Posts Tagged ‘Vascular smooth muscle’

Identification of Biomarkers that are Related to the Actin Cytoskeleton

Curator and Writer: Larry H Bernstein, MD, FCAP

This is Part I in a series of articles on Calcium and Cell motility.

The Series consists of the following articles:

Part I: Identification of Biomarkers that are Related to the Actin Cytoskeleton

Larry H Bernstein, MD, FCAP

Part II: Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility

Larry H. Bernstein, MD, FCAP, Stephen Williams, PhD and Aviva Lev-Ari, PhD, RN

Part III: Renal Distal Tubular Ca2+ Exchange Mechanism in Health and Disease

Larry H. Bernstein, MD, FCAP, Stephen J. Williams, PhD
 and Aviva Lev-Ari, PhD, RN

Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets

Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN

Part V: Ca2+-Stimulated Exocytosis:  The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter

Larry H Bernstein, MD, FCAP
Aviva Lev-Ari, PhD, RN

Part VI: Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD

Aviva Lev-Ari, PhD, RN

Part VII: Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmias and Non-ischemic Heart Failure – Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses

Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

Part VIII: Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism

Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

Part IXCalcium-Channel Blockers, Calcium Release-related Contractile Dysfunction (Ryanopathy) and Calcium as Neurotransmitter Sensor

Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

Part X: Synaptotagmin functions as a Calcium Sensor: How Calcium Ions Regulate the fusion of vesicles with cell membranes during Neurotransmission

Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

Part XI: Sensors and Signaling in Oxidative Stress

Larry H. Bernstein, MD, FCAP

Part XII: Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))

Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN


In this article the Author will cover two types of biomarker on the function of actin in cytoskeleton mobility in situ.

  • First, is an application in developing the actin or other component, for a biotarget and then, to be able to follow it as

(a) a biomarker either for diagnosis, or

(b) for the potential treatment prediction of disease free survival.

  • Second, is mostly in the context of MI, for which there is an abundance of work to reference, and a substantial body of knowledge about

(a) treatment and long term effects of diet, exercise, and

(b) underlying effects of therapeutic drugs.

1.  Cell Membrane (cytoskeletal) Plasticity

Refer to … Squeezing Ovarian Cancer Cells to Predict Metastatic Potential: Cell Stiffness as Possible Biomarker

Reporter/curator: Prabodh Kandala, PhD

New Georgia Tech research shows that cell stiffness could be a valuable clue for doctors as they search for and treat cancerous cells before they’re able to spread. The findings, which are published in the journal PLoS One, found that highly metastatic ovarian cancer cells are several times softer than less metastatic ovarian cancer cells. This study used atomic force microscopy (AFM) to study the mechanical properties of various ovarian cell lines. A soft mechanical probe “tapped” healthy, malignant and metastatic ovarian cells to measure their stiffness. In order to spread, metastatic cells must push themselves into the bloodstream. As a result, they must be highly deformable and softer. This study results indicate that cell stiffness may be a useful biomarker to evaluate the relative metastatic potential of ovarian and perhaps other types of cancer cells.

Comparative gene expression analyses indicate that the reduced stiffness of highly metastatic HEY A8 cells is associated with actin cytoskeleton remodeling and microscopic examination of actin fiber structure in these cell lines is consistent with this prediction.   The results suggest either of two approaches. Atomic Force Microscopy is not normally used by pathologists in diagnostics. Electron microscopy requires space for making and cutting the embedded specimen, and a separate room for the instrument. The instrument is large and the technique was not suitable for anything other than research initially until EM gained importance in Renal Pathology. It has not otherwise been used.  This new method looks like it might be more justified over a spectrum of cases.

A.  Atomic Force Microscopy

So the first point related to microscopy is whether AFM has feasibility for routine clinical use in the pathologists’ hands. This requires:

  1. suitable size of equipment
  2.  suitable manipulation of the specimen
  3. The question of whether you are using overnight fixed specimen, or whether the material is used unfixed
  4. Nothing is said about staining of cells for identification.
  5. Then there is the question about whether this will increase the number of Pathologist Assistants used across the country, which I am not against.   This would be the end of “house” trained PAs, and gives more credence to the too few PA programs across the country. The PA programs have to be reviewed and accredited by NAACLS (I served 8 years on the Board). A PA is represented on the Board, and programs are inspected by qualified peers.   There is no academic recognition given to this for tenure and promotion in Pathology Departments, and a pathologist is selected for a medical advisory role by the ASCP, and must be a Medical Advisor to a MLS accredited Program.   The fact is that PAs do gross anatomic dictation of selected specimens, and they do autopsies under the guidance of a pathologist. This is the reality of the profession today. The pathologist has to be in attendance at a variety of quality review conferences, for surgical morbidity and mortality to obstetrics review, and the Cancer Review. Cytopathology and cytogenetics are in the pathology domain.   In the case of tumors of the throat, cervix, and accessible orifices, it seems plausible to receive a swab for preparation. However, sampling error is greater than for a biopsy. A directed needle biopsy or a MIS specimen is needed for the ovary.

B.  identification of biomarkers that are related to the actin cytoskeleton

The alternative to the first approach is the identification of biomarkers that are related to the actin cytoskeleton, perhaps in the nature of the lipid or apoprotein isoform that gives the cell membrane deformability. The method measuring by Atomic Force Microscopy is shown with the current method of cytological screening, and I call attention to cells clustered together that have a scant cytoplasm surrounding nuclei occupying 1/2 to 3/4 of the cell radius.  The cells are not anaplastic, but the clumps are suggestive of glnad forming epithelium.

English: Animation showing 3-D nature of clust...

English: Animation showing 3-D nature of cluster. Image:Serous carcinoma 2a – cytology.jpg (Photo credit: Wikipedia)

The cell membrane, also called the plasma memb...

The cell membrane, also called the plasma membrane or plasmalemma, is a semipermeable lipid bilayer common to all living cells. It contains a variety of biological molecules, primarily proteins and lipids, which are involved in a vast array of cellular processes. It also serves as the attachment point for both the intracellular cytoskeleton and, if present, the cell wall. (Photo credit: Wikipedia)

English: AFM bema detection

AFM non contact mode

AFM non contact mode (Photo credit: Wikipedia)

