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目的:研究咖啡因对猪冠状动脉平滑肌细胞KCa通道的调控机理,以期揭示胞内钙库RyR激活后,局部钙离子浓度升高和KCa的关系。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大脑皮层神经元猪冠状动脉平滑肌细胞上KCa通道电流活动。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。实验数据应用CLAMP9.0软件系统进行数据采集及分析。结果:在cell-attached膜片上咖啡因对KCa通道有明显的作用,咖啡(0.1-5.0 mM)可以增加通道的开放概率(NPo),呈现出浓度依赖性,开放时间延长,关闭时间也随之缩短,而对电流幅值无明显影响,开放概率的增加是通过明显缩短平均关闭时间实现的(n=8,P<0.05);洗去药物后通道活性可以一定程度的恢复到对照水平,再加入一定浓度咖啡因(如1.0mM)可再次激活KCa,激活程度与洗脱前较接近。结论:在细胞贴附式构型上咖啡因浓度依赖性地激活KCa,有饱和性。可能是通过影响胞内信号转导过程而调控KCa活性。
Objective: To study the regulatory mechanism of caffeine on KCa channel in porcine coronary artery smooth muscle cells in order to reveal the relationship between local calcium concentration and KCa after intracellular calcium RyR activation. Methods: Acute patch-clamp technique was used to record the current activity of KCa channel in porcine coronary artery smooth muscle cells of rat cortical neurons. The current signal is amplified, filtered and A / D, D / A conversion input to the computer for sampling and storage. Experimental data CLAMP9.0 software system for data acquisition and analysis. RESULTS: Caffeine had a significant effect on KCa channel in cell-attached patch. Caffeine (0.1-5.0 mM) increased the open channel probability (NPo) in a concentration-dependent manner, with prolonged open time and closed time (N = 8, P <0.05). After the drug was washed away, the channel activity could be recovered to the control level to a certain extent, and the increase of open probability could be achieved by significantly shortening the average closing time Add a certain concentration of caffeine (such as 1.0mM) to reactivate KCa, the degree of activation and elution before close. CONCLUSIONS: Caffeine has a saturating effect in a concentration-dependent manner on KCa in a cell-attached configuration. It is likely that KCa activity is regulated by affecting intracellular signal transduction.