of the wasteful fermentation in the respiring cell. By looking for a chemical explanation of this economy measure on the cellular level, I was prompted into a study of the mechanism of pyruvic acid oxidation, since it is at the pyruvic stage where respiration branches off from fermentation. For this study I chose as a promising system a relatively simple looking pyruvic acid oxidation enzyme in a certain strain of Lactobacillus delbrueckii1. The decision to explore this particular reaction started me on a rather continuous journey into partly virgin territory to meet with some unexpected discoveries, but also to encounter quite a few nagging disappointments

The most important event during this whole period, I now feel, was the accidental observation that in the L. delbrueckii system, pyruvic acid oxidation was completely dependent on the presence of inorganic phosphate. This observation was made in the course of attempts to replace oxygen by methylene blue. To measure the methylene blue reduction manometrically, I had to switch to a bicarbonate buffer instead of the otherwise routinely used phosphate. In bicarbonate, to my surprise, as shown in Fig. 1, pyruvate oxidation was very slow, but the addition of a little phosphate caused a  remarkable increase in rate. The next figure, Fig. 2, shows the phosphate effect more drastically, using a preparation from which all phosphate was removed by washing with acetate buffer. Then it appeared that the reaction was really fully dependent on phosphate. In spite of such a phosphate dependence, the phosphate balance measured by the ordinary Fiske-Subbarow procedure did not at first indicate any phosphorylative step. Nevertheless, the suspicion remained that phosphate in some manner was entering into the reaction and that a phosphorylated intermediary was formed. As a first approximation, a coupling of this pyruvate

oxidation with adenylic acid phosphorylation was attempted. And, indeed, addition of adenylic acid to the pyruvic oxidation system brought out a net disappearance of inorganic phosphate, accounted for as adenosine triphosphate (Table 11). In parallel with the then just developing fermentation now concluded that the missing link in the reaction chain was acetyl phosphate. In partial confirmation it was shown that a crude preparation of acetyl phosphate, synthesized by the old method of Kämmerer and Carius2

would transfer phosphate to adenylic acid (Table 2). However, it still took quite some time from then on to identify acetyl phosphate definitely as the initial product of the pyruvic oxidation in this system3,4.

At the time when these observations were made, about a dozen years ago, there was, to say the least, a tendency to believe that phosphorylation was rather specifically coupled with the glycolytic reaction. Here, however, we had found a coupling of phosphorylation with a respiratory system. This observation immediately suggested a rather sweeping biochemical significance, of transformations of electron transfer potential, respiratory or fermentative, to phosphate bond energy and therefrom to a wide range of biosynthetic reactions7.

There was a further unusual feature in this pyruvate oxidation system in that the product emerging from the process not only carried an energy-rich phosphoryl radical such as already known, but the acetyl phosphate was even more impressive through its energy-rich acetyl. It rather naturally became a contender for the role of “active” acetate, for the widespread existence of which the isotope experience had already furnished extensive evidence. I became, therefore, quite attracted by the possibility that acetyl phosphate could serve two rather different purposes, either to transfer its phosphoryl group into the phosphate pool, or to supply its active acetyl for biosynthesis of carbon structures. Thus acetyl phosphate should be able to serve as acetyl donor as well as phosphoryl donor, transferring, as shown in Fig. 3, on either side of the oxygen center, such as indicated by Bentley’s early experiments on cleavage7a of acetyl phosphate in H2 18O.

These two novel aspects of the energy problem, namely

(1) the emergence of an energy-rich phosphate bond from a purely respiratory reaction; and

(2) the presumed derivation of a metabolic building-block through this same there towards a general concept of transfer of activated groupings by carrier as the fundamental reaction in biosynthesis8,9.

Although in the related manner the appearance of acetyl phosphate as a metabolic intermediary first

focussed attention to possible mechanisms for the metabolic elaboration of group activation, it soon turned out that the relationship between acetyl phosphate and acetyl transfer was much more complicated than anticipated. reaction, prompted me to propose

  • not only the generalization of the phosphate bond as a versatile energy distributing system,
  • but also to aim there towards a general concept of transfer of activated groupings by carrier as the fundamental reaction in biosynthesis8,9.

Although in the related manner the appearance of acetyl phosphate as a metabolic intermediary first focussed attention to possible mechanisms for the metabolic elaboration of group activation, it soon turned out that the relationship between acetyl phosphate and acetyl transfer was much more complicated than anticipated.

It appeared that as an energy source the particle bound oxidative phosphorylation of the kind observed first by Herman Kalckar14 could be replaced by ATP, as had first been observed with the acetylation of choline in brain preparations by Nachmansohn and his group15,16. Using ATP and acetate as precursors, it was possible to set up a homogeneous particle-free acetylation system obtained by extraction of acetone pigeon liver. In this extract acetyl phosphate was unable to replace the ATP acetate as acetyl precursor.

In spite of this disappointment with acetyl phosphate, our decision to turn to a study of acetylation started then to be rewarding in another way. During these studies we became aware of the participation of a heat-stable factor which disappeared from our enzyme extracts on aging or dialysis. This cofactor was present in boiled extracts of all organs, as well as in microorganisms and yeast. It could not be replaced by any other known cofactor. Therefore, it was suspected that we were dealing with a new coenzyme. From then on, for a number of years, the isolation and identification of this coenzyme became the prominent task of our laboratory. The problem now increased in volume and I had the very good fortune that a group of exceedingly able people were attracted to the laboratory; first Constance Tuttle, then Nathan O. Kaplan and shortly afterwards, G. David Novelli, and then others.

Early data on the replacement of this heat-stable factor by boiled extracts are shown in the next table (Table 3). The pigeon liver acetylation system proved to be a very convenient assay system for the new coenzyme17 since on aging for 4 hours at room temperature, the cofactor was completely autolyzed.

Fortunately, on the other hand, the enzyme responsible for the decomposition of this factor was quite unstable and faded out during the aging, while the acetylation apoenzymes were unaffected.

The next figure, Fig. 4, shows coenzyme A (CoA) assay curves obtained with acetone pigeon liver extract. Finding pig liver a good source for the coenzyme, we set out to collect a reasonably large quantity of a highly purified preparation and then to concentrate on the chemistry with this material. In this analysis we paid particular attention to the possibility of finding in this obviously novel cofactor one of the vitamins.

The subsequence finding of a B-vitamin in the preparation gave us further confidence that we were dealing here with a key substance. We still felt, however, slightly dissatisfied with the proof for pantothenic acid. Therefore, to liberate the chemically rather unstable pantothenic acid from CoA, we made use of observations on enzymatic cleavage of the coenzyme. Two enzyme preparations, intestinal phosphatase and an enzyme in pigeon liver extract, had caused independent inactivation. It then was found that through combined action of these two enzymes, pantothenic acid was liberated18,19.

The two independent enzymatic cleavages indicated early that in CoA existed two independent sites of attachment to the pantothenic acid molecule. One of these obviously was a phosphate link, linking presumably to one of a hydroxyl group in pantothenic acid. The other moiety attached to pantothenic acid, which, cleaved off by liver enzyme, remained unidentified for a long time. In addition to pantothenic acid, our sample of 40 per cent purity had been found to contain about 2 per cent sulfur by elementary analysis and identified by cyanide-nitroprusside test as a potential SH grouping 20,21. Furthermore, the coenzyme preparation contained large amounts of adenylic acid21.

Units Coenzyme

Fig. 4. Concentration-activity curves for coenzyme A preparations of different purity. The arrow indicates the point of 1 unit on the curve. (o) crude coenzyme, 0.25 unit per mg; (x) purified coenzyme, 130 units per mg.

In the subsequent elaboration of the structure, the indications by enzyme analysis for the two sites of attachment to pantothenic acid have been most helpful. The phosphate link was soon identified as a pyrophosphate bridge22; 5-adenylic acid was identified by Novelli23 as enzymatic split product and by Baddiley 24, through chemical cleavage. At the same time, Novelli made observations which indicated the presence of a third phosphate in addition to the pyrophosphate bridge. These indications were confirmed by analysis of a nearly pure preparation which was obtained by Gregoryas from Streptomyces fradiae in collaboration with the research group at the Upjohn Company26.

It was at this period that we started to pay more and more attention to the sulfur in the coenzyme. As shown in Table 5, our purest preparation contained 4.13 per cent sulfur corresponding to one mole per mole of pantothenate. We also found26 that dephosphorylation of CoA yielded a compound containing pantothenic acid and the sulfur carrying moiety, which we suspected as bound through the carboxyl. Through the work of Snell and his group27, the sulfur-containing moiety proved to be attached to pantothenic acid through a link broken by our liver enzyme. It was identified as thioethanolamine by Snell and his group, linked peptidically to pantothenic acid.

Through analysis and synthesis, Baddiley now identified the point of attachment of the phosphate bridge to pantothenic acid in 4-position24 and Novelli et al.28 completed the structure analysis by enzymatic synthesis of “dephospho-CoA” from pantetheine-4’-phosphates and ATP. Furthermore, the attachment of the third phosphate was identified by Kaplan29 to attach in s-position on the ribose of the 5-adenylic acid (while in triphosphopyridine nucleotide it happens to be in 2-position). Therefore, the structure was now

established, as shown in Fig. 5.

Fig. 5. Structure of coenzyme A


The metabolic function of CoA

Parallel with this slow but steady elaboration of the structure, all the time we explored intensively metabolic mechanisms in the acetylation field. By use of the enzymatic assay, as shown in Tables 6, 7, 8, and 9, CoA was found present in all living cells, animals, plants and microorganisms17. Furthermore,

the finding that all cellular pantothenic acid could be accounted for by CoA17 made it clear that CoA represented the only functional form of this vitamin. The finding of the vitamin furnished great impetus; nevertheless, a temptation to connect the pantothenic acid with the acetyl transfer function has

blinded us for a long time to other possibilities.

The first attempts to further explore the function of CoA were made with pantothenic acid-deficient cells and tissues. A deficiency of pyruvate oxidation in pantothenic acid-deficient Proteus morganii, an early isolated observation by Dorfman30 and Hills31, now fitted rather well into the picture. We soon became quite interested in this effect, taking it as an indication for participation of CoA in citric acid synthesis. A parallel between CoA levels and pyruvate oxidation in Proteus morganii was demonstrated32. Using panto thenic aciddeficient yeast, Novelli et al.33 demonstrated a CoA-dependence of acetate oxidation (Fig. 5a) and Olson and Kaplan34 found with duck liver a striking parallel between CoA content and pyruvic utilization, which is shown in Fig. 6.

But more important information was being gathered on -the enzymatic level. The first example of a generality of function was obtained by comparing the activation of apoenzymes for choline- and sulfonamide-acetylation respectively, using our highly purified preparations9 of CoA. As shown in Fig. 7, similar activation curves obtained for the two respective enzymes. Through these experiments, the heat-stable factor for choline acetylation that had been found by Nachmansohn and Berman35 and by Feldberg and Mann36 was identified with CoA. The next most significant step toward a generalization of CoA function for acetyl transfer was made by demonstrating its functioning in the enzymatic synthesis of acetoacetate. The CoA effect in acetoacetate synthesis was studied by Morris Soodak37, who obtained for this reaction a reactivation curve quite similar to those for enzymatic acetylation, as shown in Fig. 8.

Soon afterwards Stern and Ochoa38 showed a CoA-dependent citrate synthesis with a pigeon liver fraction similar to the one used by Soodak for acetoacetate synthesis. In our laboratory, Novelli et al. confirmed and extended this observation with extracts of Escherichia coli39.

In the course of this work, which more and more clearly defined the acetyl transfer function of CoA, Novelli once more tried acetyl phosphate. To our surprise and satisfaction, it then appeared, as shown in Table 9, that in Escherichia coli extracts in contrast to the animal tissue, acetyl phosphate was more than twice as active as acetyl donor for citrate synthesis than ATP acetate 39. Acetyl phosphate, therefore, functioned as a patent microbial acetyl donor. Acetyl transfer from acetyl phosphate, like that from ATP-acetate, was CoA-dependent, as shown in Table 9. Furthermore, a small amount of “microbial conversion factor”, as we called it first, primed acetyl phosphate for activity with pigeon liver acetylation systems40, as shown in Table 10.

Eventually the microbial conversion factor was identified by Stadtman et al.40 with the transacetylase first encountered by Stadtman and Barker in extracts of Clostridium kluyveri41 and likewise, although not clearly defined as such, in extracts of Escherichia coli and Clostridium butylicum by Lipmann and Tuttle42. The definition of such a function was based on the work of Doudoroff et al.43 on transglucosidation with sucrose phosphorylase. Their imaginative use of isotope exchange for closer definition of enzyme mechanisms has been most influential. Like glucose-I-phosphate with sucrose phosphorylase, acetyl phosphate with these various microbial preparations equilibrates its phosphate rapidly with the inorganic phosphate of the solution. As in Doudoroff et al. experiments, first a covalent substrate enzyme derivative had been proposed 43. However, then Stadtman et al.40, with the new experience of CoA dependent acetyl transfer, could implicate CoA in this equilibration between acetyl- and inorganic phosphate and thus could define the transacetylase as an enzyme equilibrating acetyl between phosphate and CoA:

In the course of these various observations, it became quite clear that there existed in cellular metabolism an acetyl distribution system centering around CoA as the acetyl carrier which was rather similar to the ATP-centered phosphoryl distribution system. The general pattern of group transfer became recognizable, with donor and acceptor enzymes being connected through the CoA —- acetyl CoA shuttle. A clearer definition of the donor-acceptor enzyme scheme was obtained through acetone fractionation of our standard system for acetylation of sulfonamide into two separate enzyme fractions, which were inactive separately but showed the acetylation effect when combined. A fraction, A-40, separating out with 40 per cent acetone, was shown by Chou44 to contain the donor enzyme responsible for the ATP-CoA-acetate reaction, while with more acetone precipitated, the acceptor function, A-60, the acetoarylamine kinase as we propose to call this type of enzyme. The need for a combination of the two for overall acetyl transfer is shown in Fig. 9. This showed that a separate system was responsible for acetyl CoA formation through interaction of ATP, CoA and acetate (cf. below) and that the overall acetylation was a two-step reaction:

These observations crystallized into the definition of a metabolic acetyl transfer territory as pictured in Fig. 10. This picture had developed from the growing understanding of enzymatic interplay involving metabolic generation of acyl CoA and transfer of the active acyl to various acceptor systems. A most important, then still missing link in the picture was supplied through the brilliant work of Feodor Lynen45 who chemically identified acetyl CoA as the thioester of CoA. Therewith the thioester link was introduced as a new energy-rich bond and this discovery added a very novel facet to our understanding of the mechanisms of metabolic energy transformation.

Enzyme Localization In The  Anaerobic Mitochondria Of Ascaris L Umbricoides


Robert S. Rew And Howard J. Saz

From the Department of Biology, University of Notre Dame, Notre Dame, Indiana 46556


Mitochondria from the muscle of the parasitic nematode Ascaris lumbricoides   var. suum function anaerobically in electron transport-associated  phosphorylations under physiological conditions. These helminth organelles have been fractionated into inner and outer membrane, matrix, and intermembrane space fractions. The distributions of enzyme systems were determined and compared with corresponding distributions reported in mammalian mitochondria.  Succinate and pyruvate dehydrogenases as well as NADH  oxidase, Mg++-dependent ATPase, adenylate kinase, citrate synthase, and cytochrome c  reductases  were  determined to be distributed  as  in mammalian mitochondria.  In contrast  with  the  mammalian systems, fumarase and NAD-linked “malic” enzyme were isolated primarily from the intermembrane  space fraction of the worm mitochondria. These enzymes required for the anaerobic  energy-generating system in Ascaris and would be expected to give rise to NADH in the intermembrane space.  The need for and possible mechanism of a proton translocation system to obtain energy generation is suggested.                                Downloaded from







David Keilin’s Respiratory Chain Concept and its Chemiosmotic Consequences

Peter Mitchell              Nobel Lecture, 8 December, 1978

Glynn Research Institute, Bodmin, Cornwall, U. K.
“for his contribution to the understanding of biological energy transfer through the formulation of the chemiosmotic theory”

Peter D. Mitchell (1920-1992) received the Nobel Prize in 1978 for developing the Chemiosmotic Theory to explain ATP synthesis resulting from membrane-associated electron transport [Ubiquinone and the Proton Pump].

Mitchell is the last of the gentleman scientists. He first proposed the chemiosmotic principle in a 1961 Nature article while he was at the University of Edinburgh. Shortly after that, ill health forced him to move to Cornwall where he renovated an old manor house and converted it into a research laboratory. From then on, he and his research colleague, Jennifer Moyle, continued to work on the chemiosmotic theory while being funded by his private research foundation. [Peter Mitchell: Wikipedia]

The Chemiosmotic Theory was controversial in 1978 and it still has not been fully integrated into some biochemistry textbooks in spite of the fact that it is now proven. The main reason for the resistance is that it overthrows much of traditional biochemistry and introduces a new way of thinking. It is a good example of a “paradigm shift” in biology.

Because he was such a private, and eccentric, scientist there are very few photos of Peter Mitchell or his research laboratory at Glynn House . The best description of him is in his biography Wandering in the Gardens of the Mind: Peter Mitchell and the Making of Glynn by John Prebble, and Bruce Weber. A Nature review by E.C. Slater [Metabolic Gardening] gives some of the flavor and mentions some of the controversy.





Many scientists believe that the Chemiosmotic Theory was the second greatest contribution to biology in the 2oth century (after the discovery of the structure of DNA). Mitchell had to overcome many critics including Hans Krebs. The case is strong.

In the 1960s, ATP was known to be the energy currency of life, but the mechanism by which ATP was created in the mitochondria was assumed to be by substrate-level phosphorylation. Mitchell’s chemiosmotic hypothesis was the basis for understanding the actual process of oxidative phosphorylation. At the time, the biochemical mechanism of ATP synthesis by oxidative phosphorylation was unknown.

Mitchell realised that the movement of ions across an electrochemical potential difference could provide the energy needed to produce ATP. His hypothesis was derived from information that was well known in the 1960s. He knew that living cells had a membrane potential; interior negative to the environment. The movement of charged ions across a membrane is thus affected by the electrical forces (the attraction of positive to negative charges). Their movement is also affected by thermodynamic forces, the tendency of substances to diffuse from regions of higher concentration. He went on to show that ATP synthesis was coupled to this electrochemical gradient.[11]

His hypothesis was confirmed by the discovery of ATP synthase, a membrane-bound protein that uses the potential energy of the electrochemical gradient to make ATP.

Growth, development and metabolism are some of the central phenomena in the study of biological organisms. The role of energy is fundamental to such biological processes. The ability to harness energy from a variety of metabolic pathways is a property of all living organisms. Life is dependent on energy transformations; living organisms survive because of exchange of energy within and without.

In a living organism, chemical bonds are broken and made as part of the exchange and transformation of energy. Energy is available for work (such as mechanical work) or for other processes (such as chemical synthesis and anabolic processes in growth), when weak bonds are broken and stronger bonds are made. The production of stronger bonds allows release of usable energy.

One of the major triumphs of bioenergetics is Peter D. Mitchell‘s chemiosmotic theory of how protons in aqueous solution function in the production of ATP in cell organelles such as mitochondria.[5] This work earned Mitchell the 1978 Nobel Prize for Chemistry. Other cellular sources of ATP such as glycolysis were understood first, but such processes for direct coupling of enzyme activity to ATP production are not the major source of useful chemical energy in most cells. Chemiosmotic coupling is the major energy producing process in most cells, being utilized in chloroplasts and several single celled organisms in addition to mitochondria.


In August 1960, Robert K. Crane presented for the first time his discovery of the sodium-glucose cotransport as the mechanism for intestinal glucose absorption.[2] Crane’s discovery of cotransport was the first ever proposal of flux coupling in biology and was the most important event concerning carbohydrate absorption in the 20th century.[3][4]

The free energy (ΔG) gained or lost in a reaction can be calculated: ΔG = ΔH – TΔS
where G = Gibbs free energy, H = enthalpy, T = temperature, and S = entropy.

How inositol pyrophosphates control cellular phosphate homeostasis?

Adolfo Saiardi*

Cell Biology Unit, Medical Research Council Laboratory for Molecular Cell Biology, Department of Cell and Developmental Biology,

University College London, Gower Street, London WC1E 6BT, United Kingdom

Advances in Biological Regulation 52 (2012) 351–359

Phosphorus in his phosphate PO43_ configuration is an essential constituent of all life forms. Phosphate diesters are at the core of nucleic acid structure, while phosphate monoester transmits information under the control of protein kinases and phosphatases. Due to these fundamental roles in biology it is not a surprise that phosphate cellular homeostasis is under tight control.

Inositol pyrophosphates are organic molecules with the highest proportion of phosphate groups, and they are capable of regulating many biological processes, possibly by controlling energetic metabolism and adenosine triphosphate (ATP) production.

Furthermore, inositol pyrophosphates influence inorganic polyphosphates (polyP) synthesis. The polymer polyP is solely constituted by phosphate groups and beside other known functions, it also plays a role in buffering cellular free phosphate [Pi] levels, an event that is ultimately necessary to generate ATP and inositol pyrophosphate.

Two distinct classes of proteins the inositol hexakisphosphates kinases (IP6Ks) and the diphosphoinositol pentakisphosphate kinases (PP-IP5Ks or IP7Ks) are capable of synthesizing inositol pyrophosphates.

IP6Ks utilize ATP as a phosphate donor to phosphorylate IP6 to IP7, generation the isomer 5PP-IP5 (Fig. 1A), and inositol pentakisphosphate I(1,3,4,5,6)P5 to PP-IP4 (Saiardi et al., 1999, 2000; Losito et al., 2009). Furthermore, at least in vitro, IP6Ks generate more complex molecules containing two or more pyrophosphate moieties, or even three-phosphate species (Draskovic et al., 2008; Saiardi et al., 2001). Three IP6K isoforms referred to as IP6K1, 2, 3 exist in mammal; however, there is a single IP6K in the yeast Saccharomyces cerevisiae called Kcs1.

The PP-IP5Ks enzymes, synthesize inositol pyrophosphate from IP6, but not from IP5, (Losito et al., 2009) generating the isomer 1PP-IP5. Kinetic studies performed in vitro suggested that IP7, the 5PP-IP5 isomer generated by IP6Ks, is the primary substrate of this new enzyme, and this finding was confirmed in vivo by analysing PP-IP5K null yeast (vip1D) that accumulate the un-metabolized substrate IP7 (Azevedo et al., 2009; Onnebo and Saiardi, 2009). Thus PP-IP5K is responsible for IP8,

isomer 1,5PP2-IP4 synthesis (Fig. 1A). Two PP-IP5K isoforms referred to as PP-IP5Ka and b exist in mammal while a single PP-IP5K called Vip1 is present in S. cerevisiae.

Inositol pyrophosphates are hydrolysed by the diphosphoinositol-polyphosphate phosphohydrolases (DIPPs) (Safrany et al., 1998). Four mammalian enzymes DIPP1,2,3,4 have been identified, while only one DIPP protein exists in S. cerevisiae called Ddp1. These phosphatases are promiscuous enzymes able to hydrolyse inositol pyrophosphate as well as nucleotide analogues, such as diadenosine hexaphosphate (Ap6A) (Caffrey et al., 2000; Fisher et al., 2002). More recently, it has been shown that DIPPs also degrade polyP (Lonetti et al., 2011). Inositol pyrophosphates control the most disparate biological processes, from telomere length to vesicular trafficking. It is conceivable that all these function can be focused on the fact that inositol pyrophosphates are controlling cellular energy metabolism and consequently, ATP production. We have recently, demonstrated that inositol pyrophosphates control glycolysis and mitochondrial oxidative phosphorylation by both inhibiting the glycolytic flux and increasing mitochondrial activity (Szijgyarto et al., 2011).

Another important molecule to briefly introduce is polyP (Fig. 1B). The interested reader is encouraged to read the following comprehensive reviews (Kornberg et al., 1999; Rao et al., 2009). The polyP polymer likely represents a phosphate buffer that is synthesized and degraded in function of the phosphate needs of the cells. Furthermore, it also functions as a chelator of metal ions, thereby regulating cellular cation homeostasis. However, polyP also possesses more classical signalling roles.

In bacteria for example, it influences pathogenicity (Brown and Kornberg, 2008) and in mammalian cells it has been proposed to regulate fibrinolysis and platelet aggregation (Caen and Wu, 2010). In prokaryotes, polyP synthesis is carried out by a family of conserved polyP kinases (PPKs), whereas degradation is mediated by several polyP phosphatases (Rao et al., 2009). In higher eukaryotes polyP synthesis remains poorly characterized.

In humans alteration of phosphate metabolism is implicated in several pathological states. Higher serum phosphate leads to vascular calcification and cardiovascular complications. Although only very small amount of phosphate circulates in the serum, its concentration is tightly regulated and it is independent from dietary phosphorus intake (de Boer et al., 2009). Therefore, it is not surprising that intense research efforts are aimed to elucidate phosphate uptake and metabolism. IP6K2 was initially cloned while searching for a novel mammalian intestinal phosphate transporter that the group of Murer identified as PiUS (Phosphate inorganic Uptake Stimulator) (Norbis et al., 1997). Once transfected into Xenopus oocytes, PiUS stimulated the cellular uptake of radioactive phosphate.

Subsequently, two groups discovered that PiUS was capable of converting IP6 to IP7 and rename it to IP6K2 (Saiardi et al., 1999; Schell et al., 1999). The ability of inositol pyrophosphate to control the uptake of phosphate is an evolutionary conserved feature; in fact, kcs1D yeast with undetectable level of IP7 exhibits a reduced uptake of phosphate from the culture medium (Saiardi et al., 2004).

In mammals, regulation of phosphate homeostasis is not restricted to IP6K2, all three mammalian IP6Ks are likely to play a role. A genome-wide study aimed at identifying genetic variations associated with changes of serum phosphorus concentration identified IP6K3 (Kestenbaum et al., 2010). This human genetic study identified two independent single nucleotide polymorphisms (SNP) at locus 6p21.31, which are localised within the first intron of the IP6K3 gene. Interestingly, this study that analysed more than 16,000 humans identified SNP variant in only seven genes. Three of which, the sodium phosphate cotransporter type IIa, the calcium sensing receptor and the fibroblast growth factor 23, are well known regulators of phosphate homeostasis. These evidences support a role for IP6K3 in controlling serum phosphate levels in humans (Kestenbaum et al., 2010).


The hypothesis


Although, inositol pyrophosphate may have acquired unique organism-specific functions, the conserved ability of this class of molecules to regulate phosphate metabolism suggests an evolutionary ancient role. In this last paragraph, I will formulate few hypotheses that I hope will stimulate further research aimed at elucidating the biological link between phosphate, inositol pyrophosphates and polyP.

Inositol pyrophosphates regulate the entry of phosphate into the cells (Norbis et al., 1997), suggesting that they could affect phosphate uptake either directly (by stimulating a transporter, for example) or a indirectly by helping ‘fixing’ free phosphates in organic molecules. The cytosolic concentration of free phosphate [Pi] cannot fluctuate widely. Therefore, cellular entry of phosphates and its utilization may well be coupled. For example, the synthesis of polyP may be linked to phosphate entry in the cell. Inositol pyrophosphate control of energy metabolism (Szijgyarto et al., 2011) affects not only ATP levels but it can also alter the entire cellular balance of adenine nucleotides. Given that phosphate transfer reactions mainly use ATP as a vehicle for the phosphate groups, inositol pyrophosphate could affect phosphate metabolism by regulating the adenylate cellular pool. Moreover, it is tempting to speculate the existence of a feedback mechanism that coordinates the metabolic balance between ATP, phosphate and inositol pyrophosphates.

Inositol pyrophosphates could either contribute to the regulation of polyP synthesis, play a role in polyP degradation, or both. The yeast polyP polymerase has been identified with the subunit four (Vtc4) of the vacuolar membrane transporter chaperone (VTC) complex (Hothorn et al., 2009). Interestingly, pyrophosphates (Pi–Pi) dramatically accelerate the polyP polymerase reaction. It would therefore be interesting to determine whether the pyrophosphate moiety of IP7 can stimulate polyP vacuolar synthesis in a similar fashion. Similarly, it would be interesting to analyse the effect of inositol pyrophosphates on controlling the activity of the actin-like DdIPK2 enzyme. It should be noted however, that the existence even in yeast or Dictyostelium of other enzymes able to synthesize different polyP pools cannot be excluded. Thus, we will be able to validate and fully appreciate the role played by inositol pyrophosphates on polyP synthesis only after the identification of higher eukaryotes polyp synthesizing peptide/s.

The most abundant form of organic phosphate on earth is IP6, or phytic acid, a molecule that is highly abundant in plant seeds from which was originally characterised. In plant seeds, IP6 represents a phosphate storage molecule that it is hydrolysed during germination, releasing phosphates and cations. It will be an astonishing twist of event if inositol pyrophosphates were controlling the levels of their own precursor IP6 (Raboy, 2003), although due to the evolutionary conserved ability of inositol pyrophosphate to control phosphate homeostasis we should not be entirely surprised.

Although it is not yet clear how inositol pyrophosphates regulate cellular metabolism, understanding how inositol pyrophosphates influence phosphates homeostasis will help to clarify this important link.

Auesukaree C, Tochio H, Shirakawa M, Kaneko Y, Harashima S. Plc1p, Arg82p, and Kcs1p, enzymes involved in inositol pyrophosphate synthesis, are essential for phosphate regulation and polyphosphate accumulation in Saccharomyces cerevisiae. J Biol Chem 2005;280:25127–33.

Azevedo C, Burton A, Ruiz-Mateos E, Marsh M, Saiardi A. Inositol pyrophosphate mediated pyrophosphorylation of AP3B1 regulates HIV-1 Gag release. Proc Natl Acad Sci U S A 2009;106:21161–6.

Bennett M, Onnebo SM, Azevedo C, Saiardi A. Inositol pyrophosphates: metabolism and signaling. Cell Mol Life Sci 2006;63:552–64.

Boer VM, Crutchfield CA, Bradley PH, Botstein D, Rabinowitz JD. Growth-limiting intracellular metabolites in yeast growing under diverse nutrient limitations. Mol Biol Cell 2010;21:198–211.

Brown MR, Kornberg A. The long and short of it – polyphosphate, PPK and bacterial survival. Trends Biochem Sci 2008;33:284–90.

Burton A, Hu X, Saiardi A. Are inositol pyrophosphates signalling molecules? J Cell Physiol 2009;220:8–15.

Caen J, Wu Q. Hageman factor, platelets and polyphosphates: early history and recent connection. J Thromb Haemost 2010;8:1670–4.

Caffrey JJ, Safrany ST, Yang X, Shears SB. Discovery of molecular and catalytic diversity among human diphosphoinositol polyphosphate phosphohydrolases. An expanding Nudt family. J Biol Chem 2000;275:12730–6.

A Mitochondrial RNAi Screen Defines Cellular Bioenergetic Determinants and Identifies an Adenylate Kinase as a Key Regulator of ATP Levels

Nathan J. Lanning,1 Brendan D. Looyenga,1,2 Audra L. Kauffman,1 Natalie M. Niemi,1 Jessica Sudderth,3

Ralph J. DeBerardinis,3 and Jeffrey P. MacKeigan1,*

Cell Reports

Altered cellular bioenergetics and mitochondrial function are major features of several diseases, including cancer, diabetes, and neurodegenerative disorders. Given this important link to human health, we sought to define proteins within mitochondria that are critical for maintaining homeostatic ATP levels.

We screened an RNAi library targeting >1,000 nuclear-encoded genes whose protein products localize to the mitochondria in multiple metabolic conditions in order to examine their effects on cellular ATP levels. We identified a mechanism by which electron transport chain (ETC) perturbation under glycolytic conditions increased ATP production through enhanced glycolytic flux, thereby highlighting the cellular potential for metabolic plasticity.

Additionally, we identified a mitochondrial adenylate kinase (AK4) that regulates cellular ATP levels and AMPK signaling and whose expression significantly correlates with glioma patient survival. This study maps the bioenergetic landscape of >1,000 mitochondrial proteins in the context of varied metabolic substrates and begins to link key metabolic genes with clinical outcome.

