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: 7620065
• 负责人: ANNIE V BEUVE
• 金额: $ 23.4万
• 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 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7620065
• 关键词: Address; Adenoviruses; Affect; Angina Pectoris; Atherosclerosis; Back; Biochemical; Blood Vessels; Cardiovascular Diseases; Cardiovascular system; Cells; Centenarian; Cheek structure; Clinical; Collaborations; 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; 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; novel therapeutics; prevent; protein function; public health relevance; research study; response; second messenger; therapeutic development; 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）通过激活可溶性圭尼环酶（SGC）诱导血管舒张，这又产生了第二个Messenger CGMP。暴露于NO之后，SGC变得脱敏，无法响应其他NO刺激。到目前为止，SGC脱敏的机理尚不清楚。我们看到SGC脱敏和硝酸盐耐受性的临床发育之间的有趣相似之处。长时间暴露于硝酸甘油（GTN）通过NO产生诱导血管舒张，导致对NO和硝酸盐的血管舒张反应的丧失。这种硝酸盐耐受性的机制尚不清楚，但它是一种主要使用的心血管疗法。除SGC激活外，NO通过S-亚硝基化调节蛋白质功能，这是对半胱氨酸残基的游离硫醇的添加。为了了解SGC脱敏的机理，我们最近发现SGC在体外和完整细胞中已被S-亚硝基化。我们进一步建立了SGC的S-亚硝基化及其脱敏之间的因果关系，其特征在于SGC活性无刺激（而基础活性仍未改变）。最近，显示GTN处理会增加组织中蛋白质的S-硝基化。重要的是，我们只是表明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处理诱导SGC的S-硝基化导致其脱敏，这又构成了硝酸盐耐受性。为了探索这一挑衅性假设，我们需要首先证明SGC的GTN依赖性S-硝基化直接引起脱敏。其次，我们将评估体内SGC的S-亚硝基化（使用S-硝基化剂）是否可以模仿硝酸盐耐受性的发展。第三，我们将确定在已知诱导硝酸盐耐受性S-硝酸盐SGC中的GTN治疗中是否会引起其对无刺激的反应。初级大鼠主动脉平滑肌细胞将用于表征GTN诱导的S-亚硝基化和使用生化工具脱敏的发展。 SGC S-亚硝基化对无依赖性血管舒张的影响的生理表征将在体内使用仓鼠脸颊袋装进行，然后在大鼠Cremaster肌肉中进行。使用腺病毒技术，将尝试使用S-硝基化的SGC突变体对体内的耐硝酸盐耐受性进行抗性。这些最初的实验应该为更完整的研究奠定基础，以解决更多问题：S-亚硝基化的SGC脱敏的分子机制是什么？在氧化应激条件下，SGC S-硝基基化是否会导致心血管硬化等心血管功能障碍的发展？ SGC S-硝基化的特定阻滞是否可以阻止硝酸盐耐受性或其他心血管疾病的发展？该建议及其未来扩展对于制定SGC及其脱敏的治疗策略可能是新目标至关重要的。公共卫生相关性：一个多世纪以来，硝酸甘油已被用于通过放松血管来治疗许多心血管疾病。不幸的是，它诱导硝酸盐耐受性，这意味着生物体对治疗不敏感。我们建议探索硝酸盐耐受性的基本机制，到目前为止，该机制仍然无法解释。