S-nitrosylation of soluble guanylyl cyclase: potential role in nitrate tolerance

可溶性鸟苷酸环化酶的 S-亚硝基化：在硝酸盐耐受性中的潜在作用

• 批准号: 7472094
• 负责人: ANNIE V BEUVE
• 金额: $ 19.5万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-07 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7472094
• 关键词: Address; Adenoviruses; Affect; Angina Pectoris; Atherosclerosis; Back; Biochemical; Blood Vessels; Cardiovascular Diseases; Cardiovascular system; Cells; Centenarian; Cheek structure; Clinical; Collaborations; Condition; Cyclic GMP; Cysteine; Development; Event; Exposure to; Failure; Foundations; Functional disorder; Future; Generations; Hamsters; Hour; Hypertension; Immigration; In Vitro; Indium; Ischemia; Lead; Link; Mass Spectrum Analysis; Memory; Molecular; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitroglycerin; Organism; Oxidative Stress; Pathology; Physiological; Physiology; Play; Preparation; Production; Proteins; Public Health; Range; Rattus; Regulation; Resistance; Role; Second Messenger Systems; Signal Transduction; Smooth Muscle Myocytes; Soluble Guanylate Cyclase; Staging; Sulfhydryl Compounds; System; Technology; Therapeutic; Tissues; Topical application; Vasodilation; cremaster muscle; desensitization; in vivo; interest; mutant; nitrate; novel therapeutics; prevent; protein function; research study; response; second messenger; therapeutic effectiveness; tool

DESCRIPTION (provided by applicant): Nitric oxide (NO) induces vasodilation by activation of soluble guanylyl cyclase (sGC), which in turn produces the second messenger cGMP. Following exposure to NO, sGC becomes desensitized and fails to respond to additional NO stimulation. Until now, the mechanism of sGC desensitization was unknown. We see an interesting parallel between sGC desensitization and clinical development of nitrate tolerance. Prolonged exposure to nitroglycerin (GTN), which induces vasodilation via NO generation, results in the loss of vasodilatory response to NO and nitrates. The mechanism of this nitrate tolerance is not understood, yet it is a major draw back to a widely used cardiovascular therapy. In addition to sGC activation, NO modulates protein function by S-nitrosylation, which is the addition of a NO moiety to the free thiols of cysteine residues. In our quest to understand the mechanism of desensitization of sGC, we discovered recently that sGC is S-nitrosylated in vitro and in intact cells. We further established a cause-effect relationship between S-nitrosylation of sGC and its desensitization, characterized by the loss of NO-stimulation of sGC activity (while the basal activity remains unaltered). Recently, it was shown that GTN treatment increases S-nitrosylation of proteins in tissues. Importantly, we just showed that GTN induces S-nitrosylation and desensitization of sGC in cells. Therefore, our hypothesis is that GTN treatment induces S-nitrosylation of sGC leading to its desensitization, which in turn underlies nitrate tolerance. To explore this provocative hypothesis, we will need to show first that GTN-dependent S-nitrosylation of sGC directly causes desensitization. Second, we will assess whether S-nitrosylation of sGC in vivo (using an S-nitrosylating agent) can mimic the development of nitrate tolerance. Third, we will determine if GTN treatment under conditions known to induce nitrate tolerance S-nitrosylates sGC in vivo thus provoking its failure to respond to NO stimulation. Primary rat aortic smooth muscle cells will be used to characterize the GTN-induced development of S-nitrosylation and desensitization of sGC with biochemical tools. Physiological characterization of the impact of sGC S-nitrosylation on NO-dependent vasodilation will be conducted in vivo with a hamster cheek pouch preparation and later in the rat cremaster muscle. Resistance to nitrate tolerance in vivo will be attempted with sGC mutants of S-nitrosylation, using adenovirus technology. These initial experiments should lay the foundations for a more complete study that will address many more questions: What is the molecular mechanism of sGC desensitization by S-nitrosylation? Is sGC S-nitrosylated under conditions of oxidative stress, and if so, does it contribute to the development of cardiovascular dysfunctions such as atherosclerosis? Could a specific blockade of sGC S-nitrosylation prevent the development of nitrate tolerance or other cardiovascular diseases? This proposal and its future extension could be critical for the development of therapeutic strategies in which sGC and its desensitization are new targets. PUBLIC HEALTH RELEVANCE: Nitroglycerin has been used for more than a century to treat many cardiovascular diseases by relaxing the vasculature. Unfortunately, it induces nitrate tolerance, which means that the organism becomes insensitive to the treatment. We propose to explore the mechanism underlying nitrate tolerance, which until now remains unexplained.