Comments to be further addressed by JES Roselino

I will add some observations or at least one single observation.
Just at the beginning, when phosphorylation of proteins is presented, I assume you must mention that some proteins are activated by phosphorylation. This is fundamental in order to present self –organization reflex upon fast regulatory mechanisms. Even from an historical point of view. The first observation arrived from a sample due to be studied on the following day of glycogen synthetase. It was unintended left overnight out of the refrigerator. The result was it has changed from active form of the previous day to a non-active form. The story could have being finished here, if the researcher did not decide to spent this day increasing substrate levels (it could be a simple case of denaturation of proteins that changes its conformation despite the same order of amino acids). He kept on trying and found restoration of maximal activity. This assay was repeated with glycogen phosphorylase and the result was the opposite it increases its activity. This lead to the discovery of cAMP activated protein kinase and the assembly of a very complex system in the glycogen granule that is not a simple carbohydrate polymer. Instead it has several proteins assembled and preserves the capacity to receive from a single event (rise in cAMP) two opposing signals with maximal efficiency, stops glycogen synthesis, as long as levels of glucose 6 phosphate are low and increases glycogen phosphorylation as long as AMP levels are high).
I did everything I was able to do by the end of 1970 in order to repeat this assays with PK I, PKII and PKIII of M. Rouxii and Sutherland route to cAMP failed in this case. I ask Leloir to suggest to my chief (SP) the idea of AA, AB, BB subunits as was observed in lactic dehydrogenase (tetramer) indicating this as his idea. The reason was my “chief”(SP) more than once, have said it to me: “Leave these great ideas for the Houssay, Leloir etc…We must do our career with small things.” However, as she also have a faulty ability for recollection she also uses to arrive some time later, with the very same idea but in that case, as her idea.
Leloir, said to me: I will not offer your interpretation to her as mine. I think it is not phosphorylation, however I think it is glycosylation that explains the changes in the isoenzymes with the same molecular weight preserved. This dialogue explains why during “What is life” reading with him he asked me if from biochemist in exile, to biochemist I talked everything to him. Since I have considered that Schrödinger did not have confronted Darlington & Haldane for being in exile. Also, may explain why Leloir could have answered a bad telephone call from P. Boyer, Editor of The Enzymes in a way that suggest the the pattern could be of covalent changes over a protein. Our FEBS and Eur J. Biochemistry papers on pyruvate kinase of M. Rouxii is wrongly quoted in this way on his review about pyruvate kinase of that year(1971).

Another aspect I think you must call attention, in my opinion, is the following, show in detail with different colors what carbons belongs to CoA a huge molecule, in comparison with the single two carbons of acetate that will produce the enormous jump in energy yield in comparison with anaerobic glycolysis. The idea is how much must have being spent in DNA sequences to build that molecule in order to use only two atoms of carbon. Very limited aspects of biology could be explained in this way. In case we follow an alternative way of thinking, it becomes clearer that proteins were made more stable by interaction with other molecules (great and small). Afterwards, it rather easy to understand how the stability of protein-RNA complexes where transmitted to RNA (vibrational +solvational reactivity stability pair of conformational energy). Latter, millions of years, or as soon as, the information of interaction leading to activity and regulation could be found in RNA, proteins like reverse transcriptase move this information to a more stable form (DNA). In this way it is easier to understand the use of CoA to make two carbon molecules more reactive.


JES Roselino


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Lipid Metabolism

Lipid Metabolism

Reporter and Curator: Larry H. Bernstein, MD, FCAP 


This is fourth of a series of articles, lipid metabolism, that began with signaling and signaling pathways. These discussion lay the groundwork to proceed in later discussions that will take on a somewhat different approach. These are critical to develop a more complete point of view of life processes.  I have indicated that many of the protein-protein interactions or protein-membrane interactions and associated regulatory features have been referred to previously, but the focus of the discussion or points made were different.  The role of lipids in circulating plasma proteins as biomarkers for coronary vascular disease can be traced to the early work of Frederickson and the classification of lipid disorders.  The very critical role of lipids in membrane structure in health and disease has had much less attention, despite the enormous importance, especially in the nervous system.

  1. Signaling and signaling pathways
  2. Signaling transduction tutorial.
  3. Carbohydrate metabolism

3.1  Selected References to Signaling and Metabolic Pathways in Leaders in Pharmaceutical Intelligence

  1. Lipid metabolism
  2. Protein synthesis and degradation
  3. Subcellular structure
  4. Impairments in pathological states: endocrine disorders; stress hypermetabolism; cancer.


Lipid Metabolism

Overview of Lipid Catabolism:

The major aspects of lipid metabolism are involved with

  • Fatty Acid Oxidationto produce energy or
  • the synthesis of lipids which is called Lipogenesis.

The metabolism of lipids and carbohydrates are related by the conversion of lipids from carbohydrates. This can be seen in the diagram. Notice the link through actyl-CoA, the seminal discovery of Fritz Lipmann. The metabolism of both is upset by diabetes mellitus, which results in the release of ketones (2/3 betahydroxybutyric acid) into the circulation.


metabolism of fats

metabolism of fats

The first step in lipid metabolism is the hydrolysis of the lipid in the cytoplasm to produce glycerol and fatty acids.

Since glycerol is a three carbon alcohol, it is metabolized quite readily into an intermediate in glycolysis, dihydroxyacetone phosphate. The last reaction is readily reversible if glycerol is needed for the synthesis of a lipid.

The hydroxyacetone, obtained from glycerol is metabolized into one of two possible compounds. Dihydroxyacetone may be converted into pyruvic acid, a 3-C intermediate at the last step of glycolysis to make energy.

In addition, the dihydroxyacetone may also be used in gluconeogenesis (usually dependent on conversion of gluconeogenic amino acids) to make glucose-6-phosphate for glucose to the blood or glycogen depending upon what is required at that time.

Fatty acids are oxidized to acetyl CoA in the mitochondria using the fatty acid spiral. The acetyl CoA is then ultimately converted into ATP, CO2, and H2O using the citric acid cycle and the electron transport chain.

There are two major types of fatty acids – ω-3 and ω-6.  There are also saturated and unsaturated with respect to the existence of double bonds, and monounsaturated and polyunsatured.  Polyunsaturated fatty acids (PUFAs) are important in long term health, and it will be seen that high cardiovascular risk is most associated with a low ratio of ω-3/ω-6, the denominator being from animal fat. Ω-3 fatty acids are readily available from fish, seaweed, and flax seed. More can be said of this later.

Fatty acids are synthesized from carbohydrates and occasionally from proteins. Actually, the carbohydrates and proteins have first been catabolized into acetyl CoA. Depending upon the energy requirements, the acetyl CoA enters the citric acid cycle or is used to synthesize fatty acids in a process known as LIPOGENESIS.

The relationships between lipid and carbohydrate metabolism are
summarized in Figure 2.





 Energy Production Fatty Acid Oxidation:

Visible” ATP:

In the fatty acid spiral, there is only one reaction which directly uses ATP and that is in the initiating step. So this is a loss of ATP and must be subtracted later.

A large amount of energy is released and restored as ATP during the oxidation of fatty acids. The ATP is formed from both the fatty acid spiral and the citric acid cycle.


Connections to Electron Transport and ATP:

One turn of the fatty acid spiral produces ATP from the interaction of the coenzymes FAD (step 1) and NAD+ (step 3) with the electron transport chain. Total ATP per turn of the fatty acid spiral is:

Electron Transport Diagram – (e.t.c.)

Step 1 – FAD into e.t.c. = 2 ATP
Step 3 – NAD+ into e.t.c. = 3 ATP
Total ATP per turn of spiral = 5 ATP

In order to calculate total ATP from the fatty acid spiral, you must calculate the number of turns that the spiral makes. Remember that the number of turns is found by subtracting one from the number of acetyl CoA produced. See the graphic on the left bottom.

Example with Palmitic Acid = 16 carbons = 8 acetyl groups

Number of turns of fatty acid spiral = 8-1 = 7 turns

ATP from fatty acid spiral = 7 turns and 5 per turn = 35 ATP.

This would be a good time to remember that single ATP that was needed to get the fatty acid spiral started. Therefore subtract it now.

NET ATP from Fatty Acid Spiral = 35 – 1 = 34 ATP

Review ATP Summary for Citric Acid Cycle:The acetyl CoA produced from the fatty acid spiral enters the citric acid cycle. When calculating ATP production, you have to show how many acetyl CoA are produced from a given fatty acid as this controls how many “turns” the citric acid cycle makes.Starting with acetyl CoA, how many ATP are made using the citric acid cycle? E.T.C = electron transport chain

 Step  ATP produced
7  1
Step 4 (NAD+ to E.T.C.) 3
Step 6 (NAD+ to E.T.C.)  3
Step10 (NAD+ to E.T.C.)  3
Step 8 (FAD to E.T.C.) 2



 ATP Summary for Palmitic Acid – Complete Metabolism:The phrase “complete metabolism” means do reactions until you end up with carbon dioxide and water. This also means to use fatty acid spiral, citric acid cycle, and electron transport as needed.Starting with palmitic acid (16 carbons) how many ATP are made using fatty acid spiral? This is a review of the above panel E.T.C = electron transport chain

 Step  ATP (used -) (produced +)
Step 1 (FAD to E.T.C.) +2
Step 4 (NAD+ to E.T.C.) +3
Total ATP  +5
 7 turns  7 x 5 = 35
initial step  -1
 NET  34 ATP

The fatty acid spiral ends with the production of 8 acetyl CoA from the 16 carbon palmitic acid.

Starting with one acetyl CoA, how many ATP are made using the citric acid cycle? Above panel gave the answer of 12 ATP per acetyl CoA.

E.T.C = electron transport chain

 Step  ATP produced
One acetyl CoA per turn C.A.C. +12 ATP
8 Acetyl CoA = 8 turns C.A.C. 8 x 12 = 96 ATP
Fatty Acid Spiral 34 ATP


Fyodor Lynen

Feodor Lynen was born in Munich on 6 April 1911, the son of Wilhelm Lynen, Professor of Mechanical Engineering at the Munich Technische Hochschule. He received his Doctorate in Chemistry from Munich University under Heinrich Wieland, who had won the Nobel Prize for Chemistry in 1927, in March 1937 with the work: «On the Toxic Substances in Amanita». in 1954 he became head of the Max-Planck-Institut für Zellchemie, newly created for him as a result of the initiative of Otto Warburg and Otto Hahn, then President of the Max-Planck-Gesellschaft zur Förderung der Wissenschaften.

Lynen’s work was devoted to the elucidation of the chemical details of metabolic processes in living cells, and of the mechanisms of metabolic regulation. The problems tackled by him, in conjunction with German and other workers, include the Pasteur effect, acetic acid degradation in yeast, the chemical structure of «activated acetic acid» of «activated isoprene», of «activated carboxylic acid», and of cytohaemin, degradation of fatty acids and formation of acetoacetic acid, degradation of tararic acid, biosynthesis of cysteine, of terpenes, of rubber, and of fatty acids.

In 1954 Lynen received the Neuberg Medal of the American Society of European Chemists and Pharmacists, in 1955 the Liebig Commemorative Medal of the Gesellschaft Deutscher Chemiker, in 1961 the Carus Medal of the Deutsche Akademie der Naturforscher «Leopoldina», and in 1963 the Otto Warburg Medal of the Gesellschaft für Physiologische Chemie. He was also a member of the U>S> National Academy of Sciences, and shared the Nobel Prize in Physiology and Medicine with Konrad Bloch in 1964, and was made President of the Gesellschaft Deutscher Chemiker (GDCh) in 1972.

This biography was written at the time of the award and first published in the book series Les Prix Nobel. It was later edited and republished in Nobel Lectures, and shortened by myself.

The Pathway from “Activated Acetic Acid” to the Terpenes and Fatty Acids

My first contact with dynamic biochemistry in 1937 occurred at an exceedingly propitious time. The remarkable investigations on the enzyme chain of respiration, on the oxygen-transferring haemin enzyme of respiration, the cytochromes, the yellow enzymes, and the pyridine proteins had thrown the first rays of light on the chemical processes underlying the mystery of biological catalysis, which had been recognised by your famous countryman Jöns Jakob Berzelius. Vitamin B2 , which is essential to the nourishment of man and of animals, had been recognised by Hugo Theorell in the form of the phosphate ester as the active group of an important class of enzymes, and the fermentation processes that are necessary for Pasteur’s “life without oxygen”

had been elucidated as the result of a sequence of reactions centered around “hydrogen shift” and “phosphate shift” with adenosine triphosphate as the phosphate-transferring coenzyme. However, 1,3-diphosphoglyceric acid, the key substance to an understanding of the chemical relation between oxidation and phosphorylation, still lay in the depths of the unknown. Never-

theless, Otto Warburg was on its trail in the course of his investigations on the fermentation enzymes, and he was able to present it to the world in 1939.


This was the period in which I carried out my first independent investigation, which was concerned with the metabolism of yeast cells after freezing in liquid air, and which brought me directly into contact with the mechanism of alcoholic fermentation. This work taught me a great deal, and yielded two important pieces of information.


  • The first was that in experiments with living cells, special attention must be given to the permeability properties of the cell membranes, and
  • the second was that the adenosine polyphosphate system plays a vital part in the cell,
    • not only in energy transfer, but
    • also in the regulation of the metabolic processes.



This investigation aroused by interest in problems of metabolic regulation, which led me to the investigation of the Pasteur effects, and has remained with me to the present day.


My subsequent concern with the problem of the acetic acid metabolism arose from my stay at Heinrich Wieland’s laboratory. Workers here had studied the oxidation of acetic acid by yeast cells, and had found that though most of the acetic acid undergoes complete oxidation, some remains in the form of succinic and citric acids.


The explanation of these observations was provided-by the Thunberg-Wieland process, according to which two molecules of acetic acid are dehydrogenated to succinic acid, which is converted back into acetic acid via oxaloacetic acid, pyruvic acid, and acetaldehyde, or combines at the oxaloacetic acid stage with a further molecule of acetic acid to form citric acid (Fig. 1). However, an experimental check on this view by a Wieland’s student Robert Sonderhoffs brought a surprise. The citric acid formed when trideuteroacetic acid was supplied to yeast cells contained the expected quantity of deuterium, but the succinic acid contained only half of the four deuterium atoms required by Wieland’s scheme.


This investigation aroused by interest in problems of metabolic regulation, which led me to the investigation of the Pasteur effects, and has remained with me to the present day. My subsequent concern with the problem of the acetic acid metabolism arose from my stay at Heinrich Wieland’s laboratory. Workers here had studied the oxidation of acetic acid by yeast cells, and had found that though most of the acetic acid undergoes complete oxidation, some remains in the form of succinic and citric acid

The answer provided by Martius was that citric acid  is in equilibrium with isocitric acid and is oxidised to cr-ketoglutaric acid, the conversion of which into succinic acid had already been discovered by Carl Neuberg (Fig. 1).

It was possible to assume with fair certainty from these results that the succinic acid produced by yeast from acetate is formed via citric acid. Sonderhoff’s experiments with deuterated acetic acid led to another important discovery.

In the analysis of the yeast cells themselves, it was found that while the carbohydrate fraction contained only insignificant quantities of deuterium, large quantities of heavy hydrogen were present in the fatty acids formed and in the sterol fraction. This showed that

  • fatty acids and sterols were formed directly from acetic acid, and not indirectly via the carbohydrates.

As a result of Sonderhoff’s early death, these important findings were not pursued further in the Munich laboratory.

  • This situation was elucidated only by Konrad Bloch’s isotope experiments, on which he reports.

My interest first turned entirely to the conversion of acetic acid into citric acid, which had been made the focus of the aerobic degradation of carbohydrates by the formulation of the citric acid cycle by Hans Adolf Krebs. Unlike Krebs, who regarded pyruvic acid as the condensation partner of acetic acid,

  • we were firmly convinced, on the basis of the experiments on yeast, that pyruvic acid is first oxidised to acetic acid, and only then does the condensation take place.

Further progress resulted from Wieland’s observation that yeast cells that had been “impoverished” in endogenous fuels by shaking under oxygen were able to oxidise added acetic acid only after a certain “induction period” (Fig. 2). This “induction period” could be shortened by addition of small quantities of a readily oxidisable substrate such as ethyl alcohol, though propyl and butyl alcohol were also effective. I explained this by assuming that acetic acid is converted, at the expense of the oxidation of the alcohol, into an “activated acetic acid”, and can only then condense with oxalacetic acid.

In retrospect, we find that I had come independently on the same group of problems as Fritz Lipmann, who had discovered that inorganic phosphate is indispensable to the oxidation of pyruvic acid by lactobacilli, and had detected acetylphosphate as an oxidation product. Since this anhydride of acetic acid and phosphoric acid could be assumed to be the “activated acetic acid”.

I learned of the advances that had been made in the meantime in the investigation of the problem of “activated acetic acid”. Fritz Lipmann has described the development at length in his Nobel Lecture’s, and I need not repeat it. The main advance was the recognition that the formation of “activated acetic acid” from acetate involved not only ATP as an energy source, but also the newly discovered coenzyme A, which contains the vitamin pantothenic acid, and that “activated acetic acid” was probably an acetylated coenzyme  A.

Fyodor Lynen

Lynen’s most important research at the University of Munich focused on intermediary metabolism, cholesterol synthesis, and fatty acid biosynthesis. Metabolism involves all the chemical processes by which an organism converts matter and energy into forms that it can use. Metabolism supplies the matter—the molecular building blocks an organism needs for the growth of new tissues. These building blocks must either come from the breakdown of molecules of food, such as glucose (sugar) and fat, or be built up from simpler molecules within the organism.

Cholesterol is one of the fatty substances found in animal tissues. The human body produces cholesterol, but this substance also enters the body in food. Meats, egg yolks, and milk products, such as butter and cheese, contain cholesterol. Such organs as the brain and liver contain much cholesterol. Cholesterol is a type of lipid, one of the classes of chemical compounds essential to human health. It makes up an important part of the membranes of each cell in the body. The body also uses cholesterol to produce vitamin D and certain hormones.

All fats are composed of an alcohol called glycerol and substances called fatty acids. A fatty acid consists of a long chain of carbon atoms, to which hydrogen atoms are attached. There are three types of fatty acids: saturated, monounsaturated, and polyunsaturated.

Living cells manufacture complicated chemical compounds from simpler substances through a process called biosynthesis. For example, simple molecules called amino acids are put together to make proteins. The biosynthesis of both fatty acids and cholesterol begins with a chemically active form of acetate, a two-carbon molecule. Lynen discovered that the active form of acetate is a coenzyme, a heat-stabilized, water-soluble portion of an enzyme, called acetyl coenzyme A. Lynen and his colleagues demonstrated that the formation of cholesterol begins with the condensation of two molecules of acetyl coenzyme A to form acetoacetyl coenzyme A, a four-carbon molecule.

Fyodor Lynen

Fyodor Lynen


SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver

Jay D. Horton1,2, Joseph L. Goldstein1 and Michael S. Brown1

1Department of Molecular Genetics, and
2Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA

J Clin Invest. 2002;109(9):1125–1131.
Lipid homeostasis in vertebrate cells is regulated by a family of membrane-bound transcription factors designated sterol regulatory element–binding proteins (SREBPs). SREBPs directly activate the expression of more than 30 genes dedicated to the synthesis and uptake of cholesterol, fatty acids, triglycerides, and phospholipids, as well as the NADPH cofactor required to synthesize these molecules (14). In the liver, three SREBPs regulate the production of lipids for export into the plasma as lipoproteins and into the bile as micelles. The complex, interdigitated roles of these three SREBPs have been dissected through the study of ten different lines of gene-manipulated mice. These studies form the subject of this review.

SREBPs: activation through proteolytic processing

SREBPs belong to the basic helix-loop-helix–leucine zipper (bHLH-Zip) family of transcription factors, but they differ from other bHLH-Zip proteins in that they are synthesized as inactive precursors bound to the endoplasmic reticulum (ER) (1, 5). Each SREBP precursor of about 1150 amino acids is organized into three domains: (a) an NH2-terminal domain of about 480 amino acids that contains the bHLH-Zip region for binding DNA; (b) two hydrophobic transmembrane–spanning segments interrupted by a short loop of about 30 amino acids that projects into the lumen of the ER; and (c) a COOH-terminal domain of about 590 amino acids that performs the essential regulatory function described below.

In order to reach the nucleus and act as a transcription factor, the NH2-terminal domain of each SREBP must be released from the membrane proteolytically (Figure 1). Three proteins required for SREBP processing have been delineated in cultured cells, using the tools of somatic cell genetics (see ref. 5for review). One is an escort protein designated SREBP cleavage–activating protein (SCAP). The other two are proteases, designated Site-1 protease (S1P) and Site-2 protease (S2P). Newly synthesized SREBP is inserted into the membranes of the ER, where its COOH-terminal regulatory domain binds to the COOH-terminal domain of SCAP (Figure 1).


Figure 1

Model for the sterol-mediated proteolytic release of SREBPs from membranes JCI0215593.f1

Model for the sterol-mediated proteolytic release of SREBPs from membranes JCI0215593.f1


Model for the sterol-mediated proteolytic release of SREBPs from membranes. SCAP is a sensor of sterols and an escort of SREBPs. When cells are depleted of sterols, SCAP transports SREBPs from the ER to the Golgi apparatus, where two proteases, Site-1 protease (S1P) and Site-2 protease (S2P), act sequentially to release the NH2-terminal bHLH-Zip domain from the membrane. The bHLH-Zip domain enters the nucleus and binds to a sterol response element (SRE) in the enhancer/promoter region of target genes, activating their transcription. When cellular cholesterol rises, the SCAP/SREBP complex is no longer incorporated into ER transport vesicles, SREBPs no longer reach the Golgi apparatus, and the bHLH-Zip domain cannot be released from the membrane. As a result, transcription of all target genes declines. Reprinted from ref. 5 with permission.

SCAP is both an escort for SREBPs and a sensor of sterols. When cells become depleted in cholesterol, SCAP escorts the SREBP from the ER to the Golgi apparatus, where the two proteases reside. In the Golgi apparatus, S1P, a membrane-bound serine protease, cleaves the SREBP in the luminal loop between its two membrane-spanning segments, dividing the SREBP molecule in half (Figure 1). The NH2-terminal bHLH-Zip domain is then released from the membrane via a second cleavage mediated by S2P, a membrane-bound zinc metalloproteinase. The NH2-terminal domain, designated nuclear SREBP (nSREBP), translocates to the nucleus, where it activates transcription by binding to nonpalindromic sterol response elements (SREs) in the promoter/enhancer regions of multiple target genes.


Figure 1


When the cholesterol content of cells rises, SCAP senses the excess cholesterol through its membranous sterol-sensing domain, changing its conformation in such a way that the SCAP/SREBP complex is no longer incorporated into ER transport vesicles. The net result is that SREBPs lose their access to S1P and S2P in the Golgi apparatus, so their bHLH-Zip domains cannot be released from the ER membrane, and the transcription of target genes ceases (1, 5). The biophysical mechanism by which SCAP senses sterol levels in the ER membrane and regulates its movement to the Golgi apparatus is not yet understood. Elucidating this mechanism will be fundamental to understanding the molecular basis of cholesterol feedback inhibition of gene expression.

SREBPs: two genes, three proteins

The mammalian genome encodes three SREBP isoforms, designated SREBP-1a, SREBP-1c, and SREBP-2. SREBP-2 is encoded by a gene on human chromosome 22q13. Both SREBP-1a and -1c are derived from a single gene on human chromosome 17p11.2 through the use of alternative transcription start sites that produce alternate forms of exon 1, designated 1a and 1c (1). SREBP-1a is a potent activator of all SREBP-responsive genes, including those that mediate the synthesis of cholesterol, fatty acids, and triglycerides. High-level transcriptional activation is dependent on exon 1a, which encodes a longer acidic transactivation segment than does the first exon of SREBP-1c. The roles of SREBP-1c and SREBP-2 are more restricted than that of SREBP-1a. SREBP-1c preferentially enhances transcription of genes required for fatty acid synthesis but not cholesterol synthesis. Like SREBP-1a, SREBP-2 has a long transcriptional activation domain, but it preferentially activates cholesterol synthesis (1). SREBP-1a and SREBP-2 are the predominant isoforms of SREBP in most cultured cell lines, whereas SREBP-1c and SREBP-2 predominate in the liver and most other intact tissues (6).

When expressed at higher than physiologic levels, each of the three SREBP isoforms can activate all enzymes indicated in Figure 2, which shows the biosynthetic pathways used to generate cholesterol and fatty acids. However, at normal levels of expression, SREBP-1c favors the fatty acid biosynthetic pathway and SREBP-2 favors cholesterologenesis. SREBP-2–responsive genes in the cholesterol biosynthetic pathway include those for the enzymes HMG-CoA synthase, HMG-CoA reductase, farnesyl diphosphate synthase, and squalene synthase. SREBP-1c–responsive genes include those for ATP citrate lyase (which produces acetyl-CoA) and acetyl-CoA carboxylase and fatty acid synthase (which together produce palmitate [C16:0]). Other SREBP-1c target genes encode a rate-limiting enzyme of the fatty acid elongase complex, which converts palmitate to stearate (C18:0) (ref.7); stearoyl-CoA desaturase, which converts stearate to oleate (C18:1); and glycerol-3-phosphate acyltransferase, the first committed enzyme in triglyceride and phospholipid synthesis (3). Finally, SREBP-1c and SREBP-2 activate three genes required to generate NADPH, which is consumed at multiple stages in these lipid biosynthetic pathways (8) (Figure 2).


Figure 2


major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides JCI0215593.f2

major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides JCI0215593.f2


Genes regulated by SREBPs. The diagram shows the major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides. In vivo, SREBP-2 preferentially activates genes of cholesterol metabolism, whereas SREBP-1c preferentially activates genes of fatty acid and triglyceride metabolism. DHCR, 7-dehydrocholesterol reductase; FPP, farnesyl diphosphate; GPP, geranylgeranyl pyrophosphate synthase; CYP51, lanosterol 14α-demethylase; G6PD, glucose-6-phosphate dehydrogenase; PGDH, 6-phosphogluconate dehydrogenase; GPAT, glycerol-3-phosphate acyltransferase.

Genes regulated by SREBPs. The diagram shows the major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides. In vivo, SREBP-2 preferentially activates genes of cholesterol metabolism, whereas SREBP-1c preferentially activates genes of fatty acid and triglyceride metabolism. DHCR, 7-dehydrocholesterol reductase; FPP, farnesyl diphosphate; GPP, geranylgeranyl pyrophosphate synthase; CYP51, lanosterol 14α-demethylase; G6PD, glucose-6-phosphate dehydrogenase; PGDH, 6-phosphogluconate dehydrogenase; GPAT, glycerol-3-phosphate acyltransferase.

Knockout and transgenic mice

Ten different genetically manipulated mouse models that either lack or overexpress a single component of the SREBP pathway have been generated in the last 6 years (916). The key molecular and metabolic alterations observed in these mice are summarized in Table 1.


Table 1
Alterations in hepatic lipid metabolism in gene-manipulated mice overexpressing or lacking SREBPs

Knockout mice that lack all nSREBPs die early in embryonic development. For instance, a germline deletion of S1p, which prevents the processing of all SREBP isoforms, results in death before day 4 of development (15, 17). Germline deletion of Srebp2 leads to 100% lethality at a later stage of embryonic development than does deletion of S1p (embryonic day 7–8). In contrast, germline deletion of Srebp1, which eliminates both the 1a and the 1c transcripts, leads to partial lethality, in that about 15–45% of Srebp1–/– mice survive (13). The surviving homozygotes manifest elevated levels of SREBP-2 mRNA and protein (Table 1), which presumably compensates for the loss of SREBP-1a and -1c. When the SREBP-1c transcript is selectively eliminated, no embryonic lethality is observed, suggesting that the partial embryonic lethality in the Srebp1–/– mice is due to the loss of the SREBP-1a transcript (16).

To bypass embryonic lethality, we have produced mice in which all SREBP function can be disrupted in adulthood through induction of Cre recombinase. For this purpose, loxP recombination sites were inserted into genomic regions that flank crucial exons in the Scap or S1p genes (so-called floxed alleles) (14, 15). Mice homozygous for the floxed gene and heterozygous for a Cre recombinase transgene, which is under control of an IFN-inducible promoter (MX1-Cre), can be induced to delete Scap or S1p by stimulating IFN expression. Thus, following injection with polyinosinic acid–polycytidylic acid, a double-stranded RNA that provokes antiviral responses, the Cre recombinase is produced in liver and disrupts the floxed gene by recombination between the loxP sites.

Cre-mediated disruption of Scap or S1p dramatically reduces nSREBP-1 and nSREBP-2 levels in liver and diminishes expression of all SREBP target genes in both the cholesterol and the fatty acid synthetic pathways (Table 1). As a result, the rates of synthesis of cholesterol and fatty acids fall by 70–80% in Scap- and S1p-deficient livers.

In cultured cells, the processing of SREBP is inhibited by sterols, and the sensor for this inhibition is SCAP (5). To learn whether SCAP performs the same function in liver, we have produced transgenic mice that express a mutant SCAP with a single amino acid substitution in the sterol-sensing domain (D443N) (12). Studies in tissue culture show that SCAP(D443N) is resistant to inhibition by sterols. Cells that express a single copy of this mutant gene overproduce cholesterol (18). Transgenic mice that express this mutant version of SCAP in the liver exhibit a similar phenotype (12). These livers manifest elevated levels of nSREBP-1 and nSREBP-2, owing to constitutive SREBP processing, which is not suppressed when the animals are fed a cholesterol-rich diet. nSREBP-1 and -2 increase the expression of all SREBP target genes shown in Figure 2, thus stimulating cholesterol and fatty acid synthesis and causing a marked accumulation of hepatic cholesterol and triglycerides (Table 1). This transgenic model provides strong in vivo evidence that SCAP activity is normally under partial inhibition by endogenous sterols, which keeps the synthesis of cholesterol and fatty acids in a partially repressed state in the liver.

Function of individual SREBP isoforms in vivo

To study the functions of individual SREBPs in the liver, we have produced transgenic mice that overexpress truncated versions of SREBPs (nSREBPs) that terminate prior to the membrane attachment domain. These nSREBPs enter the nucleus directly, bypassing the sterol-regulated cleavage step. By studying each nSREBP isoform separately, we could determine their distinct activating properties, albeit when overexpressed at nonphysiologic levels.

Overexpression of nSREBP-1c in the liver of transgenic mice produces a triglyceride-enriched fatty liver with no increase in cholesterol (10). mRNAs for fatty acid synthetic enzymes and rates of fatty acid synthesis are elevated fourfold in this tissue, whereas the mRNAs for cholesterol synthetic enzymes and the rate of cholesterol synthesis are not increased (8). Conversely, overexpression of nSREBP-2 in the liver increases the mRNAs only fourfold. This increase in cholesterol synthesis is even more remarkable when encoding all cholesterol biosynthetic enzymes; the most dramatic is a 75-fold increase in HMG-CoA reductase mRNA (11). mRNAs for fatty acid synthesis enzymes are increased to a lesser extent, consistent with the in vivo observation that the rate of cholesterol synthesis increases 28-fold in these transgenic nSREBP-2 livers, while fatty acid synthesis increases one considers the extent of cholesterol overload in this tissue, which would ordinarily reduce SREBP processing and essentially abolish cholesterol synthesis (Table 1).

We have also studied the consequences of overexpressing SREBP-1a, which is expressed only at low levels in the livers of adult mice, rats, hamsters, and humans (6). nSREBP-1a transgenic mice develop a massive fatty liver engorged with both cholesterol and triglycerides (9), with heightened expression of genes controlling cholesterol biosynthesis and, still more dramatically, fatty acid synthesis (Table 1). The preferential activation of fatty acid synthesis (26-fold increase) relative to cholesterol synthesis (fivefold increase) explains the greater accumulation of triglycerides in their livers. The relative representation of the various fatty acids accumulating in this tissue is also unusual. Transgenic nSREBP-1a livers contain about 65% oleate (C18:1), markedly higher levels than the 15–20% found in typical wild-type livers (8) — a result of the induction of fatty acid elongase and stearoyl-CoA desaturase-1 (7). Considered together, the overexpression studies indicate that both SREBP-1 isoforms show a relative preference for activating fatty acid synthesis, whereas SREBP-2 favors cholesterol.

The phenotype of animals lacking the Srebp1 gene, which encodes both the SREBP-1a and -1c transcripts, also supports the notion of distinct hepatic functions for SREBP-1 and SREBP-2 (13). Most homozygous SREBP-1 knockout mice die in utero. The surviving Srebp1–/– mice show reduced synthesis of fatty acids, owing to reduced expression of mRNAs for fatty acid synthetic enzymes (Table 1). Hepatic nSREBP-2 levels increase in these mice, presumably in compensation for the loss of nSREBP-1. As a result, transcription of cholesterol biosynthetic genes increases, producing a threefold increase in hepatic cholesterol synthesis (Table 1).

The studies in genetically manipulated mice clearly show that, as in cultured cells, SCAP and S1P are required for normal SREBP processing in the liver. SCAP, acting through its sterol-sensing domain, mediates feedback regulation of cholesterol synthesis. The SREBPs play related but distinct roles: SREBP-1c, the predominant SREBP-1 isoform in adult liver, preferentially activates genes required for fatty acid synthesis, while SREBP-2 preferentially activates the LDL receptor gene and various genes required for cholesterol synthesis. SREBP-1a and SREBP-2, but not SREBP-1c, are required for normal embryogenesis.

