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Muscular dystrophy has deficient stem cell dystrophin

Posted in Biological Networks, Ca2+ triggered activation, Ca2+ triggered activation, Cell Biology, Signaling & Cell Circuits, Clinical Genomics, Curation, Developmental biology, Disease Biology, Gene Regulation, Genetics & Innovations in Treatment, Genetics & Pharmaceutical, Genome Biology, Inflammasome, Interviews with Scientific Leaders, Investment in Technological Breakthrough, MS, Neurodegenerative Diseases, Neurohumoral Transmission, Neurological Diseases, Neuroscience, Pharmaceutical Analytics, Pharmaceutical Discovery, Population genetics, Proteins, Proteomics, Regenerative Biology and Medicine, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, Skeletal muscle regeneration, Stem Cells for Regenerative Medicine, tagged blocking cell differentiation, Ca2+-regulation, Clinical trial, dystrophin, genomic expression, Muscular dystrophy, skeletal and cardiomyocyte, Stem cell on November 21, 2015| Leave a Comment »

Muscular dystrophy has deficient stem cell dystrophin

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Dystrophin Deficient Stem Cell Pathology

Muscular Dystrophy is a Stem Cell-Based Disease

November 19, 2015 by mburatov

https://beyondthedish.wordpress.com/2015/11/19/muscular-dystrophy-is-a-stem-cell-based-disease/

Michael Rudnicki, who has done pioneering work in muscle stem cell biology and muscle regeneration, and whose work has been featured several times on this blog, has struck again. Rudnicki, who serves as director of the Regenerative Medicine Program at The Ottawa Hospital and a professor at the University of Ottawa and holds the prestigious Canada Research Chair in Molecular Genetics, teamed up with workers from the Sprott Centre for Stem Cell Research and the Sinclair Centre for Regenerative Medicine to investigate the role of muscle-specific stem cells in patients who suffer from Duchenne muscular dystrophy. This new earth-shaking study, which was published in the journal Nature Medicine (November 16, 2015), has changed the way we think about muscular dystrophy and will almost certainly force people to rethink the treatments and cures for this dreadful disease.

According to this new study, Duchenne muscular dystrophy directly affects muscle stem cells, and is, largely a disease of muscle stem cells.

Rudicki said: “For nearly 20 years, we’ve thought that the muscle weakness observed in patients with Duchenne muscular dystrophy is primarily due to problems in their muscle fibers, but our research shows that it is also due to intrinsic defects in the function of their muscle stem cells. This completely changes our understanding of Duchenne muscular dystrophy and could eventually lead to far more effective treatments.”

Muscular dystrophy comes in several different forms, but the predominant sign of muscular dystrophy is progressive muscle weakness. Altogether, muscular dystrophy refers to a group of more than 30 genetic diseases, all of which cause progressive weakness and degeneration of skeletal muscles used during voluntary movement. Approximately half of all who suffer from muscular dystrophy have Duchenne muscular dystrophy (DMD). Because muscular dystrophy results from mutations in the dystrophin gene, which is on the X chromosome, the vast majority of muscular dystrophy patients are male. Girls can be carriers of muscular dystrophy and can be mildly affected.

Interestingly, somewhere around one-third of boys who suffer from DMD have no family history of the disease. Because the dystrophin gene is so large, spontaneous mutations in it are probably relatively common.

The signs and symptoms typically appear between the ages of 2 and 3, and may include frequent falls, difficulty getting up from a lying or sitting position, trouble running and jumping, a strange, shuffling way of walking or having a tendency to walk on their toes, calf muscles that are abnormally large, muscle pain and stiffness, and some learning disabilities.

Muscular dystrophy affects all ethnic groups and occurs globally. It affects around 1 in every 3,500 to 6,000 male births each year in the United States. DMD affects approximately one in 3,600 boys.

Because DMD results from mutations in the dystrophin gene, the vast majority of muscular dystrophy research was based on a simple model in which the Dystrophin protein played a structural role in the structural integrity of muscle fibers. Abnormal versions of the Dystrophin protein caused the muscle fibers to become damaged and die as a result of contraction. Dystrophin anchors the cytoskeleton of the muscle fibers, which are essential for muscle contraction, to the muscle cell membrane, and then to the extracellular matrix outside the cell that serves as a foundation upon which the muscle cells are built.

However in this current study, Rudnicki and his team discovered that muscle stem cells also express the dystrophin protein. This is a revelation because Dystrophin was thought to be protein that ONLY appeared in mature muscle. However, in this study, it became exceedingly clear that in the absence of Dystrophin, muscle stem cells generated ten-fold fewer muscle precursor cells, and, consequently, far fewer functional muscle fibers. Dystrophin is also a component of a signal transduction pathway that allows muscle stem cells to properly ascertain if they need to replace dead or dying muscle. Muscle stem cells repair the muscle in response to injury or exercise by dividing to generate precursor cells that differentiate into muscle fibers.

Even though Rudnicki used mice as a model system in these experiments, the Dystrophin protein is highly conserved in most vertebrate animals. Therefore, it is highly likely that these results will also apply to human muscle stem cells.

Gene therapy experiments and trials are in progress and even show some promise, but Rudnicki’s work tells us that gene therapy approaches must target muscle stem cells as well as muscle fibers if they are to work properly.

“We’re already looking at approaches to correct this problem in muscle stem cells,” said Dr. Rudnicki.

This paper has received high praise from the likes of Ronald Worton, who was one of the co-discovers of the dystrophin gene with Louis Kunkel in 1987.

Early pathogenesis of Duchenne muscular dystrophy modelled in patient-derived human induced pluripotent stem cells

Emi Shoji, Hidetoshi Sakurai, Tokiko Nishino, Tatsutoshi Nakahata, Toshio Heike, Tomonari Awaya, Nobuharu Fujii, Yasuko Manabe, Masafumi Matsuo & Atsuko Sehara-Fujisawa

Scientific Reports 5, Article number: 12831 (2015) http://dx.doi.org:/10.1038/srep12831

Duchenne muscular dystrophy (DMD) is a progressive and fatal muscle degenerating disease caused by a dystrophin deficiency. Effective suppression of the primary pathology observed in DMD is critical for treatment. Patient-derived human induced pluripotent stem cells (hiPSCs) are a promising tool for drug discovery. Here, we report an in vitro evaluation system for a DMD therapy using hiPSCs that recapitulate the primary pathology and can be used for DMD drug screening. Skeletal myotubes generated from hiPSCs are intact, which allows them to be used to model the initial pathology of DMD in vitro. Induced control and DMD myotubes were morphologically and physiologically comparable. However, electric stimulation of these myotubes for in vitro contraction caused pronounced calcium ion (Ca²⁺) influx only in DMD myocytes. Restoration of dystrophin by the exon-skipping technique suppressed this Ca²⁺ overflow and reduced the secretion of creatine kinase (CK) in DMD myotubes. These results suggest that the early pathogenesis of DMD can be effectively modelled in skeletal myotubes induced from patient-derived iPSCs, thereby enabling the development and evaluation of novel drugs.

Duchenne muscular dystrophy (DMD) is characterised by progressive muscle atrophy and weakness that eventually leads to ambulatory and respiratory deficiency from early childhood¹. It is an X-linked recessive inherited disease with a relatively high frequency of 1 in 3500 males^1,2.DMD, which is responsible for DMD, encodes 79 exons and produces dystrophin, which is one of the largest known cytoskeletal structural proteins³. Most DMD patients have various types of deletions or mutations in DMD that create premature terminations, resulting in a loss of protein expression⁴. Several promising approaches could be used to treat this devastating disease, such as mutation-specific drug exon-skipping^5,6, cell therapy⁷, and gene therapy^1,2.

Myoblasts from patients are the most common cell sources for assessing the disease phenotypes of DMD^11,12. …Previous reports have shown that muscle cell differentiation from DMD patient myoblasts is delayed and that these cells have poor proliferation capacity compared to those of healthy individuals^11,12. Our study revealed that control and DMD myoblasts obtained by activating tetracycline-dependent MyoD transfected into iPS cells (iPS^tet-MyoD cells) have comparable growth and differentiation potential and can produce a large number of intact and homogeneous myotubes repeatedly.

The pathogenesis of DMD is initiated and progresses with muscle contraction. The degree of muscle cell damage at the early stage of DMD can be evaluated by measuring the leakage of creatine kinase (CK) into the extracellular space¹⁵. Excess calcium ion (Ca²⁺) influx into skeletal muscle cells, together with increased susceptibility to plasma membrane injury, is regarded as the initial trigger of muscle damage in DMD^{19,20,21,22,23,24}. Targeting these early pathogenic events is considered essential for developing therapeutics for DMD.

In this study, we established a novel evaluation system to analyse the cellular basis of early DMD pathogenesis by comparing DMD myotubes with the same clone but with truncated dystrophin-expressing DMD myotubes, using the exon-skipping technique. We demonstrated through in vitro contraction that excessive Ca²⁺ influx is one of the earliest events to occur in intact dystrophin-deficient muscle leading to extracellular leakage of CK in DMD myotubes.