C.  The diagnosis of ovarian cancer can be problematic because it can have a long period of growth undetected.

On the other hand, it is easily accessible once there is reason to suspect it. They are terrible to deal with because they metastasize along the abdominal peritoneum and form a solid cake. It is a problem of location and silence until it is late. Once they do announce a presence on the abdominal wall, there is probably a serous effusion. It was not possible to rely on a single marker, but when CA125 was introduced, Dr. Marguerite Pinto, Chief of Cytology at Bridgeport Hospital-Yale New Haven Health came to the immnunochemistry lab and we worked out a method for analyzing effusions, as we had already done with carcinoembryonic antigen.       The use of CEA and CA125 was published by Pinto and Bernstein as a first that had an impact.  This was followed by a study with the Chief of Oncology, Dr. Martin Rosman, that showed that the 30 month survival of patients post treatment is predicted by the half-life of disappearance of CA125 in serum.  At the time of this writing, I am not sure of the extent of its use 20 years later. History has taught us that adoption can be slow, depending very much on dissemination from major academic medical centers.  On the other hand, concepts can also be stuck at academic medical centers because of a rigid and unprepared mindset in the professional community.  The best example of this is the story of Ignaz Semmelweis, the best student of Rokitansky in Vienna for discovering the cause and prevention of childbirth fever at a time that nursemaids had far better results at obstetrical delivery than physicians.  Contrary to this, Edward Jenner, the best student of John Hunter (anatomist, surgeon, and physician to James Hume), discovered vaccination from the observation that milkmaids did not get smallpox (cowpox was a better alternative).
Only this year a Nobel Prize in Physics was awarded to an Israeli scientist who, working in the US, was unable to convince his associates of his discovery of PSEUDOCRYSTALS. – Diagnostic efficiency of carcinoembryonic antigen and CA125 in the cytological evaluation of effusions. M M Pinto, L H Bernstein, R A Rudolph, D A Brogan, M Rosman Arch Pathol Lab Med 1992; 116(6):626-631 ICID: 825503 Article type: Review article – Immunoradiometric assay of CA 125 in effusions. Comparison with carcinoembryonic antigen. M M Pinto, L H Bernstein, D A Brogan, E Criscuolo Cancer 1987; 59(2):218-222 ICID: 825555 Article type: Review article – Carcinoembryonic antigen in effusions. A diagnostic adjunct to cytology. M M Pinto, L H Bernstein, D A Brogan, E M Criscuolo Acta Cytologica 1987; 31(2):113-118 ICID: 825557

Predictive Modeling

Ovarian Cancer a plot of the CA125 elimination half-life vs the Kullback-Liebler distance

Ca125 half-life vs Kullback Entropy                                                          HL vs Survival KM plot 

Troponin(s) T, I, C  and the contractile apparatus  (contributed by Aviva Lev-Ari, PhD, RN)


For 2012 – 2013 Frontier Contribution in Cardiology on Gene Therapy Solutions for Improving Myocardial Contractility, see

Lev-Ari, A. 8/1/2013 Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD

For explanation of Conduction prior to Myocardial Contractility, see

Lev-Ari, A. 4/28/2013 Genetics of Conduction Disease: Atrioventricular (AV) Conduction Disease (block): Gene Mutations – Transcription, Excitability, and Energy Homeostasis

The contraction of skeletal muscle is triggered by nerve impulses, which stimulate the release of Ca2+ from the sarcoplasmic reticulum—a specialized network of internal membranes, similar to the endoplasmic reticulum, that stores high concentrations of Ca2+ ions. The release of Ca2+ from the sarcoplasmic reticulum increases the concentration of Ca2+ in the cytosol from approximately 10-7 to 10-5 M. The increased Ca2+ concentration signals muscle contraction via the action of two accessory proteins bound to the actin filaments: tropomyosin and troponin (Figure 11.25). Tropomyosin is a fibrous protein that binds lengthwise along the groove of actin filaments. In striated muscle, each tropomyosin molecule is bound to troponin, which is a complex of three polypeptides: troponin C (Ca2+-binding), troponin I (inhibitory), and troponin T (tropomyosin-binding).

  • When the concentration of Ca2+ is low, the complex of the troponins with tropomyosin blocks the interaction of actin and myosin, so the muscle does not contract.
  • At high concentrations, Ca2+ binding to troponin C shifts the position of the complex, relieving this inhibition and allowing contraction to proceed.

Figure 11.25

Association of tropomyosin and troponins with actin filaments. (A) Tropomyosin binds lengthwise along actin filaments and, in striated muscle, is associated with a complex of three troponins: troponin I (TnI), troponin C (TnC), and troponin T (TnT). In (more…)
Contractile Assemblies of Actin and Myosin in Nonmuscle Cells

Contractile assemblies of actin and myosin, resembling small-scale versions of muscle fibers, are present also in nonmuscle cells. As in muscle, the actin filaments in these contractile assemblies are interdigitated with bipolar filaments of myosin II, consisting of 15 to 20 myosin II molecules, which produce contraction by sliding the actin filaments relative to one another (Figure 11.26). The actin filaments in contractile bundles in nonmuscle cells are also associated with tropomyosin, which facilitates their interaction with myosin II, probably by competing with filamin for binding sites on actin.

Figure 11.26

Contractile assemblies in nonmuscle cells. Bipolar filaments of myosin II produce contraction by sliding actin filaments in opposite directions.

Two examples of contractile assemblies in nonmuscle cells, stress fibers and adhesion belts, were discussed earlier with respect to attachment of the actin cytoskeleton to regions of cell-substrate and cell-cell contacts (see Figures 11.13 and 11.14). The contraction of stress fibers produces tension across the cell, allowing the cell to pull on a substrate (e.g., the extracellular matrix) to which it is anchored. The contraction of adhesion belts alters the shape of epithelial cell sheets: a process that is particularly important during embryonic development, when sheets of epithelial cells fold into structures such as tubes.

The most dramatic example of actin-myosin contraction in nonmuscle cells, however, is provided by cytokinesis—the division of a cell into two following mitosis (Figure 11.27). Toward the end of mitosis in animal cells, a contractile ring consisting of actin filaments and myosin II assembles just underneath the plasma membrane. Its contraction pulls the plasma membrane progressively inward, constricting the center of the cell and pinching it in two. Interestingly, the thickness of the contractile ring remains constant as it contracts, implying that actin filaments disassemble as contraction proceeds. The ring then disperses completely following cell division.

Figure 11.27

Cytokinesis. Following completion of mitosis (nuclear division), a contractile ring consisting of actin filaments and myosin II divides the cell in two.

2.  Use of Troponin(s) in Diagnosis

Troponins T and I are released into the circulation at the time of an acute coronary syndrome (ACS).  Troponin T was first introduced by Roche (developed in Germany) for the Roche platform as a superior biomarker for identifying acute myocardial infarction (AMI), because of a monoclonal specificity to the cardiac troponin T.  It could not be measured on any other platform (limited license patent), so the Washington University Clinical Chemistry group developed a myocardiocyte specific troponin I that quickly became widely available to Beckman, and was adapted to other instruments.  This was intended to replace the CK isoenzyme MB, that is highly elevated in rhabdomyolysis associated with sepsis or with anesthesia in special cases.

The troponins I and T had a tenfold scale difference, and the Receiver Operator Curve Generated cutoff was accurate for AMI, but had significant elevation with end-stage renal disease.  The industry worked in concert to develop a high sensitivity assay for each because there were some missed AMIs just below the ROC cutoff, which could be interpreted as Plaque Rupture.  However, the concept of plaque rupture had to be reconsidered, and we are left with type1 and type 2 AMI (disregarding the case of post PCI or CABG related).   This led to the current establishment of 3 standard deviations above the lowest measureable level at 10% coefficient of variation.  This has been discussed sufficiently elsewhere.  It did introduce a problem in the use of the test as a “silver bullet” once the finer distinctions aqnd the interest in using the test for prognosis as well as diagnosis.   This is where the use of another protein associated with heart failure came into play – either the B type natriuretic peptide, or its propeptide, N-terminal pro BNP.  The prognostic value of using these markers, secreted by the HEART and acting on the kidneys (sodium reabsorption) has proved useful.  But there has not been a multivariate refinement of the use of a two biomarker approach.  In the following part D, I illustrate what can be done with an algorithmic approach to multiple markers.

