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Desensitization of soluble guanylyl cyclase, the nitric oxide receptor, by S-nitrosylation: Implications for nitrate tolerance

by
Nazish S. Sayed
YOU ARE INVITED TO ATTEND THE
DEFENSE OF THE DOCTORAL
DISSERATION

Pharmacology and Physiology

M.B.B.S, 1998, University of Bombay, India
M.S., 2003, Montclair State University

Thesis Advisor: Annie Beuve, Ph.D.
Assistant Professor
Department of Pharmacology and Physiology

Department of Pharmacology and Physiology
MSB H-609b
Conference Room

Wednesday, April 30, 2008
12:00 p.m.


Abstract

Signaling cascades initiated by nitric oxide (NO) are important in the physiology and pathophysiology of the cardiovascular system. NO signal is mediated by its receptor, soluble guanylyl cyclase (sGC), a heme containing heterodimer that produces cGMP. The mechanism by which sGC is desensitized has been the subject of intense study for decades. Elucidating the mechanism of desensitization is crucial as it could explain, in part, the mechanisms of NO/nitrate tolerance. Nitrate tolerance occurs following prolonged exposure to nitroglycerin (GTN), which is vasoactive via stimulation of sGC and considered as a major drawback to the therapeutic effectiveness of GTN.
Herein, we provide evidence that S-nitrosylation, an NO-dependent posttranslational modification of cysteines is a mechanism for sGC desensitization and could be prevented by altering the redox state of the cells. Mutational analysis of the cysteines identified by mass spectrometry, confirmed their participation in desensitization indicating a causal relationship between S-nitrosylation and desensitization of sGC. Kinetic studies suggested that S-nitrosylation of sGC induces desensitization probably by affecting the efficacy of NO-stimulation. The fact that sGC is not endogenously S-nitrosylated in isolated aortas unless exposed to high concentrations of NO via Ach stimulation, suggested S-nitrosylation as a mechanism for development of NO tolerance. Indeed, our in vivo studies showed that vascular tolerance could be induced by CSNO, an S-nitrosylating agent and that these tolerant tissues had S-nitrosylated/desensitized sGC.
We then tested whether desensitization of sGC via S-nitrosylation is a mechanism of nitrate tolerance. We established that GTN induces S-nitrosylation and desensitization of sGC in cells and that both S-nitrosylation/desensitization could be prevented by N-acetyl-cysteine, a compound known to increase intracellular thiols and reverse nitrate tolerance. Our correlative studies in an animal model of nitrate tolerance (chronic GTN treatment) showed S-nitrosylation and desensitization of sGC in GTN-tolerant tissues.
This thesis work could contribute to a better understanding of sGC modulation, whose dysfunctions could lead to various cardiovascular pathologies.


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