Generation of tetracycline-inducible MyoD-transfected DMD patient-derived iPSCs (iPS^tet-MyoD cells)

Figure 1: Generation and characterization of control and DMD patient-derived Tet-MyoD-transfected hiPS cells. Full size image

Morphologically and physiologically comparable intact myotubes differentiated from control and DMD-derived hiPSCs

Figure 2: Morphologically and physiologically comparable skeletal muscle cells differentiated from Control-iPS^tet-MyoD and DMD-iPS^tet-MyoD. Full size image

Exon-skipping with AO88 restored expression of Dystrophin in DMD myotubes differentiated from DMD-iPS^tet-MyoD cells

Figure 3: Restoration of dystrophin protein expression by AO88. Full size image

Restored dystrophin expression attenuates Ca²⁺ overflow in DMD-Myocytes

Figure 4: Restored expression of dystrophin diminishes Ca²⁺ influx in DMD muscle in response to electric stimulation. Full size image

Ca²⁺ influx provokes skeletal muscle cellular damage in DMD muscle

Figure 5: Ca²⁺ influx induces prominent skeletal muscle cellular damage in DMD-Myocytes. Full size image

Skeletal muscle differentiation in myoblasts from DMD patients is generally delayed compared to that in healthy individuals^11,36,37. Our differentiation system successfully induced the formation of myotubes from DMD patients, and the myotubes displayed analogous morphology and maturity compared with control myotubes (Fig. 2a–c). Comparing myotubes generated from patient-derived iPS cells with those derived from the same DMD clones but expressing dystrophin by application of the exon-skipping technique enabled us to demonstrate the primary cellular phenotypes in skeletal muscle solely resulting from the loss of the dystrophin protein (Fig. 4b). Our results demonstrate that truncated but functional dystrophin protein expression improved the cellular phenotype of DMD myotubes.

In DMD, the lack of dystrophin induces an excess influx of Ca²⁺ , leading to pathological dystrophic changes²². We consistently observed excess Ca²⁺ influx in DMD-Myocytes compared to Control-Myocytes (Supplementary Figure S3a and S3b) in response to electric stimulation. TRP channels, which are mechanical stimuli-activated Ca²⁺ channels⁴⁰that are expressed in skeletal muscle cells⁴¹, can account for this pathogenic Ca²⁺ influx…

In conclusion, our study revealed that the absence of dystrophin protein induces skeletal muscle damage by allowing excess Ca²⁺ influx in DMD myotubes. Our experimental system recapitulated the early phase of DMD pathology as demonstrated by visualisation and quantification of Ca²⁺ influx using intact myotubes differentiated from hiPS cells. This evaluation system significantly expands prospective applications with regard to assessing the effectiveness of exon-skipping drugs and also enables the discovery of drugs that regulate the initial events in DMD.

Duchenne muscular dystrophy affects stem cells, University of Ottawa study finds

New treatments could one day be available for the most common form of muscular dystrophy after a study suggests the debilitating genetic disease affects the stem cells that produce healthy muscle fibres.

The findings are based on research from the University of Ottawa and The Ottawa Hospital, published Monday in the journal Nature Medicine.

For nearly two decades, doctors had thought the muscular weakness that is the hallmark of the disease was due to problems with human muscle fibers, said Dr. Michael Rudnicki, the study’s senior author.

The new research shows the specific protein characterized by its absence in Duchenne muscular dystrophy normally exists in stem cells.

Dystrophin protein found in stem cells

“The prevailing notion was that the protein that’s missing in Duchenne muscular dystrophy — a protein called dystrophin — was not involved at all in the function of the stem cells.”

http://soundcloud.com/cbcottawa1

When the genetic mutations caused by Duchenne muscular dystrophy inhibit the production of dystrophin in stem cells, those stem cells produce significantly fewer precursor cells — and thus fewer properly functioning muscle fibres. Further, stem cells need dystrophin to sense their environment to figure out if they need to divide to produce more stem cells or perform muscle repair work.

Genetic repair might treat Duchenne muscular dystrophy

July 25, 2011|By Thomas H. Maugh II, Los Angeles Times

A genetic technique that allows the body to work around a crucial mutation that causes Duchenne muscular dystrophy increased the mass and function of muscles in a small group of patients with the devastating disease, paving the way for larger clinical trials of the drug. The study in a handful of boys age 5 to 15 showed that patients receiving the highest level of the drug, called AVI-4658 or eteplirsen, had a significant increase in production of a missing protein and increases in muscle fibers. The study demonstrated that the drug is safe in the short term. Results were reported Sunday in the journal Lancet.

Duchenne muscular dystrophy affects about one in every 3,500 males worldwide. It is caused by any one of several different mutations that affect production of a protein called dystrophin, which is important for the production and maintenance of muscle fibers. Affected patients become unable to walk and must use a wheelchair by age 8 to 12. Deterioration continues through their teens and 20s, and the condition typically proves fatal as muscle failure impairs their ability to breathe.

This study is designed to assess the efficacy, safety, tolerability, and pharmacokinetics (PK) of AVI-4658 (eteplirsen) in both 50.0 mg/kg and 30.0 mg/kg doses administered over 24 weeks in subjects diagnosed with Duchenne muscular dystrophy (DMD).

Condition	Intervention	Phase
Duchenne Muscular Dystrophy	Drug: AVI-4658 (Eteplirsen) Other: Placebo	Phase 2

Study Type:	Interventional
Study Design:	Allocation: Randomized Endpoint Classification: Safety/Efficacy Study Intervention Model: Parallel Assignment Masking: Double Blind (Subject, Caregiver, Investigator, Outcomes Assessor) Primary Purpose: Treatment
Official Title:	A Randomized, Double-Blind, Placebo-Controlled, Multiple Dose Efficacy, Safety, Tolerability and Pharmacokinetics Study of AVI-4658(Eteplirsen),in the Treatment of Ambulant Subjects With Duchenne Muscular Dystrophy

Resource links provided by NLM:

Genetics Home Reference related topics: Duchenne and Becker muscular dystrophy

MedlinePlus related topics: Muscular Dystrophy

Genetic and Rare Diseases Information Center resources: Becker Muscular Dystrophy Duchenne Muscular Dystrophy Muscular Dystrophy

U.S. FDA Resources

Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division

Nicolas A Dumont, Yu Xin Wang, Julia von Maltzahn, Alessandra Pasut, C Florian Bentzinger, Caroline E Brun & Michael A Rudnicki

Nature Medicine(2015) http://dx.doi.org:/10.1038/nm.3990

Dystrophin is expressed in differentiated myofibers, in which it is required for sarcolemmal integrity, and loss-of-function mutations in the gene that encodes it result in Duchenne muscular dystrophy (DMD), a disease characterized by progressive and severe skeletal muscle degeneration. Here we found that dystrophin is also highly expressed in activated muscle stem cells (also known as satellite cells), in which it associates with the serine-threonine kinase Mark2 (also known as Par1b), an important regulator of cell polarity. In the absence of dystrophin, expression of Mark2 protein is downregulated, resulting in the inability to localize the cell polarity regulator Pard3 to the opposite side of the cell. Consequently, the number of asymmetric divisions is strikingly reduced in dystrophin-deficient satellite cells, which also display a loss of polarity, abnormal division patterns (including centrosome amplification), impaired mitotic spindle orientation and prolonged cell divisions. Altogether, these intrinsic defects strongly reduce the generation of myogenic progenitors that are needed for proper muscle regeneration. Therefore, we conclude that dystrophin has an essential role in the regulation of satellite cell polarity and asymmetric division. Our findings indicate that muscle wasting in DMD not only is caused by myofiber fragility, but also is exacerbated by impaired regeneration owing to intrinsic satellite cell dysfunction.

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Update on mitochondrial function, respiration, and associated disorders

Posted in Anaerobic Glycolysis, Ca2+ triggered activation, Ca2+ triggered activation, Cell Biology, Signaling & Cell Circuits, Curation, Discovery process, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Experimental validation, Explanatory, Inferential analysis, Metabolomics, Na-K-ATPase, Nephrology, Neurohumoral Transmission, Oxidative phosphorylation, Phosphorylation, Proteomics, Translational Science, tagged 5HT1F, alpha ketoglutarate, ATP synthase, brown adipose tissue (BAT), C. Elegans, ca-uniporter (MCU), Ca2+-regulation, Cancer - General, CLL, coupled respiration, Glut4 transporter, inner membrane, K-depolarization, mitochondria, mTOR, NADPH, neurons, OCR, Oxidative phosphorylation, pyruvate -rate limiting, ROS, Sestrin2, TOR, UCP1 on July 8, 2014| Leave a Comment »

Larry H. Benstein, MD, FCAP, Gurator and writer

http://pharmaceuticalintelligence.com/7/8/2014/Update on mitochondrial function, respiration, and associated disorders

This is a condensed account of very recent published work on respiration and disturbed mitochondrail function. We know that their is an equilibrium between respiration and autophagy in eukaryotic cells. The Krebs Cycle produces 32 ATPs in oxidative phosphorylation, which is far more efficient than glycolysis. There is also a different contribution of mitochondrial metabolism, in the balance, between tissues that are synthetic and those that are catabolic. This is a subject long understood, essential for cellular energetics, and not adequately explored.