Software Agent for Diagnosis of AMI

Isaac E. Mayzlin, Ph.D., David Mayzlin, Larry H. Bernstein, M.D. The so called gold standard of proof of a method is considered the Receiver-Operating Characteristic Curve, developed for detecting “enemy planes or missiles”, and adopted first by radiologists in medicine.  This matches the correct “hits” to the actual calssification and it is generally taught as a plot of sensitivity vs (1 – specifity).  But what if you had no “training” variable?  Work inspired by Eugene Rypka’s bacterial classification led to Rosser Rudolph’s application of the Entropy of Shannon and Weaver to identify meaningful information, referring to what was Kullback-Liebler distance as “effective information”.  This allowed Rudolph and Bernstein to classify using disease biomarkers obtaing the same results as the ROC curve using an apl program.  The same data set was used by Bernstein, Adan et al. previously, and was again used by Izaak Mayzlin from University of Moscow with a new wrinkle.  Dr. Mayzlin created a neural network (Maynet), and then did a traditional NN with training on the data, and also clustered the data using geometric distance clustering and trained on the clusters.  It was interesting to see that the optimum cluster separation was closely related to the number of classes and the accuracy of classification.  An earlier simpler model using the slope of the MB isoenzyme increase and percent of total CK activity was perhaps related to Burton Sobel’s work on CK-MB disappearance rate for infarct size. The main process consists of three successive steps: (1)       clustering performed on training data set, (2)       neural network’s training on clusters from previous step, and (3)       classifier’s accuracy evaluation on testing data. The classifier in this research will be the ANN, created on step 2, with output in the range [0,1], that provides binary result (1 – AMI, 0 – not AMI), using decision point 0.5. Table  1.  Effect  of  selection  of  maximum  distance  on  the  number  of  classes  formed  and  on  the accuracy of recognition by ANN

Clustering Distance Factor F(D = F * R) Number ofClasses Number of Nodes in The Hidden Layers Number of Misrecognized Patterns inThe TestingSet of 43 Percent ofMisrecognized 2414135 1,  02,  03,  01,  02,  03,  0 3,  2 3,  2 121121 1 1 2.3 2.3

Creatine kinase B-subunit activity in serum in cases of suspected myocardial infarction: a prediction model based on the slope of MB increase and percentage CK-MB activity. L H Bernstein, G Reynoso Clin Chem 1983; 29(3):590-592 ICID: 825549 Diagnosis of acute myocardial infarction from two measurements of creatine kinase isoenzyme MB with use of nonparametric probability estimation. L H Bernstein, I J Good, G I Holtzman, M L Deaton, J Babb.  Clin Chem 1989; 35(3):444-447 ICID: 825570 – Information induction for predicting acute myocardial infarction. R A Rudolph, L H Bernstein, J Babb. Clin Chem 1988; 34(10):2031-2038 ICID: 825568

Related articles

Related articles published on this Open Access Online Scientific Journal, include the following:

Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD

Aviva Lev-Ari, PhD, RN 8/1/2013

High-Sensitivity Cardiac Troponin Assays- Preparing the United States for High-Sensitivity Cardiac Troponin Assays

Larry Bernstein, MD, FCAP 6/13/2013

Dealing with the Use of the High Sensitivity Troponin (hs cTn) Assays

Larry Bernstein and Aviva Lev-Ari  5/18/2013

Acute Chest Pain/ER Admission: Three Emerging Alternatives to Angiography and PCI – Corus CAD, hs cTn, CCTA

Aviva Lev-Ari  3/10/2013

  • Redberg’s conclusions are correct for the initial screening. The issue has been whether to do further testing for low or intermediate risk patients.
  • The most intriguing finding that is not at all surprising is that the CCTA added very little in the suspect group with small or moderate risk.
  • The ultra sensitive troponin threw the ROC out the window
  • The improved assay does pick up minor elevations of troponin in the absence of MI.

Critical Care | Abstract | Cardiac ischemia in patients with septic …
Aviva Lev-Ari  6/26/2013

  • refer to:  Cardiac ischemia in patients with septic shock randomized to vasopressin or norepinephrine

Mehta S, Granton J,  Gordon AC, Cook DJ, et al.
Critical Care 2013, 17:R117
Troponin and CK levels, and rates of ischemic ECG changes were similar in the VP and NE groups. In multivariable analysis

  • only APACHE II was associated with 28-day mortality (OR 1.07, 95% CI 1.01-1.14, p=0.033).

Assessing Cardiovascular Disease with Biomarkers

Larry H Bernstein, MD, FCAP 12/25/2012

Vascular Medicine and Biology: CLASSIFICATION OF FAST ACTING THERAPY FOR PATIENTS AT HIGH RISK FOR MACROVASCULAR EVENTS Macrovascular Disease – Therapeutic Potential of cEPCs

Aviva Lev-Ari, PhD, RN 8/24/2012

 PENDING Integration

  • ‘Ryanopathy’: causes and manifestations of RyR2 dysfunction in heart failureCardiovasc Res. 2013;98:240-247,
  • Up-regulation of sarcoplasmic reticulum Ca2+ uptake leads to cardiac hypertrophy, contractile dysfunction and early mortality in mice deficient in CASQ2Cardiovasc Res. 2013;98:297-306,
  • Myocardial Delivery of Stromal Cell-Derived Factor 1 in Patients With Ischemic Heart Disease: Safe and PromisingCirc. Res.. 2013;112:746-747,
  • Circulation Research Thematic Synopsis: Cardiovascular GeneticsCirc. Res.. 2013;112:e34-e50,
  • Gene and cytokine therapy for heart failure: molecular mechanisms in the improvement of cardiac functionAm. J. Physiol. Heart Circ. Physiol.. 2012;303:H501-H512,
  • Ryanodine Receptor Phosphorylation and Heart Failure: Phasing Out S2808 and “Criminalizing” S2814Circ. Res.. 2012;110:1398-1402,


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Telling NO to Cardiac Risk

DDAH Says NO to ADMA(1); The DDAH/ADMA/NOS Pathway(2)

Author-Writer-Reporter:  Stephen J. Williams, PhD

Endothelium-derived nitric oxide (NO) has been shown to be vasoprotective.  Nitric oxide enhances endothelial cell survival, inhibits excessive proliferation of vascular smooth muscle cells, regulates vascular smooth muscle tone, and prevents platelets from sticking to the endothelial wall.  Together with evidence from preclinical and human studies, it is clear that impairment of the NOS pathway increases risk of cardiovascular disease (3-5).

