LDL/CHOLESTEROL EFFECTS ON ARGININE TRANSPORT IN VASCULAR ENDOTHELIAL CELLS

低密度脂蛋白/胆固醇对血管内皮细胞中精氨酸转运的影响

• 批准号: 6240295
• 负责人: NELSON ESCOBALES
• 金额: $ 6.95万
• 依托单位: UNIVERSITY OF PUERTO RICO MED SCIENCES
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6240295
• 关键词: G protein; acidity /alkalinity; aminoacid transport; arginine; atherosclerosis; calcium; cholesterol; congestive heart failure; hypertension; low density lipoprotein; membrane potentials; membrane transport proteins; pathologic process; protein kinase C; radiotracer; statistics /biometry; tissue /cell culture; vascular endothelium

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.

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 血管扩张器。 最近的研究表明，高胆固醇和/或LDL 通过减少 内皮衍生的放松因子的合成； 一氧化氮（NO）。 由于船只壁的生产减少可以促进 内膜和血小板聚集的平滑肌细胞增殖， 粘附和脱粒，这似乎是重要事件 动脉粥样硬化和血管病理。 但是，精确的机制 尚未定义高胆固醇损害的合成。 大量证据支持高胆固醇和/或LDL的观念 水平会影响涉及L-精氨酸传输的步骤，前体 内皮细胞中的NO。 确实，由 申请人表明高胆固醇可显着降低L-精氨酸 这些细胞中的运输。 但是，缺乏有关 高胆固醇状态中L-精氨酸转运的状态。 这 拟议的研究计划将集中于血管中的L-精氨酸运输 内皮细胞确定其在高诱导的功能障碍中的作用 胆固醇（LDL）国家。 具体目标是； 1）提供L-的详细表征 血管内皮细胞中的精氨酸转运。 本节将 包括确定L- [3H]精氨酸转运的动力学行为的研究， 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）评估胆固醇和LDL/氧化的LDL的影响 在基础和刺激条件下精氨酸的转运以及3） 通过确定这些药物的可能作用部位 在正常情况下调节精氨酸转运的途径 条件（高胆固醇/LDL）。 为此， 受体介导的钙动员（G蛋白； insp3/Ca2+; DAG/PKC， Ca2+/钙调蛋白等）在精氨酸运输的调节中 使用有选择性抑制的药理学剂确定 磷酸肌醇级联反应的不同步骤，Ca2+/钙调蛋白依赖性 过程等。将进行类似的实验，以测量无释放 评估精氨酸转运与无合成之间的耦合。 这 膜电位变化的作用是NO激活之间的联系 将评估合成和L-精氨酸的运输。 提议 研究计划将有助于我们对细胞的基本理解 正常和改变氨基酸转运的机制 与内皮功能障碍的关系 动脉粥样硬化，高血压和充血性心力衰竭。