NO & Regulation of Cardiac Adrenergic Receptors in Heart

β-肾上腺素能受体(β-ARs)在心血管功能调节中起着重要作用。心脏β-受体激动剂的刺激作用增加了心脏收缩的速度和力量，并对心肌细胞的生长和凋亡有额外的作用，β-受体是交感神经来源的儿茶酚胺调节心血管功能的主要机制之一。心力衰竭，特征为 由于心脏不能泵出足够的血液来满足身体的代谢需求，这与β-AR信号减弱有关。β-AR功能的下降与β-AR脱敏和下调的增加直接相关，涉及G蛋白偶联受体激酶(GRKs)的受体磷酸化和随后的β-arrestin结合。大量研究表明，一氧化氮(NO)的生物利用度降低与心力衰竭有关。转基因过表达内皮型一氧化氮合酶(ENOS)是产生NO的酶的一种结构性表达的异构体，已被证明可以减轻实验性心力衰竭。另一方面，一氧化氮合酶的抑制导致对β-AR激动剂的血管扩张作用的快速反应，这可以通过添加含有NO的S亚硝硫醇(SNO)来缓解。这表明，NO生物利用度的降低会导致β-AR信号传递能力的下降，这与在心力衰竭中观察到的情况类似。这些发现提供了一致的证据，表明没有生物利用度是β-AR功能的动态平衡调节的基础。我们实验室的初步数据表明，亚硝硫醇阻止激活的β2-AR与参与脱敏和下调的细胞机制联系在一起。具体来说，我们 证明亚硝硫醇，L-SNC和GSNO，抑制GRK2介导的β2-AR的磷酸化，并阻止其随后与β-抑制素2的相互作用。我们进一步证明，增加内源性亚硝硫醇(GSNO)水平，可以防止β-AR下调，并延迟实验性心力衰竭的发生。我们的中心假设是，没有生物可用性调节β-AR功能，防止β-AR的脱敏和下调。与心力衰竭相关的β-AR受体功能改变是NO生物利用度降低的直接结果。具体目标是(1)测试是否没有生物利用度 通过评估激动剂诱导的β-AR在培养细胞中的脱敏和下调来直接调节β-AR信号；(2)确定NO/亚硝硫醇是否改变重组系统中由β-AR刺激的G蛋白偶联以及GRK和PKA介导的受体磷酸化；(3)测试内源性亚硫醇是否调节小鼠心脏β-AR功能和实验性心力衰竭(HF)的进展；(4)测试亚硫醇治疗作为一种新的改善心力衰竭的治疗手段 心肌肥厚和/或心力衰竭的小(小鼠)和大(兔和猪)动物模型的心功能。

Beta-adrenergic receptors (beta-ARs) ptay a fundamental role in the regulation of cardiovascular function. The stimulation of cardiac beta-ARs effects increases in both the rate and force of cardiac comraction, and has additional effects on cardiac cell growth and apoptosis, beta-ARs represent one of the primary mechanisms by which sympathetically derived catecholamines modulate cardiovascular function. Heart failure, characterized by the inability of the heart to pump enough blood to meet the metabolic demands of the body, is associated with attenuated beta-AR signaling. The decrease in beta-AR function is directly related to an increase in the desensitization and down-regulation of beta-ARs, involving receptor phosphorylation by G protein-coupled receptor kinases (GRKs) and the subsequent binding of beta-arrestin. Numerous studies have demonstrated decreased nitric oxide (NO) bioavailability associated with heart failure. The transgenic overexpression of endothelial nitric oxide synthase (eNOS), a constitutively expressed isoform of the enzyme that produces NO, has been shown to attenuate experimental heart failure. Conversly, the inhibition of NOS leads to the development of tachyphylaxis to the vasodilator effects of beta-AR agonists, which can be rescued by the addition of NO containing S-nitrosothiols (SNOs). This suggests that compromised NO bioavailability leads to a decrease in the ability of beta-ARs to signal, similar to that which is observed in heart failure. These findings provide converging lines of evidence that NO bioavailability is fundamental for the homeostatic regulation of beta-AR function. Preliminary data from our lab suggests that nitrosothiols prevent the activated beta2-AR from associating with the cellular machinery involved in desensitization and down-regulation. Specifically, we demonstrate that the nitrosothiols, L-SNC & GSNO, inhibit GRK2-mediated phosphorylation of the beta2-AR and prevent its subsequent interaction with beta-arrestin2. We further demonstrate that increasing levels of the endogenous nitrosothiol, GSNO, prevents beta-AR down-regulation and delays the onset of experimental heart failure. Our Central Hypothesis is that NO bioavaitability regulates beta-AR function, preventing the desensitization and down-regulation of beta-ARs. Altered beta-AR receptor function associated with heart failure is the direct result of decreased NO bioavailability. The Specific Aims are (1) To test whether NO bioavailability directly modulates beta-AR signaling by assessing agonist induced beta-AR desensitization and down-regulation in cultured cells; (2) To determine whether NO/nitrosothiols alter beta-AR stimulated G protein coupling, and GRK-& PKA-mediated receptor phosphorylation using purified proteins in a reconstituted system; (3) To test whether endogenous nitrosothiols modulate cardiac beta-AR function and the progression of experimental heart failure (HF) in the mouse; (4) To test nitrosothiol-based therapies as a novel therapeutic modality for improving cardiac function in both small (mouse) and large (rabbit & pig) animal models of cardiac hypertrophy and/or heart failure.