This post contains two articles on the physiological regulation of nitric oxide (NO) by an endogenous NO synthase inhibitor asymmetrical dimethylarginine (ADMA) and ADMA metabolism by the enzyme DDAH(1,2).  Previous posts on nitric oxide, referenced at the bottom of the page, provides excellent background and further insight for this posting. In summary plasma ADMA levels are elevated in patients with cardiovascular disease and several large studies have shown that plasma ADMA is an independent biomarker for cardiovascular-related morbidity and mortality(6-8).



Figure 1 A. Cardiac risks of ADMA B. Effects of ADMA (Photo credit: Wikipedia)

ADMA Production and Metabolism

Nuclear proteins such as histones can be methylated on arginine residues by protein-arginine methyltransferases, enzymes which use S-adenosylmethionine as methyl groups.  This methylation event is thought to regulate protein function, much in the way of protein acetylation and phosphorylation (9).  And much like phosphorylation, these modifications are reversible through methylesterases.   The proteolysis of these arginine-methyl modifications lead to the liberation of free guanidine-methylated arginine residues such as L-NMMA, asymmetric dimethylarginine (ADMA) and symmetrical methylarginine (SDMA).

The first two, L-NMMA and ADMA, have been shown to inhibit the activity of the endothelial NOS.  This protein turnover is substantial: for instance the authors note that each day 40% of constitutive protein in adult liver is newly synthesized protein. And in several diseases, such as muscular dystrophy, ischemic heart disease, and diabetes, it has been known since the 1970’s that protein catabolism rates are very high, with corresponding increased urinary excretion of ADMA(10-13).  Methylarginines are excreted in the urine by cationic transport.  However, the majority of ADMA and L-NMMA are degraded within the cell by dimethylaminohydrolase (DDAH), first cloned and purified in rat(14).

endogenous NO inhibitors from pubchem

Figure 2.  Endogenous inhibitors of NO synthase.  Chemical structures generated from PubChem.


DDAH specifically hydrolyzes ADMA and L-NMMA to yield citruline and demethylamine and usually shows co-localization with NOS. Pharmacologic inhibition of DDAH activity causes accumulation of ADMA and can reverse the NO-mediated bradykinin-induced relaxation of human saphenous vein.

Two isoforms have been found in human:

  • DDAH1 (found in brain and kidney and associated with nNOS) and
  • DDAH2 (highly expressed in heart, placenta, and kidney and associated with eNOS).

DDAH2 can be upregulated by all-trans retinoic acid (atRA can increase NO production).  Increased reactive oxygen species and possibly homocysteine, a risk factor for cardiovascular disease, can decrease DDAH activity(15,16).

  • The importance of DDAH activity can also be seen in transgenic mice which overexpress DDAH, exhibiting increased NO production, increased insulin sensitivity, and reduced vascular resistance  (17).  Likewise,
  • Transgenic mice, null for the DDAH1, showed increase in blood pressure, decreased NO production, and significant increase in tissue and plasma ADMA and L-NMMA.


Figure 3.  The DDAH/ADMA/NOS cycle. Figure adapted from Cooke and Ghebremarian (1).

As mentioned in the article by Cooke and Ghebremariam, the authors state: the weight of the evidence indicates that DDAH is a worthy therapeutic target. Agents that increase DDAH expression are known, and 1 of these, a farnesoid X receptor agonist, is in clinical trials

An alternate approach is to

  • develop an allosteric activator of the enzyme.  Although
  • development of an allosteric activator is not a typical pharmaceutical approach, recent studies indicate that this may be achievable aim(18).


1.            Cooke, J. P., and Ghebremariam, Y. T. : DDAH says NO to ADMA.(2011) Arteriosclerosis, thrombosis, and vascular biology 31, 1462-1464

2.            Tran, C. T., Leiper, J. M., and Vallance, P. : The DDAH/ADMA/NOS pathway.(2003) Atherosclerosis. Supplements 4, 33-40

3.            Niebauer, J., Maxwell, A. J., Lin, P. S., Wang, D., Tsao, P. S., and Cooke, J. P.: NOS inhibition accelerates atherogenesis: reversal by exercise. (2003) American journal of physiology. Heart and circulatory physiology 285, H535-540

4.            Miyazaki, H., Matsuoka, H., Cooke, J. P., Usui, M., Ueda, S., Okuda, S., and Imaizumi, T. : Endogenous nitric oxide synthase inhibitor: a novel marker of atherosclerosis.(1999) Circulation 99, 1141-1146

5.            Wilson, A. M., Shin, D. S., Weatherby, C., Harada, R. K., Ng, M. K., Nair, N., Kielstein, J., and Cooke, J. P. (2010): Asymmetric dimethylarginine correlates with measures of disease severity, major adverse cardiovascular events and all-cause mortality in patients with peripheral arterial disease. Vasc Med 15, 267-274

6.            Kielstein, J. T., Impraim, B., Simmel, S., Bode-Boger, S. M., Tsikas, D., Frolich, J. C., Hoeper, M. M., Haller, H., and Fliser, D. : Cardiovascular effects of systemic nitric oxide synthase inhibition with asymmetrical dimethylarginine in humans.(2004) Circulation 109, 172-177

7.            Kielstein, J. T., Donnerstag, F., Gasper, S., Menne, J., Kielstein, A., Martens-Lobenhoffer, J., Scalera, F., Cooke, J. P., Fliser, D., and Bode-Boger, S. M. : ADMA increases arterial stiffness and decreases cerebral blood flow in humans.(2006) Stroke; a journal of cerebral circulation 37, 2024-2029

8.            Mittermayer, F., Krzyzanowska, K., Exner, M., Mlekusch, W., Amighi, J., Sabeti, S., Minar, E., Muller, M., Wolzt, M., and Schillinger, M. : Asymmetric dimethylarginine predicts major adverse cardiovascular events in patients with advanced peripheral artery disease.(2006) Arteriosclerosis, thrombosis, and vascular biology 26, 2536-2540

9.            Kakimoto, Y., and Akazawa, S.: Isolation and identification of N-G,N-G- and N-G,N’-G-dimethyl-arginine, N-epsilon-mono-, di-, and trimethyllysine, and glucosylgalactosyl- and galactosyl-delta-hydroxylysine from human urine. (1970) The Journal of biological chemistry 245, 5751-5758

10.          Inoue, R., Miyake, M., Kanazawa, A., Sato, M., and Kakimoto, Y.: Decrease of 3-methylhistidine and increase of NG,NG-dimethylarginine in the urine of patients with muscular dystrophy. (1979) Metabolism: clinical and experimental 28, 801-804

11.          Millward, D. J.: Protein turnover in skeletal muscle. II. The effect of starvation and a protein-free diet on the synthesis and catabolism of skeletal muscle proteins in comparison to liver. (1970) Clinical science 39, 591-603

12.          Goldberg, A. L., and St John, A. C.: Intracellular protein degradation in mammalian and bacterial cells: Part 2. (1976) Annual review of biochemistry 45, 747-803

13.          Dice, J. F., and Walker, C. D.: Protein degradation in metabolic and nutritional disorders. (1979) Ciba Foundation symposium, 331-350

14.          Ogawa, T., Kimoto, M., and Sasaoka, K.: Purification and properties of a new enzyme, NG,NG-dimethylarginine dimethylaminohydrolase, from rat kidney. (1989) The Journal of biological chemistry 264, 10205-10209