Gain-of-Function Mutant p53 Promotes Cell Growth and Cancer Cell Metabolism via Inhibition of AMPK Activation.

Zhou G¹, Wang J², Zhao M², Xie TX², Tanaka N², et al.
Mol Cell. 2014 Jun 19;54(6):960-974. doi: 10.1016/j.molcel.2014.04.024.

Many mutant p53 proteins (mutp53s) exert oncogenic gain-of-function (GOF) properties, but the mechanisms mediating these functions remain poorly defined.

We show here that GOF mutp53s inhibit AMP-activated protein kinase (AMPK) signaling in head and neck cancer cells.

Conversely, downregulation of GOF mutp53s enhances AMPK activation under energy stress, decreasing the activity of the anabolic factors acetyl-CoA carboxylase and ribosomal protein S6 and inhibiting aerobic glycolytic potential and invasive cell growth.

Under conditions of energy stress, GOF mutp53s, but not wild-type p53, preferentially bind to the AMPKα subunit and inhibit AMPK activation.

Given the importance of AMPK as an energy sensor and tumor suppressor that inhibits anabolic metabolism, our findings reveal that direct inhibition of AMPK activation is an important mechanism through which mutp53s can gain oncogenic function. PMID:24857548

Investigating and Targeting Chronic Lymphocytic Leukemia Metabolism with the HIV Protease Inhibitor Ritonavir and Metformin.

Adekola KU, Aydemir SD, Ma S, Zhou Z, Rosen ST, Shanmugam M.
Leuk Lymphoma. 2014 May 14:1-23.

Chronic Lymphocytic Leukemia (CLL) remains fatal due to the development of resistance to existing therapies. Targeting abnormal glucose metabolism sensitizes various cancer cells to chemotherapy and/or elicits toxicity.

Examination of glucose dependency in CLL demonstrated variable sensitivity to glucose deprivation. Further evaluation of metabolic dependencies of CLL cells resistant to glucose deprivation revealed increased engagement of fatty acid oxidation upon glucose withdrawal.

Investigation of glucose transporter expression in CLL reveals up-regulation of glucose transporter GLUT4. Treatment of CLL cells with HIV protease inhibitor ritonavir, that inhibits GLUT4, elicits toxicity similar to that elicited upon glucose-deprivation.

CLL cells resistant to ritonavir are sensitized by co-treatment with metformin, potentially targeting compensatory mitochondrial complex 1 activity. Ritonavir and metformin have been administered in humans for treatment of diabetes in HIV patients, demonstrating the tolerance of this combination in humans. Our studies strongly substantiate further investigation of FDA approved ritonavir and metformin for CLL.

KEYWORDS: Basic Biology; Chemotherapeutic approaches; Lymphoid Leukemia; Signal transduction PMID: 24828872

Utilizing hydrogen sulfide as a novel anti-cancer agent by targeting cancer glycolysis and pH imbalance.

Lee ZW¹, Teo XY, Tay EY, Tan CH, Hagen T, Moore PK, Deng LW.
Br J Pharmacol. 2014 May 15. doi: 10.1111/bph.12773

Many disparate studies have reported the ambiguous role of hydrogen sulfide (H₂ S) in cell survival. The present study investigated the effect of H₂ S on viability of cancer and non-cancer cells.

Cancer and non-cancer cells were exposed to H₂ S (using sodium hydrosulfide, NaHS and GYY4137) and cell viability was examined by crystal violet assay. We then examined cancer cellular glycolysis process by in vitro enzymatic assays and pH regulator activity. Lastly, intracellular pH (pHi) was determined by ratiometric pHi measurement using BCECF staining.

Continuous, but not single, exposure to H₂ S decreased cell survival more effectively in cancer cells, as compared to non-cancer cells. Slow H₂ S-releasing donor, GYY4137, significantly increased glycolysis leading to overproduction of lactate. H₂ S also decreased anion exchanger and sodium/proton exchanger activity. The combination of increased metabolic acid production and defective pH regulation resulted in an uncontrolled intracellular acidification leading to cancer cell death. In contrast, no significant intracellular acidification or cell death was observed in non-cancer cells.

Low and continuous exposure to H₂ S targets metabolic processes and pH homeostasis in cancer cells, potentially serving as a novel and selective anti-cancer strategy.

KEYWORDS: cancer cell death; cancer glucose metabolism; hydrogen sulfide; pH homeostasis PMID: 24827113

Agonism of the 5-Hydroxytryptamine 1F Receptor Promotes Mitochondrial Biogenesis and Recovery from Acute Kidney Injury

Garrett SM, Whitaker RM, Beeson CC, and Schnellmann RG

Center for Cell Death, Injury, and Regeneration, Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, South Carolina (S.M.G., R.M.W., C.C.B., R.G.S.); and Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina (R.G.S.)
Address correspondence to: Dr. Rick G. Schnellmann, Department of Drug Discovery and Biomedical Sciences, MUSC, Charleston, SC 29425.
E-mail: schnell@musc.edu

Many acute and chronic conditions, such as acute kidney injury, chronic kidney disease, heart failure, and liver disease, involve mitochondrial dysfunction. Although we have provided evidence that drug-induced stimulation of mitochondrial biogenesis (MB) accelerates mitochondrial and cellular repair, leading to recovery of organ function, only a limited number of chemicals have been identified that induce MB.

The goal of this study was to assess the role of the 5-hydroxytryptamine 1F (5-HT_1F) receptor in MB. Immunoblot and quantitative polymerase chain reaction analyses revealed 5-HT_1F receptor expression in renal proximal tubule cells (RPTC). A MB screening assay demonstrated that two selective 5-HT_1F receptor agonists,

LY334370 (4-fluoro-N-[3-(1-methyl-4-piperidinyl)-1H-indol-5-yl]benzamide) and
LY344864 (N-[(3R)-3-(dimethylamino)-2,3,4,9-tetrahydro-1H-carbazol-6-yl]-4-fluorobenzamide; 1–100 nM)

increased carbonylcyanide-p-trifluoromethoxyphenylhydrazone–uncoupled oxygen consumption in RPTC, and

validation studies confirmed both agonists increased mitochondrial proteins in vitro.
[e.g., ATP synthase β, cytochrome c oxidase 1 (Cox1), and NADH dehydrogenase (ubiquinone) 1β subcomplex subunit 8 (NDUFB8)]

Small interfering RNA knockdown of the 5-HT_1F receptor

blocked agonist-induced MB.

Furthermore, LY344864 increased

peroxisome proliferator–activated receptor (PPAR) coactivator 1-α, Cox1, and
NDUFB8 transcript levels and
mitochondrial DNA (mtDNA) copy number

in murine renal cortex, heart, and liver.

Finally, LY344864 accelerated recovery of renal function, as indicated by

decreased blood urea nitrogen and kidney injury molecule 1 and
increased mtDNA copy number

following ischemia/reperfusion-induced acute kidney injury (AKI).

In summary, these studies reveal that

the 5-HT_1F receptor is linked to MB, 5-HT_1F receptor agonism promotes MB in vitro and in vivo, and

5-HT_1F receptor agonism promotes recovery from AKI injury.

Induction of MB through 5-HT_1F receptor agonism represents a new target and approach to treat mitochondrial organ dysfunction.

Footnotes

Portions of this work have been presented previously: Garrett SM, Wills LP, and Schnellmann RG (2012) Serotonin (5-HT) 1F receptor agonism as a potential treatment for acceleration of recovery from acute kidney injury.American Society of Nephrology Annual Meeting; 2012 Nov 1–4; San Diego, CA.
dx.doi.org/10.1124/jpet.114.214700.

Ca2+ regulation of mitochondrial function in neurons.

Rueda CB¹, Llorente-Folch I¹, Amigo I¹, Contreras L¹, González-Sánchez P¹, Martínez-Valero P¹, Juaristi I¹, Pardo B¹, Del Arco A², Satrústegui J³

Biochim Biophys Acta. 2014 May 10. pii: S0005-2728(14)00126-1.
doi: 10.1016/j.bbabio.2014.04.010.

Calcium is thought to regulate respiration but it is unclear whether this is dependent on the increase in ATP demand caused by any Ca²⁺ signal or to Ca²⁺ itself.

[Na⁺]_i, [Ca²⁺]_i and [ATP]_i dynamics in intact neurons exposed to different workloads in the absence and presence of Ca²⁺ clearly showed that

Ca²⁺-stimulation of coupled respiration is required to maintain [ATP]_i levels.

Ca²⁺ may regulate respiration by

activating metabolite transport in mitochondria from outer face of the inner mitochondrial membrane, or
after Ca²⁺ entry in mitochondria through the calcium uniporter (MCU).

