在inside-out膜片上caffeine对猪冠状动脉平滑肌细胞钙激活钾通道的调节作用及ryanodine的影响  

Effects of caffeine and ryanodine on KCa in porcine coronary artery smooth muscle cells

作　　者：仲维高[1] 杨艳[2] 曾晓荣[2] 崔跃[3] 孔天翰[1] 

机构地区：[1]广州医学院蛇毒所,广东广州510183 [2]中山大学疼痛研究中心,广东广州510080 [3]泸州医学院心血管医学研究所,四川泸州646000

出　　处：《中国病理生理杂志》2010年第1期53-58,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30370527)

摘　　要：目的:利用膜片钳技术,研究咖啡因对钙激活钾通道(calcium-activated potassium channel,KCa)的作用机制,及咖啡因存在时兰尼定(ryanodine)对KCa的影响以阐明冠状动脉平滑肌的调控机制。方法:采用急性酶分离方法,应用膜片钳单通道电流记录技术记录猪冠状动脉平滑肌钿胞上KCa电流活动。电流信号经放大、滤波及A/D、D/A转换后输入微机进行采样和储存。应用PCLAMP9.0软件系统进行数据采集及分析。结果:在内面向外式(inside-out)膜片下,咖啡因(0.1、0.5、1.0、5.0mmol/L)可浓度依赖性地增加通道开放概率,而对电流幅值无明显影响,开放概率增加是通过明显缩短平均关闭时间实现的(n=8,P<0.01);洗去药物后通道活性可以恢复到对照水平;5.0mmol/L咖啡因对KCa激活作用最大(P<0.01)。在细胞贴附式(cell-attached)膜片上,咖啡因激活KCa后,ryanodine(10-40μmol/L)浓度依赖性地抑制通道开放概率,开放时间缩短,关闭时间延长,对电流幅度无明显影响。结论:在inside-out膜片下,咖啡因能够直接激活猪冠状动脉平滑肌细胞KCa通道。在cell-atta-ched构型上,ryanodine可通过胞内一定的信号通路浓度依赖性间接抑制咖啡因对KCa激活。AIM： To study the effect of caffeine on the large conductance calcium activated potassium （KCa） channels by patch -clamp technique on smooth muscle cells enzymatically isolated from the porcine coronary artery （ PCASMC）, and to investigate the effect of ryanodine on KCa being activated by caffeine. METHODS ： Using the single channel patch - clamp technique, single PCASMC was isolated by collagenase, the activity of single KCa channel was recorded in porcine coronary artery smooth muscle cells. RESULTS： Caffeine （0. 1 - 10 mmol/L） enhanced the open probability （Po） of KCa channels in a dose - dependent manner in the intracellular side of inside - out patches and its effect was almost completely abolished by washout. Caffeine decreased the mean close time markedly, but had no effect on the amplitude of KCa channels. However, ryanodine （ 10 -40 p.mot/L） decreased Po of KCa channels activated by caffeine in a dose - dependent manner in cell - attached patches. The mean open time also decreased. CONCLUSION ： Caffeine directly activates KCa channels of porcine coronary artery smooth muscle cells in inside - out patches, the activity of single KCa channel is inhibited by ryanodine indirectly in cell -attached patches.

关 键 词：咖啡因 钾通道 钙激活 Inside—out膜片 兰尼定 猪冠状动脉平滑肌细胞 Cell-attached膜片 

分 类 号：R331.3[医药卫生—人体生理学]