15.          Ito, A., Tsao, P. S., Adimoolam, S., Kimoto, M., Ogawa, T., and Cooke, J. P.: Novel mechanism for endothelial dysfunction: dysregulation of dimethylarginine dimethylaminohydrolase. (1999) Circulation 99, 3092-3095

16.          Stuhlinger, M. C., Tsao, P. S., Her, J. H., Kimoto, M., Balint, R. F., and Cooke, J. P. : Homocysteine impairs the nitric oxide synthase pathway: role of asymmetric dimethylarginine.(2001) Circulation 104, 2569-2575

17.          Sydow, K., Mondon, C. E., Schrader, J., Konishi, H., and Cooke, J. P.: Dimethylarginine dimethylaminohydrolase overexpression enhances insulin sensitivity. (2008) Arteriosclerosis, thrombosis, and vascular biology 28, 692-697

18.          Zorn, J. A., and Wells, J. A.: Turning enzymes ON with small molecules. (2010) Nature chemical biology 6, 179-188

Other research papers on Nitric Oxide and Cardiac Risk  were published on this Scientific Web site as follows:

The Nitric Oxide and Renal is presented in FOUR parts:

Part I: The Amazing Structure and Adaptive Functioning of the Kidneys: Nitric Oxide

Part II: Nitric Oxide and iNOS have Key Roles in Kidney Diseases

Part III: The Molecular Biology of Renal Disorders: Nitric Oxide

Part IV: New Insights on Nitric Oxide donors

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Nitric Oxide has a Ubiquitous Role in the Regulation of Glycolysis – with a Concomitant Influence on Mitochondrial Function


Reporter, Editor, and Topic Co-Leader: Larry H. Bernstein, MD, FACP, Clinical Pathologist and Biochemist



Apoptosis signaling pathways

Apoptosis signaling pathways (Photo credit: AJC1)

This discussion is a followup on a series of articles elucidating the importance of NO, eNOS, iNOS, cardiovascular and vascular endothelium effects, and therapeutic targets.

This mechanism of action and signaling actions have been introduced so that we identify endocrine, paracrine, and such effects in the normal, stressed, and dysfunctional state. The size and breadth of this vital adaptive process is now further explored.

The title is short, befitting a subtitle.  The full topic may be considered “Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function that is active in endothelium, platelets, vascular smooth muscle and neural cells and the balance has a role in chronic inflammation, asthma, hypertension, sepsis and cancer”.

Vascular endothelium

Vascular endothelium (Photo credit: Wikipedia)

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Nitric Oxide Synthase

Nitric Oxide Synthase (Photo credit: Wikipedia)



Nitric Oxide has a ubiquitous role in the regulation of glycolysis with a concomitant influence on mitochondrial function that is active in endothelium, platelets, vascular smooth muscle and neural cells and the balance has a role in chronic inflammation, asthma, hypertension, sepsis and cancer.

Uncoupling of aerobic glycolysis
Potential cytotoxic mediators of endothelial cell (EC) apoptosis include increased formation of reactive oxygen and nitrogen species (ROSRNS) during the atherosclerotic process. Nitric oxide (NO) has a biphasic action on oxidative cell killing with low concentrations protecting against cell death, whereas higher concentrations are cytotoxic. High levels of NO can be produced by inducible nitric-oxide synthase in response to cytokine stimulation, primarily from macrophages, and elevated levels of NO is injurious to endothelium.Ccytochrome c release and caspase activation are involved in NO induced apoptosis. ROS also induces mitochondrial DNA damage in ECs, and this damage is accompanied by a decrease in mitochondrial RNA (mtRNA) transcripts, mitochondrial protein synthesis, and cellular ATP levels. Mitochondria have been recognized to play a pivotal role in the signaling cascade of apoptosis leading to atherosclerosis-induced damage in endothelial cells.
The processes involved in the signaling pathways leading to apoptosis are complex but have some degree of convergence between cell types including those in the vasculature. Release of cytochrome c from mitochondria is a proapoptotic signal, which activates several downstream signaling events including formation of the apoptosome and activation of caspases. Ubiquinol cytochrome c reductase (complex III) is a site for ROS formation, and cytochrome c oxidase (complex IV) is a target for the interaction of NO in mitochondria.
The impact of the inhibition of mitochondrial protein synthesis is particularly important in NO-dependent cytotoxicity, and depends also on other factors such as glycolysis. These authors examined whether the inhibition of mitochondrial protein synthesis by chloramphenicol increases the susceptibility of endothelial cells to undergo NO-dependent apoptosis in glucose-free media. Bovine aortic endothelial cells were treated with chloramphenicol, which resulted in a decreased ratio of mitochondrial complex IV to cytochrome c and increased oxidant production in the cell. Inhibition of mitochondrial protein synthesis was associated with a greater susceptibility of the cells to apoptosis induced by NO in glucose-free medium.
Inhibition of mitochondrial protein synthesis results in increased endothelial cell susceptibility to nitric oxide-induced apoptosis. A Ramachandran, DR Moellering, E Ceaser, S Shiva, J Xu, and V Darley-Usmar. PNAS May 14, 2002: 99(10): 6643–6648 http://www.pnas.orgcgidoi10.1073pnas.102019899

Nitric oxide (NO) is a ubiquitous signaling molecule whose physiological roles mediated through the activation of the soluble guanylate cyclase are now clearly recognized. At physiological concentrations, NO also inhibits the mitochondrial enzyme cytochrome c oxidase (complex IV) in competition with oxygen, and recently we have suggested that the interplay between the two gases allows this enzyme to act as an oxygen sensor in cells. In addition, NO plays a variety of patho-physiological roles, some of which also may be the consequence of its action at a mitochondrial level. We have characterized the sequence of events that follow inhibition of complex IV by continuous exposure to NO.
The mitochondrion is a key organelle in the control of cell death. Nitric oxide (NO) inhibits complex IV in the respiratory chain and is reported to possess both proapoptotic and antiapoptotic actions. We investigated the effects of continuous inhibition of respiration by NO on mitochondrial energy status and cell viability. Serum-deprived human T cell leukemia (Jurkat) cells were exposed to NO at a concentration that caused continuous and complete (;85%) inhibition of respiration. Serum deprivation caused progressive loss of mitochondrial membrane potential (Dcm) and apoptotic cell death. In the presence of NO, Dcm was maintained compared to controls, and cells were protected from apoptosis. Similar results were obtained by using staurosporin as the apoptotic stimulus. As exposure of serum-deprived cells to NO progressed (>5 h), however, Dcm fell, correlating with the appearance of early apoptotic features and a decrease in cell viability. Glucose deprivation or iodoacetate treatment of cells in the presence of NO resulted in a collapse of Dcm, demonstrating involvement of glycolytic ATP in its maintenance. Under these conditions cell viability also was decreased. Treatment with oligomycin and or bongkrekic acid indicated that the maintenance of Dcm during exposure to NO is caused by reversal of the ATP synthase and other electrogenic pumps. Thus, blockade of complex IV by NO initiates a protective action in the mitochondrion to maintain Dcm; this results in prevention of apoptosis. It is likely that during cellular stress involving increased generation of NO this compound will trigger a similar sequence of events, depending on its concentration and duration of release. (mitochondrial membrane potential ; apoptosis ; necrosis)

The effect of nitric oxide on cell respiration: A key to understanding its role in cell survival or death. B Beltra, A Mathur, MR Duchen, JD. Erusalimsky, and S Moncada. PNAS Dec 19, 2000; 97(26):4602–14607.