Transcriptional regulation of SREBP genes

Regulation of SREBPs occurs at two levels — transcriptional and posttranscriptional. The posttranscriptional regulation discussed above involves the sterol-mediated suppression of SREBP cleavage, which results from sterol-mediated suppression of the movement of the SCAP/SREBP complex from the ER to the Golgi apparatus (Figure 1). This form of regulation is manifest not only in cultured cells (1), but also in the livers of rodents fed cholesterol-enriched diets (19).

The transcriptional regulation of the SREBPs is more complex. SREBP-1c and SREBP-2 are subject to distinct forms of transcriptional regulation, whereas SREBP-1a appears to be constitutively expressed at low levels in liver and most other tissues of adult animals (6). One mechanism of regulation shared by SREBP-1c and SREBP-2 involves a feed-forward regulation mediated by SREs present in the enhancer/promoters of each gene (20, 21). Through this feed-forward loop, nSREBPs activate the transcription of their own genes. In contrast, when nSREBPs decline, as in Scap or S1p knockout mice, there is a secondary decline in the mRNAs encoding SREBP-1c and SREBP-2 (14, 15).

Three factors selectively regulate the transcription of SREBP-1c: liver X-activated receptors (LXRs), insulin, and glucagon. LXRα and LXRβ, nuclear receptors that form heterodimers with retinoid X receptors, are activated by a variety of sterols, including oxysterol intermediates that form during cholesterol biosynthesis (2224). An LXR-binding site in the SREBP-1c promoter activates SREBP-1c transcription in the presence of LXR agonists (23). The functional significance of LXR-mediated SREBP-1c regulation has been confirmed in two animal models. Mice that lack both LXRα and LXRβ express reduced levels of SREBP-1c and its lipogenic target enzymes in liver and respond relatively weakly to treatment with a synthetic LXR agonist (23). Because a similar blunted response is found in mice that lack SREBP-1c, it appears that LXR increases fatty acid synthesis largely by inducing SREBP-1c (16). LXR-mediated activation of SREBP-1c transcription provides a mechanism for the cell to induce the synthesis of oleate when sterols are in excess (23). Oleate is the preferred fatty acid for the synthesis of cholesteryl esters, which are necessary for both the transport and the storage of cholesterol.

LXR-mediated regulation of SREBP-1c appears also to be one mechanism by which unsaturated fatty acids suppress SREBP-1c transcription and thus fatty acid synthesis. Rodents fed diets enriched in polyunsaturated fatty acids manifest reduced SREBP-1c mRNA expression and low rates of lipogenesis in liver (25). In vitro, unsaturated fatty acids competitively block LXR activation of SREBP-1c expression by antagonizing the activation of LXR by its endogenous ligands (26). In addition to LXR-mediated transcriptional inhibition, polyunsaturated fatty acids lower SREBP-1c levels by accelerating degradation of its mRNA (27). These combined effects may contribute to the long-recognized ability of polyunsaturated fatty acids to lower plasma triglyceride levels.

SREBP-1c and the insulin/glucagon ratio

The liver is the organ responsible for the conversion of excess carbohydrates to fatty acids to be stored as triglycerides or burned in muscle. A classic action of insulin is to stimulate fatty acid synthesis in liver during times of carbohydrate excess. The action of insulin is opposed by glucagon, which acts by raising cAMP. Multiple lines of evidence suggest that insulin’s stimulatory effect on fatty acid synthesis is mediated by an increase in SREBP-1c. In isolated rat hepatocytes, insulin treatment increases the amount of mRNA for SREBP-1c in parallel with the mRNAs of its target genes (28, 29). The induction of the target genes can be blocked if a dominant negative form of SREBP-1c is expressed (30). Conversely, incubating primary hepatocytes with glucagon or dibutyryl cAMP decreases the mRNAs for SREBP-1c and its associated lipogenic target genes (30, 31).

In vivo, the total amount of SREBP-1c in liver and adipose tissue is reduced by fasting, which suppresses insulin and increases glucagon levels, and is elevated by refeeding (32, 33). The levels of mRNA for SREBP-1c target genes parallel the changes in SREBP-1c expression. Similarly, SREBP-1c mRNA levels fall when rats are treated with streptozotocin, which abolishes insulin secretion, and rise after insulin injection (29). Overexpression of nSREBP-1c in livers of transgenic mice prevents the reduction in lipogenic mRNAs that normally follows a fall in plasma insulin levels (32). Conversely, in livers of Scap knockout mice that lack all nSREBPs in the liver (14) or knockout mice lacking either nSREBP-1c (16) or both SREBP-1 isoforms (34), there is a marked decrease in the insulin-induced stimulation of lipogenic gene expression that normally occurs after fasting/refeeding. It should be noted that insulin and glucagon also exert a posttranslational control of fatty acid synthesis though changes in the phosphorylation and activation of acetyl-CoA carboxylase. The posttranslational regulation of fatty acid synthesis persists in transgenic mice that overexpress nSREBP-1c (10). In these mice, the rates of fatty acid synthesis, as measured by [3H]water incorporation, decline after fasting even though the levels of the lipogenic mRNAs remain high (our unpublished observations).

Taken together, the above evidence suggests that SREBP-1c mediates insulin’s lipogenic actions in liver. Recent in vitro and in vivo studies involving adenoviral gene transfer suggest that SREBP-1c may also contribute to the regulation of glucose uptake and glucose synthesis. When overexpressed in hepatocytes, nSREBP-1c induces expression of glucokinase, a key enzyme in glucose utilization. It also suppresses phosphoenolpyruvate carboxykinase, a key gluconeogenic enzyme (35, 36).

SREBPs in disease

Many individuals with obesity and insulin resistance also have fatty livers, one of the most commonly encountered liver abnormalities in the US (37). A subset of individuals with fatty liver go on to develop fibrosis, cirrhosis, and liver failure. Evidence indicates that the fatty liver of insulin resistance is caused by SREBP-1c, which is elevated in response to the high insulin levels. Thus, SREBP-1c levels are elevated in the fatty livers of obese (ob/ob) mice with insulin resistance and hyperinsulinemia caused by leptin deficiency (38, 39). Despite the presence of insulin resistance in peripheral tissues, insulin continues to activate SREBP-1c transcription and cleavage in the livers of these insulin-resistant mice. The elevated nSREBP-1c increases lipogenic gene expression, enhances fatty acid synthesis, and accelerates triglyceride accumulation (31, 39). These metabolic abnormalities are reversed with the administration of leptin, which corrects the insulin resistance and lowers the insulin levels (38).

Metformin, a biguanide drug used to treat insulin-resistant diabetes, reduces hepatic nSREBP-1 levels and dramatically lowers the lipid accumulation in livers of insulin-resistant ob/ob mice (40). Metformin stimulates AMP-activated protein kinase (AMPK), an enzyme that inhibits lipid synthesis through phosphorylation and inactivation of key lipogenic enzymes (41). In rat hepatocytes, metformin-induced activation of AMPK also leads to decreased mRNA expression of SREBP-1c and its lipogenic target genes (41), but the basis of this effect is not understood.

The incidence of coronary artery disease increases with increasing plasma LDL-cholesterol levels, which in turn are inversely proportional to the levels of hepatic LDL receptors. SREBPs stimulate LDL receptor expression, but they also enhance lipid synthesis (1), so their net effect on plasma lipoprotein levels depends on a balance between opposing effects. In mice, the plasma levels of lipoproteins tend to fall when SREBPs are either overexpressed or underexpressed. In transgenic mice that overexpress nSREBPs in liver, plasma cholesterol and triglycerides are generally lower than in control mice (Table 1), even though these mice massively overproduce fatty acids, cholesterol, or both. Hepatocytes of nSREBP-1a transgenic mice overproduce VLDL, but these particles are rapidly removed through the action of LDL receptors, and they do not accumulate in the plasma. Indeed, some nascent VLDL particles are degraded even before secretion by a process that is mediated by LDL receptors (42). The high levels of nSREBP-1a in these animals support continued expression of the LDL receptor, even in cells whose cholesterol concentration is elevated. In LDL receptor–deficient mice carrying the nSREBP-1a transgene, plasma cholesterol and triglyceride levels rise tenfold (43).

Mice that lack all SREBPs in liver as a result of disruption of Scap or S1p also manifest lower plasma cholesterol and triglyceride levels (Table 1).

In these mice, hepatic cholesterol and triglyceride synthesis is markedly reduced, and this likely causes a decrease in VLDL production and secretion. LDL receptor mRNA and LDL clearance from plasma is also significantly reduced in these mice, but the reduction in LDL clearance is less than the overall reduction in VLDL secretion, the net result being a decrease in plasma lipid levels (15). However, because

humans and mice differ substantially with regard to LDL receptor expression, LDL levels, and other aspects of lipoprotein metabolism,

it is difficult to predict whether human plasma lipids will rise or fall when the SREBP pathway is blocked or activated.

SREBPs in liver: unanswered questions

The studies of SREBPs in liver have exposed a complex regulatory system whose individual parts are coming into focus. Major unanswered questions relate to the ways in which the transcriptional and posttranscriptional controls on SREBP activity are integrated so as to permit independent regulation of cholesterol and fatty acid synthesis in specific nutritional states. A few clues regarding these integration mechanisms are discussed below.

Whereas cholesterol synthesis depends almost entirely on SREBPs, fatty acid synthesis is only partially dependent on these proteins. This has been shown most clearly in cultured nonhepatic cells such as Chinese hamster ovary cells. In the absence of SREBP processing, as when the Site-2 protease is defective, the levels of mRNAs encoding cholesterol biosynthetic enzymes and the rates of cholesterol synthesis decline nearly to undetectable levels, whereas the rate of fatty acid synthesis is reduced by only 30% (44). Under these conditions, transcription of the fatty acid biosynthetic genes must be maintained by factors other than SREBPs. In liver, the gene encoding fatty acid synthase (FASN) can be activated transcriptionally by upstream stimulatory factor, which acts in concert with SREBPs (45). The FASN promoter also contains an LXR element that permits a low-level response to LXR ligands even when SREBPs are suppressed (46). These two transcription factors may help to maintain fatty acid synthesis in liver when nSREBP-1c is low.

Another mechanism of differential regulation is seen in the ability of cholesterol to block the processing of SREBP-2, but not SREBP-1, under certain metabolic conditions. This differential regulation has been studied most thoroughly in cultured cells such as human embryonic kidney (HEK-293) cells. When these cells are incubated in the absence of fatty acids and cholesterol, the addition of sterols blocks processing of SREBP-2, but not SREBP-1, which is largely produced as SREBP-1a in these cells (47). Inhibition of SREBP-1 processing requires an unsaturated fatty acid, such as oleate or arachidonate, in addition to sterols (47). In the absence of fatty acids and in the presence of sterols, SCAP may be able to carry SREBP-1 proteins, but not SREBP-2, to the Golgi apparatus. Further studies are necessary to document this apparent independent regulation of SREBP-1 and SREBP-2 processing and to determine its mechanism.



Support for the research cited from the authors’ laboratories was provided by grants from the NIH (HL-20948), the Moss Heart Foundation, the Keck Foundation, and the Perot Family Foundation. J.D. Horton is a Pew Scholar in the Biomedical Sciences and is the recipient of an Established Investigator Grant from the American Heart Association and a Research Scholar Award from the American Digestive Health Industry.


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Structural Biochemistry/Lipids/Membrane Lipids

< Structural Biochemistry‎ | Lipids

Membrane proteins rely on their interaction with membrane lipids to uphold its structure and maintain its functions as a protein. For membrane proteins to purify and crystallize, it is essential for the membrane protein to be in the appropriate lipid environment. Lipids assist in crystallization and stabilize the protein and provide lattice contacts. Lipids can also help obtain membrane protein structures in a native conformation. Membrane protein structures contain bound lipid molecules. Biological membranes are important in life, providing permeable barriers for cells and their organelles. The interaction between membrane proteins and lipids facilitates basic processes such as respiration, photosynthesis, transport, signal transduction and motility. These basic processes require a diverse group of proteins, which are encoded by 20-30% of an organism’s annotated genes.

There exist a great number of membrane lipids. Specifically, eukaryotic cells have a very complex collection of lipids that rely on many of the cell’s resources for its synthesis. Interactions between proteins and lipids can be very specific. Specific types of lipids can make a structure stable, provide control in insertion and folding processes, and help to assemble multisubunit complexes or supercomplexes, and most importantly, can significantly affect a membrane protein’s functions. Protein and lipid interactions are not sufficiently tight, meaning that lipids are retained during membrane protein purification. Since cellular membranes are fluid arrangements of lipids, some lipids affect interesting changes to membrane due to their characteristics. Glycosphigolipids and cholesterol tend to form small islands within the membranes, called lipid rafts, due to their physical properties. Some proteins also tend to cluster in lipid raft, while others avoid being in lipid rafts. However, the existence of lipid rafts in cells seems to be transitory.

Recent progress in determining membrane protein structure has brought attention to the importance of maintaining a favorable lipid environment so proteins to crystallize and purify successfully. Lipids assist in crystallization by stabilizing the protein fold and the relationships between subunits or monomers. The lipid content in protein-lipid detergent complexes can be altered by adjusting solubilisation and purification protocols, also by adding native or non-native lipids.

There are three type of membrane lipids: 1. Phospholipids: major class of membrane lipids. 2. glycolipids. 3. Cholesterols. Membrane lipids were started with eukaryotes and bacteria.

Types of Membrane Lipids

Lipids are often used as membrane constituents. The three major classes that membrane lipids are divided into are phospholipids, glycolipids, and cholesterol. Lipids are found in eukaryotes and bacteria. Although the lipids in archaea have many features that are related to the membrane formation that is similar with lipids of other organisms, they are still distinct from one another. The membranes of archaea differ in composition in three major ways. Firstly, the nonpolar chains are joined to a glycerol backbone by ether instead of esters, allowing for more resistance to hydrolysis. Second, the alkyl chains are not linear, but branched and make them more resistant to oxidation. The ability of archaeal lipids to resist hydrolysis and oxidation help these types of organisms to withstand the extreme conditions of high temperature, low pH, or high salt concentration. Lastly, the stereochemistry of the central glycerol is inverted. Membrane lipids have an extensive repertoire, but they possess a critical common structural theme in which they are amphipathic molecules, meaning they contain both a hydrophilic and hydrophobic moiety.

Membrane lipids are all closed bodies or boundaries separating substituent parts of the cell. The thickness of membranes is usually between 60 and 100 angstroms. These bodies are constructed from non-covalent assemblies. Their polar heads align with each other and their non-polar hydrocarbon tails align as well. The resulting stability is credited to hydrophobic interaction which proves to be quite stable due to the length of their hydrocarbon tails.


Membrane Lipids

Lipid Vesicles

Lipid vesicles, also known as liposomes, are vesicles that are essentially aqueous vesicles that are surrounded by a circular phospholipid bilayer. Like the other phospholipid structures, they have the hydrocarbon/hydrophobic tails facing inward, away from the aqueous solution, and the hydrophilic heads facing towards the aqueous solution. These vesicles are structures that form enclosed compartments of ions and solutes, and can be utilized to study the permeability of certain membranes, or to transfer these ions or solutes to certain cells found elsewhere.

Liposomes as vesicles can serve various clinical uses. Injecting liposomes containing medicine or DNA (for gene therapy) into patients is a possible method of drug delivery. The liposomes fuse with other cells’ membranes and therefore combine their contents with that of the patient’s cell. This method of drug delivery is less toxic than direct exposure because the liposomes carry the drug directly to cells without any unnecessary intermediate steps.

Because of the hydrophobic interactions among several phospholipids and glycolipids, a certain structure called the lipid bilayer or bimolecular sheet is favored. As mentioned earlier, phospholipids and glycolipids have both hydrophilic and hydrophobic moieties; thus, when several phospholipids or glycolipids come together in an aqueous solution, the hydrophobic tails interact with each other to form a hydrophobic center, while the hydrophilic heads interact with each other forming a hydrophilic coating on each side of the bilayer.











Evidence Report/Technology Assessment   Number 89


Effects of Omega-3 Fatty Acids on Lipids and Glycemic Control in Type II Diabetes and the Metabolic Syndrome and on Inflammatory Bowel Disease, Rheumatoid Arthritis, Renal Disease, Systemic Lupus Erythematosus, and Osteoporosis


Prepared for:

Agency for Healthcare Research and Quality

U.S. Department of Health and Human Services

540 Gaither Road

Rockville, MD 20850

Contract No. 290-02-0003


Chapter 1. Introduction

This report is one of a group of evidence reports prepared by three Agency for Healthcare Research and Quality (AHRQ)-funded Evidence-Based Practice Centers (EPCs) on the role of omega-3 fatty acids (both from food sources and from dietary supplements) in the prevention or treatment of a variety of diseases. These reports were requested and funded by the Office of Dietary Supplements, National Institutes of Health. The three EPCs – the Southern California EPC (SCEPC, based at RAND), the Tufts-New England Medical Center (NEMC) EPC, and the University of Ottawa EPC – have each produced evidence reports. To ensure consistency of approach, the three EPCs collaborated on selected methodological elements, including literature search strategies, rating of evidence, and data table design.

The aim of these reports is to summarize the current evidence on the effects of omega-3 fatty acids on prevention and treatment of cardiovascular diseases, cancer, child and maternal health, eye health, gastrointestinal/renal diseases, asthma, immune- mediated diseases, tissue/organ transplantation, mental health, and neurological diseases and conditions. In addition to informing the research community and the public on the effects of omega-3 fatty acids on various health conditions, it is anticipated that the findings of the reports will also be used to help define the agenda for future research.

This report focuses on the effects of omega-3 fatty acids on immune- mediated diseases, bone metabolism, and gastrointestinal/renal diseases. Subsequent reports from the SCEPC will focus on cancer and neurological diseases and conditions.

This chapter provides a brief review of the current state of knowledge about the metabolism, physiological functions, and sources of omega-3 fatty acids.


The Recognition of Essential Fatty Acids

Dietary fat has long been recognized as an important source of energy for mammals, but in the late 1920s, researchers demonstrated the dietary requirement for particular fatty acids, which came to be called essential fatty acids. It was not until the advent of intravenous feeding, however, that the importance of essential fatty acids was widely accepted: Clinical signs of essential fatty acid deficiency are generally observed only in patients on total parenteral nutrition who received mixtures devoid of essential fatty acids or in those with malabsorption syndromes.

These signs include dermatitis and changes in visual and neural function. Over the past 40 years, an increasing number of physiological functions, such as immunomodulation, have been attributed to the essential fatty acids and their metabolites, and this area of research remains quite active.1, 2

Fatty Acid Nomenclature

The fat found in foods consists largely of a heterogeneous mixture of triacylglycerols (triglycerides)–glycerol molecules that are each combined with three fatty acids. The fatty acids can be divided into two categories, based on chemical properties: saturated fatty acids, which are usually solid at room temperature, and unsaturated fatty acids, which are liquid at room temperature. The term “saturation” refers to a chemical structure in which each carbon atom in the fatty acyl chain is bound to (saturated with) four other atoms, these carbons are linked by single bonds, and no other atoms or molecules can attach; unsaturated fatty acids contain at least one pair of carbon atoms linked by a double bond, which allows the attachment of additional atoms to those carbons (resulting in saturation). Despite their differences in structure, all fats contain approximately the same amount of energy (37 kilojoules/gram, or 9 kilocalories/gram).

The class of unsaturated fatty acids can be further divided into monounsaturated and polyunsaturated fatty acids. Monounsaturated fatty acids (the primary constituents of olive and canola oils) contain only one double bond. Polyunsaturated fatty acids (PUFAs) (the primary constituents of corn, sunflower, flax seed and many other vegetable oils) contain more than one double bond. Fatty acids are often referred to using the number of carbon atoms in the acyl chain, followed by a colon, followed by the number of double bonds in the chain (e.g., 18:1 refers to the 18-carbon monounsaturated fatty acid, oleic acid; 18:3 refers to any 18-carbon PUFA with three double bonds).

PUFAs are further categorized on the basis of the location of their double bonds. An omega or n notation indicates the number of carbon atoms from the methyl end of the acyl chain to the first double bond. Thus, for example, in the omega-3 (n-3) family of PUFAs, the first double bond is 3 carbons from the methyl end of the molecule. The trivial names, chemical names and abbreviations for the omega-3 fatty acids are detailed in Table 1.1.  Finally, PUFAs can be categorized according to their chain length. The 18-carbon n-3 and n-6 short-chain PUFAs are precursors to the longer 20- and 22-carbon PUFAs, called long-chain PUFAs (LCPUFAs).

Fatty Acid Metabolism

Mammalian cells can introduce double bonds into all positions on the fatty acid chain except the n-3 and n-6 position. Thus, the short-chain alpha- linolenic acid (ALA, chemical abbreviation: 18:3n-3) and linoleic acid (LA, chemical abbreviation: 18:2n-6) are essential fatty acids.

No other fatty acids found in food are considered ‘essential’ for humans, because they can all be synthesized from the short chain fatty acids.

Following ingestion, ALA and LA can be converted in the liver to the long chain, more unsaturated n-3 and n-6 LCPUFAs by a complex set of synthetic pathways that share several enzymes (Figure 1). LC PUFAs retain the original sites of desaturation (including n-3 or n-6). The omega-6 fatty acid LA is converted to gamma-linolenic acid (GLA, 18:3n-6), an omega- 6 fatty acid that is a positional isomer of ALA. GLA, in turn, can be converted to the longerchain omega-6 fatty acid, arachidonic acid (AA, 20:4n-6). AA is the precursor for certain classes of an important family of hormone- like substances called the eicosanoids (see below).

The omega-3 fatty acid ALA (18:3n-3) can be converted to the long-chain omega-3 fatty acid, eicosapentaenoic acid (EPA; 20:5n-3). EPA can be elongated to docosapentaenoic acid (DPA 22:5n-3), which is further desaturated to docosahexaenoic acid (DHA; 22:6n-3). EPA and DHA are also precursors of several classes of eicosanoids and are known to play several other critical roles, some of which are discussed further below.

The conversion from parent fatty acids into the LC PUFAs – EPA, DHA, and AA – appears to occur slowly in humans. In addition, the regulation of conversion is not well understood, although it is known that ALA and LA compete for entry into the metabolic pathways.

Physiological Functions of EPA and AA

As stated earlier, fatty acids play a variety of physiological roles. The specific biological functions of a fatty acid are determined by the number and position of double bonds and the length of the acyl chain.

Both EPA (20:5n-3) and AA (20:4n-6) are precursors for the formation of a family of hormone- like agents called eicosanoids. Eicosanoids are rudimentary hormones or regulating – molecules that appear to occur in most forms of life. However, unlike endocrine hormones, which travel in the blood stream to exert their effects at distant sites, the eicosanoids are autocrine or paracrine factors, which exert their effects locally – in the cells that synthesize them or adjacent cells. Processes affected include the movement of calcium and other substances into and out of cells, relaxation and contraction of muscles, inhibition and promotion of clotting, regulation of secretions including digestive juices and hormones, and control of fertility, cell division, and growth.3

The eicosanoid family includes subgroups of substances known as prostaglandins, leukotrienes, and thromboxanes, among others. As shown in Figure 1.1, the long-chain omega-6 fatty acid, AA (20:4n-6), is the precursor of a group of eicosanoids that include series-2 prostaglandins and series-4 leukotrienes. The omega-3 fatty acid, EPA (20:5n-3), is the precursor to a group of eicosanoids that includes series-3 prostaglandins and series-5 leukotrienes. The AA-derived series-2 prostaglandins and series-4 leukotrienes are often synthesized in response to some emergency such as injury or stress, whereas the EPA-derived series-3 prostaglandins and series-5 leukotrienes appear to modulate the effects of the series-2 prostaglandins and series-4 leukotrienes (usually on the same target cells). More specifically, the series-3 prostaglandins are formed at a slower rate and work to attenuate the effects of excessive levels of series-2 prostaglandins. Thus, adequate production of the series-3 prostaglandins seems to protect against heart attack and stroke as well as certain inflammatory diseases like arthritis, lupus, and asthma.3.

EPA (22:6 n-3) also affects lipoprotein metabolism and decreases the production of substances – including cytokines, interleukin 1ß (IL-1ß), and tumor necrosis factor a (TNF-a) – that have pro-inflammatory effects (such as stimulation of collagenase synthesis and the expression of adhesion molecules necessary for leukocyte extravasation [movement from the circulatory system into tissues]).2 The mechanism responsible for the suppression of cytokine production by omega-3 LC PUFAs remains unknown, although suppression of omega-6-derived eicosanoid production by omega-3 fatty acids may be involved, because the omega-3 and omega-6 fatty acids compete for a common enzyme in the eicosanoid synthetic pathway, delta-6 desaturase.

DPA (22:5n-3) (the elongation product of EPA) and its metabolite DHA (22:6n-3) are frequently referred to as very long chain n-3 fatty acids (VLCFA). Along with AA, DHA is the major PUFA found in the brain and is thought to be important for brain development and function. Recent research has focused on this role and the effect of supplementing infant formula with DHA (since DHA is naturally present in breast milk but not in formula).

Dietary Sources and Requirements

Both ALA and LA are present in a variety of foods. LA is present in high concentrations in many commonly used oils, including safflower, sunflower, soy, and corn oil. ALA is present in some commonly used oils, including canola and soybean oil, and in some leafy green vegetables. Thus, the major dietary sources of ALA and LA are PUFA-rich vegetable oils. The proportion of LA to ALA as well as the proportion of those PUFAs to others varies considerably by the type of oil. With the exception of flaxseed, canola, and soybean oil, the ratio of LA to ALA in vegetable oils is at least 10 to 1. The ratios of LA to ALA for flaxseed, canola, and soy are approximately 1: 3.5, 2:1, and 8:1, respectively; however, flaxseed oil is not typically consumed in the North American diet. It is estimated that on average in the U.S., LA accounts for 89% of the total PUFAs consumed, and ALA accounts for 9%. Another estimate suggests that Americans consume 10 times more omega-6 than omega-3 fatty acids.4 Table 1.2 shows the proportion of omega 3 fatty acids for a number of foods.

Syntheis and Degradation

Source of Acetyl CoA for Fatty Acid Synthesis

Source of Acetyl CoA for Fatty Acid Synthesis

step 1

step 1

condensation reaction with malonyl ACP

ACP (acyl carrier protein)

ACP (acyl carrier protein)

synthesis requires acetyl CoA from citrate shuttle

synthesis requires acetyl CoA from citrate shuttle

conversion to fatty acyl co A in cytoplasm

conversion to fatty acyl co A in cytoplasm

ACP (acyl carrier protein)

ACP (acyl carrier protein)

FA synthesis not exactly reverse of catabolism

FA synthesis not exactly reverse of catabolism


Fatty Acid Synthase

Fatty Acid Synthase

complete FA synthesis

complete FA synthesis



Elongation and Desaturation of Fatty Acids

Elongation and Desaturation of Fatty Acids

release of FAs from adiposites

release of FAs from adiposites

Fatty acid beta oxidation and Krebs cycle produce NAD, NADH, FADH2

Fatty acid beta oxidation and Krebs cycle produce NAD, NADH, FADH2

ketone bodies

ketone bodies

metabolism of ketone bodies

metabolism of ketone bodies



Arachidonate pathways

Arachidonate pathways

arachidonic acid derivatives

arachidonic acid derivatives

major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides

major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides

Model for the sterol-mediated proteolytic release of SREBPs from membrane

Model for the sterol-mediated proteolytic release of SREBPs from membrane

hormone regulation

hormone regulation

 insulin receptor and and insulin receptor signaling pathway (IRS)

insulin receptor and and insulin receptor signaling pathway (IRS)

 islet brain glucose signaling

islet brain glucose signaling









Fish source

Fish source

omega FAs

omega FAs


Excessive omega 6s

Excessive omega 6s

omega 6s

omega 6s

diet and cancer

diet and cancer

Patients at risk of FA deficiency

Patients at risk of FA deficiency

PPAR role

PPAR role

PPAR role

PPAR role

Omega 6_3 pathways

Omega 6_3 pathways

n3 vs n6 PUFAs

n3 vs n6 PUFAs

triene-teraene ratio

triene-teraene ratio

arachidonic acid, leukotrienes, PG and thromboxanes

arachidonic acid, leukotrienes, PG and thromboxanes

Cox 2 and cancer

Cox 2 and cancer

Lipidomics of atherosclerotic plaques

Lipidomics of atherosclerotic plaques
















Effect of TPN on EFAD

Effect of TPN on EFAD

benefits of omega 3s

benefits of omega 3s

food consumption

food consumption


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Summary of Translational Medicine – e-Series A: Cardiovascular Diseases, Volume Four – Part 1

Summary of Translational Medicine – e-Series A: Cardiovascular Diseases, Volume Four – Part 1

Author and Curator: Larry H Bernstein, MD, FCAP


Curator: Aviva Lev-Ari, PhD, RN


Part 1 of Volume 4 in the e-series A: Cardiovascular Diseases and Translational Medicine, provides a foundation for grasping a rapidly developing surging scientific endeavor that is transcending laboratory hypothesis testing and providing guidelines to:

  • Target genomes and multiple nucleotide sequences involved in either coding or in regulation that might have an impact on complex diseases, not necessarily genetic in nature.
  • Target signaling pathways that are demonstrably maladjusted, activated or suppressed in many common and complex diseases, or in their progression.
  • Enable a reduction in failure due to toxicities in the later stages of clinical drug trials as a result of this science-based understanding.
  • Enable a reduction in complications from the improvement of machanical devices that have already had an impact on the practice of interventional procedures in cardiology, cardiac surgery, and radiological imaging, as well as improving laboratory diagnostics at the molecular level.
  • Enable the discovery of new drugs in the continuing emergence of drug resistance.
  • Enable the construction of critical pathways and better guidelines for patient management based on population outcomes data, that will be critically dependent on computational methods and large data-bases.

What has been presented can be essentially viewed in the following Table:


Summary Table for TM - Part 1

Summary Table for TM – Part 1




There are some developments that deserve additional development:

1. The importance of mitochondrial function in the activity state of the mitochondria in cellular work (combustion) is understood, and impairments of function are identified in diseases of muscle, cardiac contraction, nerve conduction, ion transport, water balance, and the cytoskeleton – beyond the disordered metabolism in cancer.  A more detailed explanation of the energetics that was elucidated based on the electron transport chain might also be in order.

2. The processes that are enabling a more full application of technology to a host of problems in the environment we live in and in disease modification is growing rapidly, and will change the face of medicine and its allied health sciences.


Electron Transport and Bioenergetics

Deferred for metabolomics topic

Synthetic Biology

Introduction to Synthetic Biology and Metabolic Engineering

Kristala L. J. Prather: Part-1    <iBiology > iBioSeminars > Biophysics & Chemical Biology > Lecturers generously donate their time to prepare these lectures. The project is funded by NSF and NIGMS, and is supported by the ASCB and HHMI.
Dr. Prather explains that synthetic biology involves applying engineering principles to biological systems to build “biological machines”.

Dr. Prather has received numerous awards both for her innovative research and for excellence in teaching.  Learn more about how Kris became a scientist at
Prather 1: Synthetic Biology and Metabolic Engineering  2/6/14IntroductionLecture Overview In the first part of her lecture, Dr. Prather explains that synthetic biology involves applying engineering principles to biological systems to build “biological machines”. The key material in building these machines is synthetic DNA. Synthetic DNA can be added in different combinations to biological hosts, such as bacteria, turning them into chemical factories that can produce small molecules of choice. In Part 2, Prather describes how her lab used design principles to engineer E. coli that produce glucaric acid from glucose. Glucaric acid is not naturally produced in bacteria, so Prather and her colleagues “bioprospected” enzymes from other organisms and expressed them in E. coli to build the needed enzymatic pathway. Prather walks us through the many steps of optimizing the timing, localization and levels of enzyme expression to produce the greatest yield. Speaker Bio: Kristala Jones Prather received her S.B. degree from the Massachusetts Institute of Technology and her PhD at the University of California, Berkeley both in chemical engineering. Upon graduation, Prather joined the Merck Research Labs for 4 years before returning to academia. Prather is now an Associate Professor of Chemical Engineering at MIT and an investigator with the multi-university Synthetic Biology Engineering Reseach Center (SynBERC). Her lab designs and constructs novel synthetic pathways in microorganisms converting them into tiny factories for the production of small molecules. Dr. Prather has received numerous awards both for her innovative research and for excellence in teaching.



