褪黑素激活大鼠大脑中动脉BK_(Ca)通道的受体依赖和非受体依赖途径

The receptor-dependent and non-receptor-dependent mechanism of melatonin activated BK_(Ca) channels in middle cerebral artery myocyte

目的:研究褪黑素通过大电导Ca^(2+)激活K^+(BK_(Ca))通道介导大脑中动脉张力变化的作用机制。方法:8周龄雄性Wistar大鼠,麻醉后取大脑中动脉,酶消化法急性分离脑中动脉平滑肌细胞,采用膜片钳技术全细胞记录模式检测细胞外液加入褪黑素前后BK_(Ca)通道和电压门控钾(K_V)通道的电流密度,褪黑素受体抑制剂2-苯基-N-乙酰色胺(luzindole)孵育后,全细胞记录模式记录加入褪黑素后BK_(Ca)通道电流幅值和贴附式单通道记录模式记录加入褪黑素后BK_(Ca)通道Po值,内面向外记录模式检测加入褪黑素后BK_(Ca)通道电导(G),开放概率(Po),平均开放时间(To)和关闭时间(Tc)。结果:(1)褪黑素(100μmol/L)显著增加全细胞BK_(Ca)通道电流密度,但对K_V通道电流密度无显著影响;(2)luzindole (1μmol/L)显著抑制褪黑素引起的BK_(Ca)通道电流密度增加;(3)贴附式单通道记录模式下,褪黑素(100μmol/L)增加BK_(Ca)单通道Po值,luzindole (1μmol/L)显著抑制褪黑素引起的Po增加;(4)内面向外单通道记录模式下,褪黑素(1μmol/L,100μmol/L)缩短BK_(Ca)单通道的To和Tc值,且Tc较To显著缩短;结论:褪黑素通过受体依赖和非受体依赖途径激活BK_(Ca)通道,介导大脑中动脉血管舒张。

Objective: To investigate the mechanisms through which myocyte large-conductance Ca2+-activated K+( BKCa) channels mediate the vasodilation effects of melatonin on cerebral arteries( CAs). Methods: Middle cerebral arteries( MCA) were obtained from 8-week-old male Wistar rats after anaesthetized. Middle cerebral arterial smooth muscle cells were enzymatically isolated. Whole cell recording mode of patch clamp technique was used to measure the current density of BKCa channel and voltage-gated potassium( KV) channel before and after adding melatonin. Currents density of melatonin on BKCa channels with melatonin receptor inhibitor 2-phenyl-N-acetyl( luzindole) was recorded using whole cell recording mode and open probability( Po) was recorded using single-channel attached recording mode. The conductance( G) and average open time( To) and off time( Tc) of the BKCa channel were detected before and after the addition of melatonin in the internal-outward mode. Results:(1) Melatonin markedly increased the whole-cell BKCa channel current density but not the voltage-gated potassium( KV) channel current density.(2) Luzindole( 1 μmol/L) greatly suppressed melatonin-induced increase of BKCa channel current density.(3) The Po of BKCa channel was significantly increased by melatonin( 100 μmol/L) under cell attached recording mode,which was markedly inhibited by luzindole( 1 μmol/L).(4) In inside-outside recording mode,melatonin( 1 μmol/L,100 μmol/L) reduced both To and Tc of BKCa channel,and Tc was reduced much more than To. Conclusion: Melatonin mediates vasodilation of MCA through the activation of BKCa channels via both melatonin receptor dependent and independent mode.