Two Ca²⁺-regulated mitochondrial metabolite transporters are expressed in neurons,

the aspartate-glutamate exchanger ARALAR/AGC1/Slc25a12, a component of the malate-aspartate shuttle, with a Kd for Ca²⁺ activation of 300nM, and
the ATP-Mg/Pi exchanger SCaMC-3/Slc25a23, with S_0.5 for Ca²⁺ of 300nM and 3.4μM, respectively.

The lack of SCaMC-3 results in a smaller Ca²⁺-dependent stimulation of respiration only at high workloads, as caused by veratridine, whereas

the lack of ARALAR reduced by 46% basal OCR in intact neurons using glucose as energy source and the Ca²⁺-dependent responses to all workloads (veratridine, K⁺-depolarization, carbachol).

The lack of ARALAR caused a reduction of about 65-70% in the response to the high workload imposed by veratridine, and

completely suppressed the OCR responses to moderate (K⁺-depolarization) and small (carbachol) workloads,
effects reverted by pyruvate supply.

For K⁺-depolarization, this occurs in spite of the presence of large [Ca²⁺]_mit signals and increased reduction of mitochondrial NAD(P)H.

These results show that ARALAR-MAS is a major contributor of Ca²⁺-stimulated respiration in neurons

by providing increased pyruvate supply to mitochondria.

In its absence and under moderate workloads, matrix Ca²⁺ is unable to stimulate pyruvate metabolism and entry in mitochondria suggesting a limited role of MCU in these conditions.

KEYWORDS: ATP-Mg/Pi transporter; Aspartate–glutamate transporter; Calcium; Calcium-regulated transport; Mitochondrion; Neuronal respiration PMID: 24820519

Sestrin2 inhibits uncoupling protein 1 expression through suppressing reactive oxygen species.

Ro SH¹, Nam M², Jang I¹, Park HW¹, Park H¹, Semple IA¹, Kim M¹, et al.
Proc Natl Acad Sci U S A. 2014 May 27;111(21):7849-54.
doi: 10.1073/pnas.1401787111.

Uncoupling protein 1 (Ucp1), which is localized in the mitochondrial inner membrane of mammalian brown adipose tissue (BAT), generates heat by uncoupling oxidative phosphorylation. Upon cold exposure or nutritional abundance, sympathetic neurons stimulate BAT to express Ucp1 to induce energy dissipation and thermogenesis. Accordingly, increased Ucp1 expression reduces obesity in mice and is correlated with leanness in humans.

Despite this significance, there is currently a limited understanding of how Ucp1 expression is physiologically regulated at the molecular level. Here, we describe the involvement of Sestrin2 and reactive oxygen species (ROS) in regulation of Ucp1 expression. Transgenic overexpression of Sestrin2 in adipose tissues inhibited both basal and cold-induced Ucp1 expression in interscapular BAT, culminating in decreased thermogenesis and increased fat accumulation.

Endogenous Sestrin2 is also important for suppressing Ucp1 expression because BAT from Sestrin2(-/-) mice exhibited a highly elevated level of Ucp1 expression. The redox-inactive mutant of Sestrin2 was incapable of regulating Ucp1 expression, suggesting that Sestrin2 inhibits Ucp1 expression primarily through reducing ROS accumulation.

Consistently, ROS-suppressing antioxidant chemicals, such as butylated hydroxyanisole and N-acetylcysteine, inhibited cold- or cAMP-induced Ucp1 expression as well. p38 MAPK, a signaling mediator required for cAMP-induced Ucp1 expression, was inhibited by either Sestrin2 overexpression or antioxidant treatments.

Taken together, these results suggest that Sestrin2 and antioxidants inhibit Ucp1 expression through suppressing ROS-mediated p38 MAPK activation, implying a critical role of ROS in proper BAT metabolism.

KEYWORDS: aging; homeostasis; mouse; β-adrenergic signaling PMID: 24825887 PMCID: PMC4040599

Mitochondrial EF4 links respiratory dysfunction and cytoplasmic translation in Caenorhabditis elegans.

Yang F¹, Gao Y¹, Li Z², Chen L³, Xia Z⁴, Xu T⁵, Qin Y⁶Biochim Biophys Acta. 2014 May 15. pii: S0005-2728(14)00499-X.
doi: 10.1016/j.bbabio.2014.05.353.

How animals coordinate cellular bioenergetics in response to stress conditions is an essential question related to aging, obesity and cancer. Elongation factor 4 (EF4/LEPA) is a highly conserved protein that promotes protein synthesis under stress conditions, whereas its function in metazoans remains unknown.

Here, we show that, in Caenorhabditis elegans, the mitochondria-localized CeEF4 (referred to as mtEF4) affects mitochondrial functions, especially at low temperature (15°C).

At worms’ optimum growing temperature (20°C), mtef4 deletion leads to self-brood size reduction, growth delay and mitochondrial dysfunction.

Transcriptomic analyses show that mtef4 deletion induces retrograde pathways, including mitochondrial biogenesis and cytoplasmic translation reorganization.

At low temperature (15°C), mtef4 deletion reduces mitochondrial translation and disrupts the assembly of respiratory chain supercomplexes containing complex IV.

These observations are indicative of the important roles of mtEF4 in mitochondrial functions and adaptation to stressful conditions.

KEYWORDS: C. elegans; EF4(LepA/GUF1); Mitochondrial dysfunction; Retrograde pathways; Translation PMID: 24837196

The metabolite α-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR.

Chin RM¹, Fu X², Pai MY³, Vergnes L⁴, Hwang H⁵, Deng G⁶, Diep S², et al.
Nature 2014 Jun 19;509(7505):397-401. doi: 10.1038/nature13264.

Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits.

Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show that α-ketoglutarate (α-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans.

ATP synthase subunit β is identified as a novel binding protein of α-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution.

Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan.

We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells.

We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit β and is dependent on target of rapamycin (TOR) downstream.

Endogenous α-KG levels are increased on starvation and α-KG does not extend the lifespan of dietary-restricted animals, indicating that α-KG is a key metabolite that mediates longevity by dietary restriction.

Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.

PMID: 24828042

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

Posted in Ca2+ triggered activation, Ca2+ triggered activation, Calcium Signaling, Calmodulin, Cell Biology, Signaling & Cell Circuits, Cytoskeleton, Innovations in Neurophysiology & Neuropsychology, Neurohumoral Transmission, PKC, Proteomics, Synaptic vesicle, tagged Ca2+-regulation, Ca2+/calmodulin-dependent protein kinase, Calmodulin, calmodulin-dependent pathway, calmodulin-independent pathway, CaMK, exocytosis, Howard Hughes Medical Institute, PKC, Protein kinase C, SNARE, Stanford University School of Medicine, Synaptic vesicle on December 23, 2013| Leave a Comment »

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

Writer and Curator: Larry H Bernstein, MD, FCAP
and
Curator and Content Editor: Aviva Lev-Ari, PhD, RN

This article is Part V in a series of TWELVE articles, listed at the end of this article, on the

cytoskeleton,
calcium calmodulin kinase signaling,
muscle and nerve transduction, and
calcium,
Na+-K+-ATPase,
neurohumoral activity and vesicles vital and essential for all functions related to

cell movement,
migration, and
contraction.

Calmodulin and Protein Kinase C Increase Ca–stimulated Secretion by Modulating
Membrane-attached Exocytic Machinery

YA Chen, V Duvvuri, H Schulmani, and RH.Scheller‡

From the ‡Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology,
and the Department of Neurobiology, Stanford University School of Medicine, Stanford, CA

J Biol Chem 1999; 274(37): 25469-26476.

The molecular mechanisms underlying the Ca2+ regulation of hormone and neurotransmitter release
are largely unknown.

Using a reconstituted [3H]norepinephrine release assay in permeabilized PC12 cells, we found

essential proteins that support the triggering stage of Ca2+-stimulated exocytosis
are enriched in an EGTA extract of brain membranes.

Fractionation of this extract allowed purification of two factors that stimulate secretion

in the absence of any other cytosolic proteins.

These are calmodulin and protein kinase Ca (PKCa). Their effects on secretion were

confirmed using commercial and recombinant proteins.

Calmodulin enhances secretion

in the absence of ATP, whereas
PKC requires ATP to increase secretion, suggesting that
phosphorylation is involved in PKC-mediated stimulation
but not calmodulin mediated stimulation.
Both proteins modulate
- The half-maximal increase was elicited by
  3 nM PKC and 75 nM calmodulin.

These results suggest that calmodulin and PKC increase Ca2+-activated exocytosis by

directly modulating the membrane- or cytoskeleton-attached exocytic machinery
downstream of Ca2+ elevation.

The abbreviations used are:

NE, norepinephrine; PKC, protein kinase C; CaM, calmodulin; SNAP-25, synaptosome-associated protein of 25 kDa; CAPS, calcium-dependent activator protein for secretion; SNARE, SNAP (soluble N-ethylmaleimide-sensitive factor attachment proteins) receptor; CaMK, Ca2+/calmodulin-dependent protein kinase; PAGE, polyacrylamide gel electrophoresis; AMP-PNP, adenosine 59-(b,g- imido) triphosphate; HA, hydroxyapatite

*This work was supported in part by Conte Center Grant MH48108. The costs of publication of
this article were defrayed in part by the payment of page charges. This article has been marked
“advertisement” in accordance with 18 U.S.C. Section 1734.