II. Regulatory Effects of Mammalian microRNAs

Calcium Cycling in Synthetic and Contractile Phasic or Tonic Vascular Smooth Muscle Cells

Current Basic and Pathological Approaches to
the Function of Muscle Cells and Tissues – From Molecules to HumansLarissa Lipskaia, Isabelle Limon, Regis Bobe and Roger Hajjar
Additional information is available at the end of the chapter
1. Introduction
Calcium ions (Ca ) are present in low concentrations in the cytosol (~100 nM) and in high concentrations (in mM range) in both the extracellular medium and intracellular stores (mainly sarco/endo/plasmic reticulum, SR). This differential allows the calcium ion messenger that carries information
as diverse as contraction, metabolism, apoptosis, proliferation and/or hypertrophic growth. The mechanisms responsible for generating a Ca signal greatly differ from one cell type to another.
In the different types of vascular smooth muscle cells (VSMC), enormous variations do exist with regard to the mechanisms responsible for generating Ca signal. In each VSMC phenotype (synthetic/proliferating and contractile [1], tonic or phasic), the Ca signaling system is adapted to its particular function and is due to the specific patterns of expression and regulation of Ca.
For instance, in contractile VSMCs, the initiation of contractile events is driven by mem- brane depolarization; and the principal entry-point for extracellular Ca is the voltage-operated L-type calcium channel (LTCC). In contrast, in synthetic/proliferating VSMCs, the principal way-in for extracellular Ca is the store-operated calcium (SOC) channel.
Whatever the cell type, the calcium signal consists of  limited elevations of cytosolic free calcium ions in time and space. The calcium pump, sarco/endoplasmic reticulum Ca ATPase (SERCA), has a critical role in determining the frequency of SR Ca release by upload into the sarcoplasmic
sensitivity of  SR calcium channels, Ryanodin Receptor, RyR and Inositol tri-Phosphate Receptor, IP3R.
Synthetic VSMCs have a fibroblast appearance, proliferate readily, and synthesize increased levels of various extracellular matrix components, particularly fibronectin, collagen types I and III, and tropoelastin [1].
Contractile VSMCs have a muscle-like or spindle-shaped appearance and well-developed contractile apparatus resulting from the expression and intracellular accumulation of thick and thin muscle filaments [1].
Schematic representation of Calcium Cycling in Contractile and Proliferating VSMCs

Schematic representation of Calcium Cycling in Contractile and Proliferating VSMCs


Figure 1. Schematic representation of Calcium Cycling in Contractile and Proliferating VSMCs.

Left panel: schematic representation of calcium cycling in quiescent /contractile VSMCs. Contractile re-sponse is initiated by extracellular Ca influx due to activation of Receptor Operated Ca (through phosphoinositol-coupled receptor) or to activation of L-Type Calcium channels (through an increase in luminal pressure). Small increase of cytosolic due IP3 binding to IP3R (puff) or RyR activation by LTCC or ROC-dependent Ca influx leads to large SR Ca IP3R or RyR clusters (“Ca -induced Ca SR calcium pumps (both SERCA2a and SERCA2b are expressed in quiescent VSMCs), maintaining high concentration of cytosolic Ca and setting the sensitivity of RyR or IP3R for the next spike.
Contraction of VSMCs occurs during oscillatory Ca transient.
Middle panel: schematic representa tion of atherosclerotic vessel wall. Contractile VSMC are located in the media layer, synthetic VSMC are located in sub-endothelial intima.
Right panel: schematic representation of calcium cycling in quiescent /contractile VSMCs. Agonist binding to phosphoinositol-coupled receptor leads to the activation of IP3R resulting in large increase in cytosolic Ca calcium pumps (only SERCA2b, having low turnover and low affinity to Ca depletion leads to translocation of SR Ca sensor STIM1 towards PM, resulting in extracellular Ca influx though opening of Store Operated Channel (CRAC). Resulted steady state Ca transient is critical for activation of proliferation-related transcription factors ‘NFAT).
Abbreviations: PLC – phospholipase C; PM – plasma membrane; PP2B – Ca /calmodulin-activated protein phosphatase 2B (calcineurin); ROC- receptor activated channel; IP3 – inositol-1,4,5-trisphosphate, IP3R – inositol-1,4,5- trisphosphate receptor; RyR – ryanodine receptor; NFAT – nuclear factor of activated T-lymphocytes; VSMC – vascular smooth muscle cells; SERCA – sarco(endo)plasmic reticulum Ca sarcoplasmic reticulum.


Time for New DNA Synthesis and Sequencing Cost Curves

By Rob Carlson

I’ll start with the productivity plot, as this one isn’t new. For a discussion of the substantial performance increase in sequencing compared to Moore’s Law, as well as the difficulty of finding this data, please see this post. If nothing else, keep two features of the plot in mind: 1) the consistency of the pace of Moore’s Law and 2) the inconsistency and pace of sequencing productivity. Illumina appears to be the primary driver, and beneficiary, of improvements in productivity at the moment, especially if you are looking at share prices. It looks like the recently announced NextSeq and Hiseq instruments will provide substantially higher productivities (hand waving, I would say the next datum will come in another order of magnitude higher), but I think I need a bit more data before officially putting another point on the plot.




Illumina’s instruments are now responsible for such a high percentage of sequencing output that the company is effectively setting prices for the entire industry. Illumina is being pushed by competition to increase performance, but this does not necessarily translate into lower prices. It doesn’t behoove Illumina to drop prices at this point, and we won’t see any substantial decrease until a serious competitor shows up and starts threatening Illumina’s market share. The absence of real competition is the primary reason sequencing prices have flattened out over the last couple of data points.

Note that the oligo prices above are for column-based synthesis, and that oligos synthesized on arrays are much less expensive. However, array synthesis comes with the usual caveat that the quality is generally lower, unless you are getting your DNA from Agilent, which probably means you are getting your dsDNA from Gen9.

Note also that the distinction between the price of oligos and the price of double-stranded sDNA is becoming less useful. Whether you are ordering from Life/Thermo or from your local academic facility, the cost of producing oligos is now, in most cases, independent of their length. That’s because the cost of capital (including rent, insurance, labor, etc) is now more significant than the cost of goods. Consequently, the price reflects the cost of capital rather than the cost of goods. Moreover, the cost of the columns, reagents, and shipping tubes is certainly more than the cost of the atoms in the sDNA you are ostensibly paying for. Once you get into longer oligos (substantially larger than 50-mers) this relationship breaks down and the sDNA is more expensive. But, at this point in time, most people aren’t going to use longer oligos to assemble genes unless they have a tricky job that doesn’t work using short oligos.

Looking forward, I suspect oligos aren’t going to get much cheaper unless someone sorts out how to either 1) replace the requisite human labor and thereby reduce the cost of capital, or 2) finally replace the phosphoramidite chemistry that the industry relies upon.

IDT’s gBlocks come at prices that are constant across quite substantial ranges in length. Moreover, part of the decrease in price for these products is embedded in the fact that you are buying smaller chunks of DNA that you then must assemble and integrate into your organism of choice.

Someone who has purchased and assembled an absolutely enormous amount of sDNA over the last decade, suggested that if prices fell by another order of magnitude, he could switch completely to outsourced assembly. This is a potentially interesting “tipping point”. However, what this person really needs is sDNA integrated in a particular way into a particular genome operating in a particular host. The integration and testing of the new genome in the host organism is where most of the cost is. Given the wide variety of emerging applications, and the growing array of hosts/chassis, it isn’t clear that any given technology or firm will be able to provide arbitrary synthetic sequences incorporated into arbitrary hosts.

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Startup to Strengthen Synthetic Biology and Regenerative Medicine Industries with Cutting Edge Cell Products

28 Nov 2013 | PR Web

Dr. Jon Rowley and Dr. Uplaksh Kumar, Co-Founders of RoosterBio, Inc., a newly formed biotech startup located in Frederick, are paving the way for even more innovation in the rapidly growing fields of Synthetic Biology and Regenerative Medicine. Synthetic Biology combines engineering principles with basic science to build biological products, including regenerative medicines and cellular therapies. Regenerative medicine is a broad definition for innovative medical therapies that will enable the body to repair, replace, restore and regenerate damaged or diseased cells, tissues and organs. Regenerative therapies that are in clinical trials today may enable repair of damaged heart muscle following heart attack, replacement of skin for burn victims, restoration of movement after spinal cord injury, regeneration of pancreatic tissue for insulin production in diabetics and provide new treatments for Parkinson’s and Alzheimer’s diseases, to name just a few applications.

While the potential of the field is promising, the pace of development has been slow. One main reason for this is that the living cells required for these therapies are cost-prohibitive and not supplied at volumes that support many research and product development efforts. RoosterBio will manufacture large quantities of standardized primary cells at high quality and low cost, which will quicken the pace of scientific discovery and translation to the clinic. “Our goal is to accelerate the development of products that incorporate living cells by providing abundant, affordable and high quality materials to researchers that are developing and commercializing these regenerative technologies” says Dr. Rowley


Life at the Speed of Light

NHMU Lecture featuring – J. Craig Venter, Ph.D.
Founder, Chairman, and CEO – J. Craig Venter Institute; Co-Founder and CEO, Synthetic Genomics Inc.

J. Craig Venter, Ph.D., is Founder, Chairman, and CEO of the J. Craig Venter Institute (JVCI), a not-for-profit, research organization dedicated to human, microbial, plant, synthetic and environmental research. He is also Co-Founder and CEO of Synthetic Genomics Inc. (SGI), a privately-held company dedicated to commercializing genomic-driven solutions to address global needs.

In 1998, Dr. Venter founded Celera Genomics to sequence the human genome using new tools and techniques he and his team developed.  This research culminated with the February 2001 publication of the human genome in the journal, Science. Dr. Venter and his team at JVCI continue to blaze new trails in genomics.  They have sequenced and a created a bacterial cell constructed with synthetic DNA,  putting humankind at the threshold of a new phase of biological research.  Whereas, we could  previously read the genetic code (sequencing genomes), we can now write the genetic code for designing new species.

The science of synthetic genomics will have a profound impact on society, including new methods for chemical and energy production, human health and medical advances, clean water, and new food and nutritional products. One of the most prolific scientists of the 21st century for his numerous pioneering advances in genomics,  he  guides us through this emerging field, detailing its origins, current challenges, and the potential positive advances.

His work on synthetic biology truly embodies the theme of “pushing the boundaries of life.”  Essentially, Venter is seeking to “write the software of life” to create microbes designed by humans rather than only through evolution. The potential benefits and risks of this new technology are enormous. It also requires us to examine, both scientifically and philosophically, the question of “What is life?”

J Craig Venter wants to digitize DNA and transmit the signal to teleport organisms

2013 Genomics: The Era Beyond the Sequencing of the Human Genome: Francis Collins, Craig Venter, Eric Lander, et al.

Human Longevity Inc (HLI) – $70M in Financing of Venter’s New Integrative Omics and Clinical Bioinformatics



Where Will the Century of Biology Lead Us?

By Randall Mayes

A technology trend analyst offers an overview of synthetic biology, its potential applications, obstacles to its development, and prospects for public approval.

  • In addition to boosting the economy, synthetic biology projects currently in development could have profound implications for the future of manufacturing, sustainability, and medicine.
  • Before society can fully reap the benefits of synthetic biology, however, the field requires development and faces a series of hurdles in the process. Do researchers have the scientific know-how and technical capabilities to develop the field?

Biology + Engineering = Synthetic Biology

Bioengineers aim to build synthetic biological systems using compatible standardized parts that behave predictably. Bioengineers synthesize DNA parts—oligonucleotides composed of 50–100 base pairs—which make specialized components that ultimately make a biological system. As biology becomes a true engineering discipline, bioengineers will create genomes using mass-produced modular units similar to the microelectronics and computer industries.

Currently, bioengineering projects cost millions of dollars and take years to develop products. For synthetic biology to become a Schumpeterian revolution, smaller companies will need to be able to afford to use bioengineering concepts for industrial applications. This will require standardized and automated processes.

A major challenge to developing synthetic biology is the complexity of biological systems. When bioengineers assemble synthetic parts, they must prevent cross talk between signals in other biological pathways. Until researchers better understand these undesired interactions that nature has already worked out, applications such as gene therapy will have unwanted side effects. Scientists do not fully understand the effects of environmental and developmental interaction on gene expression. Currently, bioengineers must repeatedly use trial and error to create predictable systems.

Similar to physics, synthetic biology requires the ability to model systems and quantify relationships between variables in biological systems at the molecular level.

The second major challenge to ensuring the success of synthetic biology is the development of enabling technologies. With genomes having billions of nucleotides, this requires fast, powerful, and cost-efficient computers. Moore’s law, named for Intel co-founder Gordon Moore, posits that computing power progresses at a predictable rate and that the number of components in integrated circuits doubles each year until its limits are reached. Since Moore’s prediction, computer power has increased at an exponential rate while pricing has declined.

DNA sequencers and synthesizers are necessary to identify genes and make synthetic DNA sequences. Bioengineer Robert Carlson calculated that the capabilities of DNA sequencers and synthesizers have followed a pattern similar to computing. This pattern, referred to as the Carlson Curve, projects that scientists are approaching the ability to sequence a human genome for $1,000, perhaps in 2020. Carlson calculated that the costs of reading and writing new genes and genomes are falling by a factor of two every 18–24 months. (see recent Carlson comment on requirement to read and write for a variety of limiting  conditions).

Startup to Strengthen Synthetic Biology and Regenerative Medicine Industries with Cutting Edge Cell Products

Synthetic Biology: On Advanced Genome Interpretation for Gene Variants and Pathways: What is the Genetic Base of Atherosclerosis and Loss of Arterial Elasticity with Aging

Synthesizing Synthetic Biology: PLOS Collections

Capturing ten-color ultrasharp images of synthetic DNA structures resembling numerals 0 to 9

Silencing Cancers with Synthetic siRNAs

Genomics Now—and Beyond the Bubble

Futurists have touted the twenty-first century as the century of biology based primarily on the promise of genomics. Medical researchers aim to use variations within genes as biomarkers for diseases, personalized treatments, and drug responses. Currently, we are experiencing a genomics bubble, but with advances in understanding biological complexity and the development of enabling technologies, synthetic biology is reviving optimism in many fields, particularly medicine.


Michael Brooks holds a PhD in quantum physics. He writes a weekly science column for the New Statesman, and his most recent book is The Secret Anarchy of Science.

The basic idea is that we take an organism – a bacterium, say – and re-engineer its genome so that it does something different. You might, for instance, make it ingest carbon dioxide from the atmosphere, process it and excrete crude oil.

That project is still under construction, but others, such as using synthesised DNA for data storage, have already been achieved. As evolution has proved, DNA is an extraordinarily stable medium that can preserve information for millions of years. In 2012, the Harvard geneticist George Church proved its potential by taking a book he had written, encoding it in a synthesised strand of DNA, and then making DNA sequencing machines read it back to him.

When we first started achieving such things it was costly and time-consuming and demanded extraordinary resources, such as those available to the millionaire biologist Craig Venter. Venter’s team spent most of the past two decades and tens of millions of dollars creating the first artificial organism, nicknamed “Synthia”. Using computer programs and robots that process the necessary chemicals, the team rebuilt the genome of the bacterium Mycoplasma mycoides from scratch. They also inserted a few watermarks and puzzles into the DNA sequence, partly as an identifying measure for safety’s sake, but mostly as a publicity stunt.

What they didn’t do was redesign the genome to do anything interesting. When the synthetic genome was inserted into an eviscerated bacterial cell, the new organism behaved exactly the same as its natural counterpart. Nevertheless, that Synthia, as Venter put it at the press conference to announce the research in 2010, was “the first self-replicating species we’ve had on the planet whose parent is a computer” made it a standout achievement.

Today, however, we have entered another era in synthetic biology and Venter faces stiff competition. The Steve Jobs to Venter’s Bill Gates is Jef Boeke, who researches yeast genetics at New York University.

Boeke wanted to redesign the yeast genome so that he could strip out various parts to see what they did. Because it took a private company a year to complete just a small part of the task, at a cost of $50,000, he realised he should go open-source. By teaching an undergraduate course on how to build a genome and teaming up with institutions all over the world, he has assembled a skilled workforce that, tinkering together, has made a synthetic chromosome for baker’s yeast.


Stepping into DIYbio and Synthetic Biology at ScienceHack

Posted April 22, 2014 by Heather McGaw and Kyrie Vala-Webb

We got a crash course on genetics and protein pathways, and then set out to design and build our own pathways using both the “Genomikon: Violacein Factory” kit and Synbiota platform. With Synbiota’s software, we dragged and dropped the enzymes to create the sequence that we were then going to build out. After a process of sketching ideas, mocking up pathways, and writing hypotheses, we were ready to start building!

The night stretched long, and at midnight we were forced to vacate the school. Not quite finished, we loaded our delicate bacteria, incubator, and boxes of gloves onto the bus and headed back to complete our bacterial transformation in one of our hotel rooms. Jammed in between the beds and the mini-fridge, we heat-shocked our bacteria in the hotel ice bucket. It was a surreal moment.

While waiting for our bacteria, we held an “unconference” where we explored bioethics, security and risk related to synthetic biology, 3D printing on Mars, patterns in juggling (with live demonstration!), and even did a Google Hangout with Rob Carlson. Every few hours, we would excitedly check in on our bacteria, looking for bacterial colonies and the purple hue characteristic of violacein.

Most impressive was the wildly successful and seamless integration of a diverse set of people: in a matter of hours, we were transformed from individual experts and practitioners in assorted fields into cohesive and passionate teams of DIY biologists and science hackers. The ability of everyone to connect and learn was a powerful experience, and over the course of just one weekend we were able to challenge each other and grow.

Returning to work on Monday, we were hungry for more. We wanted to find a way to bring the excitement and energy from the weekend into the studio and into the projects we’re working on. It struck us that there are strong parallels between design and DIYbio, and we knew there was an opportunity to bring some of the scientific approaches and curiosity into our studio.



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Richard Lifton, MD, PhD of Yale University & Howard Hughes Medical Institute: Recipient of 2014 Breakthrough Prizes Awarded in Life Sciences for the Discovery of Genes and Biochemical Mechanisms that cause Hypertension

Curator: Aviva Lev-Ari, PhD, RN


Yale’s Lifton receives $3 million science prize at gala Silicon Valley ceremony

Friday, December 13, 2013

Bill Hathaway / 203-432-1322

Read this article on YaleNews

Richard Lifton, Sterling Professor of Genetics and chair of the Department of Genetics, has received a $3 million Breakthrough Prize in Life Sciences, created by top Silicon Valley entrepreneurs.

Lifton was one of eight scientists honored Dec. 12 with $21 million in prizes at gala ceremonies hosted by actor Kevin Spacey in Mountain View, California. Celebrities — including Conan O’Brien, Glenn Close, Rob Lowe, and Michael C. Hall — handed out awards to six winners of the life sciences prizes and two co-winners of the Breakthrough Prize in Fundamental Physics.

“Scientists should be celebrated as heroes, and we are honored to be part of today’s celebration,” said Google co-founder Sergey Brin and his wife, biologist and entrepreneur Anne Wojcicki, two of the event’s sponsors.

Lifton, who is also an investigator for the Howard Hughes Medical Institute, was recognized for his pioneering work to identify the genetic and biochemical underpinnings of hypertension, a disease that affects more than 1 billion people worldwide and that contributes to 17 million deaths annually from heart attack and stroke. Lifton and his colleagues identified patients around the world with exceptionally high or low blood pressure due to single gene mutations. They identified the mutated genes and established their role in salt reabsorption by the kidney and regulation of blood pressure. The work gave scientific rationale to limit dietary salt intake and suggested rational combinations of antihypertensive medications and development of new therapies.

Other sponsors of the event are Chinese internet entrepreneur Jack Ma and Cathy Zhang; Russian entrepreneur and venture capitalist Yuri Milner and his wife, Julia Milner; and Facebook founder Mark Zuckerberg and Priscilla Chan.

At the end of the ceremonies, which will be televised on the Science Channel at 9 p.m. on Jan. 27, Milner and Zuckerberg announced the creation of a $3 million Breakthrough Prize in Mathematics that will be awarded next year.

Additional information on the prizes can be found or



Laliotis MD, Zhang J, Volkman HM, Kahle KT, Hoffmann, KE, Toka HR, Nelson-Williams C, Ellison, DH, Flavell, R, Booth, CJ, Lu Y, Geller, DS, Lifton, RP. Wnk4 controls blood pressure and potassium homeostasis via regulation of mass and activity of the distal convoluted tubule. Nature Genetics, in press

Earlier Research Results on this discovey
Proc Natl Acad Sci U S A. 2003 Jan 21;100(2):680-4. Epub 2003 Jan 6.

Molecular pathogenesis of inherited hypertension with hyperkalemia: the Na-Cl cotransporter is inhibited by wild-type but not mutant WNK4.

Wilson FH1Kahle KTSabath ELalioti MDRapson AKHoover RSHebert SCGamba GLifton RP.


Mutations in the serine-threonine kinases WNK1 and WNK4 [with no lysine (K) at a key catalytic residue] cause pseudohypoaldosteronism type II (PHAII), a Mendelian disease featuring hypertension, hyperkalemia, hyperchloremia, and metabolic acidosis. Both kinases are expressed in the distal nephron, although the regulators and targets of WNK signaling cascades are unknown. The Cl(-) dependence of PHAII phenotypes, their sensitivity to thiazide diuretics, and the observation that they constitute a “mirror image” of the phenotypes resulting from loss of function mutations in the thiazide-sensitive Na-Cl cotransporter (NCCT) suggest that PHAII may result from increased NCCT activity due to altered WNK signaling. To address this possibility, we measured NCCT-mediated Na(+) influx and membrane expression in the presence of wild-type and mutant WNK4 by heterologous expression in Xenopus oocytes. Wild-type WNK4 inhibits NCCT-mediated Na-influx by reducing membrane expression of the cotransporter ((22)Na-influx reduced 50%, P < 1 x 10(-9), surface expression reduced 75%, P < 1 x 10(-14) in the presence of WNK4). This inhibition depends on WNK4 kinase activity, because missense mutations that abrogate kinase function prevent this effect. PHAII-causing missense mutations, which are remote from the kinase domain, also prevent inhibition of NCCT activity, providing insight into the pathophysiology of the disorder. The specificity of this effect is indicated by the finding that WNK4 and the carboxyl terminus of NCCT coimmunoprecipitate when expressed in HEK 293T cells. Together, these findings demonstrate that WNK4 negatively regulates surface expression of NCCT and implicate loss of this regulation in the molecular pathogenesis of an inherited form of hypertension.

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LISTEN TO AUDIO TAPE by Prof. Richard Lifton

January 27, 2014
Richard Lifton

Yale’s Richard Lifton is one of eight world-changing researchers whose work is celebrated during a program airing tonight (Jan. 27) on the Science Channel at 9 p.m. EST.

Lifton, Sterling Professor of Genetics and chair of the Department of Genetics, received a $3 million Breakthrough Prize in Life Sciences, created by top Silicon Valley entrepreneurs.

The Science Channel program features the Dec. 12 ceremony where Lifton and others received their prize. The festivities were hosted by actor Kevin Spacey and featured such celebrities as Conan O’Brien, Glenn Close, Rob Lowe, and Michael C. Hall, as well as tech leaders Mark Zuckerberg, Larry Page, Sergey Brin, Anne Wojcicki, Jimmy Wales, and Yuri Milner.


Yale consortium awarded $6 million to study therapies for vascular disease

Tuesday, January 21, 2014


Helen Dodson / 203-436-3984

Stacey Buba / 203-432-1333

Read this article on YaleNews

An international research team spearheaded by William C. Sessa, the Alfred Gilman Professor of Pharmacology and professor of medicine (cardiology), has been awarded a $6 million Transatlantic Networks of Excellence grant from the Fondation Leducq in France.

Sessa will be the U.S. coordinator for the consortium as it explores the mechanisms of secreted microRNAs and microRNA-based therapies for vascular disease. Sessa will be joined by a European coordinator, Dr. Thomas Thum, director of the Institute for Molecular and Translational Therapeutic Strategies at Hanover Medical School in Germany, and five investigators including recent Yale recruit, Carlos Fenandez-Hernando, associate professor of comparative medicine. The grant will be distributed over five years.

Sessa is director of the vascular biology and therapeutics program and vice chairman of pharmacology at Yale School of Medicine.

Sessa has long worked at the intersection of pharmacology and cardiovascular disease. He is on the scientific advisory board of the William Harvey Research Institute and NIHR Biomedical Research Unit in London, and also served on the joint strategy committee for the Yale-UCL collaborative in cardiovascular research.

“I am grateful to Fondation Leducq for funding this new international collaboration to find new and effective ways to treat a disease that kills millions of people each year,” Sessa said. “We have assembled a fantastic team of world class scientists to tackle the basic questions of how microRNAs are packaged and transferred between cells, and their therapeutic potential in vascular diseases.”

Fondation Leducq is a French non-profit health research foundation. Its mission is to improve human health through international efforts to combat cardiovascular disease. To this end, Fondation Leducq created the Transatlantic Networks of Excellence in Cardiovascular Research Program, which is designed to promote collaborative research involving centers in North America and Europe in the areas of cardiovascular and neurovascular disease.

Yale has had two previous Leducq grants — to Dr. Richard Lifton, chair of genetics, and Dr. Michael Simons, director of the Yale Cardiovascular Research Center.


International Activity

  • YALE-UCL Collaborative
    London, United Kingdom (2011)
    Dr. Lifton is on the Joint Strategy Committee for the Yale-UCL Collaborative, an alliance which will provide opportunities for high-level scientific research, clinical and educational collaboration across the institutions involved: Yale University, Yale School of Medicine, Yale-New Haven Hospital and UCL (University College London) and UCL Partners
  • Transatlantic Network on Hypertension-Renal Salt Handling in the Control of Blood Pressure
    France (2007)
    Drs Hebert and Lifton will join leading researchers in Switzerland, France and Mexico in a transatlantic collaboration aimed at pinpointing the kidney’s role in high blood pressure.

Education & Training

Stanford University (1982)
Stanford University (1986)

Honors & Recognition

  • National Academy of Sciences
  • The Basic Science Prize
    American Heart Association
  • Homer Smith Award
    American Society of Nephrology
  • MSD International Award
    International Society of Hypertension

Research Interests

Molecular genetics of common human diseases

Research Summary

The common human diseases that account for the vast majority of morbidity and mortality in human populations are known to have underlying inherited components. Advances in human genetics have made the identification of genetic variants contributing to these traits feasible. Such identification promises to revolutionize the diagnostic and therapeutic approaches to these disorders. We have focused on cardiovascular and renal disease. To date, we have identified mutations underlying more than 20 human diseases; these include a host of diseases that define molecular determinants of hypertension, stroke and heart attack. We have gone on from these starting points to use biochemistry and animal models to define the physiologic mechanisms linking genotype and phenotype. These findings have provided new insight into normal and disease biology, are identifying new pathways underlying disease pathogenesis, and are identifying new targets for development of novel therapeutics.

Extensive Research Description

Cardiovascular disease is the leading cause of death world-wide. Epidemiologic studies have identified hypertension, high cholesterol, diabetes and smoking as major risk factors. By investigation of rare families recruited from around the world that segregate single genes with large effect, we have identified genes that contribute to these traits, putting a molecular face on their pathogenesis. For example, we have identified mutations in 8 genes that cause high blood pressure (hypertension) and another 8 that cause low blood pressure. These mutations all converge on a final common pathway, the regulation of net salt reabsorption in the kidney. These findings have established the key role of variation in renal salt handling in blood pressure variation, and have led to changes in the approach to treatment of this disease in the general population. They have also identified new therapeutic targets that are predicted to have greater efficacy with reduced side effects. Finally, they have identified new signaling pathways involved in the regulation of blood pressure homeostasis. We have taken similar approaches to another common disease, osteoporosis, with the identification of gain of function mutations in LRP5, a component of the Wnt signaling pathway, in development of high bone density. This finding has led to intensive efforts to identify small molecules that impact this pathway to protect against and/or reverse osteoporosis in the general population. Ongoing studies use both emerging and novel approaches to identification of genes that contribute to disease burden in the population, and to understanding the pathways that link genes to disease. Mutations that affect blood pressure in humans. A diagram of a nephron, the filtering unit of the kidney, is shown. The molecular pathways mediating NaCl reabsorption in individual renal cells along the nephron are shown, along with the pathway of the renin-angiotensin system, a major regulator of renal salt reabsorption. Inherited diseases affecting these pathways are indicated, with hypertensive disorders in red and hypotensive disorders in blue. From Lifton, Gharavi, and Geller. Cell, 104:545-556, 2001.

Selected Publications

  • Mani, A., et al. (2007). LRP6 mutation in a family with early coronary disease and metabolic risk factors. Science 315:1278-82.
  • Ring, A.M., et al. (2007). An SGK1 site in WNK4 regulates Na+ channel and K+ channel activity and has implications for aldosterone signaling and K+ homeostasis. Proc. Natl. Acad. Sci. (USA) 104:4025-9.
  • Lalioti MD, Zhang J, Volkman HM, Kahle KT, Hoffmann, KE, Toka HR, Nelson-Williams C, Ellison, DH, Flavell, R, Booth, CJ, Lu Y, Geller, DS, Lifton, RP. Wnk4 controls blood pressure and potassium homeostasis via regulation of mass and activity of the distal convoluted tubule. Nature Genetics, in press.
  • Wilson FH, Hariri A, Farhi A, Zhao H, Peterson K, Toka HR, Nelson- Williams C, Raja KM, Kashgarian M, Shulman GI, Scheinman SJ, Lifton RP. A cluster of metabolic defects caused by mutation in a mitochondrial tRNA. Science, 306:1190-94, 2004.
  • Boyden LM, Mao J, Belsky J, Mitzner L, Farhi A, Mitnick MA, Wu D, Insogna K, Lifton RP. High bone density due to a mutation in LDL-receptor-related protein 5. New Engl J Med. 346:1513-1521, 2002.
  • Wilson FH, Disse-Nicodème S, Choate KA, Ishikawa K, Nelson-Williams C, Desitter I, Gunel M, Milford DV, Lipkin GW, Achard JM, Feely MP, Dussol B, Berland Y, Unwin RJ, Mayan H, Simon DB, Farfel Z, Jeunemaitre X, Lifton RP. Human Hypertension Caused by Mutations in WNK Kinases. Science, 293:1107-1112, 2001.
  • Lifton RP, Gharavi A, Geller DS. Molecular mechanisms of human hypertension. Cell, 104:545-556, 2001.
  • Geller DS, Farhi A, Pinkerton N, Fradley M, Moritz M, Spitzer A, Meinke G, Tsai TF, Sigler P, Lifton RP. Activating mineralocorticoid receptor mutation in hypertension exacerbated by pregnancy. Science, 289:119-123, 2000.
  • Simon DB, Lu Y, Choate KA, Velazquez H, Al-Sabban E, Praga M, Casari G, Bettinelli A, Colussi G, Rodriguez-Soriano J, McCredie D, Milford D, Sanjad S, Lifton RP. Paracellin-1, a renal tight junction protein required for paracellular Mg2+ reabsorption. Science, 285:103-106, 1999.


PubMed Results: 10

Select item 225138461.

Protein phosphatase 1 modulates the inhibitory effect of With-no-Lysine kinase 4 on ROMK channels.

Lin DH, Yue P, Rinehart J, Sun P, Wang Z, Lifton R, Wang WH.

Am J Physiol Renal Physiol. 2012 Jul 1;303(1):F110-9. doi: 10.1152/ajprenal.00676.2011. Epub 2012 Apr 18.



[PubMed – indexed for MEDLINE]

Free PMC Article

Related citations

Select item 165287062.

Haplotype analysis in the presence of informatively missing genotype data.

Liu N, Beerman I, Lifton R, Zhao H.

Genet Epidemiol. 2006 May;30(4):290-300.



[PubMed – indexed for MEDLINE]

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Select item 165282533.

Familial aggregation of primary glomerulonephritis in an Italian population isolate: Valtrompia study.

Izzi C, Sanna-Cherchi S, Prati E, Belleri R, Remedio A, Tardanico R, Foramitti M, Guerini S, Viola BF, Movilli E, Beerman I, Lifton R, Leone L, Gharavi A, Scolari F.

Kidney Int. 2006 Mar;69(6):1033-40.



[PubMed – indexed for MEDLINE]

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Select item 127823554.

Mice lacking the B1 subunit of H+ -ATPase have normal hearing.

Dou H, Finberg K, Cardell EL, Lifton R, Choo D.

Hear Res. 2003 Jun;180(1-2):76-84.



[PubMed – indexed for MEDLINE]

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Select item 113430495.

Glucocorticoid-remediable aldosteronism is associated with severe hypertension in early childhood.

Dluhy RG, Anderson B, Harlin B, Ingelfinger J, Lifton R.

J Pediatr. 2001 May;138(5):715-20.



[PubMed – indexed for MEDLINE]

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Select item 102327426.

Elevated ambulatory blood pressure in 20 subjects with Williams syndrome.

Broder K, Reinhardt E, Ahern J, Lifton R, Tamborlane W, Pober B.