描述（由申请人提供）：一氧化氮（NO）通过激活可溶性鸟苷酸环化酶（sGC）诱导血管舒张，进而产生第二信使cGMP。暴露于 NO 后，sGC 变得不敏感并且无法对额外的 NO 刺激做出反应。迄今为止，sGC 脱敏的机制尚不清楚。我们发现 sGC 脱敏和硝酸盐耐受的临床发展之间存在有趣的相似之处。长期接触硝酸甘油（GTN）会通过生成一氧化氮诱导血管舒张，导致对一氧化氮和硝酸盐的血管舒张反应丧失。这种硝酸盐耐受性的机制尚不清楚，但它是广泛使用的心血管治疗的一个主要缺点。除了 sGC 激活之外，NO 还通过 S-亚硝基化调节蛋白质功能，S-亚硝基化是在半胱氨酸残基的游离硫醇上添加 NO 部分。在我们探索 sGC 脱敏机制的过程中，我们最近发现 sGC 在体外和完整细胞中是 S-亚硝基化的。我们进一步建立了 sGC 的 S-亚硝基化与其脱敏之间的因果关系，其特征是 sGC 活性的 NO 刺激丧失（而基础活性保持不变）。最近，研究表明 GTN 治疗会增加组织中蛋白质的 S-亚硝基化。重要的是，我们刚刚证明 GTN 诱导细胞中 sGC 的 S-亚硝基化和脱敏。因此，我们的假设是 GTN 治疗诱导 sGC 的 S-亚硝基化，导致其脱敏，进而导致硝酸盐耐受。为了探索这一令人兴奋的假设，我们首先需要证明 sGC 的 GTN 依赖性 S-亚硝基化直接导致脱敏。其次，我们将评估体内 sGC 的 S-亚硝基化（使用 S-亚硝基化剂）是否可以模拟硝酸盐耐受性的发展。第三，我们将确定在已知的条件下进行 GTN 处理是否会在体内诱导硝酸盐耐受性 S-亚硝基化物 sGC，从而导致其无法对 NO 刺激做出反应。原代大鼠主动脉平滑肌细胞将用于通过生化工具表征 GTN 诱导的 S-亚硝基化发展和 sGC 脱敏。 sGC S-亚硝基化对 NO 依赖性血管舒张的影响的生理学特征将通过仓鼠颊囊制剂在体内进行，随后在大鼠提睾肌中进行。将使用腺病毒技术，尝试使用 S-亚硝基化的 sGC 突变体来抵抗体内硝酸盐耐受性。这些初步实验应该为更完整的研究奠定基础，从而解决更多问题：S-亚硝基化使 sGC 脱敏的分子机制是什么？ sGC 在氧化应激条件下是否会发生 S-亚硝基化？如果是，它是否会导致动脉粥样硬化等心血管功能障碍的发生？特异性阻断 sGC S-亚硝基化能否预防硝酸盐耐受或其他心血管疾病的发生？该提案及其未来的扩展可能对于以 sGC 及其脱敏为新目标的治疗策略的开发至关重要。公众健康相关性：一个多世纪以来，硝酸甘油一直被用来通过放松脉管系统来治疗许多心血管疾病。不幸的是，它会诱导硝酸盐耐受，这意味着生物体对治疗变得不敏感。我们建议探索硝酸盐耐受性的潜在机制，但迄今为止仍无法解释。