ANGIOTENSINS, PROSTAGLANDINS-ADRENERGIC INTERACTIONS

描述（由申请人提供）：我们的长期目标是研究肾上腺素能神经系统、血管紧张素II （Ang II）和类二十烷酸在调节心血管功能和高血压发生中的相互作用机制。在过去的资助期内，我们已经证明去甲肾上腺素(NE)-和Ang II刺激钙/钙调素依赖性蛋白激酶II （CaMKII）磷酸化细胞质磷脂酶A2 （cPLA2）并释放血管平滑肌细胞（VSMC）中的AA；通过细胞色素P450 （CYP45O）和脂氧合酶（LO）形成的AA代谢物激活磷脂酶D (PLD)，增强cPLA2活性，促进VSMC增生和/或肥厚和高血压。本研究是这些研究的延伸，并将验证以下假设：1)CaMKII对cPLA2 Ser-515位点的磷酸化负责其激活，转运到核膜和响应NE-和Ang II的AA释放。2) cPLA2向核膜的易位依赖于vimentin磷酸化和肌动蛋白丝的组织，这是通过ERK1/2磷酸化热休克蛋白27 （HSP27）来维持的。3)通过活化表皮生长因子受体（EGFR）和/或NAD(P)H氧化酶，通过CYP45O和LO衍生的AA代谢物激活ERK1/2，导致cPLA2的Ser505/727位点和PLD2的tyr1残基位点进一步磷酸化，促进VSMC增殖和/或肥大。具体内容包括：1 . ne诱导cPLA2和PLD激活的信号机制及其功能意义。目的1。为了确定ne刺激CaMKII是否通过磷酸化Ser-515上的cPLA2，促进AA释放，Ras/ERK1/2和PLD激活和VSMC增殖。目标2。探讨CaMKII、Ras-ERK1/2、actin、vimentin和热休克蛋白（HSP27）在ne诱导的cPLA2向核膜易位中的作用及其与Aim 3相关的潜在机制。研究表皮生长因子受体（EGFR）反式活化和NAD(P)H氧化酶对AA代谢物诱导的Ras-ERK1/2和PLD2活化、cPLA2活性扩增和NE对VSMC增殖的影响。目标4。阐明cPLA2衍生的AA代谢物通过ERK1/2和p38 MAPK介导酪氨酸磷酸化激活PLD的机制。2。angii诱导cPLA2和PLD激活的信号机制及其功能意义我们还将使用类似于NE的方法研究Ang II对VSMC中上述信号机制的影响。我们将把Ang II和NE纳入同一协议。这些研究将有助于我们进一步了解ne和Ang II诱导cPLA2活化和AA释放的细胞机制，以及AA代谢物引起PLD活化、cPLA2活性扩增和VSMC增殖和肥大的机制。

DESCRIPTION (provided by the applicant): Our long-term goal is to investigate the mechanism of interaction of the adrenergic nervous system, angiotensin II (Ang II) and eicosanoids in the regulation of cardiovascular function and in the development of hypertension. Over the past grant period, we have shown that norepinephrine (NE)- and Ang II stimulate calcium/calmodulin dependent protein kinase II (CaMKII) phosphorylate cytosolic phospholipase A2 (cPLA2) and release AA in vascular smooth muscle cells (VSMC); AA metabolltes formed via cytochrome P450 (CYP45O) and lipoxygenase (LO) activate phosphoilpase D (PLD), amplify cPLA2 activity and contribute to VSMC hyperplasia and/or hypertropby and hypertension. This proposal is an extension of these studies and will test the hypotheses that: 1) Phosphorylation of cPLA2 at Ser-515 by CaMKII is responsible for its activation, translocation to the nuclear envelope and AA release in response to NE- and Ang II. 2) Translocation of cPLA2 to the nuclear envelope is dependent upon vimentin phosphorylation and actin filament organization, which is maintained by phosphorylation of heat shock protein 27 (HSP27) by ERK1/2. 3) AA metabolites derived via CYP45O and LO by transactivating epidermal growth factor receptor (EGFR) and/or NAD(P)H oxidase, activate ERK1/2, which causes further phosphorylation of cPLA2 at Ser505/727, and of PLD2 at a Tyr-residue(s), and promotes VSMC proliferation and/or hypertrophy. The specific alms of the proposal are: I. Signaling Mechanisms Involved in NE-induced cPLA2 and PLD Activation and their Functional Significance. Aim 1. To determine if NE-stimulated CaMKII, by phosphorylating cPLA2 on Ser-515, promotes AA release, Ras/ERK1/2 and PLD activation and VSMC proliferation. Aim 2. To investigate the contribution of CaMKII, Ras-ERK1/2, actin, vimentin and heat shock protein (HSP27) to NE-induced cPLA2 translocation to the nuclear envelope and the underlying mechanism involved Aim 3. To examine the contribution of epidermal growth factor receptor (EGFR) transactivation and NAD(P)H oxidase to AA metabolite-induced activation of Ras-ERK1/2 and PLD2, amplification of cPLA2 activity and VSMC proliferation in response to NE. Aim 4. To elucidate the mechanism of PLD activation by tyrosine phosphorylation caused by cPLA2 derived AA metabolites via ERK1/2 and p38 MAPK in response to NE. II. Signaling Mechanisms Involved in Ang II-Induced cPLA2 and PLD Activation and their Functional Significance. We will also investigate the effects of Ang II on the above signaling mechanisms in VSMC using an approach similar to that described for NE. We will include Ang II and NE in the same protocols. These studies should further our understanding of the cellular mechanisms involved in NE-and Ang II- induced cPLA2 activation and AA release and the mechanism by which AA metabolites cause PLD activation, amplification of cPLA2 activity and VSMC proliferation and hypertrophy.