NPY & B-ADRENERGIC RECEPTORS IN VASCULAR REMODELING

神经肽Y(NPY)是一种中枢交感神经递质，具有多种功能，从食欲控制到心血管功能。在过去的十年里，我们的实验室专注于NPY对血管紧张性的调节研究，并取得了一些重要发现，例如NPY是一种应激激活的睾酮依赖的血管收缩因子，通过Y1受体(RS)发挥作用-这一概念现在被支持通过过度表达NPY而在雄性大鼠中发生高血压。在之前的赠款期间，我们已经确定了NPY的另一种生物活性--作为一种血管生长因子。在体内，NPY可刺激血管平滑肌(VSMCs)和内皮细胞(对血管生成)的增殖和新生内膜的增殖，浓度低于血管收缩的浓度，并通过多个R：Y2/Y5或Y1/Y5或Y1/Y5 Rs，取决于NPY的浓度。这些受体中的每一个都是Gi偶联的，但可能与不同的腺酰环化酶(AC)亚型相连。由于VSMCs和大鼠颈动脉具有极低的NPY受体丰度，NPY的作用需要它们的上调-这一过程由NPY本身和β-肾上腺素能受体(β-AR)激动剂介导。在这一更新应用中，我们建议重点研究BetaAR和NPY诱导的血管生长之间的相互作用。我们发现，预先给予异丙肾上腺素可诱导VSMC产生Y1、Y2和Y5受体，并显著增强NPY的促有丝分裂作用。总的假设是，通过β-AR激活-脱敏对血管壁进行预适应，上调NPY受体和/或受体后cAMP依赖的机制，并增强由NPY和损伤引起的新生内膜增殖。我们将确定1)损伤是否上调NPY Rs和下调BetaAR，以及它们与AC异构体和Gs/Ipha的偶联；2)BetaAR的这种允许作用是否是NPY-R所特有的，并依赖于BetaAR的激活或脱敏；以及3)转录和/或转录后机制是否参与了NPY R表达的BetaAR增强。这些研究将在体外的大鼠主动脉VSMCs中进行：损伤的、未损伤的、转βAR基因的大鼠、体内的大鼠和接受血管成形术的特异性NPY受体缺陷的小鼠。β受体激活或脱敏可通过预先暴露于β-激动剂、β-ARK操作或应激(在动物中)而诱导，并被β-受体拮抗剂和cAMP途径调节剂改变。用NPY受体激动剂或拮抗剂或反义D-寡核苷酸处理细胞和动物，测定NPY受体、β受体、Gs/I、βARK和β-arrestin(mRNA和蛋白质)的表达，以及βAR刺激的cAMP水平。这些研究将解决与临床相关的问题，即在应激、心力衰竭和心绞痛中，β-AR的激活和/或脱敏是否通过激活特定的NPY受体和cAMP依赖的信号来启动血管生长，如果是这样的话，可能会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.