ANGIOTENSINS, PROSTAGLANDINS--ADRENERGIC INTERACTIONS

本提案的长期目标是研究 去甲肾上腺素（NE）和血管紧张素II（AII）在释放 花生四烯酸（AA）用于血管中前列腺素（PG）的合成。 兔主动脉血管平滑肌细胞的研究进展 表明NE和AII通过激活钙离子促进AA释放， 钙调蛋白依赖性蛋白激酶（CaMKII），其直接和 通过丝裂原活化蛋白激酶（MAPK），刺激细胞质 磷脂酶A2（cPLA2）。 现时的建议是澄清 NE和AII刺激CaMKII激活MAPK和cPLA2的机制。 基于AA抑制剂的初步研究， 代谢和外源性类花生酸，是NE和AII刺激 CaMKII直接激活cPLA2并释放AA;生成AA的产物 通过细胞色素P-450（CYP-450）和脂氧合酶（LO）激活MAPK， 进一步刺激cPLA2和磷脂酶D（PLD）。 以下是 具体目标。 I. NE诱导AA的信号转导机制 release. 目标1。 为了研究CaMK II是否诱导MAPK的活化， 进一步激活cPLA2，由AA产生的产物介导 VSMC中NE的响应。 目标2. 确定 谷胱甘肽激活蛋白（GAP）、磷脂酰肌醇3- PI3激酶和Syk激酶介导AA的作用 NE诱导的Ras和Rho/MAPK及MAPK通路中通过ERK-450和LO衍生的代谢产物 VSMC中的cPLA2活化。 目标3。 研究CaMKII是否直接 激活cPLA2及其激活的潜在机制。 目标4。 到 研究PLD对NE刺激的AA释放的贡献以及 血管平滑肌细胞PLD激活的机制。 二. 参与的信号机制 血管紧张素诱导AA释放。 先前的工作和初步数据 提示AII刺激血管平滑肌细胞AA释放的机制 这与NE相似。 因此，一种方法 将使用与针对NE描述的相同的方法。 将进行研究 使用的是私家侦探实验室目前使用的技术 活动 脂肪酶、激酶和GTP结合蛋白的含量将通过 既定程序。 HPLC和GCMS程序将用于鉴别 AA代谢物。 建议的研究应进一步了解 NE和AII如何激活cPLA_2和PLD及AA释放以合成PG VSMC。 此外，从这些研究中产生的信息应允许 制定合理的方法来开发新的药物， 对血管疾病的治疗具有更大的选择性。

The long term objective of this proposal is to investigate the signaling mechanisms of norepinephrine (NE), and angiotensin II (AII) in releasing arachidonic acid (AA) for prostaglandin (PG) synthesis in blood vessels. Recent findings in rabbit aortic vascular smooth muscle cells (VSMC) indicate that NE and AII promote AA release by activating calcium- calmodulin dependent protein kinase (CaMKII), which, both directly and through mitogen activated protein kinase (MAPK), stimulates Cytosolic phospholipase A2 (cPLA2). The present proposal is to elucidate the mechanism by which NE and AII stimulated CaMKII activates MAPK and cPLA2. The overall hypothesis, based on preliminary studies with inhibitors of AA metabolism and exogenous eicosanoids, is that NE- and AII- stimulated CaMKII directly activates cPLA2 and releases AA; products of AA generated via cytochrome P-450 (CYP-450) and lipoxygenase (LO) activate MAPK, which further stimulates cPLA2 and also phospholipase D (PLD). The following are the specific aims. I. Signaling Mechanisms Involved in NE-Induced AA Release. Aim 1. To Investigate Whether CaMKII-Induced Activation of MAPK, which further Activates cPLA2, is Mediated by the Products of AA Generated via CYP-450 and LO in Response to NE in VSMC. Aim 2. To Determine the Contribution of GTPase Activating Proteins (GAP), Phosphatidylinositol 3- Kinase (PI3-Kinase) and Syk Kinase in Mediating the Action of AA Metabolites Derived via CYP-450 and LO in NE Induced Ras and Rho/MAPK and cPLA2 Activation in VSMC. Aim 3. To Investigate Whether CaMKII directly Activates cPLA2 and the Underlying Mechanism of its Activation. Aim 4. To Investigate the Contribution of PLD to NE-Stimulated AA Release and the Mechanism of PLD Activation in VSMC. II. Signaling Mechanisms Involved in Angiotensins Induced AA Release. Previous work and preliminary data suggest that the mechanisms involved in AII stimulated AA release in VSMC is similar to that of NE. Therefore, an approach identical to that described for NE will be used. Studies will be conducted using techniques currently operative in the P.I'S laboratory. The activity of the lipases, kinases and GTP binding proteins will be determined by established procedures. HPLC and GCMS, procedures will be used to identify AA metabolites. The proposed studies should further our understanding of how NE and AII activate cPLA2 and PLD and AA release for PG synthesis in VSMC. Furthermore, information generated from these studies should allow formulation of rational approaches to the development of novel agents with greater selectivity for the treatment of vascular diseases.