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

说明(申请人提供)：一氧化氮(NO)通过激活可溶性鸟苷酸环化酶(SGC)诱导血管扩张，sGC继而产生第二信使cGMP。暴露于NO后，sGC变得不敏感，对额外的NO刺激没有反应。到目前为止，sGC的脱敏机制尚不清楚。我们发现sGC脱敏和硝酸盐耐受的临床发展之间有一个有趣的平行关系。长期暴露在硝酸甘油(GTN)中，通过产生NO而诱导血管扩张，导致对NO和硝酸盐的血管扩张反应丧失。这种硝酸盐耐受的机制尚不清楚，但这是广泛使用的心血管治疗的一个重大退步。除了sGC的激活，NO还通过S亚硝化来调节蛋白质的功能，这是在半胱氨酸残基的游离硫醇上增加了一个NO部分。为了了解sGC的脱敏机制，我们最近发现sGC在体外和在完整细胞中都是S硝化的。我们进一步建立了sGC的S亚硝化与其脱敏之间的因果关系，其特征是失去对sGC活性的刺激(在基础活性保持不变的情况下)。最近有研究表明，GTN处理可增加组织中蛋白质的S亚硝化。重要的是，我们刚刚证明了GTN在细胞内诱导了S-亚硝化和sGC的脱敏。因此，我们的假设是，GTN处理诱导SGc的S亚硝化，导致其脱敏，这反过来又奠定了硝酸盐耐受性的基础。要探索这一挑衅性假说，我们首先需要证明，SGCGTN依赖的S亚硝化直接导致脱敏。其次，我们将评估S-体内SGC亚硝化(使用S-亚硝化剂)是否能够模拟硝酸盐耐受性的发展。第三，我们将确定GTN是否在已知条件下诱导体内的硝酸盐耐受S-亚硝酸盐sGC从而引起其对无刺激的失败反应。我们将利用原代培养的大鼠主动脉平滑肌细胞，用生化工具研究GTN诱导的S亚硝化和SGC的脱敏作用。生理特性的影响，SGCS-亚硝酰化对NO依赖的血管扩张将在活体进行与仓鼠颊囊制剂，稍后在大鼠提睾肌。利用腺病毒技术，将用S-亚硝化的sgc突变体尝试体内对硝酸盐耐受性的抗性。这些初步实验应该会为更完整的研究奠定基础，从而解决更多的问题：S-亚硝化脱敏的分子机制是什么？SGC S是在氧化应激条件下被亚硝化的吗？如果是这样的话，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SGGc S-亚硝化能阻止硝酸盐耐受或其他心血管疾病的发生吗？这一建议及其未来的扩展可能对发展治疗策略至关重要，其中sGC及其脱敏是新的靶点。与公众健康相关：一个多世纪以来，硝酸甘油一直被用来通过放松血管系统来治疗许多心血管疾病。不幸的是，它会导致硝酸盐耐受，这意味着机体对治疗变得不敏感。我们建议探索硝酸盐耐受的潜在机制，到目前为止，这一机制仍然不清楚。

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.