The molecular mechanisms of presynaptic vesicle release have been extensively examined
by a combination of

biochemical,
genetic, and
electrophysiological techniques.

A series of protein-protein interaction cascades have been proposed to lead to vesicle
docking and fusion (1–3). The SNARE protein family, including

syntaxin, SNAP-25, and vesicle-associated membrane protein
(VAMP, also called synaptobrevin),
plays an essential role in promoting membrane fusion, and
is thought to comprise the basic fusion machinery (4, 5).

In Ca2+-stimulated exocytosis, many additional proteins are important in the Ca2+ regulation
of the basic membrane trafficking apparatus. Calcium

not only triggers rapid fusion of release-competent vesicles, but is also involved in
earlier processes which replenish the pool of readily releasable vesicles (6).

Furthermore, it appears to be critical in initiating several forms of synaptic plasticity including

post-tetanic potentiation (7).

The molecular mechanisms by which Ca2+ regulates these processes is not well understood.

PC12 cells have often been utilized to study Ca2+-activated exocytosis, as

they offer a homogeneous cell population that possesses the same basic exocytic machinery as neurons (8).

In this study, we used an established cracked cell assay, in which

[3H]norepinephrine (NE)1 labeled PC12 cells are
permeabilized by mechanical “cracking” and
then reconstituted for secretion of NE in the presence of test proteins (9).

Transmitter-filled vesicles and intracellular cytoskeletal structures

remain intact in these cells,
while cytosolic proteins leak out (10).

These cracked cells readily release NE upon addition of

ATP,
brain cytosol, and
1 mM free Ca2+
- at an elevated temperature.

We term this a “composite assay,” as

all essential components are added into one reaction mixture.

Alternatively, cracked cells can be

first primed with cytosol and ATP, washed, then
reconstituted for NE release with cytosol and Ca2+ (11).

This sequential priming-triggering protocol is useful

for determining whether a protein acts early or late in the exocytic pathway, and
whether its effect is dependent on Ca2+ or ATP.

This semi-intact cell system serves as

a bridge between an in vitro system comprised of purified components, and
electro-physiological systems that monitor release in vivo.
It provides information on protein functions in a cell with an intact membrane infrastructure while being easily manipulatable.

Ca2+ regulation by membrane depolarization is no longer a concern, as
intra-cellular Ca2+ concentration can be controlled by a buffered solution.

Indirect readout of neurotransmitter release using a postsynaptic cell is replaced by
direct readout of [3H]NE released into the buffer.

Complications associated with interpreting overlapping

exo- and endocytotic signals are also eliminated as only one round of exocytosis is measured.

Finally, concentration estimates are likely to be accurate, since

added compounds do not need to diffuse long distances along axons and dendrites to their sites of action.

Using this assay, several proteins required for NE release have been purified from rat brain cytosol, including

phosphatidyl-inositol transfer protein (12),
phosphatidylinositol-4-phosphate 5-kinase (13), and
calcium-dependent activator protein for secretion (CAPS) (9).

The validity of the cracked cell system is confirmed by the finding that

phosphatidylinositol transfer protein and CAPS are mammalian homologues of
- yeast SEC14p (12) and
- nematode UNC31p, respectively (14),
both proteins involved in membrane trafficking (15, 16).

Calmodulin is the most ubiquitous calcium mediator in eukaryotic cells, yet its involvement in membrane trafficking has not been well established. Some early studies showed

that calmodulin inhibitors (17–19), anti-calmodulin antibodies (20,21),

calmodulin binding inhibitory peptides (22) inhibited Ca2+-activated exocytosis.

However, in other studies, calmodulin-binding peptides and an anti-calmodulin antibody led to the conclusion that

calmodulin is only involved in endocytosis,
not exocytosis (23).

More recently, it was reported that

Ca2+/ calmodulin signals the completion of docking and
triggers a late step of homotypic vacuole fusion in yeast,

thus suggesting an essential role for Ca2+/calmodulin in constitutive intracellular membrane fusion (24).

If calmodulin indeed plays an important role in exocytosis,

a likely target of calmodulin is
Ca2+/calmodulin-dependent protein kinase II (CaMKII),
- a multifunctional kinase that is found on synaptic vesicles (25) and
- has been shown to potentiate neurotransmitter release (26, 27).

Another Ca2+ signaling molecule, PKC, has also been implicated in regulated exocytosis.
In various cell systems, it has been shown that

the phorbol esters stimulate secretion (28, 29).

It is usually assumed that phorbol esters effect on exocytosis is

through activation of PKC,
but Munc13-1 was recently shown to be a presynaptic phorbol ester receptor that enhances neurotransmitter release (30, 31),

which complicates the interpretation of some earlier reports. The mode of action of PKC remains controversial. There is evidence

that PKC increases the intracellular Ca2+ levels by modulating plasma membrane Ca2+ channels (32, 33),
that it increases the size of the release competent vesicle pool (34, 35), or
that it increases the Ca2+ sensitivity of the membrane trafficking apparatus (36).

no consensus on these issues has been reached.

PKC substrates that have been implicated in exocytosis include

SNAP-25 (37),
synaptotagmin (28),
CAPS (38), and
nsec1 (39).

It is believed that upon phosphorylation, these PKC substrates might

interact differently with their binding partners, which, in turn,
leads to the enhancement of exocytosis.

In addition, evidence is accumulating that PKC and calmodulin interfere with each others actions, as

PKC phosphorylation sites are embedded in the calmodulin-binding domains of substrates such as
neuromodulin and
neurogranin (40).

It is therefore possible that PKC could modulate exocytosis via

a calmodulin-dependent pathway by synchronously releasing calmodulin from storage proteins.

In this study, we fractionated an EGTA extract of brain membranes in order to identify active components that could reconstitute release in the cracked cell assay system. We identified calmodulin and PKC as two active factors. Thus, we demonstrate that

calmodulin and PKC play a role in the Ca2+ regulation of exocytosis, and provide further insight into the mechanisms of their action.

DISCUSSION

In this study, we first identified an EGTA extract of brain membranes as a protein source

capable of reconstituting Ca2+- activated exocytosis in cracked PC12 cells.

EGTA only extracts a small pool of Ca2+-dependent membrane-associating proteins,

it served as an efficient initial purification step.

Further protein chromatography led to the identification of two active factors in the starting extract,

calmodulin and PKC,
which together accounted for about half of the starting activity.

Upon confirmation with commercially obtained proteins, this result unambiguously demonstrated

that calmodulin and PKC mediate aspects of Ca2+-dependent processes in exocytosis.

The finding that brain membrane EGTA extract alone is able

to replace cytosol in supporting Ca2+-triggered NE secretion

in PC12 cells is somewhat surprising. We suggest that the likely explanation is 2-fold.

some cytosolic proteins essential for exocytosis have a membrane-bound pool
within permeabilized cells, whose activity might be sufficient for a normal level of exocytosis.
although the 100,000 3 g membrane pellet was washed to remove as many cytosolic proteins as possible,

some cytosolic proteins that associate with membranes in a
- Ca2+-independent manner are probably present in the membrane EGTA extract.
these proteins likely constitute only a small percentage of the proteins in the extract, as
- the characteristics of the activity triggered by the membrane extract
- are quite different to that of cytosol (Fig. 2).

Using an unbiased biochemical purification method, we demonstrated that

calmodulin and PKC directly modulate the exocytotic machinery downstream of Ca2+ entry
they signal through membrane-attached molecules to increase exocytosis.

These targets include integral and peripheral membrane proteins, and cytosolic proteins that have a significant
membrane-bound pool. The modest stimulation by calmodulin and PKC on secretion might suggest a regulatory

role. However, it is also possible that some intermediates in their signaling pathways are in limiting amounts in the
cell ghosts, so that their full effects were not observed. Half-maximal stimulation was obtained at

about 3 nM for PKC and
at about 75 nM for calmodulin.

This is consistent with an enzymatic role for PKC, and predicts a high-affinity interaction between

calmodulin and its substrate protein.

Ca2+ regulates exocytosis at many different levels. Prior studies indicated that Ca2+ signaling occurs in

the priming steps as well
as in triggering steps (49, 50).

Our priming triggering protocol

does not allow Ca2+-dependent priming events to be assayed, as EGTA is present in the priming reaction.
a different approach revealed the existence of both high and low Ca2+-dependent processes (Fig. 2).
this analysis indicated that late triggering events require high [Ca2+], whereas
early priming events require low [Ca2+]. If, as proposed, there is

a pronounced intracellular spatial and temporal [Ca2+] gradient from

the point of Ca2+ entry during depolarization (51),
perhaps triggered events occur closer to the point of Ca2+ entry,
while Ca2+-dependent priming events occur further away from the point of Ca2+ entry.