Am J Med Genet. 1999 Apr 23;83(5):356-60.



[PubMed – indexed for MEDLINE]

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Select item 97986657.

Coincident acute myelogenous leukemia and ischemic heart disease: use of the cardioprotectant dexrazoxane during induction chemotherapy.

Woodlock TJ, Lifton R, DiSalle M.

Am J Hematol. 1998 Nov;59(3):246-8.



[PubMed – indexed for MEDLINE]

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Select item 95012578.

In vivo phosphorylation of the epithelial sodium channel.

Shimkets RA, Lifton R, Canessa CM.

Proc Natl Acad Sci U S A. 1998 Mar 17;95(6):3301-5.



[PubMed – indexed for MEDLINE]

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Select item 91562619.

Autotransplantation for relapsed or refractory non-Hodgkin’s lymphoma (NHL): long-term follow-up and analysis of prognostic factors.

Rapoport AP, Lifton R, Constine LS, Duerst RE, Abboud CN, Liesveld JL, Packman CH, Eberly S, Raubertas RF, Martin BA, Flesher WR, Kouides PA, DiPersio JF, Rowe JM.

Bone Marrow Transplant. 1997 May;19(9):883-90.



[PubMed – indexed for MEDLINE]

Free Article

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Physiologist, Professor Lichtstein, Chair in Heart Studies at The Hebrew University elected Dean of the Faculty of Medicine at The Hebrew University of Jerusalem

Reporter: Aviva Lev-Ari, PhD, RN

Professor David Lichtstein Elected Dean of Hebrew University’s Faculty of Medicine

December 2, 2013

Jerusalem — Professor David Lichtstein has been elected dean of the Faculty of Medicine at The Hebrew University of Jerusalem. Professor Lichtstein is the Walter & Greta Stiel Chair in Heart Studies at The Hebrew University. He replaces Professor Eran Leitersdorf, who recently completed his four-year term as dean.

According to Professor Lichtstein, “The Hebrew University’s Faculty of Medicine is devoted to creating innovative teaching, research and patient care programs that will meet the demands of 21st century health care. As global health care moves towaProfessor David Lichtsteinrd prevention, wellness and cost effectiveness, we are adapting how we train the next generation of physicians, nurses, pharmacists and biomedical researchers. Through fruitful collaborations between preclinical and clinical faculty, we are also translating basic biomedical insights into clinical treatments. Thus, the Faculty of Medicine is well-positioned to maintain its leading role in the scientific community of Israel and the world.”

Professor Lichtstein was born in Lodz, Poland, and immigrated to Israel with his family in 1957. As a student at The Hebrew University, he completed a Bachelor’s degree in Physiology and Zoology in 1970, followed by a Master’s degree in Physiology in 1972 and a Ph.D. in Physiology in 1977. He joined the Department of Physiology of The Hebrew University-Hadassah Medical School in 1980 as a lecturer, and received full professorship in 1994. Prof. Lichtstein has held many roles at The Hebrew University and its Faculty of Medicine, including Chairman of the Neurobiology Teaching Division, Chairman of the Department of Physiology, Chairman of the Institute for Medical Sciences and, until recently, Chairman of the Faculty of Medicine. From 2007 to 2011, Professor Lichtstein was the Jacob Gitlin Chair in Physiology at The Hebrew University. In 2011 he was named the Walter & Greta Stiel Chair in Heart Studies at The Hebrew University. He also served as the President of the Israel Society for Physiology and Pharmacology from 1996 to 1999.

From 1977-1979 Professor Lichtstein was a Postdoctoral Fellow at the Roche Institute of Molecular Biology in New Jersey. He was a visiting scientist at the National Institute of Child Health and Human Development (1985-1986) and the Eye Institute (1997-1998) at the National Institutes of Health in Maryland, and a visiting professor at the Toledo School of Medicine in Ohio (2007).

Professor. Lichtstein’s main research focus is the regulation of ion transport across the plasma membrane of eukaryotic cells. His work led to the discovery that specific steroids that were known to be present in plants and amphibians are actually normal constituents of the human body and have crucial roles, such as the regulation of cell viability, heart contractility, blood pressure and brain function. His research has implications for the fundamental understanding of body functions, as well as for several pathological states such as heart failure, hypertension and neurological and psychiatric diseases.


Field of Study

Regulation of ion transport across the plasma membrane:
The primary focus of the research in my laboratory is the regulation of ion transport across the plasma membrane of eukaryotic cells. In particular, we study the main transport system for sodium and potassium, the sodium-potassium-ATPase, and its regulation by cardiac steroids.
Specific areas of interest:
Identification of endogenous cardiac steroids in mammalian tissue; The biological consequences of the interaction of cardiac steroids with the sodium-potassium-ATPase; Biosynthesis of the cardiac steroids in the adrenal gland; Effects of endogenous sodium-potassium-ATPase inhibitors on cell differentiation; Determination of the levels of endogenous sodium-potassium-ATPase inhibitors in pathological states, including hypertension, preeclampsia; malignancies (cancer) and manic depressive illnesses; Involvement of the sodium-potassium–ATPase/cardiac steroids system in depressive disorders; Involvement of the sodium-potassium-ATPase/cardiac steroids system in cardiac function; Involvement of intestinal signals in the regulation of phosphate homeostasis; Volume regulation and its involvement in the mitogenic response.
Cardiac Steroids and the Na+, K+-ATPase and Cardiac Steroids
Cardiac steroids, such as ouabain, digoxin and bufalin are hormones synthesized by and released from the adrenal gland and the hypothalamus. These compounds, the structure of which resembles that of plant and amphibian and butterfly steroids, interact only with the plasma membrane Na+, K+-ATPase (Figure 1). This interaction elicits numerous specific biological responses affecting the function of cells and organs.
Topics Currently under investigation include
Cardiac Steroids
  • Ouabain
  • Bufalin
  • Dogoxin
Involvement of the sodium-potassium–ATPase/cardiac steroids system in depressive disorders
Depressive disorders, including major depression, dysthymia and bipolar disorder, are a serious and devastating group of diseases that have a major impact on the patients’ quality of life, and pose a significant concern for public health. The etiology of depressive disorders remains unclear. The Monoaminergic Hypothesis, suggesting that alterations in monoamine metabolism in the brain are responsible for the etiology of depressive disorders, is now recognized as insufficient to explain by itself the complex etiology of these diseases. Data from our and other laboratories has provided initial evidence that endogenous cardiac steroids and their only established receptor, the Na+, K+-ATPase, are involved in the mechanism underlining depressive disorders, and BD in particular. Our study (Biol. Psychiatry. 60:491-499, 2006) has proven that Na+, K+-ATPase and DLC are involved in depressive disorders particularly in manic-depression. We have also shown that specific genetic alterations in the Na+, K+-ATPase α isoforms are associated with bipolar disorders (Biol. Psychiatry, 65:985-991, 2009). Our recent study in this project (Eur. Neuropsychopharmacol. 22:72-729, 2012) showed that drugs affecting the Na+, K+-ATPase/cardiac steroids system are beneficial for the treatment of depression. Hence our work is in accordance to the proposition that mal functioning of the Na+, K+-ATPase/cardiac steroids system may be involved in manifestation of depressive disorders and identify new compounds as potential drug for the treatment of these maladies.
Involvement of the sodium-potassium-ATPase/cardiac steroids system in cardiac function
The classical and best documented effect of cardiac steroids, as their name implies, is to increase the force of contraction of heart muscle. Indeed, cardiac steroids were widely used in Western and Eastern clinical practices for the treatment of heart failure and atrial fibrillation. Despite extensive research, the mechanism underlying cardiac steroids actions have not been fully elucidated. The dogmatic explanation for cardiac steroids-induced increase in heart contractility is that the inhibition of Na+, K+-ATPase by the steroids causes an increase in intracellular Na+ which, in turn, attenuates the Na+/Ca++ exchange, resulting in an increased intracellular Ca++ concentration, and hence greater contractility. However, recent observations led to the hypothesis that the ability of cardiac steroids to modulate a number of intracellular signaling processes may be responsible for both short- and long-term changes in CS action on cardiac function. We are addressing this hypothesis using the zebrafish model and our ability to quantify heart function in-vivo. Heart contractility measurements were performed using a series of software tools for the analysis of high-speed video microscopic images, allowing the determination of ventricular heart diameter and perimeter during both diastole and systole. The ejection fraction (EF) and fractional area changes (FAC) were calculated from these measurements, providing two independent parameters of heart contractility (see attached movie bellow). We are currently testing the effect of cardiac steroids in the presence and absence of intracellular signaling pathways (MAP, AKT, IP3R) inhibitors. Reduction in the steroids ability to increase the force of contraction will serve as the first evidence, in-vivo, for the participation of the signaling processes in the molecular mechanisms responsible for the action of cardiac steroids on heart muscle.
Laboratory Techniques
We employ a broad range of preparations and techniques. These include isolated organs (arterial rings, smooth and cardiac muscle strips) and isolated nerve endings, as well as primary and established tissue-cultured cells. Our studies involve the application of biochemical and immunological techniques (transport and enzymatic activity measurements, RIA, ELISA), molecular biological techniques (e.g., Western and Northern blotting, and PCR), protein purification (HPLC), cellular techniques muscle contractility, cell proliferation and differentiation’ in-vivo measurements of heart contractility and blood flow in Zebrafish and behavior measurements in rodents.


B.Sc. in Physiology and Zoology, The Hebrew University, Jerusalem, Israel
1970-1972 M.Sc. in Physiology, Department of Physiology, The Hebrew University, Hadassah Medical School, Jerusalem, Israel.
Ph.D., Department of Physiology, Hebrew University Hadassah Medical School, Jerusalem, Israel. (Thesis: “Increased Production of Gamma Aminobutyryl choline in Cerebral Cortex Caused by Afferent Electrical Stimulation” (Thesis Advisors: Prof. J. Dobkin and Prof. J. Magnes).
Postdoctoral Fellow, Department of Physiological Chemistry and Pharmacology, Roche Institute of Molecular Biology, Nutley, New Jersey, U.S.A.
Positions held

Teaching and Research Assistant, Department of Physiology, The Hebrew University, Hadassah Medical School, Jerusalem, Israel
1972-1974 Assistant Instructor, Department of Physiology, The Hebrew University, Hadassah Medical School, Jerusalem, Israel
1975-1977 Instructor, Department of Physiology, The Hebrew University, Hadassah Medical School, Jerusalem, Israel
Postdoctoral Fellow, Department of Physiological Chemistry and Pharmacology, Roche Institute of Molecular Biology, Nutley, New Jersey, U.S.A.
Lecturer, (REVSON fellowship) Department of Physiology, The Hebrew University, Hadassah Medical School, Jerusalem, Israel
1981 (summer)
Visiting Scientist, Department of Physiological Chemistry and Pharmacology, Roche Institute of Molecular Biology, Nutley, New Jersey, USA
1983-1987 Senior Lecturer, Department of Physiology, The Hebrew University Hadassah Medical School, Jerusalem, Israel.
Visiting Scientist, Laboratory of Theoretical and Physical Biology, NICHD, National Institutes of Health, Bethesda, Maryland, USA
1988-1994 Associate Professor, Department of Physiology, The Hebrew University Hadassah Medical School, Jerusalem, Israel
1994-present Professor of Physiology, Department of Physiology, The Hebrew University Hadassah Medical School, Jerusalem, Israel
1997-1998 Visiting Scientist, Laboratory of Mechanisms of Ocular Diseases, NEI, National Institutes of Health, Bethesda, Maryland, USA
2007 (summer)
Visiting Professor, Department of Physiology, Pharmacology, Metabolism and cardiovascular Sciences, Medical Center University of Toledo, Toledo, Ohio, USA
2007-2011 Jacob Gitlin Chair in Physiology, The Hebrew University, Jerusalem, Israel
2011-present ​Walter & Greta Stiel Chair in Heart Studies, The Hebrew University, Jerusalem
Professional Membership
1979-present International Society of Neurochemistry
1979-present Israel Society for Physiological and Pharmacological
1980-present Society of Neurosciences (Europe)
1986-present The American Society of Hypertension
1992-present Israeli Society for Neurosciences
1999-present The American Physiological Society
Editorial Tasks
Serving as a Reviewer for the scientific journals:
American Journal of Hypertension Journal of Neural Transmission
American Journal of Physiology Journal of Neurochemistry
Apoptosis Journal of Pharmacology and Experimental Therapeutics
Biochemical and Biophysical Research Communications Life Sciences
Basic Journal of Physiology and Pharmacology NANO
Brain Research Neurochemistry International
Bioconjugate Chemistry Neuroscience
Cell Calcium Neurotoxicity Research
Clinical Science Pathophysiology
Endocrinology Physiology and Behavior
European Neuropsychopharmacology PNAS
General and Comparative Endocrinology Psychiatry Research
Hypertension Translational Research
Journal of Cell Sciences
University and Other Activities
1982-1985 Chairman of the Neurobiology Teaching Division, The Hebrew University, Jerusalem
1988-1994 Elected representative of the Senior Lecturers and Associate Professors for the University Senate
1989-1997 Member of the admission committee of the Medical School, The Hebrew University, Jerusalem
1990-1996 Member of the Committee for cellular biology of the graduate studies, The Hebrew University, Jerusalem
1992-1996 Member of the Teaching Committee, Faculty of Medicine, The Hebrew University, Jerusalem
Chairman, Department of Physiology, The Hebrew University, Hadassah Medical School, Jerusalem
1994-1997 Member of the Committee for graduate studies, The Hebrew University, Jerusalem
Member of the Management Committee of The Institute for Medical Sciences, Faculty of Medicine, The Hebrew University, Jerusalem
President of the Israel Society for Physiology and Pharmacology
1998- 2002 Chairman, Institute of Medical Sciences, The Hebrew University, Hadassah Medical School, Jerusalem
1999-2002 Member of the Planning and Development Committee of the Faculty of Medicine, The Hebrew University, Jerusalem
2007–Present Elected representative of the Professors for the executive University Senate
2008-2012 Member of the Planning and Development Committee of the Faculty of Medicine, The Hebrew University, Jerusalem
2008-2012 Chairman, Institute for Medical Research Israel-Canada, The Hebrew University, Hadassah Medical School, Jerusalem
2009 – Present Elected member of the Senate to the Executive Committee of the Hebrew University

PUBLICATIONS 2006 – 2012

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Dvela, M., Rosen, H., Ben-Ami, H. C., Lichtstein, D.
American journal of physiology. Cell physiology, 302(2), C442-52, 2012
Goldstein, I., Lax, E., Gispan-Herman, I., Ovadia, H., Rosen, H., Yadid, G., Lichtstein, D.
European neuropsychopharmacology : the journal of the European College of Neuropsychopharmacology, 22(1), 72-9, 2012
Nesher, M., Shpolansky, U., Viola, N., Dvela, M., Buzaglo, N., Cohen Ben-Ami, H., Rosen, H., Lichtstein, D.
British journal of pharmacology, 160(2), 346-54, 2010
Guttmann-Rubinstein, L., Lichtstein, D., Ilani, A., Gal-Moscovici, A., Scherzer, P., Rubinger, D.
Hormone and metabolic research = Hormon- und Stoffwechselforschung = Hormones et metabolisme, 42(4), 230-6, 2010
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Journal of sleep research, 19(1 Pt 2), 183-91, 2010
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Biological psychiatry, 65(11), 985-91, 2009
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Pathophysiology : the official journal of the International Society for Pathophysiology / ISP, 14(3-4), 159-66, 2007
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American journal of physiology. Cell physiology, 293(3), C885-96, 2007
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Clinical rheumatology, 26(4), 590-5, 2007
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Biological psychiatry, 60(5), 491-9, 2006
Chirinos, J. A., Garcia, J., Alcaide, M. L., Toledo, G., Baracco, G. J., Lichtstein, D. M.
American journal of cardiovascular drugs : drugs, devices, and other interventions, 6(1), 9-14, 2006
Rosen, H., Glukmann, V., Feldmann, T., Fridman, E., Lichtstein, D.
Cellular and molecular biology (Noisy-le-Grand, France), 52(8), 78-86, 2006



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The Role of Tight Junction Proteins in Water and Electrolyte Transport


Reviewer and Curator: Larry H. Bernstein, MD, FCAP 


This article is Part II of a series that explores the physiology, genomics, and the proteomics of water and electrolytes in human and mammalian function in health and disease.  In this portion of curation, we examine the role of special proteins at the tight junctions of cells, including the claudins.  Consistent with the exploration of cation homeostasis, the last featured article is one of the altered handling of calcium (Ca2+) in CHF, and the closely regulated calcium efflux by the sodium-calcium exchanger (NCX).

The Role of Aquaporin and Tight Junction Proteins in the Regulation of Water Movement in Larval Zebrafish (Danio rerio).

Kwong RW, Kumai Y, Perry SF.
Department of Biology, University of Ottawa, Ottawa, Ontario, Canada.
PLoS One. 2013 Aug 14;8(8):e70764.   eCollection 2013.

Teleost fish living in freshwater are challenged by passive water influx; however the molecular mechanisms regulating water influx in fish are not well understood. The potential involvement of aquaporins (AQP) and epithelial tight junction proteins in the regulation of transcellular and paracellular water movement was investigated in larval zebrafish (Danio rerio).

We observed that the half-time for saturation of water influx (K u) was 4.3±0.9 min, and reached equilibrium at approximately 30 min. These findings suggest a high turnover rate of water between the fish and the environment. Water influx was reduced by the putative AQP inhibitor phloretin (100 or 500 μM). Immunohistochemistry and confocal microscopy revealed that AQP1a1 protein was expressed in cells on the yolk sac epithelium. A substantial number of these AQP1a1-positive cells were identified as ionocytes, either H(+)-ATPase-rich cells or Na(+)/K(+)-ATPase-rich cells. AQP1a1 appeared to be expressed predominantly on the basolateral membranes of ionocytes, suggesting its potential involvement in regulating ionocyte volume and/or water flux into the circulation.

Additionally, translational gene knockdown of AQP1a1 protein reduced water influx by approximately 30%, further indicating a role for AQP1a1 in facilitating transcellular water uptake. On the other hand, incubation with the Ca(2+)-chelator EDTA or knockdown of the epithelial tight junction protein claudin-b significantly increased water influx. These findings indicate that the epithelial tight junctions normally act to restrict paracellular water influx. Together, the results of the present study provide direct in vivo evidence that water movement can occur through transcellular routes (via AQP); the paracellular routes may become significant when the paracellular permeability is increased.

PMID:  23967101  PMCID: PMC3743848

The tight junction protein claudin-b regulates epithelial permeability and sodium handling in larval zebrafish, Danio rerio.

Kwong RW, Perry SF.
Department of Biology, University of Ottawa, Ottawa, Ontario, Canada.
Am J Physiol Regul Integr Comp Physiol. Apr 1, 2013; 304(7):R504-13.  Epub 2013 Jan 30.

The functional role of the tight junction protein claudin-b in larval zebrafish (Danio rerio) was investigated. We showed that claudin-b protein is expressed at epithelial cell-cell contacts on the skin. Translational gene knockdown of claudin-b protein expression caused developmental defects, including edema in the pericardial cavity and yolk sac.

Claudin-b morphants exhibited an increase in epithelial permeability to the paracellular marker polyethylene glycol (PEG-4000) and fluorescein isothiocyanate-dextran (FD-4). Accumulation of FD-4 was confined mainly to the yolk sac and pericardial cavity in the claudin-b morphants, suggesting these regions became particularly leaky in the absence of claudin-b expression.

Additionally, Na(+) efflux was substantially increased in the claudin-b morphants, which contributed to a significant reduction in whole-body Na(+) levels. These results indicate that claudin-b normally acts as a paracellular barrier to Na(+). Nevertheless, the elevated loss of Na(+) in the morphants was compensated by an increase in Na(+) uptake.

Notably, we observed that the increased Na(+) uptake in the morphants was attenuated in the presence of the selective Na(+)/Cl(-)-cotransporter (NCC) inhibitor metolazone, or during exposure to Cl(-)-free water. These results suggested that the increased Na(+) uptake in the morphants was, at least in part, mediated by NCC. Furthermore, treatment with an H(+)-ATPase inhibitor bafilomycin A1 was found to reduce Na(+) uptake in the morphants, suggesting that H(+)-ATPase activity was essential to provide a driving force for Na(+) uptake. Overall, the results suggest that claudin-b plays an important role in regulating epithelial permeability and Na(+) handling in zebrafish.
PMID: 23364531

Evidence for a role of tight junctions in regulating sodium permeability in zebrafish (Danio rerio) acclimated to ion-poor water.

Kwong RW, Kumai Y, Perry SF.
Department of Biology, University of Ottawa, Ottawa, ON, Canada.
J Comp Physiol B. Feb 2013 ;183(2):203-13.  Epub 2012 Jul 29.

Freshwater teleosts are challenged by diffusive ion loss across permeable epithelia including gills and skin. Although the mechanisms regulating ion loss are poorly understood, a significant component is thought to involve paracellular efflux through pathways formed via tight junction proteins. The mammalian orthologue (claudin-4) of zebrafish (Danio rerio) tight junction protein, claudin-b, has been proposed to form a cation-selective barrier regulating the paracellular loss of Na(+).

The present study investigated the cellular localization and regulation of claudin-b, as well as its potential contribution to Na(+) homeostasis in adult zebrafish acclimated to ion-poor water. Using a green fluorescent protein-expressing line of transgenic zebrafish, we found that claudin-b was expressed along the lamellar epithelium as well as on the filament in the inter-lamellar regions. Co-localization of claudin-b and Na(+)/K(+)-ATPase was observed, suggesting its interaction with mitochondrion-rich cells. Claudin-b also appeared to be associated with other cell types, including the pavement cells. In the kidney, claudin-b was expressed predominantly in the collecting tubules. In addition,

exposure to ion-poor water caused a significant increase in claudin-b abundance as well as a decrease in Na(+) efflux, suggesting a possible role for claudin-b in regulating paracellular Na(+) loss. Interestingly, the whole-body uptake of a paracellular permeability marker, polyethylene glycol-400, increased significantly after prolonged exposure to ion-poor water, indicating that an increase in epithelial permeability is not necessarily coupled with an increase in passive Na(+) loss. Overall, our study suggests that in ion-poor conditions, claudin-b may contribute to a selective reduction in passive Na(+) loss in zebrafish.
PMID: 22843140

Claudin-16 and claudin-19 function in the thick ascending limb.

Hou J, Goodenough DA.
Washington University School of Medicine, Div Renal Diseases, St Louis, Missouri
Curr Opin Nephrol Hypertens. Sep 2010; 19(5):483-8.

The thick ascending limb (TAL) of the loop of Henle is responsible for reabsorbing 25–40% of filtered Na+, 50–60% of filtered Mg2+ and 30–35% of filtered Ca2+. The dissociation of salt and water reabsorption in the TAL serves both to dilute the urine and to establish the corticomedullary osmolality gradient. Active transcellular salt reabsorption results in a lumen-positive transepithelial voltage that drives passive paracellular reabsorption of divalent cations. Claudins are the key components of the paracellular channel. The paracellular channels in the tight junction have properties of ion selectivity, pH dependence and anomalous mole fraction effects, similar to conventional transmembrane channels. Genetic mutations in claudin-16 and claudin-19 cause an inherited human renal disorder, familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC).

In the TAL of Henle’s loop, the epithelial cells form a water-impermeable barrier, actively transport Na+ and Cl− via the transcellular route, and provide a paracellular pathway for the selective absorption of cations. Na+ K+ and Cl− enter the cell through the Na-K-2Cl cotransporter (NKCC2) in the luminal membrane. Na+ exits the cell through the Na+/K+-ATPase, in exchange for K+ entry. K+ is secreted into the lumen through the renal outer medullary potassium channel. Cl− leaves the cell through the basolateral Cl− channel, made up of two subunits, ClCKb and barttin. The polarized distribution of luminal K+ versus basolateral Cl− conductance generates a spontaneous voltage source (Vsp) of +7−8mV , depending on active transcellular NaCl reabsorption. With continuous NaCl reabsorption along the axis of the TAL segment, the luminal fluid is diluted to 30–60mmol/l  and a large NaCl transepithelial chemical gradient develops at the end of the TAL. Because the paracellular permeability of the TAL is cation-selective (with a PNa/PCl value between 2 and 4), the diffusion voltage (Vdi) is superimposed onto the active transport voltage (Vsp) and becomes the major source of the transepithelial voltage (Vte), which now increases up to +30mV.

Early in-vivo micropuncture studies have shown that approximately 50–60% of the filtered Mg2+ is reabsorbed in the TAL. The flux–voltage relationship indicates that Mg2+ is passively reabsorbed from the lumen to the peritubular space through the paracellular pathway in this segment, driven by a lumen positive Vte.  Vte is made of the sum of Vsp and Vdi. There are two prerequisites required for the paracellular Mg2+ reabsorption in the TAL: the lumen-positive Vte as the driving force and the paracellular permeability for the divalent cation Mg2+.

Claudin-16 and claudin-19 underlie familial hypercalciuric hypomagnesemia with nephrocalcinosis

Claudin-16 and claudin-19 play a major role in the regulation of magnesium reabsorption in the thick ascending limb (TAL). This review describes recent findings of the physiological function of claudin-16 and claudin-19 underlying normal transport function for magnesium reabsorption in the TAL. Mutations in the genes encoding the tight junction proteins claudin-16 and claudin-19 cause the inherited human renal disorder familial hypomagnesemia with hypercalciuria and nephrocalcinosis. FHHNC, OMIM #248250, is a rare autosomal recessive tubular disorder. As a consequence of excessive renal Mg2+ and Ca2+ wasting, patients develop the characteristic triad of hypomagnesemia, hypercalciuria and nephrocalcinosis. Recurrent urinary tract infections and polyuria/polydipsia are frequent initial symptoms. Other clinical symptoms include nephrolithiasis, abdominal pain, convulsions, muscular tetany, and failure to thrive. Additional laboratory findings include elevated serum parathyroid hormone levels before the onset of chronic renal failure, incomplete distal tubular acidosis, hypocitraturia, and hyperuricemia. In contrast to hypomagnesemia and secondary hypocalcemia (HSH, OMIM #602014), FHHNC is generally complicated by end-stage renal failure in early childhood or adolescence.

Simon et al. used the positional cloning strategy and identified claudin-16 (formerly known as paracellin-1), which is mutated in patients with FHHNC. Most mutations reported to date in claudin-16 are missense mutations clustering in the first extracellular loop composing the putative ion selectivity filter. Konrad et al. have found mutations in another tight junction gene encoding claudin-19 from new cohorts of FHHNC patients (OMIM #248190). The renal tubular phenotypes are indistinguishable between patients with mutations in claudin-16 and those with mutations in claudin-19. Although claudin-16 and claudin-19 underlie FHHNC and paracellular Mg2+ reabsorption in the TAL, the transient receptor potential channel melastatin 6 (TRPM6) regulates the apical entry of Mg2+ into the distal convoluted tubule epithelia. Mutations in TRPM6 cause the HSH syndrome.

These above data suggested the hypothesis that claudin-16 and/or claudin-19 forms a selective paracellular Mg2+/Ca2+ channel, which was tested in a number of in-vitro studies. Ikari et al. transfected low-resistance Madin-Darby canine kidney (MDCK) cells with claudin-16 and reported that the Ca2+ flux in these cells was increased in the apical to basolateral direction, whereas the Ca2+ flux in the opposite direction remained unchanged. The Mg2+ flux was without any noticeable change. Kausalya et al.  transfected the high-resistance MDCK-C7 cell line and found that claudin-16 only moderately increased Mg2+ permeability without any directional preference. The effects of claudin-16 on Mg2+/Ca2+ permeation appeared so small (<50%) that the Mg2+/Ca2+ channel theory incompletely explains the dramatic effect of mutations in claudin-16 on Mg2+ and Ca2+ homoeostasis in FHHNC patients. However, , Hou et al.  transfected the anion-selective LLC-PK1 cell line with claudin-16 and found a large increase in Na+ permeability (PNa) accompanied by a moderately enhanced Mg2+ permeability (PMg). The permeability of claudin-16 to other alkali metal cations was found to be: K+ > Rb+ > Na+.  Yu et al. emphasized that these residue replacements can influence protein structures that may have impacts on ion permeability independent of amino acid charge.

The cation selectivity of the tight junction is vital for generating the lumen positive transepithelial potential in the TAL, which drives paracellular absorption of magnesium. Claudin-16 and claudin-19 require each other for assembly into tight junctions in the TAL. Heteromeric claudin-16 and claudin-19 interaction forms a cation selective tight junction paracellular channel. Loss of either claudin-16 or claudin-19 in the mouse kidney abolishes the cation selectivity for the TAL paracellular pathway, leading to excessive renal wasting of magnesium.

Claudins interact with each other both intracellularly and intercellularly: they copolymerize linearly within the plasma membrane of the cell, together with the integral protein occludin, to form the classical intramembrane fibrils or strands visible in freeze-fracture replicas. These intramembrane interactions (side-to-side) can involve one claudin protein (homomeric or homopolymeric) or different claudins (heteromeric or heteropolymeric). In the formation of the intercellular junction, claudins may interact head-to-head with claudins in an adjacent cell, generating both homotypic and heterotypic claudin–claudin interactions. Using the split-ubiquitin yeast 2-hybrid assay, Hou et al. found strong claudin-16 and claudin-19 heteromeric interaction. The point mutations in claudin-16 (L145P, L151F, G191R, A209T, and F232C) or claudin-19 (L90P and G123R) that are known to cause human FHHNC disrupted the claudin-16 and claudin-19 heteromeric interaction. In mammalian cells such as the human embryonic kidney 293 cells, claudin-16 can be coimmunoprecipitated with claudin-19. Freeze-fracture replicas revealed the assembly of tight junction strands in L cells coexpressing claudin-16 and claudin-19, supporting their heteromeric interaction.

Coexpression of claudin-16 and claudin-19 in LLC-PK1 cells resulted in a dramatic upregulation of PNa and down-regulation of PCl, generating a highly cation-selective paracellular pathway. Certain FHHNC mutations in claudin-16 (L145P, L151F, G191R, A209T, and F232C) or claudin-19 (L90P and G123R) that disrupted their heteromeric interaction abolished this physiological change. As claudin-16 colocalizes with claudin-19 in the TAL epithelia of the kidney, claudin-16 and claudin-19 association through heteromeric interactions confers cation selectivity to the tight junction in the TAL. Human FHHNC mutations in claudin-16 or claudin-19 that abolish the cation selectivity diminish the lumen-positive Vdi as the driving force for Mg2+ and Ca2+ reabsorption, readily explaining the devastating phenotypes in FHHNC patients.  Hou et al. generated claudin-16 deficient mouse models using lentiviral transgenesis of siRNA to knock down claudin-16 expression by more than 99% in mouse kidneys. Claudin-16 knockdown mice show significantly reduced plasma Mg2+ levels and excessive urinary excretions (approximately four-fold) of Mg2+ and Ca2+. Calcium deposits are observed in the basement membranes of the medullary tubules and the interstitium in the kidney of claudin-16 knockdown mice. These phenotypes of claudin-16 knockdown mice recapitulate the symptoms in human FHHNC patients.

The paracellular reabsorption of Mg2+ and Ca2+ is driven by a lumen-positive Vte made up of two components: Vsp and Vdi. When isolated TAL segments were perfused ex vivo with symmetrical NaCl solutions, there was no difference in Vsp between claudin-16 knockdown and wild-type mice, indicating Vsp was normal in claudin-16 knockdown. Blocking the NKCC2 channel with furosemide (thus dissipating VSP), the cation selectivity (PNa/PCl) was significantly reduced from3.1 ± 0.3 in wild type to 1.5 ± 0.1 in claudin-16 knockdown, resulting in the loss of Vdi. When perfused with a NaCl gradient of 145mmol/l (bath) versus 30mmol/l (lumen), the resulting Vdi was +18mV in wild type, but only +6.6mV in claudin-16 knockdown. Thus, the reduction in Vdi accounted for a substantive loss of the driving force for Mg2+ and Ca2+ reabsorption.

Renal handling of Na+ in claudin-16 knockdown mice is more complex. In the early TAL segment, the transcellular and paracellular pathways form a current loop in which the currents traversing the two pathways are of equal size but opposite direction. Net luminal K+ secretion and basolateral Cl− absorption polarize the TAL epithelium and generate Vsp. As the paracellular pathway is cation selective (PNa/PCl=2–4 , the majority of the current driven by Vsp through the paracellular pathway is carried by Na+ moving from the lumen to the interstitium. Hebert et al. estimated that, for each Na+ absorbed through the trans-cellular pathway, one Na+ is absorbed through the paracellular pathway. With the loss of claudin-16 and the concomitant loss of paracellular cation selectivity, Na+ absorption through the paracellular pathway is reduced.  In the late TAL segment, dilution of NaCl in the luminal space creates an increasing chemical transepithelial gradient; back diffusion of Na+ through the cation-selective tight junction generates a lumen-positive Vdi across the epithelium. The paracellular absorption of Na+ will be diminished when Vdi equals Vsp, and reversed when Vdi exceeds Vsp. As an equilibrium potential, Vdi blocks further Na+ backleak into the lumen. Without claudin-16, Vdi will be markedly reduced well below normal, providing a driving force for substantial Na+ secretion. Indeed, claudin-16 knockdown mice had increased fractional excretion of Na+ (FENa) and developed hypotension and secondary hyperaldosteronism. The observed Na+ and volume loss are consistent with human FHHNC phenotypes. For example, polyuria and polydipsia are the most frequently reported symptoms from FHHNC patients.

