OXIDANT STRESS AND ENDOTHELIAL CELL CA2+ SIGNALING.

这个项目的长期目标是通过以下方式理解分子事件 氧化应激改变内皮细胞对血管反应性的调节。 血管内皮细胞胞浆钙是关键的第二信使 有助于血管活性旁分泌物质的分泌。 本研究旨在探讨氧化应激对细胞内钙信号转导的影响。 VEC。模型氧化剂为叔丁基氢过氧化氢(t-Bu-OHO)。缓激肽 将被用作模型激动剂。 T-BU-OHO抑制Fura-2负载血管内皮细胞的钙信号转导(Elliott和 希林，1990)。T-BU-OHY最初抑制缓激肽刺激的钙离子 内流，对基础胞浆钙无影响。经过更长时间的孵化 通过氧化剂、激动剂刺激的内部储存的钙释放 是减少的。 T-BU-OHO产生膜电位的渐进性损失，以吉卡- Seal，以及细胞K的净损失和细胞Na的净获得 使用放射性同位素。 这项建议探讨了导致上述现象的分子机制。 调查结果。具体目标我将描述氧化剂的作用机理 应激抑制激动剂刺激的胞浆钙变化。其影响 氧化胁迫对1)多聚磷酸肌醇(IP)产生和2)钙离子的影响 IP3和/或GTP从内部商店发布将使用以下工具进行调查 完整和皂素渗透的血管内皮细胞中的放射性标记技术。这个 氧化应激对ATP依赖的钙泵动力学的影响 也是具有特征的。 特殊目的II将描述氧化应激对离子的影响 浓度梯度和跨细胞膜的离子电导。这个 假设氧化剂应激：1)通过一种非 选择性阳离子通道；2)通过钙依赖钾刺激钾外流 通道；以及3)抑制Na/K-ATPase，将使用 放射性同位素示踪剂和直接的千兆海豹测量。两者之间的联系 氧化剂抑制激动剂刺激的钙内流和氧化剂诱导的钙内流 膜去极化的研究将在血管内皮细胞双重负载 钙敏感荧光指示剂Fura-2和电势敏感 染料，di-4-ANEPPS。 因此，这些实验将描述氧化应激如何改变。 内皮细胞钙信号转导。这项工作将构成以下工作的基础 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。

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.