OXIDANT STRESS AND ENDOTHELIAL CELL CA2+ SIGNALING.

氧化应激和内皮细胞 CA2 信号传导。

• 批准号: 3083023
• 负责人: STEPHEN J ELLIOTT
• 金额: $ 7.61万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-01 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3083023
• 关键词: adenosine triphosphate; biological signal transduction; bradykinin; calcium flux; calcium indicator; calcium transporting ATPase; electrolyte balance; electrophysiology; endoplasmic reticulum; fluorescent dye /probe; guanosine triphosphate; hyperoxia; immunofluorescence technique; inositol phosphates; ion transport; membrane potentials; oxidizing agents; peroxides; physiologic stressor; potassium channel; scintillation counter; second messengers; sodium potassium exchanging ATPase; tissue /cell culture; vascular endothelium; voltage /patch clamp; voltage gated channel

The long-term goal of this project is to understand the molecular events by which oxidant stress alters endothelial regulation of vascular reactivity. Vascular endothelial cell (VEC) cytosolic Ca2+ is a key second messenger which contributes to the secretion of vasoactive paracrine substances. This proposal focuses on the effect of oxidant stress on Ca2+ signaling in VECs. The model oxidant is ter-butyl-hydroperoxide (t-bu-OOH). Bradykinin will be employed as the model agonist. t-bu-OOH inhibits Ca2+ signaling in fura-2-loaded VECs (Elliott and Schilling, 1990). t-bu-OOH initially inhibits bradykinin-stimulated Ca2+ influx, with no effect on basal cytosolic Ca2+. After longer incubations with the oxidant, agonist-stimulated release of Ca2+ from internal stores is decreased. t-bu-OOH produces progressive loss of membrane potential, measured by giga- seal, and net loss of cellular K+ and net gain of cellular Na+, determined using radioisotopes. This proposal explores the molecular mechanisms responsible for the above findings. Specific Aim I will characterize the mechanism by which oxidant stress inhibits agonist-stimulated changes in cytosolic Ca2+. The effects of oxidant stress on 1) inositol polyphosphate (IP) production, and 2) Ca2+ release from internal stores by IP3 and/or GTP will be investigated using radiolabeling techniques in intact and saponin-permeabilized VECs. The effect of oxidant stress on the kinetics of ATP-dependent Ca2+ pumps will also be characterized. Specific Aim II will characterize the effect of oxidant stress on ion concentration gradients and ion conductance across the cell membrane. The hypotheses that oxidant stress: 1) stimulates Na+ influx via a non- selective cation channel; 2) stimulates K+ efflux via Ca2+-dependent K+ channels; and 3) inhibits Na+/K+-ATPase, will be investigated using radioisotopic tracers and direct, giga-seal measurements. The link between oxidant inhibition of agonist-stimulated Ca2+ influx and oxidant-induced membrane depolarization will be investigated in VECs dually loaded with the Ca2+-sensitive fluorescent indicator, fura-2, and the potential-sensitive dye, di-4-ANEPPS. Thus, these experiments will characterize how oxidant stress alters endothelial cell Ca2+ signaling. This work will form the basis for the long-term objective which is to understand how oxidant stress alters the regulation of vasoreactivity by VECs.

该项目的长期目标是通过以下方式了解分子事件： 该氧化应激改变血管反应性的内皮调节。 血管内皮细胞（VEC）胞浆Ca ~（2+）是一种重要的第二信使 其有助于血管活性旁分泌物质的分泌。 本研究的重点是氧化应激对细胞内Ca 2+信号转导的影响。 VEC 模型氧化剂是叔丁基过氧化氢（t-bu-OOH）。 缓激肽 将被用作模型激动剂。 t-bu-OOH抑制负载fura-2的VEC中的Ca 2+信号传导（Elliott和 Schilling，1990）。 t-bu-OOH最初抑制缓激肽刺激的Ca 2 + 内流，对基础胞质Ca 2+无影响。 经过更长时间的孵化 与氧化剂、激动剂刺激的Ca 2+从内部存储的释放 减少了。 t-bu-OOH产生膜电位的进行性损失，通过giga- 密封，以及细胞K+的净损失和细胞Na+的净增加，测定 使用放射性同位素。 该提案探讨了负责上述的分子机制 调查结果。 具体目标我将描述的机制，氧化剂 应激抑制激动剂刺激的胞质Ca 2+变化。 的影响 氧化胁迫对1）肌醇多磷酸（IP）的生产，和2）Ca 2 + 将使用以下方法研究IP 3和/或GTP从内部存储中释放的情况 放射性标记技术在完整的和皂苷渗透的血管内皮细胞。 的 氧化应激对ATP依赖性钙泵动力学的影响将 也被定性。 特定目标II将表征氧化剂强制降解对离子 浓度梯度和跨细胞膜的离子电导。 的 假设氧化应激：1）刺激Na+内流通过非- 选择性阳离子通道; 2）通过Ca 2+依赖性K+刺激K+流出 通道; 3）抑制Na+/K+-ATP酶，将使用 放射性同位素示踪剂和直接的千兆密封测量。 之间的联系 激动剂刺激的Ca 2+内流和氧化剂诱导的 膜去极化将在VEC中进行研究， 钙敏感荧光指示剂Fura-2和电位敏感荧光指示剂 染料，二-4-ANEPPS。 因此，这些实验将描述氧化应激如何改变 内皮细胞Ca 2+信号转导。 这项工作将构成基础， 长期目标是了解氧化应激如何改变 血管内皮细胞对血管反应性的调节。