Fig 2A. measurements of range of [Ca2+]total - average [Ca2+]free values._page_004

Fig. 2B. measurements of range of [Ca2+] total - average [Ca2+]free values_edited-1

Distinct Ca2+ sensors at these stages might be appropriately tuned to different [Ca2+] to handle different tasks.

By analyzing the Ca2+ sensitivity of calmodulin-and PKC-stimulated release, we addressed the question of

whether calmodulin and PKC plays an early or a late role in vesicle release.
they both require relatively high [Ca2+] (Fig. 8B),
implying that calmodulin and PKC both mediate late triggering events, consistent with some earlier reports
(34, 52, 53).

In addition, it is interesting to note that PKC does not alter the calcium sensitivity of release in cracked cells, in contrast

to observations from the chick ciliary ganglion (36). Therefore, in contrast to previous electrophysiological studies (28),
we are able to limit the possible modes of PKC action in our system to an increase in the readily releasable vesicle pool or
release sites, or an enhancement of the probability of release of individual vesicles upon Ca2+ influx.

The experiments assaying the calcium sensitivity of release (Figs. 2, 5, and 8) demonstrated

a drop in release at very high [Ca2+].

FIG. 8. PKC and calmodulin stimulate... the late triggering reaction_page_006

This decline in release at high [Ca2+] has been previously reported (49, 51), and may represent

the true Ca2+ sensitivity of the Ca2+-sensing mechanism inside cells.

However, in our system, it could also be due to the activation of a variety of Ca2+ -activated proteases, as experiments are usually performed in the presence of crude extracts, which include unsequestered proteases.

What might the molecular targets of PKC and calmodulin be? An obvious calmodulin target molecule is CaMKII.

but calmodulin’s effect on exocytosis is ATP-independent, rendering the involvement of a kinase unlikely.

Calmodulin has also been shown to associate with

synaptic vesicles in a Ca2+-dependent fashion through synaptotagmin (54),
probably by binding to its C-terminal tail (55), and to promote Rab3A dissociation from synaptic vesicles (56).
However, there was little calcium-dependent binding of calmodulin to synaptotagmin
- either on synaptic vesicles, in a bead binding assay with recombinant proteins,
- or in a calmodulin overlay (data not shown).

In addition, using immobilized calmodulin, we did not see

significant Ca2+-dependent pull-down of synaptotagmin or Rab3A from rat brain extract (data not shown).

Recent work has suggested three other candidate targets for calmodulin, Munc13, Pollux, and CRAG (57).

Pollux has similarity to a portion of a yeast Rab GTPase-activating protein, while
CRAG is related to Rab3 GTPase exchange proteins.

Further work is required to investigate the role of their interactions with calmodulin in vivo.

The recent report that calmodulin mediates yeast vacuole fusion (24) is intriguing, as it raises the possibility that

calmodulin, a highly conserved ubiquitous molecule,
- may mediate many membrane trafficking events.

It is not yet known if

the effector molecule of calmodulin is conserved or variable across species and different trafficking steps.

It is enticing to propose a model for Ca2+ sensing whereby

calmodulin is a high affinity Ca2+ sensor for both constitutive and regulated membrane fusion.

In the case of constitutive fusion, calmodulin may be the predominant Ca2+ sensor.
In the case of slow, non-local exocytosis of large dense core granules, an additional requirement for
the concerted actions of other molecule(s) that are better tuned to intermediate rises in [Ca2+] might exist.

At the highly localized sites of fast exocytosis of small clear vesicles where high [Ca2+] is reached,

specialized low affinity sensor(s) are likely required
in addition to calmodulin to achieve membrane fusion.

Therefore, although calmodulin participates in multiple types of vesicle fusion,

the impact of Ca2+ sensing by calmodulin on vesicle release likely varies.

Due to the fact that calmodulin binding to some proteins can be modulated by PKC phosphorylation, one might suspect

PKC action on exocytosis proceeds through a calmodulin-dependent pathway.
but the effects of calmodulin and PKC are additive within our system,
- suggesting that PKC does not act by releasing calmodulin from a substrate
  - that functions as a calmodulin storage protein.

How Ca2+ regulates presynaptic vesicle release has been an open question for many years. By

identifying calmodulin and PKC as modulators of Ca21-regulated exocytosis and clarifying their functions,
we have extended our knowledge of the release process.

While the basic machinery of membrane fusion is becoming better understood,

the multiple effects of Ca2+ on exocytosis remain to be elucidated at the molecular level.

In addition, the ways that Ca2+ regulation may be important to

the mechanisms of synaptic plasticity in the central nervous system

EXPERIMENTAL PROCEDURES

Materials
Rat Brain Cytosol Preparation
Membrane EGTA Extract Preparation

Cracked Cell Assay

PC12 cells were maintained and [3H]NE labeled as described previously (11). Labeled cells were harvested by pipetting with ice-cold potassium glutamate buffer (50 mM Hepes, pH 7.2, 105 mM potassium glutamate, 20 mM potassium acetate, 2 mM EGTA) containing 0.1% bovine serum albumin. Subsequent manipulations were carried out at 0–4 °C. Labeled cells (1–1.5 ml/dish) were mechanically permeabilized passage through a stainless steel homogenizer. The cracked cells were adjusted to 11 mM EGTA and

incubated on ice for 0.5–3 h, followed by three washes in which
the cells were centrifuged at 800 3 g for 5 min and
resuspended in potassium glutamate buffer containing 0.1% bovine serum albumin.

Composite Assay

Each release reaction contains 0.5–1 million cracked cells, 1.5 mM free Ca2+, 2 mM MgATP,
and the protein solution to be tested in potassium glutamate buffer. Release reactions were initiated
by incubation at 30 °C and terminated by returning to ice. The supernatant of each reaction was
isolated by centrifugation at 2,500 3 g for 30 min at 4 °C, and the

released [3H]NE was quantified by scintillation counting (Beckman LS6000IC).

Cell pellets were dissolved in 1% Triton X-100, 0.02% azide and similarly counted. NE release

was calculated as a percentage of total [3H] in the supernatant.

Priming Assay

A priming reaction contains about

1–2 million cracked cells,
2 mM MgATP, and
the protein solution to be tested.
Ca2+ is omitted.

The primed cells were spun down, washed once with fresh potassium glutamate buffer, and

distributed into two triggering reactions, each containing
rat brain cytosol and free Ca2+
The triggering reaction was performed at 30 °C for 3 min, and
the NE release was measured
- as in a composite assay.

Triggering Assay

Cracked cells were primed …, centrifuged, washed …, and

distributed into triggering reactions containing

1.5 mM free Ca2+ and the protein solution

To inhibit any ATP dependent activity in the triggering reaction, an

ATP depletion system of

1. hexokinase
2. MgCl2,
3. glucose or

a non-hydrolyzable ATP analogue AMPPNP

was added into the triggering reaction. NE release was measured as above.

Free Ca2+ Concentration Determination

The range of Ca2+free in the release reaction (Fig. 2B) was achieved

by adding Ca2+ into potassium glutamate buffer to reach final [Ca2+] total values of
- 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 1.9, and 2.0 mM.
The pH of the reaction was 7.24 when no Ca2+ was added and
7.04 when 2.0 mM Ca2+ was added,
- in the absence of protein extracts or cracked cells.

Fig. 2B. The range of [Ca21]free in the release reaction (Fig. 2B)

Free Ca2+ concentrations were determined using video microscopic
measurements of fura-2 fluorescence (41). [Ca2+]free was calculated from the equation

[Ca2+]free 5 Kd*3 (R 2 Rmin)/(Rmax 2 R) (42).

The values of Rmin, Rmax, and Kd* were determined in the following solutions:

potassium glutamate buffer (PGB) containing

8 x 3 10^6 cracked cells/ml, 2 mM MgATP (PGB+CC)

1) Rmin: PGB+CC and 10 mM additional EGTA;

2) Rmax: PBG+CC, and 10 mM total Ca2+;

3) Kd*: PGB+CC, 28 mM additional EGTA, and 18 mM total Ca2+, pH 7.2
([Ca2+]free 5 = 169 nM, determined in the absence of cells and MgATP

based on fura-2 calibration in cell-free solutions).

These solutions were

incubated at 37 °C ,
mixed with fura-2 pentapotassium salt
(100 mM; Molecular Probes, Eugene, OR), and
imaged.

This procedure allowed us to take into account

changes in fura-2 properties
caused by the presence of
- permeabilized cells.

Duplicate measurements of the above range of [Ca2+] total gave

the following average [Ca2+] free values:
106, 146, 277, 462, 971, 1468, 1847, and 2484 nM.

Purification of Active Proteins

All procedures were carried out at 4 °C or on ice. Membrane
EGTA extract of one or two bovine brain(s) was

filtered through cheesecloth and
loaded overnight onto a column packed with DEAE-Sepharose
CL-6B beads (Amersham Pharmacia Biotech).