Another study by this group shows that inhibition of respiration by exogenous nitric oxide (NO) in Jurkat cells leads to mitochondrial membrane hyperpolarization dependent on the utilization of glycolytic ATP by the F1Fo-ATPase and other transporters acting in reverse mode. This process also occurs in astrocytes, which are highly glycolytic cells, but not in neurons , which do not invoke glycolysis to maintain ATP concentrations. In addition, this hyperpolarization correlates with protection against apoptotic cell death. Others found an early phase of mitochondrial hyperpolarization after treatment of a variety of cells with different pro-apoptotic stimuli, which precedes the generation of free. At present, no satisfactory explanation has been proposed to explain the mechanism of hyperpolarization, the reasons why free radicals are released from the mitochondrion, or the connection of these phenomena with apoptosis.
The authors surmise that a pro-apoptotic stimulus, anti-Fas Ab, leads to release of endogenous NO from Jurkat cells in sufficient amounts to inhibit cell respiration and cause a hyperpolarization dependent on the reversal of the F1Fo-ATPase. Moreover, the reduction of the mitochondrial electron transport chain, after inhibition of cytochrome oxidase by NO, leads to generation of superoxide anion (O2). They suggest the process is a cellular defense response that may be overcome by pro-apoptotic mechanisms that occur in parallel.

Inhibition of mitochondrial respiration by endogenous nitric oxide: A critical step in Fas signaling. B Beltran, M Quintero, E Garcıa-Zaragoza, E O’Connor, JV. Esplugues, and Salvador Moncada. PNAS June 25, 2002 99(13): 8892–8897. http://www.pnas.orgcgidoi10.1073pnas.092259799

Nitric oxide has been shown to render cells resistant to oxidative stress. Mechanisms proposed for the ability of nitric oxide to protect cells against oxidative stress include reactions of nitric oxide and the induction of adaptive responses that require protein synthesis. Nitric oxide forms iron complexes preventing the formation of strong oxidants. In addition, reactions of nitric oxide with lipid and or organic radicals protect against membrane peroxidation and peroxidative chemistry-induced cell injury. Exposure to low, nonlethal doses of nitric oxide induces adaptive responses that render cells resistant to lethal concentrations of nitric oxide and or peroxides, such as, the induction of hemoxygenase-1 (HO-1) and Mn superoxide dismutase. The up-regulation of HO-1 was accompanied by an increase in ferritin to account for the release of iron from HO-1, indicating a role of both iron heme and nonheme iron for peroxide-mediated cellular injury. Further, nitric oxide, by regulating critical mitochondrial functions such as respiration, membrane potential, and release of cytochrome c, is able to trigger defense mechanisms against cell death induced by pro-apoptotic stimuli.
This study investigates the potential contribution of nitric oxide’s ability to protect cells from oxidative stress, low steady state levels of nitric oxide generated by endothelial nitric oxide synthase (eNOS) and the mechanisms of protection against H2O2. Spontaneously transformed human ECV304 cells, which normally do not express eNOS, were stably transfected with a green fluorescent-tagged eNOS cDNA. The eNOS-transfected cells were found to be resistant to injury and delayed death following a 2-h exposure to H2O2 (50–150 mM). Inhibition of nitric oxide synthesis abolished the protective effect against H2O2 exposure. The ability of nitric oxide to protect cells depended on the presence of respiring mitochondria. ECV3041 eNOS cells with diminished mitochondria respiration are injured to the same extent as non-transfected ECV304 cells, and recovery of mitochondrial respiration restores the ability of nitric oxide to protect against H2O2-induced death. Nitric oxide had a profound effect in cell metabolism, because ECV3041eNOS cells had lower steady state levels of ATP and higher utilization of glucose via the glycolytic pathway than ECV304 cells. However, the protective effect of nitric oxide against H2O2 exposure is not reproduced in ECV304 cells after treatment with azide and oligomycin suggesting that the dynamic regulation of respiration by nitric oxide represent a critical and unrecognized primary line of defense against oxidative stress.

Dynamic regulation of metabolism and respiration by endogenously produced nitric oxide protects against oxidative stress. E Paxinou, M Weisse, Q Chen, JM Souza, et al. PNAS Sept 25, 2001; 98( 20): 11575–11580. http://www.pnas.orgycgiydoiy10.1073ypnas.201293198.

Nitric oxide (NO) mediates a variety of biological effects including relaxation of blood vessels, cytotoxicity of activated macrophages, and formation of cGMP by activation of glutamate receptors of neurons. NO has also been implicated for such pathophysiological conditions as destruction of tumor cells by macrophages, rheumatoid arthritis, and focal brain ischemia. Some of these effects of NO are associated with hypoxic conditions. O2 radicals and ions that result from reactivity of NO are presumed to be involved in NO cytotoxicity. These investigators report that adaptive cellular response controlled by the transcription factor hypoxia-inducible factor 1 (HIF-1) in hypoxia is suppressed by NO. Induction of erythropoietin and glycolytic aldolase A mRNAs in hypoxically cultured Hep3B cells, a human hepatoma cell line, was completely and partially inhibited, respectively, by the addition of sodium nitroprusside (SNP), which spontaneously releases NO. A reporter plasmid carrying four hypoxia-response element sequences connected to the luciferase structural gene was constructed and transfected into Hep3B cells. Inducibly expressed luciferase activity in hypoxia was inhibited by the addition of SNP and two other structurally different NO donors, S-nitroso-Lglutathione and 3-morpholinosydnonimine, giving IC50 values of 7.8, 211, and 490 mM, respectively. Inhibition by SNP was also observed in Neuro 2A and HeLa cells, indicating that the inhibition was not cell-type-specific. The vascular endothelial growth factor promoter activity that is controlled by HIF-1 was also inhibited by SNP (IC50 5 6.6 mM). Induction generated by the addition of cobalt ion (this treatment mimics hypoxia) was also inhibited by SNP (IC50 5 2.5 mM). Increased luciferase activity expressed by cotransfection of effector plasmids for HIF-1a or HIF-1a-like factor in hypoxia was also inhibited by the NO donor. We also showed that the inhibition was performed by blocking an activation step of HIF-1a to a DNA-binding form.
Inhibition of hypoxia-inducible factor 1 activity by nitric oxide donors in hypoxia. K Sogawa, K Numayama-Tsuruta, M Ema, M Abe, et al. Proc. Natl. Acad. Sci. USA (Biochemistry) June 1998; 95:7368–7373. 1998. The National Academy of Sciences 0027-8424.98.957368-6.

