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Inactivation mechanisms of voltage-gated Ca channels by CaィイD12+ィエD1 in smooth muscle cells.

平滑肌细胞中 CaiD12+D1 的电压门控 Ca 通道失活机制。

基本信息

批准号：
10660283
负责人：
OHASHI Hidenori
金额：
$ 1.86万
依托单位：
Gifu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

Voltage-gated Ca channels in smooth muscle cells are the most important pathway for allowing CaィイD12+ィエD1 to flow down a concentration gradient into the cell. In the present study, we explored the inactivation mechanisms of voltage-gated Ca channels using the whole-cell patch clamp technique. Outlines of the obtained results are as follows.1. Carbachol (acting at muscarinic receptors) or histamine (acting at H1 histamine receptors) suppressed voltage-gated Ca channel currents (IィイD2CaィエD2) evoked by depolarizing pulses in a biphasic manner, an initial transient component followed by a sustained component. The possibility that phospholipid metabolisms are implicated in the biphasic suppression of IィイD2CaィエD2 was examined using inhibitors of phospholipases, such as wortmannin and D609. The results show that phospholipase C is involved in the transient component of IィイD2CaィエD2 suppression, and phospholipase C or D in the sustained component of IィイD2CaィエD2 suppression.2. To see if cytoskeletons, such as actin microfilaments and microtubules, are involved in the biphasic suppression of IィイD2CaィエD2 induced by carbachol, effects of cytoskeletal depolymerizers and polymerizers on the carbachol action were investigated. The results sugget that the bipbasic suppression of IィイD2CaィエD2 is mediated by microtubule polymerization.3. The possibility thet phosphorylation of Ca channels is implicated in the biphasic suppression of IィイD2CaィエD2 was examined using inhibitors and activators of protein kinase A, protein kinase G, protein kinase C and calmodulin dependent myosin light chain kinase. The results suggest that phosphorylation process is not involved in the biphasic suppression of IィイD2CaィエD2.The above results have been published as 2 papers in the Britishi Journal of Pharmacology. How muscarinic receptor stimulation polymerizes microtubules, and how microtubule polymerization suppress voltage-gated Ca channel activity will be examined.

平滑肌细胞中的电压门控Ca通道是允许Ca ~（2+）D12+ Ca ~（2+）D1以浓度梯度向下流入细胞的最重要途径。本研究采用全细胞膜片钳技术探讨电压门控性钙通道失活的机制。所得结果概述如下：1.卡巴胆碱（作用于毒蕈碱受体）或组胺（作用于H1组胺受体）抑制电压门控钙通道电流（I钙通道D2钙通道D2）引起的去极化脉冲在一个双相的方式，一个初始的瞬态组件，然后持续的组件。使用磷脂酶的抑制剂，如渥曼青霉素和D 609，检查磷脂代谢与I型钙通道D2的双相抑制有关的可能性。结果表明，磷脂酶C参与了I型钙依赖性D2Ca依赖性D2抑制的瞬时成分，而磷脂酶C或D参与了I型钙依赖性D2Ca依赖性D2抑制的持续成分.为了了解细胞骨架，如肌动蛋白微丝和微管，是否参与卡巴胆碱诱导的I β D2Ca β D2的双相抑制，研究了细胞骨架解聚剂和聚合剂对卡巴胆碱作用的影响。结果提示，I β D2 Ca β D2的双碱基抑制作用是通过微管聚合介导的.用蛋白激酶A、蛋白激酶G、蛋白激酶C和钙调蛋白依赖性肌球蛋白轻链激酶的抑制剂和激活剂，研究了钙通道磷酸化参与I β D2钙通道双相抑制的可能性。上述结果已在英国药理学杂志上发表2篇论文。毒蕈碱受体刺激如何聚合微管，以及微管聚合如何抑制电压门控钙通道活性将被检查。