Regulatory Mechanism of Biosynthesis and Cellular Action of Renal Vasoactive Hormones.

肾血管活性激素生物合成和细胞作用的调节机制。

基本信息

批准号：
63480221
负责人：
ABE Keishi
金额：
$ 3.9万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1990
项目状态：
已结题

项目摘要

1. Effect of Atrial Natriuretic Peptide on Cytosolic Free CalciumWe developed the method of measuring intracellular calcium using fluorescent calcium indicators (fura-2 & indol). Atrial natriuretic peptide (ANP) decreased either the resting level of intracellular calcium or sustained increases in cytosolic calcium induced by vasoconstrictive hormones, angiotensin II and vasopressin. ANP also decreased an increase in intracellular calcium evoked by high potassium depolarization. On the other hand, the initial increases in cytosolic free calcium induced by the hormones were not inhibited by ANP. Calcium antagonists, nicardipine and nifedipine, inhibited the increase in cytosolic free calcium induced by high potassium depolarization, whereas they did not affect increases in intracellular calcium brought about by angiotensin II and vasopressin. The results indicate that ANP can decrease intracellular calcium level and it is suggested that ANP stimulates a calcium-extrusion active transport … More in vascular smooth muscle cells.2. Intracellular Compartmetalization of Fura-2 Demonstrated by Laser-Excitation Fluorescence MicroscopyWhen we examined cytosolic free calcium level in individual cell using laser-excitation fluorescence microscopy, we observed inhomogenous distribution of fura-2 dye. Subcellurar fluorescence of fura-2 dye appeared spotty or filamentous and resembled in shapes the intracellular organelles. Using either an analysis by high performance liquid chromatography or fluorescence spectra analysis, we examined fura-2/AM metabolism in smooth muscle cells. It has been shown that no other than fura-2 was found in the soluble fraction of the cell lysates, whereas membrane fraction contains fura2/AM or its unidentified lipophilic metabilites, In conclusion, we should take into account these problems ( intracellular compartmentalization of fura-2 or fura-2 metabolism ) in measuring intracellular calcium level in individual vascular smooth muscle cell.3. Separation of Inositol phosphates by High Performance Liqu Chromatography : Effect of Bradykinin.We observed bradykinin-induced increase in cytosolic free vascular smooth muscle cells. In order to examine the mechanis we examined the effect of bradykinin on phosphoinositide hydrol sis. We newly developed the method of separation of inosit phosphates using high pressure liquid chromatography. By t method, we separated isomers of inositol monophosphate, isome of inositol bisphosphate, inositol (1,3,4)trisphosphate a inositol ( 1, 4, 5 ) trisphosphate, inositol (1, 3, 4, 5 ) tetrakisphosphate, inositol pentakisphosphate and inosit hexakisphosphate, Bradykinin stimulated rapid formation inositol (1, 4, 5) trisphosphate, followed by formation of inosit tetrakisphosphate and inositol (1, 3, 4) trisphosphate. It idicat that bradykinin induces an increase in cytosolic free calci mediated by inositol (1, 4, 5) trisphosphate4. Relationship between Bradykinin-Induced Increase in Calci and Prostaglandin Synthesis.Bradykinin stimulates an increase in cytosolic free calcium vascular smooth muscle cells. Bradykinin also stimulate prostacyclin synthesis in the cells. To clarify the ti courserelationship between prostacyclin synthesis and calc increase induced by bradykinin, we developed a perfused monolay system which enables to monitor simulataneously the level cytosolic calcium ( using fura-2 method ) and proxtacycl production in the perfusion solution. The result clearly show that bradykinin simultaneously induces an increase in cytosol free calcium and prostacyclin synthesis. It is suggested the calcium is supplied by bradykinin (mediated by phoshpholipase pathway) to phospholipase A which is ready to be activated by t bradykinin-receptor complex, resulting in prostacycin synthesi Less

1. 心房钠尿肽对细胞质游离钙的影响我们开发了使用荧光钙指示剂（fura-2 和吲哚）测量细胞内钙的方法。心房钠尿肽（ANP）可降低细胞内钙的静息水平或使细胞质钙持续增加。由血管收缩激素、血管紧张素 II 和加压素诱导的 ANP 也能降低高钾引起的细胞内钙的增加。另一方面，由激素诱导的胞质游离钙的初始增加不受ANP钙拮抗剂尼卡地平和硝苯地平的抑制，但它们不影响高钾去极化诱导的胞质游离钙的增加。血管紧张素 II 和加压素引起的细胞内钙含量。结果表明，ANP 可以降低细胞内钙水平，表明 ANP 会刺激激光激发荧光显微镜证实 Fura-2 的细胞内钙挤压主动转运…更多当我们使用激光激发荧光显微镜检查单个细胞中的胞质游离钙水平时，我们观察到 fura 的不均匀分布-2 染料。fura-2 染料的亚细胞荧光呈斑点状或丝状，并且与细胞内细胞器的形状相似。通过高效液相色谱或荧光光谱分析，我们检查了平滑肌细胞中的fura-2/AM代谢，结果表明，在细胞裂解物的可溶部分中没有发现除fura-2以外的物质，而膜部分则含有fura2。 /AM或其未鉴定的亲脂性代谢物，总之，在测量个体血管平滑肌细胞内钙水平时，我们应该考虑这些问题（fura-2或fura-2代谢的细胞内区室化） 3.通过高效液相色谱法分离肌醇磷酸：缓激肽的作用。为了研究缓激肽对磷酸肌醇水解的作用，我们观察了缓激肽诱导的细胞质游离血管平滑肌细胞的增加。开发了高压液相色谱法分离肌醇磷酸酯的方法，我们分离了肌醇的异构体。单磷酸酯，肌醇二磷酸酯异构体，肌醇（1,3,4）三磷酸酯，肌醇（1,4,5）三磷酸酯，肌醇（1,3,4,5）四磷酸酯，肌醇五磷酸酯和肌醇六磷酸酯，缓激肽刺激快速形成肌醇(1,4,5)三磷酸，随后形成肌醇四磷酸和肌醇 (1, 3, 4) 三磷酸表明缓激肽可诱导肌醇 (1, 4, 5) 三磷酸介导的胞质游离钙增加4。缓激肽诱导的钙增加与前列腺素合成之间的关系。血管平滑肌细胞胞质游离钙的增加也刺激前列环素的合成。为了阐明前列环素合成与缓激肽诱导的钙增加之间的关系，我们开发了一种灌注单层系统，该系统能够同时监测灌注溶液中的胞质钙水平（使用 fura-2 方法）和前列环素产生。结果清楚地表明，缓激肽同时诱导胞质游离钙和前列环素合成的增加，这表明钙是由缓激肽提供的（由缓激肽介导）。磷脂酶途径）转化为磷脂酶 A，磷脂酶 A 已准备好被 t 缓激肽受体复合物激活，从而导致前列环素合成 Less