LDL/CHOLESTEROL EFFECTS ON ARGININE TRANSPORT IN VASCULAR ENDOTHELIAL CELLS

动物和人类的动脉粥样硬化的特征是 动脉和小动脉对内皮依赖性的无反应性 血管扩张剂。最近的研究表明，高胆固醇和/或低密度脂蛋白 可以通过减少 内皮源性松弛因子的合成；一氧化氮(NO)。 因为减少血管壁产生的NO可以促进 血管内膜的平滑肌细胞增殖和血小板聚集， 粘连和脱颗粒，这似乎是 动脉粥样硬化和血管病理学。然而，精确的机制 高胆固醇导致NO合成受阻的机制尚未确定。 大量证据支持高胆固醇和/或低密度脂蛋白 水平影响涉及前体L-精氨酸运输的一步 血管内皮细胞中NO的含量。事实上，美国政府获得的初步证据 申请人指出，高胆固醇显著降低L-精氨酸 在这些细胞中运输。然而，缺乏关于这一事件的证据 高胆固醇血症状态下L-精氨酸转运状况。这个 拟议的研究计划将集中在L-精氨酸在血管中的转运 血管内皮细胞在高脂诱导的功能障碍中的作用 胆固醇(低密度脂蛋白)状态。 具体目的是：1)对L进行详细的刻画-- 精氨酸在血管内皮细胞中的运输。本节将介绍 包括确定L-[~3H]精氨酸转运动力学行为的研究， 膜电位依赖性，外部pH变化的影响，以及 竞争研究，以确定运输的选择性 机制。2)评价胆固醇和低密度脂蛋白/氧化低密度脂蛋白的影响 关于基础和刺激条件下的精氨酸转运和3)，至 通过确定这些毒剂可能的作用部位 正常和异常状态下精氨酸转运的调控途径(S) 条件(高胆固醇/低密度脂蛋白)。为此目的， 受体介导的钙动员(G蛋白；InsP3/钙离子；DAG/PKC， Ca2+/钙调蛋白等)在精氨酸运输的调节中 使用选择性地抑制血管紧张素转换酶 磷酸肌醇级联的不同步骤，依赖于钙/钙调素 过程等。将进行类似的实验，以测量NO释放 评估精氨酸转运和NO合成之间的偶联作用。这个 膜电位变化在NO激活之间的联系作用 将对其合成和L-精氨酸转运进行评价。建议数 研究计划将有助于我们对细胞的基本了解 正常和改变氨基酸转运的机制 血管内皮细胞功能障碍与血管内皮细胞损伤的关系 动脉粥样硬化、高血压和充血性心力衰竭。

Atherosclerosis in animals and humans is characterized by an unresponsiveness of arteries and arterioles to endothelium-dependent vasodilators. Recent studies indicate that high cholesterol and/or LDL could play a significant role in inducing such alteration by reducing the synthesis of the endothelial-derived relaxing factor; nitric oxide (NO). AS reduction in the production of NO by the vessel wall could promote smooth muscle cell proliferation in the intima and platelet aggregation, adhesion, and degranulation, which appear to be important events in atherosclerosis and vascular pathologies. However, the precise mechanism by which NO synthesis is impaired by high cholesterol has not been defined. Substantial evidence support the notion that high cholesterol and/or LDL levels affect a step involving the transport of L-arginine, the precursor of NO in endothelial cells. Indeed, preliminary evidence obtained by the applicant indicates that high cholesterol significantly reduces L-arginine transport in these cells. However, evidence is lacking concerning the status of l-arginine transport in hypercholesterolemic states. The proposed research plan will focus on L-arginine transport in vascular endothelial cells to determine its role in the dysfunction induced by high cholesterol (LDL) states. The specific aims are; 1) To provide a detailed characterization of L- arginine transport in vascular endothelial cells. This section will include studies to define the kinetic behavior of L-[3H]arginine transport, membrane potential dependence, the effect of external pH changes, and competition studies to determine the selectivity of the transport mechanism. 2) To evaluate the effect of cholesterol and LDL/oxidized LDL on arginine transport in basal and stimulated conditions and 3), to identify possible sites of action of these agents by determining the pathway(s) regulating arginine transport under normal and altered conditions (high cholesterol/LDL). For this purpose, the role of the receptor-mediated calcium mobilization (G-proteins; InsP3/Ca2+; DAG/PkC, Ca2+/calmodulin, etc.) in the regulation of arginine transport will be determined using pharmacological agents that selectively inhibit the different steps of the phosphoinositol cascade, Ca2+/calmodulin-dependent processes, etc. Similar experiments will be conducted measuring NO release to assess the coupling between arginine transport and NO synthesis. The role of membrane potential changes as a link between the activation of NO synthesis and L-arginine transport will be evaluated. The proposed research program will contribute to our basic understanding of the cellular mechanisms underlying normal and altered amino acid transport in relationship to endothelial dysfunction int he pathogenesis of atherosclerosis, hypertension and congestive heart failure.