Epithelial paracellular channels are increasingly understood to be formed from claudin oligomeric complexes. In the mouse TAL, claudin-16 and claudin-19 cooperate to form cation-selective paracellular channels required for normal levels of magnesium reabsorption. Different subsets of the claudin family of tight junction proteins are found distributed throughout the nephron, and understanding their roles in paracellular ion transport will be fundamental to understanding renal ion homeostasis.

Keywords: claudin; hypomagnesemia; thick ascending limb; tight junction; transepithelial voltage.     PMID: 20616717  PMCID: PMC3378375

Function and regulation of claudins in the thick ascending limb of Henle.

Günzel D, Yu AS.
Depart Clin Physiol, Charité, Campus Benjamin Franklin, Berlin, Germany.
Pflugers Arch. May 2009; 458(1):77-88.  Epub 2008 Sep 16.

The thick ascending limb (TAL) of Henle mediates transcellular reabsorption of NaCl while generating a lumen-positive voltage that drives passive paracellular reabsorption of divalent cations. Disturbance of paracellular reabsorption leads to Ca(2+) and Mg(2+) wasting in patients with the rare inherited disorder of familial hypercalciuric hypomagnesemia with nephrocalcinosis (FHHNC). Recent work has shown that the claudin family of tight junction proteins form paracellular pores and determine the ion selectivity of paracellular permeability. Importantly, FHHNC has been found to be caused by mutations in two of these genes, claudin-16 and claudin-19, and mice with knockdown of claudin-16 reproduce many of the features of FHHNC. Here, we review the physiology of TAL ion transport, present the current view of the role and mechanism of claudins in determining paracellular permeability, and discuss the possible pathogenic mechanisms responsible for FHHNC.

Tight junctions form the paracellular barrier in epithelia. Claudins are ~22 kDa proteins that were first identified by Mikio Furuse in the laboratory of the late Shoichiro Tsukita as proteins that copurified in a tight junction fraction from the chicken liver [23]. The observation that they were transmembrane proteins with 4 predicted membrane domains and 2 extracellular domains raised early on the possibility that they could play a key role in intercellular adhesion and formation of the paracellular barrier. In 1999, Richard Lifton’s group identified mutations in a novel gene, which they called paracellin, as the cause of familial hypercalciuric hypomagnesemia, an inherited disorder believed to be due to failure of paracellular reabsorption of divalent cations in the thick ascending limb of the renal tubule. Paracellin turned out to be a claudin family member (claudin-16). This suggested that claudins in general might be directly involved in regulating paracellular transport in all epithelia. This is now supported by numerous studies demonstrating that overexpressing or ablating expression of various claudin isoforms in cultured cell lines or in mice affects both the degree of paracellular permeability and its selectivity (vide infra). Furthermore, in mammals alone there are ~24 claudin genes and each exhibits a distinct tissue-specific, pattern of expression. Thus, the specific claudin isoform(s) expressed in each tissue might explain its paracellular permeability properties.

Each nephron segment expresses a unique set of multiple claudin isoforms, and each isoform is expressed in multiple segments, thus making a complicated picture which even varies between different species. The role of combinations of claudins in determining paracellular permeability properties has hardly been studied yet. In mouse, rabbit and cattle, the thick ascending limb of Henle’s loop is thought to express claudins 3, 10, 11, 16  in adulthood, and, at least in mouse, additionally claudin-6 during development. In addition, claudin-4 has been found in cattle and claudin-8 in rabbit. To date, the distribution of Claudin-19 has been investigated in mouse, rat, and man where its presence in the TAL was demonstrated.

The thick ascending limb (TAL) of Henle’s loop, working as “diluting segment” of the nephron, is characterized by two major properties: high transepithelial, resorptive transport of electrolytes and low permeability to water. Major players to achieve electrolyte transport are the apical Na+-K+-2Cl−symporter (NKCC2), the apical K+ channel ROMK, the basolateral Cl− channel (CLC-Kb) together with its subunit barttin and the basolateral Na+/K+-ATPase . The combined actions of these transport systems have been extensively reviewed and are therefore only briefly summarized here. Na+ and Cl− are resorbed by entering the cells apically through NKCC2 and leaving the cells basolaterally through the Na+/K+-ATPase and CLC-Kb, respectively. In contrast, K+ is either recycled across the apical membrane as it is entering through NKCC2 and leaving through ROMK, or even secreted, as it is also entering the cells basolaterally through the Na+/K+-ATPase. Taking these ion movements together, there is a net movement of positive charge from the basolateral to the apical side of the epithelium, giving rise to a lumen positive voltage (3 – 9 mV [11]; about 5 – 7 mV [30,31]; 7 – 8 mV [57]). Over the length of the TAL, luminal NaCl concentration decreases gradually to concentrations of 30 – 60 mM at the transition to the distal tubule, depending on the flow rate within the tubule (low flow rates resulting in low concentrations).

To keep up such a high gradient, the TAL epithelium has to be tight to water and various studies summarized by Burg and Good report water permeability values from 28 µm/s down to values indistinguishable from zero. Tight junctions of the TAL are, however, highly permeable to cations with PNa being about 2 – 2.7 fold, 2.5 fold or even up to 6 fold that of PCl. Amongst the monovalent cations, a permeability sequence of PK > PNa > PRb = PLi > PCs > Porganic cation was observed which is similar to Eisenman sequence VIII or IX, indicating a strong interaction between the permeating ion and the paracellular pore that enables at least partial removal of the hydration shell (see below). As reviewed by Burg and Good, the transepithelial sodium and chloride permeabilities, estimated from radioisotope fluxes, are high (in the range of 10 – 63·10−6 cm/s) and the transepithelial electrical resistance is correspondingly low (21 – 25 W cm2 ; 30 – 40 W cm2 ; 11 – 34 W cm2 . Blocking active transport by the application of furosemide or ouabain increases transepithelial resistance only slightly, indicating that the low values are primarily due to a very high paracellular permeability. Due to these properties of TAL epithelial cells, Na+ ions leak back into the lumen of the tubule, creating a diffusion (dilution) potential that adds another 10 – 15 mV to the lumen positive potential, so that considerable potential differences (25 mV ; 30 mV; cTAL 23 mV, mTAL 17 mV ) may be reached at very slow flow rates.

Considerable proportions of the initially filtrated Mg2+ (50 – 60%; 50 – 70%; 65 – 75%) and Ca2+ (20%; 30 – 35% are resorbed in the TAL. The transepithelial potential is considered to provide the driving force for the predominantly paracellular resorption of Mg2+ and Ca2+ as in many studies, transport of both divalent ions in the TAL has been found to be strictly voltage dependent (resorbtive at lumen positive potentials, zero at 0 mV and secretory at lumen negative potentials) and permeability considerable (PCa 7.7·10−6 cm/s, i.e. approximately 25% of PNa). There is however, some conflicting evidence, e.g. by Suki et al. and Friedman. Both studies used cTAL (cortical TAL) and found that decreasing the transepithelial potential by applying furosemide did either not alter the unidirectional lumen to bath Ca2+ flux (rabbit) or left a substantial net Ca2+ resorption (mouse). Similarly, Rocha et al. found that bath application of ouabain almost abolished the transepithelial potential, but hardly affected net Ca2+ resorption and conclude that (a) all segments of Henle’s loop are relatively impermeable to calcium and (b) net calcium resorption occurs in the thick ascending limb which cannot be explained by passive mechanisms.  Mandon et al. conclude that both Mg2+ and Ca2+ are transported in the cTAL but not in the mTAL (medullary TAL) of rat and mouse, although transepithelial potential differences were similar in both segments, and even if the transepithelial potential was experimentally elevated to values above 20 mV. Wittner et al. even found evidence that in mouse mTAL the passive permeability to divalent cations is very low and that Ca2+ and Mg2+ can be secreted into the luminal fluid under conditions which elicit large lumen-positive transepithelial potential differences. They conclude that this Ca2+ and Mg2+ transport is most probably of cellular origin. In contrast, in rabbit, both ions are transported along the whole length of the TAL.

Both, Mg2+ and Ca2+ resorption are modulated through the action of the basolateral Ca (and Mg) sensing receptor (CaSR) which is found along the entire nephron but especially in the loop of Henle, distal convoluted tubule (DCT) and the inner medullary collecting duct. Different modes of action on Ca2+ and Mg2+ homeostasis exist, such as an indirect action through the modulation of PTH secretion or direct effects on the cells expressing CaSR. In the TAL the latter model is based on the assumptions depicted above, i.e. that Mg2+ and Ca2+ are resorbed paracellularly, driven by the lumen positive potential, so that a reduction in NaCl resorption causes a reduction in driving force for Mg2+ and Ca2+ resorption. As reviewed by Hebert and Ward, CaSR is activated through an increase in basolateral Ca2+ and/or Mg2+ concentration which triggers an increase in the intracellular Ca2+ concentration. This reduces the activity of the adenylate cyclase which, in turn, inhibits transcellular transport of Na+ and Cl−. In addition, the increase in intracellular Ca2+ activates phospholipase A2 (PLA2) and thus increases the intracellular concentration of arachidonic acid and its derivative, 20-HETE. 20-HETE inhibits NKCC2, ROMK and the Na+/K+-ATPase and by this Mg2+ and Ca2+ resorption. In keeping with this hypothesis, mutations in CaSR affect Ca/Mg resorption. Inactivating mutations cause hypercalcemia, hypocalciuria, hypomagnesiuria and, in some patients hypermagnesemia. Conversely, activating mutations (gain of function mutations) lead to hypocalcemia, hypercalciuria, hypermagnesiuria and in up to 50% of the patients mild hypomagnesemia.

Bartter syndrome type I (mutations in NKCC2), and type II (mutations in ROMK) lead to hypercalciuria and thus cause nephrocalcinosis, but no hypomagnesemia is observed. Reports on hypermagnesiuria are conflicting: while Kleta and Bockenhauer link it to nephrocalcinosis seen in these patients, Rodriguez-Soriano states that patients with neonatal Bartter syndrome (i.e. type I or II) show a lack of hypermagnesiuria that may be explained by compensation in the DCT. Hypomagnesemia is occationally present in Bartter type III (CLC-Kb). However, here it is believed to be mainly due to effects on DCT, where CLC-Kb shows highest expression. Patients with Bartter syndrome IV (CLC-Kbsubunit barttin) may or may not present nephrocalcinosis, while Mg2+ homeostasis appears undisturbed. Interestingly, the largest effects on Mg2+-homeostasis are observed in Gitelman syndrome, a defect in the Na+/Cl− symport (NCC) predominantly found in the DCT, where Mg2+ is transported along the transcellular route. Affected patients suffer from hypomagnesemia, hypermagnesuria and hypocalciuria. The effect on Mg2+ in Gitelman syndrome is still poorly understood and possibly due to a concomitant down-regulation of TRPM6, the apical Mg2+ uptake channel in DCT.

More than 30 different claudin-16 mutations have now been reported in families with FHHNC. Because of the large number of unique mutations, it has not been possible to identify any clear qualitative correlation between the phenotype and individual mutations, although certain mutations are associated with greater severity of disease. In 2006, a second locus was identified, CLDN19, which encodes claudin-19. In the initial report, the phenotype appeared similar to that due to claudin-16 mutations, with the exception that there was a high prevalence of ocular abnormalities, including macular colobomata, nystagmus and myopia. Claudin-19 is normally expressed at high levels in the retina, but why it causes these ocular disorders is unknown.

In vitro studies of claudin function comprise inducible or non-inducible transfection of various cells lines with cDNA for claudins that are not endogenously expressed by the cell line used. Alternatively, cells can be transfected with siRNA directed against an endogenous claudin. In both cases, cells are then grown to confluence on permeable filter supports that allow measurement of transepithelial permeabilities. Before the results of permeability studies can be interpreted, however, several parameters have to be controlled.

First, special care has to be taken to make sure that the exogenous claudin is correctly inserted into the tight junction. This can be achieved e.g. by confocal laser scanning microscopy, colocalizing the claudin of interest  with a tight junction marker protein such as occludin.

Second, it has to be ensured that endogenous claudin expression remains unaffected, as permeability changes always result from the combined effects of alterations in endogenous and exogenous claudins.

Third, it has to be kept in mind that, typically, epithelia express several different claudins that act together to produce tissue specific permeability properties. Thus, ideally, a cell line should be chosen that provides a claudin background resembling that usually experienced by the claudin investigated. The latter two points may be the reason for contradicting results obtained in permeability studies expressing a specific claudin in different cell lines.

Studies of paracellular permeabilities can be divided into two groups, those employing electrophysiological measurements (e.g. determination of diffusion potentials), and those measuring ion or solute flux, using either radioactive isotopes or various analytical methods to determine the amount transported.

Although transepithelial conductances depend on paracellular permeabilities of the predominant ions in the bath solution, conductance changes alone cannot be used to predict ion permeabilities.

Firstly, conductances always depend on both ion and counter-ion, not on one ion species alone.

Secondly, transepithelial conductances are the sum of the conductances of the transcellular and paracellular pathways.

Thus, they only reflect paracellular permeability, if paracellular conductance dominates transepithelial conductance and if transcellular conductance remains constant throughout the experiment. This, however, is often not the case, as concentration changes of the ions investigated may affect transcellular conductance, e.g. by activating ion transporters or by inhibiting ion channels. Thirdly, the specific conductance of each solution employed may differ and has therefore to be assessed and taken into account. Thus, comparison of results from diffusion potential measurements or flux studies and conductance measurements may even yield contradicting results. For the same reasons, other methods based on pure conductance/resistance measurements, including the more sophisticated conductance scanning method or one-path impedance spectroscopy are not ion specific and do not allow the measurement of paracellular permeabilities to single ions.

In contrast to electrophysiological measurements, flux measurements are not limited to ions but can also be extended to uncharged molecules.  Flux measurements per se do not distinguish between transcellular or paracellular transport. Therefore, to estimate paracellular permeabilities, inhibition or at least estimation of the transcellular flux is necessary. Assuming that transcellular flux for energetic reasons is not easily reversible while paracellular flux is passive and thus generally assumed to be symmetric, the transcellular proportion is often estimated by calculating the difference between apical to basolateral and basolateral to apical fluxes. All flux measurements are very sensitive to the development of diffusion zones (“unstirred layers”) near the cells. These layers are depleted/enriched in the compound transported and thus alter the driving forces acting on these compounds, if bath solutions are not continually circulated. If ionic fluxes are investigated, transepithelial potentials may develop that diminish or completely inhibit the flux investigated.

All the techniques described above have been employed to investigate the function of claudin-16 and -19, especially with respect to their ability to increase paracellular permeability to divalent cations. The hypothesis, that claudin-16 (then called paracellin-1) may be a paracellular Mg2+ and Ca2+ pore was originally expressed by Simon et al. It was based on the findings that mutations in claudin-16 were the cause of the severe disturbance in Mg2+ and Ca2+ homeostasis in FHHNC patients together with the observations that claudin-16 is a tight junction protein located in the TAL, i.e. the nephron segment responsible for bulk Mg2+ resoption along the paracellular pathway. When, recently, it was found that claudin-19 mutations were underlying hitherto unexplained cases of FHHNC and that claudin-19 co-localized with claudin-16, the hypothesis was extended to claudin-19.

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PMID: 18795318  PMCID:  PMC2666100

Deletion of claudin-10 (Cldn10) in the thick ascending limb impairs paracellular sodium permeability and leads to hypermagnesemia and nephrocalcinosis.

Breiderhoff T, Himmerkus N, Stuiver M, Mutig K, Will C, Meij IC et al.
Max Delbrück Center for Molec Med, Berlin, Germany.

Erratum in Proc Natl Acad Sci. 2012 Sep 11;109(37):15072.
Proc Natl Acad Sci. Aug 28, 2012; 109(35):14241-6. Epub 2012 Aug 13.

In the kidney, tight junction proteins contribute to segment specific selectivity and permeability of paracellular ion transport. In the thick ascending limb (TAL) of Henle’s loop, chloride is reabsorbed transcellularly, whereas sodium reabsorption takes transcellular and paracellular routes. TAL salt transport maintains the concentrating ability of the kidney and generates a transepithelial voltage that drives the reabsorption of calcium and magnesium. Thus, functionality of TAL ion transport depends strongly on the properties of the paracellular pathway. To elucidate the role of the tight junction protein claudin-10 in TAL function, we generated mice with a deletion of Cldn10 in this segment. We show that claudin-10 determines paracellular sodium permeability, and that its loss leads to hypermagnesemia and nephrocalcinosis. In isolated perfused TAL tubules of claudin-10-deficient mice, paracellular permeability of sodium is decreased, and the relative permeability of calcium and magnesium is increased. Moreover, furosemide-inhibitable transepithelial voltage is increased, leading to a shift from paracellular sodium transport to paracellular hyperabsorption of calcium and magnesium. These data identify claudin-10 as a key factor in control of cation selectivity and transport in the TAL, and deficiency in this pathway as a cause of nephrocalcinosis.

Whereas regulation of transporters and channels involved in trans-cellular ion transport has been characterized in much detail, the functional and molecular determinants of paracellular ion trans­port in the kidney remain incompletely understood. In the thick ascending limb (TAL) of Henle’s loop, both trans-cellular and paracellular ion transport pathways contribute to reabsorption of Na+, Cl, Mg2+, and Ca2+. Na+ and Clare reabsorbed mostly transcellularly by the concerted action of chan­nels and transporters. Mutations in five of the genes involved lead to Bartter syndrome, a disorder characterized by salt wasting and polyuria. Whereas Clis transported exclusively transcellularly, 50% of the Na+ load, as well as Ca2+ and Mg2+, are reabsorbed via paracellular pathways. In the TAL, this paracellular route is highly cation-selective. The paracellular passage is largely controlled by the tight junction (TJ), a supramolecular structure of membrane-spanning proteins, their intracellular adapters, and scaffolding proteins. Claudins, a family comprising 27 members, are the main components of the TJ defining the permeability properties. They interact via their extracellular loops with corre­sponding claudins of the neighboring cell to allow or restrict pas­sage of specific solutes (5, 6). In the kidney, their expression pattern is closely related to the corresponding segment-specific solute reabsorption profile. Several claudins are expressed in the TAL, including claudin-16, -19, -10, -3, and -18 The importance of claudin-16 and -19 in this tissue is documented by mutations in CLDN16 and CLDN19, which cause familial hypomagnesemia, hypercalciuria, and nephrocalcinosis, an autosomal recessive dis­order that leads to end-stage renal disease. The relevance of CLDN16 for paracellular reabsorption of Mg2+ and Ca2+ was confirmed in mouse models with targeted gene disruption. In addition, claudin-14, expressed in the TAL of mice on a high-calcium diet, was identified as negative regulator of claudin-16 function (15), and sequence variants in CLDN14 have been asso­ciated with human kidney stone disease. The functional significance of claudin-10, which is also ex­pressed in the TAL, remains unclear. This TJ protein is expressed in two isoforms, claudin-10a and claudin-10b, which differ in their first extracellular loop. In cultured epithelial cells, heter-ologous expression of claudin-10a increases paracellular anion transport, whereas claudin-10b expression increases paracellular cation transport. Both isoforms are expressed differentially along the nephron, with claudin-10a found predominantly in cortical segments, whereas claudin-10b is enriched in the medullary region.  In the present study we generated a mouse model with a TAL-specific Cldn10 gene defect to query the role of this protein in renal paracellular in transport in vivo. We found that claudin-10 is crucial to paracellular Na+ handling in the TAL, and that its absence leads to a shift from paracellular sodium transport to paracellular hyperreabsorption of Ca2+ and Mg2+.

Analysis of claudin-10 expression in the kidney. (A) Western blot analysis of kidney membrane extracts from control (ctr) and cKO mice. A dramatic reduction in claudin-10 protein can be seen in kidneys of cKO mice. Levels of the TJ marker occludin are unchanged. (B) Gene expression analysis of Cldn10 variants on cDNA from isolated segments of the nephron. (C) Immunohistological detection of claudin-10 and markers for PCT (NHE3) and TAL (NKCC2) on sections from control mice (ctr) and cKO mice demonstrates no difference in the signal for claudin-10 in the PCT between WT and cKO. Claudin-10 is expressed in TAL tubules positive for NKCC2. No specific clau-din-10 staining is evident in the TAL of cKO mice. Claudin-10 is detected in TJs positive for ZO-1. This signal is absent in cKO mice, whereas ZO-1 staining is unchanged. (Scale bar: 25 μm.)

In control animals, claudin-10 is located mainly in the TAL, as documented by coimmunostaining with the Na+K+2Clcotrans-porter (NKCC2). In this segment, a large portion of the claudin-10 immunofluorescence signal is located outside of the TJ; however, claudin-10 is present in the TJ, as demonstrated by colocalization with the TJ protein ZO-1. PCTs positive for the sodium-proton exchanger NHE-3 showed a considerably weaker signal restricted to the TJ area. Claudin-10 immunoreactivity was virtually absent in NKCC2-positive tubules of cKO mice, in line with the activity of Cre recombinase in this cell type. The immu-noreactivity of claudin-10 in PCTs of cKOs remained unchanged, however. ZO-1 staining in TAL sections of cKOs was unchanged compared with controls, indicating no unspecific effect on TJ structures. The TJ localization of claudin-16 and claudin-19 in medullary rays was similar in cKOs and controls.   To investigate the phenotypic consequences of renal claudin-10 deficiency, we per­formed a histological examination of the kidneys of 10-wk-old cKO mice and their respective controls. Kidneys from cKO mice contained extensive medullary calcium deposits, as revealed by von Kossa and alizarin red S staining. The deposits were found along the outer stripe of the outer medulla. The detection of extensive calcification suggests alterations in renal ion homeostasis in mice deficient for claudin-10.  Serum Na+ and Cllevels and their renal FE excretion rates were not different be­tween genotypes. In addition, serum creatinine and glomerular filtration rate were not altered compared with controls. Taken together, these findings indicate that calcium deposition does not nonpecifically affect overall glomerular or tubular function.

Fig 4. Gene expression analysis of renal claudins (A) and representative renal ion transporters and channels (B) by real-time PCR. Cldn10 deficiency results in differential gene expression of several genes. Values from cKO animals are shown relative to control mice (mean ± SEM). Wnk1, Wnk1-KS, Kcnj1, and Trpm6, n = 5/4; all other genes, n = 10/10. *P < 0.05; **P < 0.01; ***P < 0.001.    The thiazide-sensitive NaCl cotransporter NCC (Slc12a3), the protein involved in NaCl absorption in the DCT, and the respective inhibitory, kidney-specific kinase-defective KS-WNK1 were expressed at lower levels in the cKO mice. Taken together, these data suggest specific compensatory alterations in components of both paracellular and transcellular renal ion transport mechanisms in mice deficient in claudin-10 in the TAL.

Urinalysis demonstrated that the inhibition of TAL tubular transport by furosemide resulted in a completely differ­ent pattern of tubular Ca2+ and Mg2+ handling that identifies the TAL as the major nephron segment affected by claudin-10 deficiency.  Interestingly, the different effects on plasma Mg2+ and Ca2+ levels reflect the different major reabsorption sites of these ions. Some 60% of the filtered Mg2+ is reabsorbed in the TAL, compared with only 20% of the filtered Ca2+ load (20). Ca2+ hyperreabsorption in TAL seems to be balanced by reduced (proximal and) distal tubular Ca2+ transport. The hyperreabsorption of divalent cations in mice deficient in claudin-10 is in opposition to the loss of divalent cations seen in mouse models of claudin-16 deficiency and in human patients with mutation in CLDN16 or CLDN19. This finding indicates that claudins in the TAL have functions that differentially affect paracellular cation transport in this segment. Mice deficient for claudin-10b in the TAL exhibit decreased permeability for Na+ and increased permeability for Ca2+ and Mg2+, whereas in mice with claudin-16 or claudin-19 deficiency, decreased sodium per­meability in the TAL is paralleled by decreased reabsorption of Ca2+ and Mg2+.

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4.  Simon DB, et al. (1999) Paracellin-1, a renal tight junction protein required for par-acellular Mg2+ resorption. Science 285:103–106.
5. Hou J, et al. (2007) Transgenic RNAi depletion of claudin-16 and the renal handling of magnesium. J Biol Chem 282:17114–17122.
6. Himmerkus N, et al. (2008) Salt and acid-base metabolism in claudin-16 knockdown mice: Impact for the pathophysiology of FHHNC patients. Am J Physiol Renal Physiol 295:F1641–F1647.

 PMID: 22891322  PMCID: PMC3435183

Paracellin-1 is critical for magnesium and calcium reabsorption in the human thick ascending limb of Henle.

Blanchard A, Jeunemaitre X, Coudol P, Dechaux M, Froissart M, et al.
Université Pierre et Marie Curie, INSERM and Laboratoire de Génétique Moléculaire, Hôpital Universitaire Européen Georges Pompidou, Paris, France.
Kidney Int. 2001 Jun; 59(6):2206-15.

A new protein, named paracellin 1 (PCLN-1), expressed in human thick ascending limb (TAL) tight junctions, possibly plays a critical role in the control of magnesium and calcium reabsorption, since mutations of PCLN-1 are present in the hypomagnesemia hypercalciuria syndrome (HHS).
No functional experiments have demonstrated that TAL magnesium and calcium reabsorption were actually impaired in patients with HHS.
Genetic studies were performed in the kindred of two unrelated patients with HHS.

We found two yet undescribed mutations of PCLN-1 (Gly 162 Val, Ala 139 Val). In patients with HHS, renal magnesium and calcium reabsorptions were impaired as expected; NaCl renal conservation during NaCl deprivation and NaCl tubular reabsorption in diluting segment were intact. Furosemide infusion in CS markedly increased NaCl, Mg, and Ca urinary excretion rates. In HHS patients, furosemide similarly increased NaCl excretion, but failed to increase Mg and Ca excretion. Acute MgCl(2) infusion in CS and ERH patient provoked a dramatic increase in urinary calcium excretion without change in NaCl excretion. When combined with MgCl(2) infusion, furosemide infusion remained able to induce normal natriuretic response, but was unable to increase urinary magnesium and calcium excretion further. In HHS patients, calciuric response to MgCl(2) infusion was blunted.

In patients with HHS, levels of circulating renin and aldosterone were normal, suggesting normal blood and extracellular volume. In addition, HHS patient 2 was normally able to lower her sodium excretion below 10 mmol/day during sodium deprivation, and in HHS pa­tient 1, sodium reabsorption in the diluting segment was normal as assessed by hypotonic saline infusion.  After oral NH4Cl load: Minimal urinary pH was 5.8 (normal value <5.4), and maximal net acid excretion reached only 24 pmol/min (normal value >80). Both subjects had hypocitraturia. The latter data suggested in the two probands distal defect of urinary acidification, probably related to nephrocalcinosis.

Because the filtered load of calcium but not the filtered load of magnesium remains unchanged during acute magnesium infusion in humans, the increase in calcium excretion is a better index of the inhibitory effect of peritubular magnesium on renal tubular divalent cation transport.  Urinary sodium excretion remained almost constant in both subjects during MgCl2 infusion (data not shown). Accordingly, the FECa/FENa ratio, which should remain constant if sodium reabsorption was primarily affected, increased in the CS and EHR patient.  Before the furosemide infusion, serum ultrafilterable (UF) Ca concentrations were similar in patients with HHS and the controls. However, Ca excretion markedly differed and was approximately five times higher in HHS patients than in controls.

In the two patients with homozygous mutations in the PCLN-1 gene, an impairment in renal tubular magne­sium and calcium reabsorption with normal NaCl recla­recla­mation was demonstrated. Accordingly, comparative studies performed under baseline condition in one pa­tient with ERH and in HHS patients demonstrated that the magnesium and calcium excretion in HHS patients were inappropriately high when compared with serum magnesium and calcium concentrations. However, renal NaCl reabsorption in HHS patients was intact. There was no clinical evidence of extracellular fluid volume  contraction. Furthermore, basal circulating renin and aldosterone concentrations were normal and adapted to the normal Na intake. Finally, abnormal NaCl reclama­tion in the diluting segment of the nephron was excluded in one patient, while the other was able to adapt normally to a sodium deprived diet.

This study is the first to our knowledge to demonstrate that homozygous mutations of PCLN-1 result in a selective defect in paracellular Mg and Ca reabsorption in the TAL, with intact NaCl reabsorption ability at this site. In addition, the study supports a selective physiological effect of basolateral Mg(2+) and Ca(2+) concentration on TAL divalent cation paracellular permeability, that is, PCLN-1 activity.   PMID: 11380823

Development of a Novel Sodium-Hydrogen Exchanger Inhibitor for Heart Failure

Elizabeth Juneman*, Reza Arsanjani, Hoang M Thai, Jordan Lancaster, Jeffrey B Madwed, Steven Goldman
Citation: Elizabeth Juneman, et al. (2013) Development of a Novel Sodium-Hydrogen Exchanger Inhibitor for Heart Failure. J Cardio Vasc Med 1: 1-6

This study was designed to determine the potential therapeutic effects of a new sodium-hydrogen exchanger (NHE-1) inhibitor in the rat coronary artery ligation model of chronic heart failure. After the induction of acute myocardial infarction, rats were entered randomly dose dranging from 0.3 mg/kg, 1.0 mg/kg, and 3.0 mg/kg. Solid state micrometer hemodynamics, echocardiographic, and pressure-volume relationships were measured after 6 weeks of treatment. Treatment with this NHE- 1 inhibitor at 3 mg/kg increased (P< 0.05) ejection fraction from 23±3% (N=6) to 33±2% (N=13) while the 1 mg/kg dose decreased (P< 0.05) the infarct size in CHF rats from 21.7±1.4% (N=7) to 15.9±0.7% (N=3) and prevented (P< 0.05) dilatation of the left ventricle in CHF rats in diastole (1.0±0.1 cm, N=6) to 0.9±0.1 cm, N=10) and in systole (0.9±0.1 cm, N=6) to 0.8±0.1, N=10). These study results suggest that this new NHE-1 inhibitor may be potentially useful in treating CHF with an improvement in maladaptive left ventricule remodeling. Because the mechanism of action of this agent is entirely different than the currently applied approach in treating CHF that focuses on aggressive neurohormonal blockade and because this agent does not adversely affect important hemodynamic variables, further investigations with this agent may be warranted.

Keywords: Congestive heart failure; Sodium/hydrogen exchange; Cardiovascular disease; Cardiovascular drugs; CHF: Chronic Heart Failure; NHE-1: Sodium-Hydrogen Exchanger; NCX: Sodium-Calcium Exchanger; Ca2+: Calcium; Na+: Sodium; Na+-K+ATPase: Sodium-Potassium ATPase; NKCC: Sodium-Potassium-Chloride co-transporter; MI: Myocardial Infarction; BI: Boehringer Ingelheim; LV: left Ventricle; EF: Ejection Fraction; LVD: left ventricular dysfunction; PV: Pressure-Volume; SE: Standard Error; ARB: Angiotensin Receptor Blocker; ACE: Angiotensin Converting Enzyme

Without reviewing the pathophysiology of CHF here, altered calcium (Ca2+) handling is a hallmark of CHF. Intracellular Ca2+ concentration is closely regulated by sodium-calcium exchanger (NCX) and Ca2+ efflux is dependent on the intracellular sodium (Na+) concentration and trans-sarcolemmal Na gradient. Multiple channels including sodium-potassium ATPase (Na+-K+ ATPase), sodium-hydrogen exporter (NHE), sodium-bicarbonate co-transporter, sodium-potassium-chloride co-transporter (NKCC), and sodium-magnesium exchanger are responsible for regulation of intracellular sodium in cardiac myocytes. The intracellular concentration of Na+ is significantly increased in heart failure, primarily due to influx of Na+. The NHE plays an integral part in rise of intracellular Na+ concentration and development of hypertrophy in heart failure. Because of its multifaceted role in myocardial function, there has been interest in examining the effects of NHE-1 inhibitors in heart failure.

