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 血管扩张剂 最近的研究表明，高胆固醇和/或低密度脂蛋白 在诱导这种改变方面可以发挥重要作用， 内皮衍生舒张因子的合成;一氧化氮（NO）。 AS减少血管壁NO的产生， 内膜中平滑肌细胞增殖和血小板聚集， 粘附和脱粒，这似乎是重要的事件， 动脉粥样硬化和血管病变。 然而，精确的机制 NO合成受高胆固醇损害的原因尚未确定。 大量证据支持高胆固醇和/或低密度脂蛋白 水平影响一个步骤，涉及运输的L-精氨酸，前体 NO在内皮细胞中的表达。 事实上， 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，缺乏证据表明， 高胆固醇血症状态下l-精氨酸转运状态 的 拟议的研究计划将集中在血管中的L-精氨酸转运 内皮细胞，以确定其在高浓度诱导的功能障碍中的作用。 胆固醇（LDL）状态。 具体目标是：1）提供L- 血管内皮细胞精氨酸转运。 本节将 包括确定L-[3 H]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LDL/氧化LDL的作用 基础和刺激条件下精氨酸转运的影响，以及3）， 通过确定这些药剂的作用部位， 在正常和改变的条件下调节精氨酸转运的途径 条件（高胆固醇/LDL）。 为此目的， 受体介导的钙动员（G蛋白; InsP 3/Ca 2 +; DAG/PkC， Ca 2 +/钙调素等）在调节精氨酸转运方面， 使用选择性抑制肿瘤细胞增殖的药理学试剂测定。 磷酸肌醇级联的不同步骤，Ca 2 +/钙调素依赖性 类似的实验将进行测量NO释放 以评估精氨酸转运和NO合成之间的耦合。 的 膜电位变化的作用作为NO激活之间的联系 合成和L-精氨酸转运。 拟议 研究计划将有助于我们对细胞的基本理解， 正常和改变的氨基酸转运机制 血管内皮功能障碍与脑血管病发病机制的关系 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.