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，sGC进而产生第二信使cGMP。暴露于NO后，sGC变得脱敏，并且不能对额外的NO刺激作出反应。迄今为止，sGC脱敏的机制尚不清楚。我们看到sGC脱敏和硝酸盐耐受性的临床发展之间存在有趣的平行关系。长期暴露于硝酸甘油（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主要障碍。除了sGC活化之外，NO还通过S-亚硝基化调节蛋白质功能，S-亚硝基化是将NO部分添加到半胱氨酸残基的游离巯基上。在我们寻求理解sGC的脱敏机制的过程中，我们最近发现sGC在体外和完整细胞中被S-亚硝基化。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，其特征在于sGC活性的NO刺激的丧失（而基础活性保持不变）。最近，它表明，GTN处理增加组织中蛋白质的S-亚硝基化。重要的是，我们刚刚表明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-亚硝基化，导致其脱敏，这反过来又是硝酸盐耐受性的基础。为了探索这一挑衅性的假设，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sGC在体内的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，我们将确定在已知诱导硝酸盐耐受性的条件下的GTN处理是否在体内使sGC S-亚硝基化，从而引起其对NO刺激的响应失败。将使用原代大鼠主动脉平滑肌细胞来表征GTN诱导的S-亚硝基化的发展和sGC的脱敏（使用生物化学工具）。sGC S-亚硝基化对NO依赖性血管舒张的影响的生理学表征将在仓鼠颊囊制备物体内进行，随后在大鼠提睾肌中进行。将使用腺病毒技术，用S-亚硝基化的sGC突变体尝试体内对硝酸盐耐受性的抗性。这些初步实验应该为更完整的研究奠定基础，该研究将解决更多问题：S-亚硝基化导致sGC脱敏的分子机制是什么？sGC在氧化应激条件下是否发生S-亚硝基化，如果是，是否有助于心血管功能障碍如动脉粥样硬化的发展？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？这一提议及其未来的扩展可能对开发sGC及其脱敏是新靶点的治疗策略至关重要。公共卫生相关性：硝酸甘油已被用于世纪以上，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变得不敏感。我们建议探索硝酸盐耐受性的机制，这到目前为止仍然无法解释。