In this study we report the physiologic responses of a new NHE-1 inhibitor, in a rodent model of heart failure. Previous evaluation of the pharmacokinetic properties of this agent in rat and dog revealed low clearance and robust oral bioavailability, suggesting a potential for once daily oral administration. This new compound was found to be potentially effective in preventing ischemic injury in isolated cells systems and in ischemic injury in isolated cells systems and in a Langendorff isolated heart preparation. Based on these encouraging a pharmacokinetic data, and the established preclinical roof of principle, the next step in new drug development was to test this inhibitor in an appropriate disease-relevant animal model. For this, we chose the rat coronary ligation model of CHF, which is the established model of chronic ischemic heart failure and well performed in our laboratory. The model with permanent occlusion of the left coronary artery is important because this model a similar to the clinical syndrome of CHF. This rat coronary artery model of CHF is the same model used in the classic study defining the beneficial use of angiotensin converting enzyme inhibition with captopril in the treatment of CHF. Thus results in this model have the potential to be predictive of the clinical response seen in patients.


In vivohemodynamic effect of NHE1: As noted previously by our laboratory, rats with severe CHF compared to Sham had changes (P< 0.05) in right ventricular weight, mean arterial pressure, tau, the time constant of LV relaxation, LV systolic pressure, LV end-diastolic pressure, +LVdP/dt, -LVdP/dt, dead volume and peak developed pressure. In this study, treatment resulted in no changes in body weight, chamber weight or hemodynamics. Because we stopped the lowest dose (0.3 mg/kg) there are only hemodynamic data with this dose in rats with CHF.

Echocardiographic changes in LV function and Dimensions with NHE1: Rats with CHF have decreases in EF accompanied by increases in LV systolic and diastolic dimensions. There was no change in anterior wallsystolic displacement. These data are consistent with other reports in this model showing that at 6 weeks after left coronary artery ligation, rats with large MIs have dilated left ventricles with LV remodeling and poor LV function (14,15). Treatment with the highest dose of 3 mg/kg increased (P< 0.05) ejection fraction from 23±3% (N=6) to 33±2% (N=13). Treatment with 1 mg/kg prevented maladaptive LV remodeling, it prevented (P< 0.05) dilatation of the LV in CHF rats in diastole (1.0±0.1 cm, N=6) to 0.9±0.1 cm, N=10) and in systole (0.9±0.1 cm, N=6) to 0.8±0.1, N=10) with no change anterior wall thickening.

Pressure-Volume relationships:  Although there are no significant changes in the PV relationships for either the Sham or CHF rats, there is a trend for the PV loop in CHF to be shifted toward the pressure axis with treatment. These data are consistent with the trend toward decreases in LV dimensions seen with treatment in CHF rats.


This study can be viewed as a corollary of a pilot Phase II clinical trial to look for a signal of a beneficial physiologic effect of this new NHE-1 inhibitor in CHF. In terms of drug development, this is an appropriate approach, i.e., take an agent with a therapeutic focus, with an acceptable toxicology profile, alter its pharmacokinetics to improve its oral delivery and bioavailability and then study the drug in an appropriate animal model. The administration of this agent to rats with CHF after left coronary artery ligation resulted in a therapeutic benefit with an increase in EF and a decrease in infarct size in rats with the largest infarcts. There is a suggestion of the prevention of LV remodeling with decreases in LV end-diastolic and end-systolic dimensions accompanied by a similar trend in the PV loop with a shift toward the pressure axis. There were no changes in hemodynamics.

Importantly, the decrease in infarct size with no changes in hemodynamicswould positively affect LV remodeling by minimizing LV dilatation without changes in LV afterload. From a therapeutic perspective, an agent like this may be advantageous in the treatment of heart failure after MI. The lack of hemodynamic changes is not a clinical problem because we already have agents that decrease afterload and lower LV end-diastolic pressure such as angiotensin converting enzyme (ACE) inhibitors and angiotens in receptor blockers (ARBs). In treating CHF, we also have diuretics to control blood volume, which in turn reduces LV end-diastolic pressure. The other potential advantage of an NHE-1 inhibitor is that as opposed to our current use of aggressive neurohormonal blockade, this represents a different approach to treating CHF. This is attractive because we essentially have exhausted or maximized our effects of neurohumoral blockade and with no real new treatments for CHF introduced in the last 10-15 years, need to look for other approaches to treat CHF.

Drug development is obviously a complicated and expensive undertaking. In exploring this agent, we would proposea stepwise approach. In this case with an agent whose analogs have been studied extensively, our thought would be to perform a larger dose ranging study in CHF rats to define dose response curves for systolic function as well as obtain more information on pharmacokinetics, as well as diastolic function and structural changes.

An attractive aspect of this work is that we are examining an agent with a different mechanisms of action that current treatments for heart failure. The stimulus to study the agent in an animal model of heart failure was based on multifaceted roles of sodium-hydrogen exchangers on myocardial function. Nine isoforms of NHE have currently been identified, with NHE- 1 being the predominant isoform in the plasma membrane of the myocardium [3,24]. Because NHE is activated by intracellular acidosis, angiotensin II, and catecholamines, its activity is expectedly increased in heart failure. Inhibition of NHE-1 has previously been associated with decreased fibrosis, apoptosis, preserved contractility, and attenuation of hypertrophy and development of heart failure.

1. Baartscheer A, van Borren MMGJ (2008) Sodium Ion Transporters as New Therapeutic Targets in Heart Failure. Cardiovasc Hematol Agents Med Chem 6: 229-236.
2. Murphy E, Eisner DA (2009) Regulation of Intracellular and Mitochondrial Sodium in Health and Disease. Circ Res 104: 292-303
3. Despa S, Islam MA, Weber CR, Pogwizd SM, Bers DM (2002) Intracellular Na(+) Concentration is Elevated in Heart Failure but Na/K Pump Function is Unchanged. Circulation 105: 2543-2548.
4. Baartscheer A, Schumacher CA, van Borren MMGJ, Belterman CNW, Coronel R, et al. (2003) Increased Na+/H+-Exchange Activity is the Cause of Increased [Na+]i and Underlies Disturbed Calcium Handling in the Rabbit Pressure and Volume Overload Heart Failure Model. Cardiovasc Res 57: 1015-1024.
5.  Pieske B, Houser SR (2003) [Na+]i Handling in the Failing Human Heart. Cardiovasc Res 57: 874-886.
6.  Engelhardt S, Hein L, Keller U, Klämbt K, Lohse MJ (2002) Inhibition of Na(+)-H(+) Exchange Prevents Hypertrophy, Fibrosis, and Heart Failure in Beta(1)- Adrenergic Receptor Transgenic Mice. Circ Res 90: 814-819.
7.  Chen L, Chen CX, Gan XT, Beier N, Scholz W, et al. (2004) Inhibition and Reversal of Myocardial Infarction-Induced Hypertrophy and Heart Failure by NHE-1 Inhibition. Am J Physiol Heart Circ Physiol 286: 381-387.
8.  Marano G, Vergari A, Catalano L, Gaudi S, Palazzesi S, et al. (2004) Na+/ H+ Exchange Inhibition Attenuates Left Ventricular Remodeling and Preserves Systolic Function in Pressure-Overloaded Hearts. Br J Pharmacol 141: 526-532.
9. Goldman S, Raya TE (1995) Rat Infarct Model of Myocardial Infarction and Heart Failure. J Card Fail 1: 169-177.
10. Gaballa MA, Goldman S (2002) Ventricular Remodeling in Heart Failure. J Card Fail. 8: 476-485.
11. Pfeffer MA, Pfeffer JM, Steinberg C, Finn P (1985) Survival After Experimental Myocardial Infarction: Beneficial Effects of Long-Term Therapy with Captopril. Circulation 72: 406-412.
12. Raya TE, Gay RG, Aguirre M, Goldman S (1989) Importance of Venodilatation in Prevention of Left Ventricular Dilatation after Chronic Large Myocardial Infarction in Rats: A Comparison of Captopril and Hydralazine. Circ Res 64: 330-337.

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pone.0070764.g006  Morpholino knockdown of aquaporin-1a1 reduces water influx.       NIHMS262281.html

nihms81087f1  Localization of claudin proteins in mammalian kidney.      F1.medium  intracellular Mg2+ in normal and Mg2+ depleted immortalized mouse distal convoluted tubule (MDCT) cells

F2.small  membrane voltage influences Mg2+ uptake in MDCT cells    pnas.1203834109fig04  Gene expression analysis of renal claudins

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Translational Research on the Mechanism of Water and Electrolyte Movements into the Cell

Reviewer and Curator: Larry H. Bernstein, MD, FACP


This article is the first in a three part curation covering work that has great importance to our understanding of hydration and possibly the effects of dehydration in cell physiology, and studied effects on renal function and brain, with possible implications for heart failure, myocardial contraction, heart rate, and arrhythmiagenesis.  The discovery of aquaporins and the elucidation of potassium channels and selective ion conduction was jointly awarded the Nobel Prize in Chemistry in 2003 to Peter Agre, at the Johns Hopkins School of Medicine, Baltimore, and Roderick Mac Kinnon, at the Howard Hughes Medical Institute, Laboratory of Molecular Neurobiology and Biophysics, Rockefeller University, New York, NY.  The transport of water, it was assumed, is associated with the movements of Na(+), K(+), Ca(2+), Mg(2+).  The calmodulin kinase, rhyanodine, and calcium sparks in the Ca(2+) release from sarcolemma is covered elsewhere in cardiac contraction, skeletal muscle, smooth muscle, and neural stimulation of muscle and adrenergic release.  The sodium/potassium exchange is depicted in diagrams, but not discussed.  In traditional chemistry we would think in terms of a cationic and anionic balance that has to be maintained in charge equivalents on both sides of a membrane.  However, the intricacies of membrane structure as well as active transporters has been delineated and has been a transformative factor in our understanding of organ function in health and disease.

Aquaporin Water Channels

AQUAPORIN WATER CHANNELS: Nobel Lecture, Dec 8, 2003, by Peter Agre. Fig1 Membrane orientation of AQP1

We have studied the aquaporin water channels for several years, and we now understand that they explain how water crosses biological membranes. Our bodies are 70% water, and all other vertebrates, invertebrates, microbes, and plants are also primarily water. The organization of water within biological compartments is fundamental to life, and the aquaporins serve as the plumbing systems for cells. Aquaporins explain how our
brains secrete and absorb spinal fluid, how we can generate aqueous humor within our eyes, how we can secrete tears, saliva, sweat, and bile, and how our kidneys can concentrate urine so effectively. These proteins are fundamental to mammalian physiology, but they are also very important in the lives of microorganisms and plants.
It was correctly proposed  in the 1920’s that water could move through the cell membrane simply by diffusing through the lipid bilayer. The current view is that the lipid bilayer has a finite permeability for water, but, in addition, a set of proteins exists that we now refer to as “aquaporins.” Their existence was suggested by a group of pioneers in the water transport field who preceded us by decades – people including Arthur K. Solomon in Boston, Alan Finkelstein in New York, Robert Macey in Berkeley, Gheorghe Benga in Romania, Guillermo Whittembury in Venezuela, Mario Parisi in Argentina – who by biophysical methods predicted that water channels must exist in certain cell types with high water permeability such as renal tubules, salivary glands, and red cells (reviewed by Finkelstein, 1987).
The difference between diffusional and channel-mediated water perme-ability is fairly distinct. Diffusion is a low capacity, bidirectional movement of water that occurs in all cell membranes, whereas the membranes of a subset of cells with aquaporin proteins have very high capacity for permeation by water.
This permeability is selective, since water (H O) crosses through the membranes with almost no resistance, while acid, the hydronium ion (H O ) does not permeate the proteins. This distinction is essential to life. The movement of water is directed by osmotic gradients, so aquaporins are not pumps or exchangers. They form a simple pore that allows water to rapidly pass through membranes by osmosis. There are also other differences between diffusion and channel-mediated water transport. No inhibitors are known for simple diffusion. In contrast, mercurials were discovered by Robert Macey to inhibit water transport in red cells but water permeability was restored by treatment with reducing agents (Macey and Farmer, 1970). These observations predicted that water channels must be proteins with sulfhydryls and characteristically low Arrhenius activation energy.
A number of investigators using ver y logical approaches attempted to identify the water channel molecule; identification proved a very difficult prolem. Isotopic mercurials labeled several membrane proteins – the anion exchanger (band 3). Solomon and a group of several proteins (band 4.5) by Benga. None of the proteins were isolated, reconstituted, and shown  to transport watter (reviewed by Agre et al., 1993a).


The field was essentially stuck, but following the well known scientific approach known as “sheer blind luck,” we stumbled upon the protein. Looking through our notebooks for the earliest studies that showed there was such a protein water channel. We were at that time attempting to raise antibodies in rabbits to the denatured partially purified Rh polypeptide.  The rabbits vigorously produced antibodies, but we failed to recognize initially that our antibody did not react with the core Rh polypeptide that migrated at 32 kDa, seen clearly by silver staining of sodium dodecyl sulfate polyacryamide electrophoresis gels (SDS-PAGE). Instead, our antibodies reacted only with a 28 kDa polypeptide. The 28 kDa was an unrelated protein.  Silver staining of SDS-PAGE migration of the isolated protein revealed a discrete band of 28 kDa in detergent insoluble extracts (it failed to stain with the conventional protein stains such as Coomassie blue). The protein was then purified in large amounts from human red cell membranes (Denker et al., 1988; Smith and Agre, 1991).  The 28 kDa protein was strikingly abundant. With approximately  copies per red cell, it was one of the major proteins in the membrane. The protein had features suggesting that it was a tetrameric membrane-spanning protein – suggesting that it was a channel, but a channel for what? The purified protein also provided us the N-terminal amino acid sequence that we used for cDNA cloning. Using our antibody, we looked at several other tissues and found the protein is also strikingly abundant in human kidney. We observed staining over the apical and basolateral membranes of proximal renal tubules and the descending thin limb of the loops of Henle, but we were still frustrated by our failure to recognize what the protein’s function might be.  My clinical mentor, John C. Parker (1935–1993) at the University of North Carolina at Chapel Hill, was the first to suggest to me that red cells and renal tubules were exceedingly permeable to water. He recommended that we consider a role in membrane water transport. While John did not live to see our later studies, he did live to see our initial discovery and we celebrated together.
Postdoctoral fellow Gregor y Preston cloned the cDNA from an erythroid brary (Preston and Agre, 1991). The coding region corresponded to a 269 amino acid polypeptide, predicted by hydropathy analysis to have six bilayer-spanning domains. Interestingly, the amino terminal half (repeat-1) and the carboxy terminal half of the molecule (repeat-2) were genetically related – about 20% identical. Two loops B and E were more highly related to each other, and each contained the signature motif – asparagine, proline, alanine (NPA) [Fig. 1]. Examining the genetics database, we recognized several sequence-related DNAs from diverse sources: lens of cow eyes, brains of fruit flies, bacteria, and plants. Nevertheless, none was functionally defined.
Figure 1. Membrane orientation of AQP1 predicted from primary amino acid sequence. Two tandem repeats each have three bilayer-spanning domains; the repeats are oriented 180˚ with respect to each other. The loops B and E each contain the conser ved motif, Asn- Pro-Ala (NPA)
These clues heightened our suspicion that the 28 kDa protein was a transporter, so we tested for possible water transport function with our colleague Bill Guggino at Johns Hopkins. We used oocytes the frog Xenopus laevis, a useful model, since frog oocytes have very low water permeability. Control oocytes were injected with water alone; oocytes were injected with 2 ng of cRNA encoding our protein. After days of protein synthesis, the oocytes appeared essentially identical. Then we stressed the oocytes by transferring them to distilled water, and an amazing difference was immediately apparent. Having exceedingly low water perme-
ability, the control oocytes failed to swell. In contrast, the test oocytes were highly permeable to water and exploded like popcorn [Fig. 2] (Preston et al., 1992).  The protein was christened “aquaporin” and is now officially designated “AQP1,” the first functionally defined water channel protein (Agre et al., 1993b).
Figure 2. Functional expression of AQP1 water channels in Xenopus laevis oocytes. Control oocyte (left) was injected with water; AQP1 oocyte (right) was injected with cRNA. The oocytes were transferred to hypotonic buffer. After 30 seconds (top) the AQP1 oocyte has begun to swell; after 3 minutes (bottom), the AQP1 oocyte has exploded. Modified and reprinted from Science with permission (Preston et al., 1992).
We  confirmed the function of this protein by studying the purified AQP1 reconstituted into synthetic lipid vesicles of ~0.1 micron diameter prepared by our colleague Suresh Ambudkar at Johns Hopkins (Zeidel et al., 1992). These simple membrane vesicles were examined by freeze fracture electron microscopy by our colleague Arvid Maunsbach, from the University of Aarhus. When lipid was reconstituted without protein, the membrane surfaces were smooth; however, membranes reconstituted with AQP1 contained many intramembraneous particles 0.01 micron diameter (Zeidel et al., 1994). We tested the membranes for water permeability in collaboration with Mark Zeidel at Har vard Medical School. Using stopped flow transfer to hypertonic buffer, the simple liposomes shrank, reaching equilibrium in about one half
second; this is believed to represent the baseline water permeability. When membranes reconstituted with AQP1 were examined, the shrinking occurred much more rapidly, reaching equilibrium in about 20 milliseconds. The channel-mediated flow of water was confirmed, since it was inhibited with mercurials. We calculated the Arrhenius activation energy (<5 kcal/mol), and we determined the unit permeability to be ~3×10 water molecules per subunit per second. Importantly, we attempted to measure proton permeation of AQP1, but despite massive water permeability, acid permeation was not detected. These studies verified that we had, in fact, isolated the long-sought water channel protein.


Subsequent efforts were devoted to identifying the mercurial inhibitory site predicted by the studies of Macey. Mercurials react with free sulfhydryls in the amino acid cysteine. Four cysteines are found in the AQP1 polypeptide, but only the residue in loop E (Cys-189 proximal to the second NPA motif) is inhibited by mercurials. We altered the AQP1 sequence by site-directed mutagenesis and expressed the recombinants in oocytes for water permeability studies. Mutation of this residue to serine (Cys-189-Ser) resulted in full water permeability without mercurial inhibition. When we then replaced the alanine in the corresponding position of loop B with a cysteine (Ala-73-Cys), the protein exhibited mercurial sensitive water permeability (Preston et al., 1993). Substitutions elsewhere in the AQP1 failed to produce this behavior. This suggested to us that loops B and E in opposite parts of the molecule must somehow form the aqueous pore. The model that we concocted turned out to be schematically correct and was termed “the hourglass.” The ancient timepiece allows sand to run from upper chamber to lower chamber; if inverted, the sand will flow in the opposite direction. Six bilayer spanning domains were predicted to surround a central domain containing loop B, dipping into the membrane from the cytoplasmic surface, and loop E, dipping into the membrane from the extracellular surface [Fig. 3 left and right].
Figure 3. Hourglass model for membrane topology of AQP1 subunit.
Left panel – Schematic folding of loops B and E overlap within the lipid bilayer to form a single aqueous pathway.
Right panel – Ribbon model of three dimensional structure of AQP1 subunit confirms hourglass with single aqueous pathway. Modified and reprinted with permission from Jour-
nal of Biological Chemistr y (Jung et al., 1994b) and Journal of Clinical Investigation (Kozono et al., 2002).
The overlap of loops B and E was predicted to form a single aqueous pore through the center of the molecule with the NPA motifs juxtaposed and mercurial inhibitory site alongside (Jung et al., 1994b). The AQP1 protein tetrameric with a central pore in each subunit. Thus, AQP1 is structurally like ion channel proteins where four subunits surround a single central as discussed by Rod MacKinnon in his lecture.
We  then sought to establish the high resolution structure of AQP1 in collaboration with Andreas Engel and his group at the Biozentrum in Basel. We were later joined with Yoshinori Fujiyoshi and his group at Kyoto University. Human red cell AQP1 protein was purified by Barb Smith in our lab; Andreas’s student Tom Walz reconstituted it into synthetic membranes at very high protein concentrations. Under these conditions, the AQP1 protein forms remarkably symmetrical arrays referred to as membrane crystals. By measuring the water permeability, we confirmed that the function was 100% retained, giving us confidence that the structure we deduced would be the biologically relevant structure (Walz et al., 1994).
Figure 4. Functional representation for selective water flow through AQP1 subunit and residues involved in human disease.
Left panel – Schematic of sagittal cross-section of AQP1 reveals bulk water in extracellu- lar and intracellular vestibules of hourglass. These are separated by a 20Å span where water passes in single file with transient interactions with pore-lining residues that prevent hy- drogen bonding between water molecules (bold colors). Two structures are believed to pre- vent permeation by protons (H O ): electrostatic repulsion is created by a fixed positive
charge from pore-lining arginine (R195) at a 2.8Å narrowing in the channel; water dipole reorientation occurs from simultaneous hydrogen bonding of water molecule with side chains of two asparagines residues in NPA motifs (N192 and N76). Two partial positive charges at the center of the channel result from orientation of two non-membrane span- ning alpha helices distal to the NPA motifs


While we were pursuing studies of AQP1, several other research groups from around the world became interested in what is now known to be a large family of related proteins. The combined efforts of these labs have led to the molecular identification of 12 mammalian aquaporin homologs, and several hundred related proteins have been recognized in other vertebrates as well as invertebrates, plants, and unicellular micro-organisms. The mammalian homologs may be loosely clustered into two subsets [Fig. 5]. The first is referred to as “classical aquaporins”, since they were initially considered to be exclusive water pores. The second is referred to as “aquaglyceroporins”, since they are permeated by water plus glycerol. Interestingly, E. coli has one member of
each – AqpZ (Calamita et al., 1995), and GlpF, isolated by other investigators much earlier. Together, the mammalian aquaporins and aquaglyceroporins are now known to contribute to multiple physiological processes that occur during our daily lives.
Figure 5. Human aquaporin gene family contains two subsets. Homologs freely permeated by water (classical aquaporins, blue) or water and glycerol (aquaglyceroporins, yellow) are represented. E. coli has one aquaporin (AqpZ) and one aquaglyceroporin (GlpF). Reprinted with permission from Journal of Physiology (Agre et al., 2002)
 The remainder of the Nobel Lecture (2003) can be found at the Nobel Prize site.  This portion is sufficient to cover the genesis and advancement of the water transport discovery.

Urinary Excretion of Aquaporin-2 Water Channel Differentiates Psychogenic Polydipsia from Central Diabetes Insipidus

T Saito, San-e Ishikawa, T Ito, H Oda, F Ando, … and T Saito Division of Endocrinology and Metabolism (Ta.S., S.I., F.A., Mi.H., S.N., To.S.), Department of Medicine, Jichi Medical School, Tochigi 329-0498; and Departments of Medicine and Psychiatry (T.I., H.O., Ma.H.), Tokyo Metropolitan Matsuzawa Hospital, Tokyo, Jp 
correspondence to: San-e Ishikawa, M.D., Division of Endocrinology and Metabolism, Department of Medicine, Jichi Medical School, Tochigi 329-0498, Japan. E-mail:
The present study was undertaken to determine whether urinary excretion of aquaporin-2 (AQP-2) water channel under ad libitum water intake is of value to differentiate polyuria caused by psychogenic polydipsia from central diabetes insipidus. A 30-min urine collection was made at 0900 h in 3 groups of: 11 patients with central diabetes insipidus (22–68 yr old), 10 patients with psychogenic polydipsia (28–60 yr old), and 15 normal subjects (21–38 yr old). In the patients with central diabetes insipidus, the plasma arginine vasopressin level was low despite hyperosmolality, resulting in hypotonic urine. Urinary excretion of AQP-2 was 37 ± 15 fmol/mg creatinine, a value one-fifth less than that in the normal subjects. In the patients with psychogenic polydipsia, plasma arginine vasopressin and urinary osmolality were as low as those in the patients with central diabetes insipidus. However, urinary excretion of AQP-2 of 187 ± 45 fmol/mg creatinine was not decreased, and its excretion was equal to that in the normal subjects. These results indicate that urinary excretion of AQP-2, under ad libitum water drinking, participates in the differentiation of psychogenic polydipsia from central diabetes insipidus. 
PSYCHOGENIC polydipsia causes a marked polyuria with hypotonic urine (1, 2). Arginine vasopressin (AVP) secretion is suppressed by hypoosmolality caused by excess intake of water. Suppression of AVP release obliges us to differentiate psychogenic polydipsia from central diabetes insipidus. Osmotic stimulation tests have been carried out to determine the reserve function of the posterior pituitary gland. Plasma AVP levels increase in response to an increase in plasma osmolality (Posm) in patients with psychogenic polydipsia but not in those with central diabetes insipidus.
In response to AVP, concentrated urine is produced by water reabsorption across the renal collecting duct (3, 4). Aquaporin-2 (AQP-2) is an AVP-regulated water channel of the collecting duct; it is translocated from the cytoplasmic vesicles to the apical plasma membranes by shuttle trafficking when the cells are stimulated by AVP (5, 6, 7), and it is again redistributed into the cytoplasmic vesicles after removal of AVP stimulation (8). Also, AQP-2 is, in part, excreted into the urine (9, 10). We demonstrated that urinary excretion of AQP-2 is of great value in diagnosing central diabetes insipidus in the hypertonic saline infusion test and impaired water excretion in the acute oral water load test (11, 12).   The present study was undertaken to determine whether urinary excretion of AQP-2, under ad libitum water intake, is a useful tool for diagnosing psychogenic polydipsia.

Subjects and study design

Three groups of subjects were examined in the present study.
[1]  11 patients who had been diagnosed as having idiopathic central diabetes insipidus. They had taken 1-deamino-8-D-AVP (DDAVP) intranasally, twice a day, and discontinued the DDAVP therapy 24 h before the study.
[2] 10 patients were diagnosed as having psychogenic polydipsia. They had been treated for psychiatric disorders, including schizophrenia, atypical psychiatric disorder, and chronic alcoholism.
[3] 15 normal volunteers, with ages ranging from 21–38 yr. (the age range of [1] and [2] reached 60)
All the subjects drank water ad libitum, and 30-min urine collection was made and blood drawn at 0900 h. Urine samples were subjected to measurements of urinary osmolality (Uosm) and urinary excretion of creatinine and AQP-2. Blood samples were used to measure Posm and plasma AVP levels. Uosm and Posm were measured by freezing-point depression (Model 3W2, Advanced Instruments, Needham Height, MA). Urinary creatinine was measured with an automatic clinical analyzer (Model 736, Hitachi Co., Tokyo, Jp). Plasma AVP levels were determined by RIA using AVP RIA kits (Mitsubishi Chemistry, Tokyo, Jp) (13). Urinary excretion of AQP-2 was measured as described below.

RIA of AQP-2

The RIA of urinary AQP-2 was performed by the method described in our previous reports (11, 12). Urinary AQP-2-like immunoreactivity was measured by a specific RIA that used the polyclonal antibody against a synthetic portion (Tyr0-AQP-2[ V257-A271]) of the C-terminal of human AQP-2 raised in rabbits. A synthetic peptide [Tyr0-AQP-2 (V257-A271)] was radioiodinated with iodine-125 (New England Nuclear, Boston, MA) by the chloramine-T method.  All samples were analyzed in duplicate. The intra- and interassay coefficients of variation were less than 10%. The minimal detectable quantity of AQP-2 was 0.86 pmol/tube, and an amount equivalent to 6.9 pmol/tube caused 50% inhibition of binding of the radiolabeled ligand.


In the patients with central diabetes insipidus, the plasma AVP level was low despite hyperosmolality of 297.8 ± 3.4 mosmol/kg H2O, resulting in hypotonic urine (Fig. 1⇓). Urinary excretion of AQP-2 was one-fifth less in the patients with central diabetes insipidus than in the normal subjects. AQP-2 is the AVP-dependent water channel of collecting duct cells and is recycling between the cytoplasmic vesicles and the apical plasma membranes in the cells (5, 6, 7, 8). AQP-2 is partly excreted into the urine, which is approximately 3% of AQP-2 in the collecting duct cells (14). In normal subjects, urinary excretion of AQP-2 is changeable in a wide range in physiological conditions (11). Because urinary excretion of AQP-2 has a positive correlation with plasma AVP levels in normal subjects (11), the reduced urinary excretion of AQP-2 was in concert with the impaired secretion of AVP in central diabetes insipidus.
Figure 1.
Posm, plasma AVP (Pavp), Uosm, and urinary excretion of AQP-2 (UAQP-2), under ad libitum water drinking, in 15 normal subjects (NL, •), 11 patients with central diabetes insipidus (CDI, ○) and 10 patients with psychogenic polydipsia (PP, □). *, P < 0.01; **, P < 0.05 vs. the normal subjects. Value are means ± sem.
In the patients with psychogenic polydipsia, Uosm was as low as that in the patients with central diabetes insipidus (Fig. 1⇑). The plasma AVP level was low because of the reduced Posm, which was derived from an exaggerated intake of water. Urinary excretion of AQP-2, however, was not decreased; and rather, its excretion kept the normal range. The relationship between plasma AVP levels and urinary excretion of AQP-2 is shown in Fig. 2⇓. The urinary excretion of AQP-2 in the patients with psychogenic polydipsia was dissociated from the positive correlation between plasma AVP and urinary excretion of AQP-2 in the normal subjects.
Figure 2.
Relationship between plasma AVP levels and UAQP-2. •, Normal subjects (n = 15); ○, patients with central diabetes insipidus (n = 11); □, patients with psychogenic polydipsia (n = 10). Values are means ± sem.


The present study demonstrated the clinical tool, of urinary excretion of AQP-2, in differentiating psychogenic polydipsia from central diabetes insipidus. What is involved in the marked difference in urinary excretion of AQP-2 in these two disorders? There is a possibility that, as patients with psychogenic polydipsia reduce water intake during sleep, antidiuresis may occur periodically at night and the production of AQP-2 be somewhat restored. Because approximately 3% of AQP-2 in collecting duct cells is excreted into the urine, urinary excretion of AQP-2 may keep relatively high, despite hypotonic urine. The difference may come from the periodicity of water intake in a day, in the patients with psychogenic polydipsia. As a whole, these changes may disrupt the positive relationship between urinary excretion of AQP-2 and plasma AVP levels. At the present time, however, other factors involved in urinary excretion of AQP-2 remain undetermined.
In conclusion, urinary excretion of AQP-2, under ad libitum water drinking, participates in the differentiation of polyuria caused by psychogenic polydipsia from central diabetes insipidus.


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Comparison of cardiovascular aquaporin-1 changes during water restriction between 25- and 50-day-old rats.

Netti VA, Vatrella MC, Chamorro MF, Rosón MI, Zotta E, Fellet AL, Balaszczuk AM.
Cátedra de Fisiología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, IQUIMEFA, CONICET, Junín 956, C1113AAD, Buenos Aires, Argentina,
Eur J Nutr. Apr 27, 2013
Aquaporin-1 (AQP1) is the predominant water channel in the heart, linked to cardiovascular homeostasis. Our aim was to study cardiovascular AQP1 distribution and protein levels during osmotic stress and subsequent hydration during postnatal growth.
Rats aged 25 and 50 days were divided in: 3d-WR: water restriction 3 days; 3d-WAL: water ad libitum 3 days; 6d-WR+ORS: water restriction 3 days + oral rehydration solution (ORS) 3 days; and 6d-WAL: water ad libitum 6 days. AQP1 was evaluated by immunohistochemistry and western blot in left ventricle, right atrium and thoracic aorta.
Water restriction induced a hypohydration state in both age groups (40 and 25 % loss of body weight in 25- and 50-day-old rats, respectively), reversible with ORS therapy. Cardiac AQP1 was localized in the endocardium and endothelium in both age groups, being evident in cardiomyocytes membrane only in 50-day-old 3d-WR group, which presented increased protein levels of AQP1; no changes were observed in the ventricle of pups. In vascular tissue, AQP1 was present in the smooth muscle of pups; in the oldest group, it was found in the endothelium, increasing after rehydration in smooth muscle. No differences were observed between control groups 3d-WAL and 6d-WAL of both ages.
Our findings suggest that cardiovascular AQP1 can be differentially regulated in response to hydration status in vivo, being this response dependent on postnatal growth. The lack of adaptive mechanisms of mature animals in young pups may indicate an important role of this water channel in maintaining fluid balance during hypovolemic state.

 Clinical application of aquaporin research: aquaporin-1 in the peritoneal membrane

Nishino T, Devuyst O.
Division of Renal Care Unit, Second Department of Internal Medicine, Nagasaki University School of Medicine, Nagasaki, Jp
Peritoneal dialysis (PD) is an established mode of renal replacement therapy based on the exchange of fluid and solutes between blood and a dialysate that has been instilled in the peritoneal cavity. The dialysis process involves osmosis, as well as diffusive and convective transports through the highly vascularized peritoneal membrane. The membrane contains ultrasmall pores responsible for the selective transport of water across the capillary endothelium. The distribution of the water channel aquaporin-1 (AQP1), as well as its molecular structure ensuring an exquisite selectivity for water, fit with the characteristics of the ultrasmall pore. Peritoneal transport studies using AQP1 knockout mice demonstrated that the osmotic water flux across the peritoneal membrane is mediated by AQP1. This water transport accounts for 50% of the ultrafiltration during PD. Treatment with high-dose corticosteroids upregulates the expression of AQP1 in peritoneal capillaries, resulting in increased water transport and ultrafiltration in rats. These data illustrate the potential of the peritoneal membrane as an experimental model in the investigation of the role of AQP1 in the endothelium. They emphasize the critical role of AQP1 during PD and suggest that manipulating AQP1 expression could be clinically useful in PD patients.

