ANGIOTENSINS, PROSTAGLANDINS-ADRENERGIC INTERACTIONS

描述（由申请方提供）：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）和类花生酸在心血管功能调节和高血压发生中的相互作用机制。在过去的研究期间，我们发现去甲肾上腺素（NE）和血管紧张素II（Ang II）刺激血管平滑肌细胞（VSMC）中钙/钙调蛋白依赖性蛋白激酶II（CaMK II）磷酸化胞浆磷脂酶A2（cPLA 2）并释放AA;通过细胞色素P450（CYP 450）和脂氧合酶（LO）形成的AA代谢物激活磷脂酶D（PLD），增强cPLA 2活性并促进VSMC增生和/或肥大和高血压。本研究是这些研究的延伸，并将验证以下假设：1）CaMK II对cPLA 2的Ser-515磷酸化负责其激活，易位到核膜和AA释放以响应NE-和Ang II。2)cPLA 2向核膜的转运依赖于波形蛋白磷酸化和肌动蛋白丝组织，其通过ERK 1/2对热休克蛋白27（HSP 27）的磷酸化来维持。3)通过反式激活表皮生长因子受体（EGFR）和/或NAD（P）H氧化酶经由CYP 45 O和LO衍生的AA代谢物激活ERK 1/2，其引起cPLA 2在Ser 505/727处的进一步磷酸化和PLD 2在Tyr残基处的进一步磷酸化，并促进VSMC增殖和/或肥大。该建议的具体施舍是：一。NE诱导cPLA 2和PLD激活的信号机制及其功能意义目标1.为了确定NE刺激的CaMK II是否通过磷酸化Ser-515上的cPLA 2促进AA释放、Ras/ERK 1/2和PLD激活以及VSMC增殖。目标2.探讨CaMK Ⅱ、Ras-ERK 1/2、actin、vimentin和热休克蛋白（HSP 27）在NE诱导的cPLA 2向核膜转位中的作用及其机制。研究表皮生长因子受体（EGFR）反式激活和NAD（P）H氧化酶在AA代谢物诱导的Ras-ERK 1/2和PLD 2激活、cPLA 2活性扩增和VSMC增殖中的作用。目标4。探讨NE诱导cPLA 2的AA代谢产物通过ERK 1/2和p38 MAPK引起的酪氨酸磷酸化激活PLD的机制。 二.血管紧张素II诱导cPLA 2和PLD激活的信号机制及其功能意义我们也将研究血管紧张素II对上述信号机制的影响，在VSMC中使用的方法类似于所描述的NE。我们将在相同的方案中包括Ang II和NE。这些研究有助于我们进一步了解NE和Ang Ⅱ诱导cPLA 2活化和AA释放的细胞机制，以及AA代谢产物引起PLD活化、cPLA 2活性增强和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.