The role of nitrogen metabolism in the survival of prolonged periods of waterlogging was investigated in highly flood-tolerant, nodulated Lotus japonicus plants. Alanine production revealed to be a critical hypoxic pathway. Alanine is the only amino acid whose biosynthesis is not inhibited by nitrogen deficiency resulting from RNA interference silencing of nodular leghemoglobin. The metabolic changes that were induced following waterlogging can be best explained by the activation of alanine metabolism in combination with the modular operation of a split tricarboxylic acid pathway. The sum result of this metabolic scenario is the accumulation of alanine and succinate and the production of extra ATP under hypoxia. The importance of alanine metabolism is discussed with respect to its ability to regulate the level of pyruvate, and this and all other changes are discussed in the context of current models concerning the regulation of plant metabolism.
Glycolysis and the Tricarboxylic Acid Cycle Are Linked by Alanine Aminotransferase during Hypoxia Induced by Waterlogging of Lotus japonicus[W][OA]. M Rocha, F Licausi, WL Arau´ jo, A Nunes-Nesi, et al. Plant Physiology Mar 2010; 152: 1501–1513. 2010 Amer Soc Plant Biologists

DNA damage occurs in ischemia, excitotoxicity, inflammation, and other disorders that affect the central nervous system (CNS). Extensive DNA damage triggers cell death and in the mature CNS, this occurs primarily through activation of the poly(ADP-ribose) polymerase-1 (PARP-1) cell death pathway. PARP-1 is an abundant nuclear enzyme that, when activated by DNA damage, consumes nicotinamide adenine dinucleotide (NAD)+ to form poly(ADP-ribose) on acceptor proteins. The PARP-1 activation leads to cell death. We used mouse astrocyte cultures to explore the bioenergetic effects of NAD+ depletion by PARP-1 and the role of NAD+ depletion in this cell death program. PARP-1 activation led to a rapid but incomplete depletion of astrocyte NAD+, a near-complete block in glycolysis, and eventual cell death. Repletion of intracellular NAD restored glycolytic function and prevented cell death. The addition of non-glucose substrates to the medium, pyruvate, glutamate, or glutamine, also prevented astrocyte death after PARP-1 activation.
These findings suggest a sequence of events in which NAD+ depletion is a key event linking DNA damage to metabolic impairment and cell deathm. A similar scenario has been proposed by Zong et al. (2004), based on the finding that cell types that depend on aerobic glycolysis for ATP production exhibit a particularly high sensitivity to DNA damage and PARP-1 activation. In mature brain, glucose is normally the dominant metabolic substrate due to relatively slow transport of other metabolites across the blood– brain barrier. Oncein brain, glucose may be metabolized directly by neurons and glia or may be metabolized to lactate in glia and thelactate subsequently shuttled to neurons for oxidative metabolism (Dringen et al., 1993; Pellerin and Magistretti,1994; Wender et al., 2000; Dienel and Cruz, 2004). In either case, a block in glycolytic flux produced by NAD depletion will block energy metabolism in both neurons and glia in brain. Interestingly, the lactate shuttle hypothesis raises the possibility that activation of PARP-1 selectively in astroglia might also block energy metabolism in neurons.

These studies suggest PARP-1 activation leads to rapid depletion of the cytosolic but not the mitochondrial NAD+ pool. Depletion of the cytosolic NAD+ pool renders the cells unable to utilize glucose as a metabolic substrate. Under conditions where glucose is the only available metabolic substrate, this leads to cell death. This cell death pathway is particularly germane to brain because glucose is normally the only metabolic substrate that is transported rapidly across the blood–brain barrier. © 2004 Wiley-Liss, Inc.
Key words: mitochondria; permeability transition; poly(ADP-ribose) polymerase; ischemia; peroxynitrite
NAD+as a metabolic link between DNA damage and cell death. DNA damage induced by alkylating agents, oxidative stress, or other agents causes PARP-1 activation. PARP-1 activation leads to depletion in cytosolic NAD with, initially, a relative preservation of mitochondrial NAD and mitochondrial function. The depletion in cytosolic NAD+ blocks glycolysis, and in cells in which glucose is the primary energy substrate, this in turn leads to a block in substrate flux to mitochondria. The resulting mitochondrial dysfunction leads to mitochondrial permeability transition (MPT) and subsequent downstream events culminating in cell death.
NAD+ as a Metabolic Link Between DNA Damage and Cell Death. W Ying, CC Alano, P Garnier, and RA Swanson. Journal of Neuroscience Research 2005;79:216–223
Key words: glycolysis, mitochondrial energy production, nitric oxide
Abbreviations: NO, nitric oxide; SNAP, S-nitroso-N-acetylpenicyllamine; SNP, sodium nitroprusside.
The results indicate that: 1) in porcine platelets NO is able to diminish mitochondrial energy production through the inhibition of cytochrome oxidase, 2) the inhibitory effect of NO on platelet secretion (but not aggregation) can be attributed to the reduction of mitochondrial energy production.
Nitric oxide (NO) has been increasingly recognized as an important intra- and intercellular messenger molecule with a physiological role in vascular relaxation, platelet physiology, neurotransmission and immune responses (Moncada et al., 1991; Radomski et al., 1996; Szabó, 1996; Riedel et al., 1999; Titheradge 1999). In vitro NO is a strong inhibitor of platelet adhesion and aggregation (Radomski et al., 1996; Riedel et al., 1999;nSogo et al., 2000). In the blood stream, platelets remain in contact with NO that is permanently released from the endothelial cells and from activated macrophages (Moncada et al., 1991; Riedel et al., 1999; Titheradge 1999). It has been suggested that the activated platelet itself is able to produce NO (Lantoine et al., 1995; Zhou et al., 1995; Radomski et al., 1996). The mechanism responsible for the inhibitory effect of NO on platelet responses is not entirely clear. It is believed that the main intracellular target for NO in platelets is soluble cytosolic guanylate cyclase (Waldman & Walter 1989; Schmidt et al., 1993; Wang et al., 1998). NO activates the enzyme (Schmidt et al., 1993). Thus, elevated intracellular cGMP level inhibits platelet activation. There are suggestions, however, that elevated cGMP may not be the only intracellular factor directly involved in the inhibition of platelet activation (Gordge et al., 1998; Sogo et al., 2000; Beghetti et al., 2003).
Platelets are fairly active metabolically and have a total ATP turnover rate of about 3–8 times that of resting mammalian muscle (Akkerman, 1978; Akkerman et al., 1978; Holmsen, 1981; Niu et al., 1996). Platelets contain mitochondria which enable these cells to produce energy both in the oxidative and anaerobic way (Holmsen, 1981). Under aerobic conditions, ATP is produced by aerobic glycolysis using glucose or glycogen which can account for 30–50% of total ATP production, and by oxidative metabolism using glucose and glycogen (6–11%), amino-acids (7%) or free fatty acids (20–40%) (Holmsen 1981; Guppy et al., 1990; Niu et al., 1996).
The inhibition of mitochondrial respiration by removing oxygen or by respiratory chain blockers (antimycin A, cyanide, rotenone) results in the stimulation of glycolytic flux (Guppy et al., 1990). This phenomenon is known as Pasteur effect and indicates that in platelets glycolysis and mitochondrial respiration are tightly functionally connected (Akkerman, 1978; Holmsen, 1981; Guppy et al., 1995; Niu et al., 1996). It has been reported that the activation of human platelets by high concentration of thrombin is accompanied by an acceleration of lactate production and an increase in oxygen consumption (Akkerman & Holmsen, 1981; Niu et al., 1996).
The results presented here suggest that also porcine blood platelets stimulated by collagen produce more lactate. This indicates that both glycolytic and oxidativeATP production supports platelet responses. This also indicates that blocking of energy production in platelets may decrease their responses. It is well established that platelet responses have different metabolic energy (ATP) requirements increasing in the order: aggregation< dense and alfa granule secretion < acid hydrolase secretion (Holmsen et al., 1982; Verhoeven et al., 1984; Morimoto & Ogihara, 1996).
The present results indicate that exogenously added NO (in the form of NO donors)stimulates glycolysis in intact porcine platelets. Since in platelets glycolysis and mitochondrial respiration are tightly functionally connected, this can be interpreted to mean that the stimulatory effectof NO on glycolysis in intact platelets may be produced by non-functional mitochondria.This can be really the case since NO donors are able to inhibit both mitochondrial respiration and platelet cytochrome oxidase. Interestingly, the concentrations of NO donors inhibiting mitochondrial respiration and cytochrome oxidase were similar to those stimulating glycolysis in intact platelets.
Studies performed on intact J774 cells have shown that mitochondrial complex I is inhibited only after a prolonged (6–18 h) exposure to NO and that this inhibition appears to result from S-nitrosylation of critical thiols in the enzyme complex (Clementi et al., 1998). Further studies are needed to establish whether long term exposure of platelets to NO affects Mitochondrial complexes I and II.
Comparison of the concentrations of SNP and SNAP affecting cytochrome oxidase activityand mitochondrial respiration with those reducing the platelet responses indicates that NO cannot significantly reduce platelet aggregation through the inhibition of oxidative energy production. By contrast, the concentrations of the NO donors inhibiting platelet secretion, mitochondrial respiration and cytochrome oxidase were similar. This and the fact that the platelet release reaction strongly depends on the oxidative energy production may suggest that in porcine platelets NO can affect platelet secretion through the inhibition of mitochondrial energy production at the step of cytochrome oxidase.