Corticosteroids induce expression of aquaporin-1 and increase transcellular water transport in rat peritoneum

Stoenoiu MS, Ni J, Verkaeren C, Debaix H, Jonas JC, Lameire N, Verbavatz JM, Devuyst O.
Division of Nephrology and ENDO Unit, Université Catholique de Louvain Medical School, Brussels, Belgium
J Am Soc Nephrol. Mar 2003; 14(3):555-565.
The water channel aquaporin-1 (AQP1) is the molecular counterpart of the ultrasmall pore responsible for transcellular water permeability during peritoneal dialysis (PD). This water permeability accounts for up to 50% of ultrafiltration (UF) during a hypertonic dwell, and its loss can be a major clinical problem for PD patients. By analogy with the lung, the hypothesis was tested that corticosteroids may increase AQP1 expression in the peritoneal membrane (PM) and improve water permeability and UF in rats. First, the expression and distribution of the glucocorticoid receptor (GR) in the PM and capillary endothelium was documented. Time-course and dose-response analyses showed that a daily IM injection of dexamethasone (1 or 4 mg/kg) for 5 d induced an approximately twofold increase in the expression of AQP1 at the mRNA and protein levels. The GR antagonist RU-486 completely inhibited the dexamethasone effect. The functional counterpart of the increased AQP1 expression was a significant increase in sodium sieving and net UF across the PM, contrasting with a lack of effect on the osmotic gradient and permeability for small solutes. The latter observation reflected the lack of effect of corticosteroids on nitric oxide synthase (NOS) activity and endothelial NOS isoform expression in the PM. In conclusion, corticosteroids induce AQP1 expression in the capillary endothelium of the PM, which is reflected by increased transcellular water permeability and UF. These data emphasize the critical role of AQP1 during PD and suggest that pharmacologic regulation of AQP1 may provide a target for manipulating water permeability across the PM.

Aquaporins: relevance to cerebrospinal fluid physiology and therapeutic potential in hydrocephalus

Owler BK, Pitham T, Wang D.
Kids Neurosurgical Research Unit, Children’s Hospital at Westmead, Westmead NSW 2145, Australia.
Cerebrospinal Fluid Res.  Sep 22, 2010; 7:15.
The discovery of a family of membrane water channel proteins called aquaporins, and the finding that aquaporin 1 was located in the choroid plexus, has prompted interest in the role of aquaporins in cerebrospinal fluid (CSF) production and consequently hydrocephalus. While the role of aquaporin 1 in choroidal CSF production has been demonstrated, the relevance of aquaporin 1 to the pathophysiology of hydrocephalus remains debated. This has been further hampered by the lack of a non-toxic specific pharmacological blocking agent for aquaporin 1. In recent times aquaporin 4, the most abundant aquaporin within the brain itself, which has also been shown to have a role in brain water physiology and relevance to brain oedema in trauma and tumours, has become an alternative focus of attention for hydrocephalus research. This review summarises current knowledge and concepts in relation to aquaporins, specifically aquaporin 1 and 4, and hydrocephalus. It also examines the relevance of aquaporins as potential therapeutic targets in hydrocephalus and other CSF circulation disorders.
PMID: 20860832  PMCID:  PMC2949735

Pathophysiology of the aquaporin water channels

King LS, Agre P.
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Annu Rev Physiol. 1996; 58:619-48.
Discovery of aquaporin water channel proteins has provided insight into the molecular mechanism of membrane water permeability. The distribution of known mammalian aquaporins predicts roles in physiology and disease.
Aquaporin-1 mediates proximal tubule fluid reabsorption, secretion of aqueous humor and cerebrospinal fluid, and lung water homeostasis.
Aquaporin-2 mediates vasopressin-dependent renal collecting duct water permeability; mutations or downregulation can cause nephrogenic diabetes insipidus.
Aquaporin-3 in the basolateral membrane of the collecting duct provides an exit pathway for reabsorbed water.
Aquaporin-4 is abundant in brain and probably participates in reabsorption of cerebrospinal fluid, osmoregulation, and regulation of brain edema.
Aquaporin-5 mediates fluid secretion in salivary and lacrimal glands and is abundant in alveolar epithelium of the lung.
Specific regulation of membrane water permeability will likely prove important to understanding edema formation and fluid balance in both normal physiology and disease.

Discovery of aquaporins: a breakthrough in research on renal water transport

van Lieburg AF, Knoers NV, Deen PM.
Department of Pediatrics, University of Nijmegen, The Netherlands.
Pediatr Nephrol. Apr 1995; 9(2):228-34.
Several membranes of the kidney are highly water permeable, thereby enabling this organ to retain large quantities of water. Recently, the molecular identification of water channels responsible for this high water permeability has finally been accomplished. At present, four distinct renal water channels have been identified, all members of the family of major intrinsic proteins.
Aquaporin 1 (AQP1), aquaporin 2 (AQP2) and the mercury-insensitive water channel (MIWC) are water-selective channel proteins, whereas the fourth,
Aquaporin 3 (AQP3), permits transport of urea and glycerol as well. Furthermore, a putative renal water channel (WCH3) has been found.
AQP1 is expressed in apical and basolateral membranes of proximal tubules and descending limbs of Henle,
AQP2 predominantly in apical membranes of principal and inner medullary collecting duct cells and
AQP3 in basolateral membranes of kidney collecting duct cells.
MIWC is expressed in the inner medulla of the kidney and has been suggested to be localised in the vasa recta.
The human genes encoding AQP1 and AQP2 have been cloned, permitting deduction of their amino acid sequence, prediction of their two-dimensional structure by hydropathy analysis, speculations on their way of functioning and DNA analysis in patients with diseases possibly caused by mutant aquaporins. Mutations in the AQP1 gene were recently detected in clinically normal individuals, a finding which contradicts the presumed vital importance of this protein. Mutations in the AQP2 gene were shown to cause autosomal recessive nephrogenic diabetes insipidus. The renal unresponsiveness to arginine vasopressin, which characterises this disease, is in accordance with the assumption that AQP2 is the effector protein of the renal vasopressin pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Selectivity of the renal collecting duct water channel aquaporin-3

Echevarría M, Windhager EE, Frindt G.
Depart Physiol Biophys, Cornell University Medical College, New York, NY
J Biol Chem. Oct 11, 1996; 271(41):25079-82.
Aquaporin-3 (AQP3) is a water channel found in the basolateral cell membrane of principal cells of the renal collecting tubule as well as in other epithelia. To examine the selectivity of AQP3, the permeability to water (Pf), urea (Pur), and glycerol (Pgly) of Xenopus oocytes injected with cRNA encoding AQP3 was measured. Oocytes injected with cRNA encoding either human or rat aquaporin-1 (AQP1) were used as controls. Although both aquaporins permit water flow across the cell membrane, only AQP3 was permeable to glycerol and urea (Pgly > Pur). The uptake of glycerol into oocytes expressing AQP3 was linear up to 165 mM. For AQP3 the Arrhenius energy of activation for Pf was 3 kcal/mol, whereas for Pgly and Pur it was >12 kcal/mol. The sulfhydryl reagent p-chloromercuriphenylsulfonate (1 mM) abolished Pf of AQP3, whereas it did not affect Pgly. In addition, phloretin (0.1 mM) inhibited Pf of AQP3 by 35%, whereas it did not alter Pgly or Pur. We conclude that water does not share the same pathway with glycerol or urea in AQP3 and that this aquaporin, therefore, forms a water-selective channel.

The aquaporin family of water channels in kidney

Agre P, Nielsen S.
Depart of Med, Johns Hopkins University School of Medicine, Baltimore, MD
Nephrologie. 1996;17(7):409-15.
The longstanding puzzle of membrane water-permeability was advanced by discovery of a new class of proteins known as the “aquaporins” (AQPs). First identified in red blood cells, AQP1 was shown to function as a water channel when expressed in Xenopus oocytes or when pure AQP1 protein was reconstituted into synthetic membranes. Analysis of the primary sequence revealed that the two halves of the AQP1 polypeptide are tandem repeats; site directed mutagenesis studies indicate that the repeats may fold into an obversely symmetric structure which resembles an hourglass. Electron crystallography elucidated the tetrameric organization of AQP1, and functional studies suggest that each tetramer contains multiple functionally independent aqueous pores.
AQP1 is abundant in the apical and basolateral membranes of renal proximal tubules and descending thin limbs, and is also present in multiple extra renal tissues.
AQP2 is expressed only in the principal cells of renal collecting duct where it is the predominant vasopressin (ADH, antidiuretic hormone) regulated water channel. AQP2 is localized in the apical membrane and in intracellular vesicles which are targeted to the apical plasma membranes when stimulated by ADH. Humans with mutations in genes encoding AQP1 and AQP2 exhibit contrasting clinical phenotypes.
AQP3 resides in the basolateral membranes of renal collecting duct principal cells providing an exit pathway for water;
AQP4 is abundant in brain where it may function as the hypothalamic osmoreceptor responsible for secretion of ADH. Continued analysis of the aquaporins is providing detailed molecular insight into the fundamental physiological problems of water balance and disorders of water balance.

Aquaporins in the kidney: from molecules to medicine

Nielsen S, Frøkiaer J, Marples D, Kwon TH, Agre P, Knepper MA.
The Water and Salt Res Center, Anatomy and Exper Clin Res Institutes, University of Aarhus, Aarhus, Denmark.
Physiol Rev. Jan 2002; 82(1):205-44.

The molecular identity of membrane water channels long-standing biophysical question of how water crosses long remained elusive until the pioneering discovery of biological membranes specifically, and provided insight, at the molecular level, of AQP1 by Agre and colleagues around 1989 –1991,  The discovery of aquaporin-1 (AQP1) answered the long-standing biophysical question of how water specifically crosses biological membranes. In the kidney, at least seven aquaporins are expressed at distinct sites. AQP1 is extremely abundant in the proximal tubule and descending thin limb and is essential for urinary concentration. AQP2 is exclusively expressed in the principal cells of the connecting tubule and collecting duct and is the predominant vasopressin-regulated water channel. AQP3 and AQP4 are both present in the basolateral plasma membrane of collecting duct principal cells and represent exit pathways for water reabsorbed apically via AQP2. Studies in patients and transgenic mice have demonstrated that both AQP2 and AQP3 are essential for urinary concentration.

Since the discovery of aquaporins, major efforts have been aimed at elucidating their structural organization. Hydropathy analysis of the deduced amino acid sequence of AQP1 led to the prediction that the protein resides primarily within the lipid bilayer (191), consistent with the initial studies of AQP1 in red cell membranes (46). AQP1 contains an internal repeat with the NH – and the first provided a molecular answer to the long-standing COOH-terminal halves being sequence related and each
containing the signature motif Asn-Pro-Ala (NPA) (181,252). This is consistent with earlier observations on the homologous major intrinsic protein from lens, (MIP, nowreferred to as AQP0). When evaluated by hydropathy analysis, six bilayer-spanning domains are apparent (Fig.1); however, the apparent interhelical loops B and E also exhibit significant hydrophobicity. Critical to the topology is the location of loop C which connects the two halves of the molecule. Preston et al. (194) demonstrated that loop C resides at the extracellular surface of the oocytes, confirming the obverse sym-metry of the NH – and COOH-terminal halves of the mol-lar surface of the oocytes, confirming the obverse symmetry of the NH – and COOH-terminal halves of the mol-ecule.The structural organization of other aquaporins such as bacterial aquaporin-Z and plant aquaporins have also been deduced. How can water channels avoid passage of protons (H O )? As predicted, loops B and E are associated by Van der Waals interactions between the two NPA motifs. Free hydrogen bonding occurs in the column of water within the pore, except at the very center where a single water molecule transiently reorients to bond with the two asparagines residues of the NPA motif. This results in minimum resistance to the flow of water, thus permitting kidneys to perform their important physiological roles of reabsorbing water while excreting acid.

FIG. 1. A: schematic representation of the structural organization of aquaporin-1 (AQP1) monomers in the membrane (top and bottom). Aquaporins have six membrane-spanning regions, both intracellular NH and COOH termini, and internal tandem repeats that, presumably, are due to an ancient gene duplication (top). The topology is consistent with an obverse symmetry for the two similar NH – and COOH- 2 terminal halves (bottom). The tandem repeat structure with two asparagine-proline-alanine (NPA) sequences has been proposed to form tight turn structures that interact in the membrane to form the pathway for translocation of water across the plasma membrane. Of the five loops in AQP1, the B and E loops dip into the lipid bilayer, and it has been proposed that they form “hemichannels” that connect between the leaflets to form a single aqueous pathway within a symmetric structure that resembles an “hourglass.” B: AQP1 is a multisubunit oligomer that is organized as a tetrameric assembly of four identical polypeptide subunits with a large glycan attached to only one.

Discovery and Biophysical Characterization of the First Molecular Water Channel AQP1 Expression of AQP1 in X. laevis oocytes by Preston et al. (192) demonstrated that AQP1-expressing oocytes exhibited remarkably high osmotic water permeability (P
cm/s), causing the cells to swell rapidly and explode in hypotonic buffer. The osmotically induced swelling of oocytes expressing AQP1 occurs with a low activation energy and is reversibly inhibited by HgCl or other mercurials. Only inward water flow (swelling) was examined, but it was predicted that the direction of water flow through AQP1 is determined by the orientation of the osmotic gradient. Consistent with this, it was later demonstrated that AQP1-expressing oocytes swell in hyposmolar buffers but shrink in hyperosmolar buffers (160).  Swelling of oocytes expressing AQP1 occurs with a low activation energy and is reversibly inhibited by HgCl or other mercurials. Only inward water flow (swelling) was examined, but it was predicted that the direction of water flow through AQP1 is determined by the orientation of the osmotic gradient. Consistent with this, it was later demonstrated that AQP1-expressing oocytes swell in hyposmolar buffers but shrink in hyperosmolar buffers (160).

Over the past 4 years a series of studies have explored the issues of selectivity and polytransport function of aquaporins. This has led to a division of aquaporins (4) into a group that transports water relatively selectively (the “orthodox” set or “aquaporins”) and a group of water channels that also conduct glycerol and other small solutes in addition to water (the “cocktail” set or aquaglyceroporins). This appears to represent an ancient phylogenetic divergence between glycerol transporters and pure water channels (185). Recently, it has become clear that transport properties are even more diverse, since AQP6 has been demonstrated to conduct anions as well (263), and it has also been demonstrated that aquaporins can be regulated by gating, as discussed below.

The signal transduction pathways have been de­scribed thoroughly in previous reviews. cAMP levels in collecting duct principal cells are in­creased by binding of vasopressin to V2 receptors. The synthesis of cAMP by adenylate cyclase is stim­ulated by a V2 receptor-coupled heterotrimeric GTP-bind-ing protein, Gs. Gs interconverts between an inactive responses to vasopressin. In this study it was demon­strated that changes in AQP2 labeling density of the apical plasma membrane correlated closely with the water per­meability in the same tubules, while there were reciprocal changes in the intracellular labeling for AQP2. In vivo studies using normal rats or vasopressin-deficient Brattleboro rats also showed a marked increase in apical plasma membrane labeling of AQP2 in response to vasopressin or dDAVP treatment.  The acute treatment of rats with vasopressin V2-receptor antagonist or acute water loading (to reduce endogenous vasopressin levels, both re­ducing vasopressin action, resulted in a prominent inter­nalization of AQP2 from the apical plasma membrane to small intracellular vesicles further underscoring the role of AQP2 trafficking in the regulation of collecting duct water permeability.

PGE2 inhibits vasopressin-induced water permeabil­ity by reducing cAMP levels. In preliminary studies, Zelenina et al. investigated the effect of PGE2 on PKA phosphorylation of AQP2 in kidney papilla, and the results suggest that the action of prostaglandins is associated with retrieval of AQP2 from the plasma membrane, but that this appears to be independent of AQP2 phosphorylation by PKA.  Phosphorylation of AQP2 by other kinases, e.g., pro­tein kinase C or casein kinase II, may potentially partici­pate in regulation of AQP2 trafficking (Fig. 9C). Phosphorylation of other cytoplasmic or vesicular regulatory proteins may also be involved. These issues remain to be investigated directly.

Since the fundamentals of the shuttle hypothesis have been confirmed, interest has turned to the cellular mechanisms mediating the vasopressin-induced transfer of AQP2 to the apical plasma membrane. The shuttle hypothesis has a number of features whose molecular basis remains poorly understood. First, AQP2 is delivered in a relatively rapid and coordinated fashion, and vesicles move from a distribution throughout the cell to the apical region of the cell in response to vasopressin stimulation. Furthermore, AQP2 is delivered specifically to the apical plasma membrane. Finally, AQP2-bearing vesicles fuse with the apical plasma membrane in response to vasopressin, but not to a significant degree in the absence of stimulation (e.g., in vasopressin-deficient Brattleboro rats where < 5% of total AQP2 is present in the apical plasma membrane. Thus there must be some kind of a “clamp” preventing fusion in the unstimulated state and/or a “trigger” when activation occurs.

The coordinated delivery of AQP2-bearing vesicles to the apical part of the cell appears to depend on the translocation of the vesicles along the cytoskeletal ele­ments. In particular, the microtubular network has been implicated in this process, since chemical disruption of microtubules inhibits the increase in permeability both in the toad bladder and in the mammalian collecting duct. Because microtubule-disruptive agents inhibit the development of the hydrosmotic response to vaso-pressin, but have no effect on the maintenance of an established response, and because they have been re­ported to slow the development of the response without affecting the final permeability in toad bladders , it has been deduced that microtubules appear to be involved in the coordinated delivery of water channels, without being involved in the actual insertion process.

In addition to increasing cAMP levels in collecting duct principal cells, vasopressin acting through the V2 receptor has also been demonstrated to transiently in­crease intracellular Ca2+. The increase occurs in the absence of activation of the phosphoinositide signaling pathway and has recently been dem­onstrated to be due to activation of ryanodine-sensitive calcium release channels in the collecting duct cells. Buffering intracellular calcium with BAPTA or inhibition of calmodulin completely blocked the water permeability response to vasopressin in isolated perfused inner med­ullary collecting ducts, suggesting a critical role for cal­cium at some step in the process of AQP2 vesicle traffick­ing.

In addition to the acute regulation of collecting duct water permeability brought about by the trafficking of AQP2 described above, it is now clear that there are longer term adaptational changes that modulate this acute response. These occur during prolonged changes in body hydration status and form an appropriate physiolog­ical response to such challenges. However, similar long ­term changes also appear to be important in a wide variety of pathological conditions,  and an understanding of the mechanisms involved in these adaptational responses may provide the basis both for a better understanding of, and for potential therapeutic ap­proaches to, pathological disorders of water balance.  Microtubules are polar structures, arising from microtubule organizing centers (MTOCs), at which their minus ends are anchored, and with the plus ends growing away “into” the cell. In fibroblastic cells, there is a single MTOC in the perinuclear region, and the plus ends project to the periphery of the cell. However, there is increasing evidence that in polarized epithelia microtubules arise from multiple MTOCs in the apical region, with their plus ends projecting down toward the basolateral membrane. If this is the case in collecting duct cells, and there is some evidence that it is , then a minus end-directed motor protein such as dynein would be expected to be involved in the movement of vesicles toward the apical plasma membrane.  Recently, it has been shown that dynein is present in the kidney of several mammalian spe­cies and that both dynein and dynactin, a protein complex believed to mediate the interaction of dynein with vesicles, associate with AQP2-bearing vesicles. It seems likely that dynein may drive the microtubule-dependent delivery of AQP2-bearing vesicles toward the apical plasma mem­brane.

The apical part of the collecting duct principal cells contains a prominent terminal web made up of actin filaments. These also appear to be involved in the hydrosmotic response, since disruption of microfilaments with cytochalasins inhibits the response in the toad bladder. Cytochalasins can also inhibit an estab­lished response, and even the offset of the response. From this it has been concluded that microfilaments are probably involved in the final movement of vesicles through the terminal web, their fusion with the plasma membrane, and the subsequent endocytic retrieval of the water channels. Interestingly, vasopressin itself causes actin depolymerization, suggesting that reor­ganization of the terminal web is an important part of the cellular response to vasopressin, a conclusion reached on morphological grounds by DiBona.

The problem of delivering vesicles to a particular domain and allowing them to fuse when, and only when, a signal arrives is conceptually very similar to the situa­tion in the neuronal synapse. It therefore seemed possible that a molecular apparatus similar to the SNAP/SNARE system described there might be present in the collecting duct principal cells.  There are specific proteins on the vesicles (vSNAREs) and the target plasma membrane (tSNAREs) that interact with components of a fusion complex to induce fusion of the vesicles only with the required target membrane. The process is thought to be regulated by other protein com­ponents that sense the signal for fusion (i.e., increased calcium in the synapse). Several groups have now shown that vSNAREs such as VAMP-2 are present in the collect­ing duct principal cells and colocalize with AQP2 in the same vesicles .

A putative tSNARE, SNAP23, has been found in collecting duct principal cells both in the apical plasma membrane and in AQP2-bearing vesicles. Some soluble components of the fusion complex, including NEM-sensitive factor (NSF) and a-soluble NSF-associated protein (SNAP), have also been identified in these cells. Thus it seems likely that the exocytic insertion of AQP2 is indeed controlled by a set of proteins similar to those involved in synaptic transmission, al­though considerable work remains to be done in isolating and characterizing the components, their regulation, and prime physiological function.

 Body water balance is tightly regulated by vasopressin, and multiple studies now have underscored the essential roles of AQP2 in this.
Vasopressin regulates acutely the water permeability of the kidney collecting duct by trafficking of AQP2 from intracellular vesicles to the apical plasma membrane.
The long-term adaptational changes in body water balance are controlled in part by regulated changes in AQP2 and AQP3 expression levels. Lack of functional AQP2 is seen in primary forms of diabetes insipidus, and reduced expression and targeting are seen in several diseases associated with urinary concentrating defects such as acquired nephrogenic diabetes insipidus, postobstructive polyuria, as well as acute and chronic renal failure. In contrast, in conditions with water retention such as severe congestive heart failure, pregnancy, and syndrome of inappropriate antidiuretic hormone secretion, both AQP2 expression levels and apical plasma membrane targetting are increased, suggesting a role for AQP2 in the development of water retention. Continued analysis of the aquaporins is providing detailed molecular insight into the fundamental physiology and pathophysiology of water balance and water balance disorders.
Three additional aquaporins are present in the kidney. AQP6 is present in intracellular vesicles in collecting duct intercalated cells, and AQP8 is present intracellularly at low abundance in proximal tubules and collecting duct principal cells, but the physiological function of these two channels remains undefined. AQP7 is abundant in the brush border of proximal tubule cells and is likely to be involved in proximal tubule water reabsorption.

Fluid transport across leaky epithelia: central role of the tight junction and supporting role of aquaporins.

Fischbarg J.
Institute of Cardiology Research , A. C. Taquini, University of Buenos Aires and National Council for Scientific and Technical Investigations, Buenos Aires, Argentina.
Physiol Rev. Oct 2010; 90(4):1271-90.
The mechanism of epithelial fluid transport remains unsolved, which is partly due to inherent experimental difficulties. However, a preparation with which our laboratory works, the corneal endothelium, is a simple leaky secretory epithelium in which we have made some experimental and theoretical headway. As we have reported, transendothelial fluid movements can be generated by electrical currents as long as there is tight junction integrity. The direction of the fluid movement can be reversed by current reversal or by changing junctional electrical charges by polylysine. Residual endothelial fluid transport persists even when no anions (hence no salt) are being transported by the tissue and is only eliminated when all local recirculating electrical currents are.   The notion that transepithelial movement of water depends on the movement of electrolytes arises from a finding by Peter Curran and Arthur K. Solomon that transintestinal water flow (“solvent” flow) depended on the transport of NaCl (“solute” flux) by that layer. That gave birth to the question of how the flow of solute (or “salt”) is linked to the movement of solvent (or “fluid”), or in the short jargon of the field, how solute-solvent cou­pling arises. 
To be noted, gradientless flow is different from transepithelial osmosis a` la Dutrochet. In this last one, in the presence of an osmotic gradient across an epithelial layer, water obligingly traverses the layer. This is well exempli­fied by the kidney collecting duct, a tight epithelium for which we accept nowadays that the water goes across both cell plasma membranes in series, traversing their aquaporins.  There is also the special case of the anuran skin epithelia, whose intercellular junctions are tight, and which water also appears to traverse through cell membrane aquaporins. As a rule, epithelia specialized to transport fluid do so in the absence of any external osmotic gradient across their layers; that is, fluid is transported between compartments of similar osmolarity.  That gave birth to the question of how the flow of solute (or “salt”) is linked to the movement of solvent (or “fluid”), or in the short jargon of the field, how solute-solvent cou­pling arises.
The progression of the ideas on fluid transport is linked to those in a parallel field, that of water channels.  After early advances in their characterization and isolation, they were molecularly identified by Peter Agre and co-workers in the early 1990s, who termed them aquaporins (AQPs). It was subsequently de­termined that AQPs were present in many fluid transport­ing epithelia  and were also present in water-perme­able kidney segments while absent in relatively water-impermeable ones . By then, the measurements of osmotic permeabilities of epithelial cell membranes had been refined using video microscopy techniques. The lab­oratories of Kenneth Spring (working on gallbladders)  and of the Welling brothers (working on kidney proximal tubule) found rather high osmotic perme­ability (or “filtration” permeability, Pf) values (Persson and Spring: 550 and 1,200 pm/s for the apical and baso-lateral membranes, respectively; Welling: -300 pm/s). Both laboratories suggested that, given such high Pf values, a few milliosmoles of osmotic pressure difference across the cell boundaries would suffice to drive the transported fluids through the cells.

There had been all along experimental evidence for the diverging view that fluid transport across leaky epi­thelia took place via paracellular, transjunctional water flow. That contrary evidence came from the laboratories of Adrian Hill using gallbladder, John Pappenheimer and his fellow James Madara using intestine, and Guillermo Whittembury and Gerhard Malnic using kidney proximal tubule. The contrary view of paracellular flow had remained a minority opinion. Still and all, these “rebels” stood their ground, led by an utterly unconvinced Adrian Hill. Con­sidering the divergent views, Kenneth Spring and col­leagues decided to take the bull by the horns and use confocal microscopy to look for evidence for or against transjunctional water flow in epithelia.
Paracellular, transjunctional fluid flow in an absorbing epithelium would lead to significant dilution of a paracellular fluorescent marker trapped in the inter­cellular spaces, which in turn would be detectable by the optical sectioning methods they mastered; all very ele­gant, for sure.

And so we come to the paper Spring and colleagues published in May of 1998  reporting that they had found no transjunctional water flow in cultured Madin-Darby canine kidney (MDCK) cell layers. Understandably, their statement had a very large impact. And yet, only some months afterwards, this notion had to be revised as it became clear that the preparation they had chosen presumably transported little if any water. By Spring’s own admission in October of the same 1998, “ . . . the fluid transport rate of MDCK cells is only about 1% of that of the renal proximal tubule… ”  To spell out the obvious, little or no fluid transport means no transjunctional (or trans-cellular) water flow either, so in perspective, the findings of Spring and colleagues (“absence of junctional flow”) bring no surprise and have no bearing on the issue of the route of fluid flow in general.

After the demise of the 1998 paper above, doubts about local osmosis continued to be fueled. Adrian Hill had been joined in his criticism of it by Thomas Zeuthen and Ernest Wright. In particular, Zeuthen and co-workers had developed an alternative model for transcellular water transfer based on molecular cotransport through transporters. Predictably, Hill’s views were newly sought out. In a thorough review written with his wife and colleague Bruria Shachar-Hill, they restated the evidence from theirs and collaborating laboratories for junctional flow for Necturus and rabbit gallbladder, Necturus intestine, Rhodnius Malpighian tubule, and rat and rabbit salivary gland. In addition, they gave a convincing account of the evidence consistent with junctional water flow for renal proximal tubule, exocrine gland (salivary, lacrimal), and small intestine. Here we will simply call attention to those arguments and will concentrate on other arguments plus additional evidence of our own.

By the end of the 1990s, Alan Verkman’s laboratory had been investigating the physiological effects of knock­ing out AQPs in mice.  The dele­tion of AQPs resulted in drastic decreases of cell mem­brane osmotic permeability, but only in rather mild decreases in rates of fluid transport, and this last to boot only in tissues that transported fluid at high rates. Verkman and colleagues generally discuss those results in a guarded manner, underlining the role of aquaporins as routes for cell water permeability without making pro­nouncements on the mechanism of transtissue fluid trans­port. Yet, paraphrasing the comments by Hill and col­leagues in another cogent review, the effects seen in the AQP knockouts are sometimes difficult to explain, and not commensurate with the deletion of what would be hypothetically a major route for transcellular transtissue water transfer.

Perhaps the existence and the location of electrogenic transporters and channels are telling us something very fundamental about the function of these layers. There does not seem to be an explanation of why epithelia in general, and specifically leaky epithelia, would have evolved to have an electrical potential difference across the layer. In principle, salts could simply be transported neutrally. In a similar vein, apical Na channels that allow Na to leak back into the cell would not make sense if the task of an epithelial cell would be to transport salt from the serosal (basal) to the luminal (apical) side. However, both of these apparent incongruencies suddenly make sense if the raison d’être of these epithelia is to perform tasks such as electro-osmosis. The electrical potential might not be an evolutionary leftover but a central fea­ture. The Na channel would not be apical by accident but to help build up the local current meant for electro-osmosis. As mentioned above, aside from the corneal endothelium , there is evidence for electro-osmosis in small intestine, kidney proximal tubule, and frog skin glands. Hence, it would be desirable if the presence of electro-osmosis would be explored in other fluid-transporting epithelia.

Electro-osmotic coupling would result in somewhat (perhaps 30%) hypotonic emerging fluid. This entails that the fluid left behind at the intercellular spaces might be correspondingly hypertonic. Such osmolarity difference in turn might be sensed by the cell and trigger mechanisms that would affect sites for regulation at basolateral and apical sites for HCO3  and Na transports, and perhaps also at the junction so as to modify the characteristics of the coupling. It is conceivable that such regulation might take place with some degree of period­icity. There may be a role for AQP1 in this regulation, which would explain the mild effects seen on fluid trans­port in this and other preparations in experiments done with AQP1 null cells. This would explain what has been noted by Verkman and colleagues, namely, that effects of AQP deletion are more pronounced in epithelia that gen­erate higher rates of fluid transport. Thus AQP deletion reduced near-isosmolar fluid transport in kidney proximal tubule and salivary gland, where fluid transport is rapid, but not in lung, lacrimal gland, sweat gland, or corneal endothelium where fluid trans­port is relatively slow.

Aquaporin (AQP) 1 is the only AQP present in these cells, and its deletion in AQP1 null mice significantly affects cell osmotic permeability (by > 40%) but fluid transport much less ( > 20%), which militates against the presence of sizable water movements across the cell. In contrast, AQP1 null mice cells have reduced regulatory volume decrease (only 60% of control), which suggests a possible involvement of AQP1 in either the function or the expression of volume-sensitive membrane channels/transporters. A mathematical model of corneal endothelium we have developed correctly predicts experimental results only when paracellular electro-osmosis is assumed rather than transcellular local osmosis. Our evidence therefore suggests that the fluid is transported across this layer via the paracellular route by a mechanism that we attribute to electro-osmotic coupling at the junctions. From our findings we have developed a novel paradigm for this preparation that includes

1) paracellular fluid flow;
2) a crucial role for the junctions;
3) hypotonicity of the primary secretion; and
4) an AQP role in regulation rather than as a significant water pathway.
These elements are remarkably similar to those proposed by the laboratory of Adrian Hill for fluid transport across other leaky epithelia.

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 1743-8454-7-15-1  Distribution in brain of aquaporin-1 (AQP1, blue) and AQP4 (orange), schematically illustrated on a sagittal section of a human brain
centralpore-small  Tetrameric Pore                     AQP-highlight
Created with The GIMP                           Gating of aquaporins
AQP-thumbnail  Gas Molecules Commute into Cell      aqpz-glpf  water channels
GlpF-ABF  Molecular Obstacle Course              nihms365271f1   Roles of water-selective aquaporins (AQPs, shown in purple).
building_a_model-02-full     nihms365271f2  Roles of water-glycerol-transporting aquaporins (aquaglyceroporins).

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