The column was then

washed with
(20 mM Hepes, pH 7.5, 0.25 mM sucrose, 2 mM EGTA, 1 mM dithiothreitol) and
step eluted with 10 column volumes of elution buffer
(20 mM Hepes, pH 7.5, 2 mM EGTA, 400 mM KCl, 1 mM dithiothreitol).
100 ml of every other fraction was
dialyzed overnight into PGB, and
tested in a composite release assay for activity.

The active fractions were pooled and dialyzed into zero salt buffer
(20 mM Hepes, pH 7.5, 2 mM EGTA) and
batch bound to 10 ml of Affi-Gel Blue beads (Bio-Rad) or DyeMatrex-Green A beads (Amicon)

Blue beads were used in earlier experiments, and Green beads were used later to

specifically deplete CAPS, which was known to bind to Green beads (9).

The unbound material was

collected,
concentrated to about 2 ml using a Centriprep-10 (Amicon), and
loaded onto a 120-ml HiPrep Sephacryl S-200 gel filtration column
(Amersham Pharmacia Biotech).

Samples were run on the S-200 column in PGB at a flow rate of 7 ml/h.

10–50 ml of every other fraction was tested for
- activity in the cracked cell composite assay, and
two peaks of activity were observed (Fig. 3).

The first peak of activity had a predicted molecular mass of 85 kDa.
The corresponding material was

adjusted to 10 mM potassium phosphate concentration (pH 7.2) and
loaded onto a 1-ml column packed with hydroxyapatite Bio-Gel HT
(Bio-Rad).

The bound material was

eluted with a linear K-PO4 gradient from 10 to 500 mM (pH 7.2)
at a flow rate of about 0.1 ml/min, and
0.4–0.5-ml fractions were collected.
each fraction was dialyzed into PGB and
tested for activity.

The fractions were also analyzed by

SDS-PAGE and silver staining (Sigma silver stain kit).

The active material was concentrated and resolved

on an 8% poly-acrylamide gel.

Two Coomassie-stained protein bands that matched the activity profile (Fig. 6)

were excised from the gel,
sequenced by the Stanford PAN facility.

The two polypeptide sequences obtained from the upper band were:

LLNQEEGEYYNVPIXEGD
IRSTLNPRWDESFT.

The only bovine protein that contains both polypeptides is PKCa.
The four polypeptide sequences obtained from the lower band were:

YELTGKFERLIVGLMRPPAY,
LIEILASRTNEQIHQLVAA,
MLVVLLQGTREEDDVVSEDL, and
EMSGDVRDVFVAIVQSVK.

Based on these sequences, the protein band was

unambiguously identified to be bovine annexin VI.

The second S-200 peak has a predicted molecular mass of 25 kDa.
The corresponding material was

dialyzed into zero salt buffer
(20 mM Tris, pH 7.5, 1 mM EGTA) and
injected onto a Mono-Q HR 5/5 FPLC column
(Pharmacia).

The FPLC run was performed at 18 °C at 1 ml/min and

1-ml fractions were collected
with a linear salt gradient from 0 to 1 M KCl over 71 ml.

The fractions containing proteins (determined by A280) were

dialyzed into PGB and
tested in the cracked cell assay.

Western Blot

Anti-calmodulin antibody and anti-PKC antibody were used, and

ECL (Amersham) was used for detection.

RESULTS

A Membrane EGTA Extract Supports NE Release

Brain cytosol, prepared as the supernatant of the brain homogenate,

effectively stimulates NE release
in the cracked cell assay (Fig. 1)
as previously shown (9).

We wondered whether crude extracts other than cytosol

could support NE release, and we focused on
extractable peripheral membrane proteins.

We found that a salt or EGTA extract of brain membranes,
membranes defined as the

100,000 3 g pellet of the crude homogenate,
reconstituted secretion in the absence of cytosol.
the salt extract only slightly enhanced NE release
above background (data not shown), the

EGTA extract not only stimulated NE release to a high level,

similar to that supported by cytosol, but also
had a higher specific activity than cytosol (Fig. 1).

Fig. 1 EGTA extract can support NE release_page_003_edited-3

FIG. 1. The EGTA extract of brain membranes can support NE release in the absence of cytosol. Rat brain membrane EGTA extract (closed triangles) and rat brain cytosol (closed squares) were prepared as described under “Experimental Procedures.” NE release was measured in a composite reaction mixture of cracked cells, MgATP, Ca2+, and the indicated amount of crude extracts.

The ability of the membrane EGTA extract to support secretion is consistent with the fact that

following cracking, the cells are immediately extracted with EGTA, and are presumably
devoid of most membrane EGTA-extractable factors.

This also suggests that these factors, some of which are probably

Ca2+-dependent membrane-associating proteins,
participate in Ca2+- triggered exocytosis.

The Membrane EGTA Extract Is Enriched in Triggering Fators

NE release in cracked cells can be resolved into two sequential stages,

an ATP-dependent priming stage and
an ATP-independent Ca21-dependent triggering stage (11), and
proteins can be tested for activity in either stage.

An effect in priming indicates

an early role for the protein, and
an effect in triggering a late ATP-independent role.

Since the protein composition of the

membrane EGTA extract and cytosol are different,

we tested whether they had different activities

in the priming stage versus the triggering stage.

We found that the membrane EGTA extract is enriched in factors that

act during triggering stage of NE release, as
the same amount of protein from the membrane EGTA extract as cytosol
gave a higher stimulation in the triggering assay, but
not in the priming assay (Fig. 2A).

Regular cytosol is prepared in a buffer containing 2 mM EGTA, and thus

presumably contains some of the proteins present in the membrane EGTA extract.

Cytosol prepared in the absence of EGTA showed an even lower specific activity

in the triggering assay compared with regular cytosol (Fig. 2A).

Identification of Calmodulin as an Active Triggering Factor in the EGTA Extract

Biochemical fractionation of the bovine brain membrane EGTA extract was carried out

to identify the active components capable of reconstituting NE release.

Activity was assayed in a composite reaction mixture containing

cracked cells,
ATP,
Ca2+, and
the test protein(s).

Except for the presence of bovine serum albumin in the basal buffer,

no other proteins were added to the cell ghosts except for the test protein(s).

Initial tests indicated that at least

part of the activity in the membrane EGTA extract binds to and
can be efficiently eluted from an anion exchanger and hydroxyapatite resin,
but does not bind to Amicon color resins.

The starting material was, therefore, sequentially purified using

DEAE, Affi-Gel Blue (or Matrex Green-A), and gel filtration chromotography.

Gel filtration fractionation indicated the presence of two peaks of activity with

predicted molecular masses of 25 and 85 kDa, respectively (Fig. 3).

FIG. 3. Gel filtration chromatography reveals two stimulatory factors in the membrane EGTA extract.

In order to purify the active component(s) in the membrane EGTA extract, the crude extract from one bovine brain was fractionated chromatographically (see Experimental Procedures” for details). Fractions from a Sephacryl S-200 gel filtration column were tested for their activity in stimulating NE release in the composite assay. The two activity peaks have predicted molecular masses of 85 and 25 kDa, respectively. The arrows indicate the retention volume of standard proteins run on the same column.

The low molecular weight active factor was purified to homogeneity, as judged by a

Coomassie-stained SDS-PAGE gel, after a subsequent Mono-Q fractionation (Fig. 4).

FIG. 4. The low molecular wen.ight active factor is calmodulin

A, the membrane EGTA extract from one bovine brain (Start) was subjected to sequential fractionation on DEAE, Blue A, and
Sephacryl S-200 columns. The pooled material containing the activity after each chromotographic step was analyzed by SDS-
PAGE and Coomassie staining. The arrowheads indicate the presence of calmodulin in all the lanes. Calmodulin shows a
mobility shift depending on whether or not Ca2+ is present during electrophoresis (see panel C).
B, the active material pooled from Sephacryl S-200 was fractionated on a Mono-Q FPLC column and the fractions
(5 ml/fraction) were tested for activity in a composite assay. The activity peak is shown.
C, the active Mono-Q fractions (5 ml/fraction) were subjected to SDS-PAGE in the presence of 1 mM EGTA or 0.1 mM Ca2+,
and the gels stained with Coomassie Blue.
D, fraction 47 (1 ml) was probed by Western blotting with a monoclonal anti-calmodulin antibody. No Ca2+ or EGTA was
added during SDS-PAGE.

We reasoned that the protein might be calmodulin (43) based on the following:

1) It is a relatively small protein (14–18 kDa) that is abundant in the
starting extract (Fig. 4A).
2) It elutes at a very high salt concentration (0.41 M KCl) on the
Mono-Q column.
3) It stains negatively in silver stain (data not shown).
4) Its electrophoretic mobility shifts depending on the presence or
absence of Ca21 (Fig. 4C).

A Western blot with an anti-calmodulin monoclonal antibody gave a
positive signal (Fig. 4D), confirming our prediction.

Properties of Calmodulin-stimulated Exocytosis

We used commercial calmodulin or bacterially expressed recombinant calmodulin to confirm our purification result; both sources of authentic calmodulin stimulated NE release as expected. Moreover, we found that calmodulin stimulates secretion in a triggering assay as well as in a composite assay (Fig. 5A).

