不同浓度二十二碳六烯酸对正常大鼠冠状动脉平滑肌细胞BK通道的激活作用及其机制

Activation effects and mechanisms of docosahexaenoic acid with different concentrations on BK channels in coronary smooth muscle cells

目的:探讨不同浓度二十二碳六烯酸(DHA)对正常大鼠冠状动脉平滑肌细胞大电导钙激活钾离子通道(BK通道)的激活作用及其机制。方法:酶消化法分离正常大鼠冠状动脉平滑肌细胞。采用全细胞膜片钳实验技术记录在未孵育与孵育细胞色素P450环氧化酶抑制剂SKF525A并灌流不同浓度DHA条件下BK通道电流密度变化;采用单通道膜片钳实验技术记录灌流不同浓度DHA时BK单通道开放概率的变化。结果:0.01~1.00μmol/L(定义为低浓度)DHA激活BK通道的半效浓度(EC50)为(0.24±0.05)μmol/L,但经SKF525A孵育后激活作用消失;3.00~10.00μmol/L(定义为高浓度)DHA激活BK通道的EC50为(2.38±0.22)μmol/L,经SKF525A孵育后,激活作用仍存在,且EC50无明显变化。在电极外液钙离子浓度为1μmol/L和刺激电位60 m V条件下,DHA浓度为0、0.01、0.03、0.10、0.30、1.00μmol/L时,BK通道开放概率分别为(0.095 2±0.009 5)、(0.093 9±0.012 6)、(0.098 6±0.016 9)、(0.099 5±0.014 7)、(0.097 5±0.010 4)和(0.102 3±0.020 6)(P>0.05,n=5),提示低浓度DHA不能激活BK单通道;继续增加DHA浓度,当浓度为3、10μmol/L时,BK单通道开放概率分别为(0.700 3±0.013 2)和(0.892 7±0.052 3)(P<0.05,n=5),提示高浓度DHA呈浓度依赖性激活BK单通道,EC50为(3.37±0.10)μmol/L。结论:不同浓度DHA激活BK通道的机制不同,低浓度DHA通过细胞色素P450环氧化酶途径激活BK通道,高浓度DHA可能通过与BK通道结合后直接激活。

Objective：To investigate the effects and mechanisms of docosahexaenoie acid（DHA） with different concentrations on large conductance calcium-activated potassium channels（BK channels）in normal rat coronary smooth muscle cells（SMCs）. Methods：Coronary SMCs were isolated by enzyme digestion. Effects of different concentrations of DHA on BK channels in absence and presence of cytochrome P450（CYP） epoxygenase inhibitor SKF525 A were studied by patch clamp in whole-cell configuration. Effects of DHA with different concentrations on BK single channels were studied by patch clamp in inside-out single channel configuration. Results：0.01-1.0 μmol/L（defined as low concentrations） of DHA activated whole-cell BK currents with a half effect concentration（EC50） of（0.24 ±0.05）μmol/L. This effect could be abolished by pre-incubation with SKF525 A. Concentration of 3-10 μmol/L（defined as high concentrations）of DHA activated whole-cell BK channels with an EC50of（2.38 ± 0.22）μmol/L. However,this effect could not be abolished by pre-incubation with SKF525 A. In the presence of 0,0.01,0.03,0.1,0.3,and 1 μmol/L DHA,the open probabilities of BK channels at 1 μmol/L calcium in external solution and test potentials at 60 m V were（0.095 2 ± 0.009 5）,（0.093 9 ± 0.012 6）,（0.098 6 ± 0.016 9）,（0.099 5 ± 0.014 7）,（0.097 5 ± 0.010 4）,and（0.102 3 ± 0.020 6）respectively（P〈0.05,n=5）. Low concentrations of DHA could not activate BK single channels. On the setting of 3 and 10 μmol/L DHA,the open probabilities of BK channels at 1μmol/L calcium in external solution and test potentials at 60 m V were（0.700 3 ± 0.013 2）,and（0.892 7 ± 0.053 2）,respectively（P〉0.05,n =5）. High concentrations of DHA could directly activate BK channels. Conclusion：BK channels can be activated by DHA in multiple mechanisms. Low concentration DHA can activate BK channel through CYP epoxygenase metabolites,while high concentration DHA can directly activate BK channel by binding to the channel proteins.