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
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摘要
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 Ca~(2+)-activated K~+( BK_(Ca)) 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 BK_(Ca) channel and voltage-gated potassium( K_V) channel before and after adding melatonin. Currents density of melatonin on BK_(Ca) 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 BK_(Ca) channel were detected before and after the addition of melatonin in the internal-outward mode. Results:(1) Melatonin markedly increased the whole-cell BK_(Ca) channel current density but not the voltage-gated potassium( K_V) channel current density.(2) Luzindole( 1 μmol/L) greatly suppressed melatonin-induced increase of BK_(Ca) channel current density.(3) The Po of BK_(Ca) 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 BK_(Ca) channel,and Tc was reduced much more than To. Conclusion: Melatonin mediates vasodilation of MCA through the activation of BK_(Ca) channels via both melatonin receptor dependent and independent mode.
Objective: To investigate the mechanisms through which myocyte large-conductance Ca~(2+)-activated K~+( BK_(Ca)) 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 BK_(Ca) channel and voltage-gated potassium( K_V) channel before and after adding melatonin. Currents density of melatonin on BK_(Ca) 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 BK_(Ca) channel were detected before and after the addition of melatonin in the internal-outward mode. Results:(1) Melatonin markedly increased the whole-cell BK_(Ca) channel current density but not the voltage-gated potassium( K_V) channel current density.(2) Luzindole( 1 μmol/L) greatly suppressed melatonin-induced increase of BK_(Ca) channel current density.(3) The Po of BK_(Ca) 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 BK_(Ca) channel,and Tc was reduced much more than To. Conclusion: Melatonin mediates vasodilation of MCA through the activation of BK_(Ca) channels via both melatonin receptor dependent and independent mode.
引文
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[16]Anwar MM,Meki AR,Rahma HH.Inhibitory effects of melatonin on vascular reactivity:possible role of vasoactive mediators[J].Comp Biochem Physiol C Toxicol Pharmacol,2001,130(3):357-367.
[17]Das R,Balonan L,Ballard HJ,et al.Chronic hypoxia inhibits the antihypertensive effect of melatonin on pulmonary artery[J].Int J Cardiol,2008,126(3):340-345.
[18]Yu H,Dickson EJ,Jung SR,et al.High membrane permeability for melatonin[J].J Gen Physiol,2016,147(1):63-76.
[19]Pandi-Perumal SR,Bahammam AS,Ojike NI,et al.Melatonin and human cardiovascular disease[J].J Cardiovasc Pharmacol Ther,2017,22(2):122-132.
[2]Paulis L,Simko F.Blood pressure modulation and cardiovascular protection by melatonin:potential mechanisms behind[J].Physiol Res,2007,56(6):671-684.
[3]景会锋.有氧运动和褪黑素对2型糖尿病大鼠抗氧化运动功能的影响[J].中国应用生理学杂志,2014,30(5):426-428.
[4]Tunstall RR,Shukla P,Grazul-Bilska A,et al.MT2 receptors mediate the inhibitory effects of melatonin on nitric oxide-induced relaxation of porcine isolated coronary arteries[J].J Pharmacol Exp Ther,2011,336(1):127-133.
[5]邱方,熊开宇,石丽君.褪黑素在有氧运动改善自发性高血压大鼠血压和肠系膜动脉功能中的作用[J].中国动脉硬化杂志,2015,23(12):1229-1235.
[6]杨玉雯,阚敬保,孔祥权.BKCa增龄变化及其与血压的相关性[J].中国应用生理学杂志,2009,(4):511-515.
[7]Suh EY,Yin MZ,Lin H,et al.Maxi-K channel(BK-Ca)activity veils the myogenic tone of mesenteric artery in rats[J].Physiol Rep,2017,5(14):e13330.
[8]李先明,秦涛.褪黑素在心血管疾病中的研究进展[J].中西医结合心血管病电子杂志,2016,4(14):7-9.
[9]Geary GG,Krause DN,Duckles SP.Melatonin directly constricts rat cerebral arteries through modulation of potassium channels[J].Am J Physiol,1997,273(3 Pt2):H1530-1536.
[10]Regrigny O,Delagrange P,Scalbert E,et al.Effects of melatonin on rat pial arteriolar diameter in vivo[J].Br JPharmacol,1999,127(7):1666-1670.
[11]Geary GG,Duckles SP,Krause DN.Effect of melatonin in the rat tail artery:role of K+channels and endothelial factors[J].Br J Pharmacol,1998,123(8):1533-1540.
[12]Lew MJ,Flanders S.Mechanisms of melatonin-induced vasoconstriction in the rat tail artery:a paradigm of weak vasoconstriction[J].Br J Pharmacol,1999,126(6):1408-1418.
[13]Girouard H,Chulak C,Lejossec M,et al.Vasorelaxant effects of the chronic treatment with melatonin on mesenteric artery and aorta of spontaneously hypertensive rats[J].J Hypertens,2001,19(8):1369-1377.
[14]Fish JE,Marsden PA.Endothelial nitric oxide synthase:insight into cell-specific gene regulation in the vascular endothelium[J].Cell Mol Life Sci,2006,63(2):144-162.
[15]Forstermann U,Munzel T.Endothelial nitric oxide synthase in vascular disease:from marvel to menace[J].Circulation,2006,113(13):1708-1714.
[16]Anwar MM,Meki AR,Rahma HH.Inhibitory effects of melatonin on vascular reactivity:possible role of vasoactive mediators[J].Comp Biochem Physiol C Toxicol Pharmacol,2001,130(3):357-367.
[17]Das R,Balonan L,Ballard HJ,et al.Chronic hypoxia inhibits the antihypertensive effect of melatonin on pulmonary artery[J].Int J Cardiol,2008,126(3):340-345.
[18]Yu H,Dickson EJ,Jung SR,et al.High membrane permeability for melatonin[J].J Gen Physiol,2016,147(1):63-76.
[19]Pandi-Perumal SR,Bahammam AS,Ojike NI,et al.Melatonin and human cardiovascular disease[J].J Cardiovasc Pharmacol Ther,2017,22(2):122-132.