Taking into account that platelets may contain NO synthase and are able to produce significant amounts of NO (Berkels et al., 1997)it seems possible that nitric oxide can function in these cells as a physiological regulator of mitochondrial energy production.
Nitric oxide and platelet energy metabolism. M Tomasiak, H Stelmach, T Rusak and J Wysocka. Acta Biochimica Polonica 2004; 51(3):789–803

These authors previously investigated the bioenergetic consequences of activating J774.A1 macrophages (MФ) with interferon (IFN)γ and lipopolysaccharide (LPS) and found that there is a nitric oxide (NO)-dependent mitochondrial impairment and stabilization of hypoxia inducible factor (HIF)-1α, which synergize to activate glycolysis and generate large
quantities of ATP. We now demonstrate, using TMRM fluorescence and time-lapse confocal microscopy, that these cells maintain a high mitochondrial membrane potential (ΔΨm) despite the complete inhibition of respiration. The maintenance of high ΔΨm is due to the utilization of a significant proportion of glycolytically generated ATP as a defence mechanism against cell death. This is achieved by the reverse functioning of FoF1-ATP synthase and adenine nucleotide translocase (ANT). Treatment of activated MФ with inhibitors of either of these enzymes, but not with inhibitors of the respiratory chain complexes I to IV, led to a collapse in ΔΨm and to an immediate increase in intracellular [ATP], due to the prevention of ATP hydrolysis by the FoF1-ATP synthase. This collapse in ΔΨm was followed by translocation of Bax from cytosol to the mitochondria, release of cytochrome c into the cytosol, activation of caspase 3 and 9 and subsequent apoptotic cell death. Our results indicate that during inflammatory activation “glycolytically competent cells” such as MФ utilize significant amounts of the glycolytically-generated ATP to maintain ΔΨm and thereby prevent apoptosis.

Activated macrophages utilize glycolytic ATP to maintain mitochondrial membranepotential and prevent apoptotic cell death. A Garedew, SO Henderson, S Moncada. Cell Death and Differentiation. 2010. DOI : 10.1038/cdd.2010.27
The effects of the sodium nitroprusside (SNP), a nitric oxide (NO) donor clinically used in the treatment of hypertensive emergencies on the energy production of rat reticulocytes were investigated. Rat reticulocyte-rich red blood cell suspensions were aerobically incubated without (control) or in the presence of different concentrations of SNP (0.1, 0.25, 0.5, 1.0 mM). SNP decreased total and coupled, but increased uncoupled oxygen consumption. This was accompanied by the stimulation of glycolysis, as measured by increased glucose consumption and lactate accumulation. Levels of all glycolytic intermediates indicate stimulation of hexokinase-phosphofructo kinase (HK-PFK), glyceraldehyde 3-phosphate dehydrogenase (GAPD) and pyruvate kinase (PK) activities in the presence of SNP. Due to the decrease of coupled oxygen consumption in the presence of SNP, ATP production via oxidative phosphorylation was significantly diminished. Simultaneous increase of glycolytic ATP production was not enough to provide constant ATP production. In addition, SNP significantly decreased ATP level, which was accompanied with increased ADP and AMP levels. However, the level of total adenine nucleotides was significantly lower, which was the consequence of increased catabolism of adenine nucleotides (increased hypoxanthine level). ATP/ADP ratio and adenylate energy charge level were significantly decreased. In conclusion, SNP induced inhibition of oxidative phosphorylation, stimulation of glycolysis, but depletion of total energy production in rat reticulocytes. These alterations were accompanied with instability of energy status.

Effects of Exogenous Donor of Nitric Oxide – Sodium Nitroprusside on Energy Production of Rat Reticulocytes. SD MALETIĆ, L M DRAGIĆEVIĆ-DJOKOVIĆ, BI OGNJANOVIĆ, RV ŽIKIĆ, AŠ ŠTAJN, MB SPASIĆ.
Physiol. Res. 2004;53: 439-447.

Key points to take from this:
1. The role of NO in regulating cellular death is in many organs and central to this function is the stabilization of mitochondria through sufficient levels of NO. High levels of eNO leads to mitochondrial dysfunction that increases the dependence of ATP generated from glycolysis.
2. This is accompanied by inhibition of oxidative phosphorylation and stimulation of glycolysis, which brings the discussion to a different domain – cancer growth and Warburgh Effect.
3. This is accompanied by PPAR activation, cytoplasmic NAD+ depletion, and inhibition of glycolysis (critical in cells dependent on aerobic glycolysis), depletion of total energy production, and apoptosis.
4. Maintenance of high glycolytic generation of ATP is essential for cellular defense, but the oxygen consumption is uncoupled.
5. NO donors inhibiting mitochondrial respiration and cytochrome oxidase are similar to those stimulating glycolysis

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