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。

• 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
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-63480221/
• 关键词: Atrial Natriuretic Peptide; Intracellular Calcium; Fura-2; Laser-excitation fluorescence microscopy; HPLC; Inostol Metabolites; Bradykinin; Prostaglandin; 腎髄質乳頭部集合管; cAMP; cGMP; 腎ネフロン; 血管平滑筋; イノシト-ル隣酸; カルシウム; アンギオランシンII; バソプレッシン; 心房性利尿ペプチ

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. 心房利钠肽对胞质游离钙的影响我们建立了用荧光钙指示剂（fura-2和吲哚）测定细胞内钙的方法。心房利钠肽（ANP）降低细胞内钙的静息水平或持续增加的细胞质钙由血管收缩激素，血管紧张素II和血管加压素引起。ANP还能降低高钾去极化引起的细胞内钙的增加。另一方面，激素引起的胞质游离钙的初始增加不受ANP的抑制。钙拮抗剂尼卡地平和硝苯地平抑制高钾去极化引起的胞质游离钙的增加，而不影响血管紧张素II和血管加压素引起的细胞内钙的增加。结果表明，ANP可降低细胞内钙水平，提示ANP刺激血管平滑肌细胞的钙挤压主动转运。当我们使用激光激发荧光显微镜检测单个细胞的胞质游离钙水平时，我们观察到Fura-2染料的不均匀分布。fura-2染料的细胞下荧光呈点状或丝状，形状与胞内细胞器相似。采用高效液相色谱分析或荧光光谱分析，我们检测了平滑肌细胞中fura-2/AM的代谢。研究表明，在细胞裂解物的可溶部分中除了fura-2外没有发现其他成分，而膜部分则含有fur2 /AM或其未知的亲脂代谢产物。总之，在测量单个血管平滑肌细胞内钙水平时，我们应该考虑到这些问题（fura-2的细胞内区隔化或fura-2代谢）。高效液相色谱法分离磷酸肌醇：缓激肽的作用。我们观察到缓激素诱导的细胞质游离血管平滑肌细胞的增加。为了探讨其作用机制，我们考察了缓激肽对磷酸肌醇水解的影响。建立了高压液相色谱法分离乙醇磷酸盐的新方法。用t方法分离了肌醇单磷酸异构体、肌醇二磷酸异构体、肌醇（1,3,4）三磷酸异构体、肌醇（1,4,5）三磷酸异构体、肌醇（1,3,4,5）四磷酸异构体、肌醇五磷酸异构体和肌醇六磷酸异构体，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。结果表明，缓激肽可诱导肌醇（1,4,5）三磷酸介导的胞质游离钙的增加。缓激肽诱导钙升高与前列腺素合成的关系。缓激肽刺激血管平滑肌细胞胞质游离钙的增加。缓激肽也刺激细胞内的前列环素合成。为了阐明缓激肽诱导的前列环素合成与钙升高之间的关系，我们开发了一种灌注单分子系统，可以同时监测灌注液中胞质钙水平（采用fura-2方法）和前列环素的产生。结果清楚地表明，缓激肽同时诱导细胞质游离钙和前列环素合成的增加。提示钙是由缓激肽（由磷脂酶途径介导）供给磷脂酶A，磷脂酶A被缓激肽受体复合物激活，导致前列腺素合成减少

项目成果

K.TAKEUCH,K.ABE,K.MAEYAMA et al: "DIFFERENCE BETWEEN THE EFFECTS OF ATRIAL NATRIURETIC PEPTIDE AND CALCIUM ANTAGONIST ON CYTOSOLIC FREE CALCIUMIN CULTURED VASCULAR SMOOTH MUSCLE CELLS" JOURNAL OF CARDIOVASCULAR PHARMACOLOGY. 13. 13-16 (1989)

K.TAKEUCH、K.ABE、K.MAEYAMA 等人：“心房钠尿肽和钙拮抗剂对胞质游离钙培养的血管平滑肌细胞的影响之间的差异”心血管药理学杂志。

M.SATO,K.ABE,M.YASUJIMA et al.: "INHIBITORY EFFECT OF CICLETANINE ON VASCULAR SMOOTH MUSCLE CELL PROLIFERATION" ARCH.MAL.COEUR.82. 63-66 (1989)

M.SATO、K.ABE、M.YASUJIMA 等人：“环己宁对血管平滑肌细胞增殖的抑制作用” ARCH.MAL.COEUR.82。

K.KUDO,K.ABE,S.CHIBA et al.: "ROLE OF THROMBOXANE A_2 IN THE HYPERTENSIVE EFFECT OF CAPTOPRIL IN ESSENTIAL HYPEPTENSION" HYPERTENSION. 11. 147-152 (1988)

K.KUDO、K.ABE、S.CHIBA 等人：“THROMBOXANE A_2 在卡托普利对原发性高血压的高血压作用中的作用”高血压。

K.Takeuchi,K.Yoshinaga H.Inaba et al.: "INTRACELLULAR COMPARTMENTALIZATION OF fura-2 DYE DEMONSTRATED BY LASER EXCITATION FLUORESCECE MICROSCOPY:A PROBLEM IN MEASURING CYTOSOLIC FREE CALCIUM CONCENTRATION USING fura-2 FLUORESCENE IN VASCULAR SMOOTH MUSCLE

K.Takeuchi、K.Yoshinaga H.Inaba 等人：“通过激光激发荧光显微镜演示的 fura-2 染料的细胞内区室化：使用 fura-2 荧光在血管平滑肌中测量细胞溶质游离钙浓度的问题

K,Abe;K.Yoshinaga;et al.: Renal Function,Hypertension and Kallikrein-Kinin System,ed.by H.S.Margolius and O.Iimura,Unibersity of Tokyo Press,Tokyo. 117-121 (1988)

K，Abe；K.Yoshinaga；等人：肾功能、高血压和激肽释放酶-激肽系统，由 H.S.Margolius 和 O.Iimura 编辑，东京出版社大学，东京。