NPY & B-ADRENERGIC RECEPTORS IN VASCULAR REMODELING

神经肽Y （NPY）是一种中枢和交感神经递质，具有多种功能，从食欲控制到心血管功能。在过去的十年中，我们的实验室专注于研究NPY对血管张力的调节，并取得了一些重要的发现，例如NPY是一种应激激活的睾酮依赖性血管收缩剂，通过Y1受体（RS）起作用-这一概念现在通过过度表达NPY支持雄性大鼠高血压的发展。在之前的资助期间，我们已经确定了npy作为血管生长因子的另一种生物活性。NPY刺激血管平滑肌（VSMCs）和内皮细胞（其具有血管生成性）的增殖，以及体内新生内膜的增殖，浓度低于血管收缩，并通过多个Rs: Y2/Y5或Y1/Y5或Y1/Y5 Rs，取决于NPY的浓度。这些Rs都是gi偶联的，但可能与不同的腺苷酸环化酶（AC）异构体偶联。由于VSMCs和大鼠颈动脉具有极低的NPY Rs含量，NPY的作用需要它们的上调，这是一个由NPY本身和β -肾上腺素能R （β - aar）激动剂介导的过程。在这篇更新的应用中，我们建议将重点放在betaARs与npy诱导的血管生长之间的相互作用上。我们发现异丙肾上腺素预先暴露于VSMCs可诱导Y1、Y2和Y5 Rs，并显著增强NPY的有丝分裂作用。总的假设是，通过β - aar激活脱敏对血管壁进行预处理，上调NPY Rs和/或受体后cAMP依赖机制，并增加NPY和损伤引起的新内膜增殖。我们将确定1)损伤是否上调NPY Rs并下调β - ars，以及它们与AC异构体和Gs/ α的耦合，以及2)β - ars的这种允许作用是否具有NPY- r特异性，取决于β - aar的激活或脱敏；3)转录和/或转录后机制是否参与NPY R表达的β - aar增强。研究将在体外大鼠主动脉VSMCs中进行：损伤的，未损伤的，β - aar转染的，以及在体内进行血管成形术的大鼠和特异性NPY Rs缺乏的小鼠。β -受体激活或脱敏可通过预先暴露于β -受体激动剂、β -受体激酶（betaARK）操作或应激（动物）诱导，并可通过β -拮抗剂和camp通路调节剂改变。将用NPY R激动剂或拮抗剂或反义d寡核苷酸处理细胞和动物，并测定NPY Rs、β - ars、Gs/i、β - aark和β -抑制素（mRNA和蛋白）的表达以及β -ar刺激的cAMP水平。这些研究将解决β - aar激活和/或脱敏的临床相关问题，如在应激、心力衰竭和心绞痛中，β - aar激活和/或脱敏是否通过激活特定的NPY Rs和camp依赖性信号来促进血管生长，如果是这样，可能为预防血管疾病开辟新的途径。

Neuropeptide Y (NPY) is a central and sympathetic neurotransmitter with pleiotropic activities ranging from appetite control to cardiovascular function. Over the last decade our lab focused its research on NPY regulation of vascular tone and made some important discoveries e.g. that NPY is a stress-activated testosterone-dependent vasoconstrictor acting via Y1 receptors (RS)- a notion now supported with development of hypertension in male rats by over-expressing NPY. During the previous grant period we have identified yet another bioactivity of NPY-as a vascular growth factor. NPY stimulates proliferation of vascular smooth muscle (VSMCs) and endothelial cells (for which it is angiogenic), and neointimal proliferation in vivo, at concentrations below vasoconstrictive and via multiple Rs: Y2/Y5 or Y1/Y5 or Y1/Y5 Rs, depending on NPY concentration. Each of these Rs is Gi-coupled but possibly to different adenylyl cyclase (AC) isoforms. Since VSMCs and rat carotid arteries possess extremely low abundance of NPY Rs, NPY's actions require their up-regulation-a process mediated by NPY itself and beta-adrenergic R (betaAR) agonists. In this renewal application, we propose to focus on interactions between betaARs and NPY-induced vascular growth. We found that pre-exposure of VSMCs to isoproterenol induces Y1, Y2 and Y5 Rs, and markedly augments NPY's mitogenic effect. The overall hypothesis is that pre-conditioning of vessel wall by betaAR activation- desensitization up-regulates NPY Rs and/or post-receptor cAMP- dependent mechanisms and augments neointimal proliferation due to NPY and injury. We will determine 1) if injury up-regulates NPY Rs and down-regulates betaARs, and their coupling to AC isoforms and Gs/ialpha, and 2) if this permissive effect of betaARs is NPY-R-specific and depends on betaAR activation or desensitization; and 3) whether transcriptional and/or post-transcriptional mechanisms are involved in the betaAR potentiation of the NPY R expression. The studies will be performed in vitro in rat aortic VSMCs: injured, uninjured, betaAR- transfected, and in vivo in rats, and mice deficient in specific NPY Rs, subjected to angioplasty. BetaAR activation or desensitization will be induced by pre-exposure to beta-agonist, betaAR kinase (betaARK) manipulations or stress (in animals) and altered by beta antagonist and cAMP-Pathway modulators. Cells and animals will be treated with NPY R agonists or antagonists or antisense D-oligonucleotides, and expression of NPY Rs, betaARs, Gs/i, betaARK and beta-arrestin (mRNA and protein), and betaAR-stimulated cAMP levels will be determined. These studies will address clinically relevant issue of whether or not betaAR activation and/or desensitization, such as in stress, heart failure and angina, primes vessels for vascular growth by activating specific NPY Rs and cAMP-dependent signaling, and if so, could open new avenues for prevention ov vascular diseases.