The half-maximal increase was at 75 nM (250 ng/200 ml) final calmodulin concentration. This is within the broad
range of affinities between calmodulin and its various targets and suggests that the interaction between
calmodulin and its target molecule in exocytosis is in the physiological range. When the triggering reaction was
performed at different Ca2+ concentrations, calmodulin increased NE release only at high [Ca2+] (0.4 – 2 mM)
similar to the crude EGTA extract (Fig. 5B),

suggesting that calmodulin contributes to the triggering activity of the membrane EGTA extract. Calmodulin’s affinity for Ca2+ has
been reported to be around 1 mM (25),

consistent with the Ca2+ requirement for
calmodulin-stimulated secretion that we observed.

FIG. 5. Calmodulin stimulates NE release in the triggering stage.
A, calmodulin (obtained from Sigma) increased NE release in the
triggering assay in a dose-dependent fashion, in the absence of ATP
or any other cytosolic proteins. In this particular experiment, the
maximal release achieved by addition of rat brain cytosol was 46.5%.

B, the triggering assay was performed with different concentrations
of free Ca2+. Calmodulin (3 mg bacterially expressed recombinant
protein; closed squares) increased NE release with a similar Ca2+
sensitivity to rat brain membrane EGTA extract (10 mg; closed
triangles), as compared with conditions in which no protein was
added (open squares).

Western analysis with commercial protein as standards indicated that calmodulin

constitutes about 5% of total proteins in the rat brain membrane EGTA extract
and about 2% of total proteins in the rat brain cytosol (data not shown).

In addition, a significant amount of calmodulin appears to be left

in the washed cell ghosts (data not shown).

Based on the activity of saturating levels of

pure calmodulin (releasing 6–10% of total [3H]NE)
and crude EGTA extract (releasing ;45% of total [3H]NE),

we estimated that

calmodulin accounts for 13–22% of total activity of the extract.

Consistent with this,

a high affinity calmodulin-binding peptide
(CaMKIIa(291–312) (44), used at 5 mM) and
an anti-calmodulin antibody (2 mg/200 ml)
inhibited about 20% of the membrane EGTA extract-stimulated release
(6.7 mg of extract added; data not shown).

We showed that calmodulin increased NE release

in the triggering stage.

Since regular triggering reactions were performed

in the absence of any added ATP,

this suggests that

calmodulin enhanced secretion in an ATP-independent fashion.

Furthermore, residual ATP in the cell ghosts did not play a role, since

addition of a hexokinase ATP depletion system that
can deplete millimolar concentrations of ATP
- within a few minutes (11) had little effect, as did
- addition of 5 mM AMPPNP,
which blocks ATP-dependent enzymatic activity (Fig.8A).

Therefore, we ruled out the possibility that a kinase mediates calmodulin’s effect.

FIG. 8. PKC and calmodulin stimulate the late triggering reaction in
an ATP-dependent and ATP-independent manner respectively.
A, triggering assays were performed to test the activity of calmodulin
(recombinant; black bars) and PKC (purified rat brain PKC from
Calbiochem; shaded bars) in the absence of ATP. A regular triggering
assay is done in the absence of ATP (2ATP). To deplete residual ATP
in the cells, hexokinase-based ATP depletion was employed (1Hexo).
Alternatively, 5 mM AMP-PNP (1AMP-PNP) was added in the triggering
reaction. Under all three conditions, calmodulin increased release
as compared with the background (buffer only; white bars), whereas
PKC did not.
B, NE release in a composite assay was measured with varying
concentrations of free Ca2+ in the presence of 10 mg of calmodulin
(recombinant; closed triangles), 70 ng of PKC (purified rat brain PKC
from Calbiochem; closed squares), or buffer only (open squares).

A series of calmodulin mutants from Paramecium and chicken were tested

for their ability to enhance Ca2+-stimulated secretion, and
none of the mutations abolished the calmodulin effect (data not shown).

These mutations include

S101F, M145V, E54K, G40E/D50N, V35I/D50N within Paramecium
calmodulin (45), and M124Q, M51A/V55A, and M51A/V55A/L32A
within chicken calmodulin (46, 47).

The Paramecium calmodulin mutants are the result of

naturally occurring mutations that result in aberrations in their behavior.

These mutants can be grouped into two categories according to their
behavior, reflecting their loss of either

a Ca2+-dependent Na1 current
(calmodulin N-terminal lobe mutants: E54K, G40E/D50N, and
V35I/D50N) or
a Ca21-dependent K1 current
(calmodulin C-terminal lobe mutants: S101F and M145V) (45).

The chicken calmodulin mutants have been shown to

differentially activate myosin light chain kinase
(M124Q, M51A/V55A, and M51A/V55A/L32A),
CaMKII (M124Q), and CaMKIV (M124Q),

and the mutated residues are thought to be important in

defining calmodulin’s binding specificity (46, 47).

Our finding that these mutant calmodulins can stimulate exocytosis suggests that

calmodulin-binding domains similar to those of Paramecium Ca2+/calmodulin-dependent
ion channels, myosin light chain kinase, CaMKII, and CaMKIV,
are unlikely to mediate release utilizing the conserved SNARE fusion machinery, as they
could be completely abolished by addition of exogenous syntaxin H3 domains (data not shown).
the same molecular pathway was not activated, since their effects were additive (data not shown).

Acknowledgments—

We thank Diana Bautista and Dr. Richard S.Lewis for generous help

with [Ca21]free determination; Dr. Ching Kung for providing the Paramecium calmodulin

mutants, and Dr. Anthony R. Means for providing the chicken calmodulin mutants. We also

thank Dr. Jesse C. Hay for the initial setup of the cracked cell assay, and Dr. Suzie J.

Scales for helpful comments on the manuscript.

REFERENCES

1. Calakos, N., and Scheller, R. H. (1996) Physiol. Rev. 76, 1–29

2. Su¨ dhof, T. C. (1995) Nature 375, 645–653

3. Zucker, R. S. (1996) Neuron 17, 1049–1055

4. Hanson, P. I., Heuser, J. E., and Jahn, R. (1997) Curr. Opin. Neurobiol. 7, 310–315

5. Chen, Y. A., Scales, S. J., Patel, S. M., Doung, Y.-C., and Scheller, R. H. (1999) Cell 97, 165–174

6. Neher, E., and Zucker, R. S. (1993) Neuron 10, 21–30

7. Kamiya, H., and Zucker, R. S. (1994) Nature 371, 603–606

SOURCE

J Biol Chem 1999; 274(37): 25469-26476.

ION CHANNEL and Cardiovascular Diseases

http://pharmaceuticalintelligence.com/?s=Ion+Channel

Calcium Role in Cardiovascular Diseases

Part I: Identification of Biomarkers that are Related to the Actin Cytoskeleton
Larry H Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2012/12/10/identification-of-biomarkers-
that-are-related-to-the-actin-cytoskeleton/

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
http://pharmaceuticalintelligence.com/2013/08/26/role-of-calcium-the-actin-
skeleton-and-lipid-structures-in-signaling-and-cell-motility/

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
http://pharmaceuticalintelligence.com/2013/09/02/renal-distal-tubular-ca2-
exchange-mechanism-in-health-and-disease/

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
http://pharmaceuticalintelligence.com/2013/09/08/the-centrality-of-ca2-signaling-and-cytoskeleton-
involving-calmodulin-kinases-and-ryanodine-receptors-in-cardiac-failure-arterial-smooth-muscle-
post-ischemic-arrhythmia-similarities-and-differen/

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 and Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2013/12/23/calmodulin-and-protein-kinase-c-drive-the-ca2-regulation-of-hormone-and-neurotransmitter-release-that-triggers-ca2-stimulated-exocytosis/

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
http://pharmaceuticalintelligence.com/2013/08/01/calcium-molecule-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/

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
http://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-
and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/

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
http://pharmaceuticalintelligence.com/2013/09/12/disruption-of-calcium-homeostasis-cardiomyocytes-and-vascular-smooth-
muscle-cells-the-cardiac-and-cardiovascular-calcium-signaling-mechanism/

Part IX: Calcium-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
http://pharmaceuticalintelligence.com/2013/09/16/calcium-channel-blocker-calcium-as-neurotransmitter-sensor-
and-calcium-release-related-contractile-dysfunction-ryanopathy/

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
http://pharmaceuticalintelligence.com/2013/09/10/synaptotagmin-functions-as-a-calcium-sensor-how-calcium-ions-
regulate-the-fusion-of-vesicles-with-cell-membranes-during-neurotransmission/

Part XI: Sensors and Signaling in Oxidative Stress
Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2013/11/01/sensors-and-signaling-in-oxidative-stress/

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

http://pharmaceuticalintelligence.com/2013/12/21/genetic-polymorphisms-of-ion-channels-have-a-role-in-the-pathogenesis-of-coronary-microvascular-dysfunction-and-ischemic-heart-disease/