PKC和ROCK对硝苯地平舒张血管作用的影响
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摘要
目的:探讨蛋白激酶C(PKC)及Rho相关激酶(ROCK)对硝苯地平舒张大鼠离体胸主动脉血管环作用的影响。方法:采用离体血管环灌流装置观察硝苯地平对基础状态血管环及去甲肾上腺素(NE,10~(-6)mol/L)或KCl(60mmol/L)预收缩血管环的张力变化,并利用PKC抑制剂和ROCK抑制剂等工具药观察对硝苯地平舒血管作用的影响并探讨可能机制。结果:系列浓度硝苯地平对基础状态血管环张力无明显影响,对NE和KCl预收缩的血管环具有浓度依赖性舒张作用(P <0. 05),去内皮组舒张作用与内皮完整组比较无明显差异。预孵PKC抑制剂星孢菌素(STA,10~(-8)mol/L)及激动剂佛波酯(PMA,10~(-7)mol/L)后,STA能增强硝苯地平对血管的舒张作用,而PMA能减弱硝苯地平对血管的舒张作用(P <0. 05);预孵ROCK抑制剂法舒地尔(fasudil,10~(-6)mol/L)及激动剂血管紧张素Ⅱ(Ang-Ⅱ,10-9mol/L)后,fasudil能增强硝苯地平对血管的舒张作用,而Ang-Ⅱ能减弱硝苯地平对血管的舒张作用(P <0. 05);钾通道阻滞剂BaCl_2(10~(-4)mol/L)、四乙胺(10~(-3)mol/L)、格列本脲(10~(-5)mol/L)和4-氨基吡啶(10~(-3)mol/L)对硝苯地平的舒张血管作用无明显影响。在无钙且含高浓度KCl的溶液中,硝苯地平可浓度依赖性地抑制累积浓度的CaCl_2对大鼠离体主动脉环的收缩作用(P <0. 05)。在无钙液中,硝苯地平对NE所引起的收缩无明显影响。结论:硝苯地平能够呈浓度依赖性地舒张大鼠主动脉,其舒张血管作用为非内皮依赖性,且与抑制细胞外钙内流密切相关,其舒张血管作用部分与抑制PKC和ROCK作用相关。
AIM:To investigate the effects of Rho-associated kinase(ROCK)and protein kinase C(PKC)on the relaxation of isolated rat aortic rings induced by nifedipine and the mechanisms.METHODS:The changes of tension in vascular rings induced by nifedipine under the basic condition and pre-contracted by norepinephrine(NE,10~(-6)mol/L)or KCl(60 mmol/L)were observed.The effects of ROCK and PKC on the vasodilation induced by nifedipine were studied using the vascular ring perfusion device.RESULTS:Nifedipine(10~(-10)mol/L,10~(-9)mol/L,10~(-8)mol/L,10~(-7)mol/L,10~(-6)mol/L and 10~(-5)mol/L)had no significant relaxation effect on isolated aortic rings under basic condition.Nifedipine induced dose-dependent relaxation in both endothelium-intact and endothelium-denuded aortic rings pre-contracted by 10~(-6)mol/L NE and 60 mmol/L KCl(P<0.05).No obvious difference between endothelium-intact group and endothelium-denuded group was observed.After incubation of the PKC inhibitor staurosporine(STA,10~(-8)mol/L)and PKC agonist phorbol 12-myristate 13-acetate(PMA,10~(-7)mol/L),STA increased the relaxation induced by nifedipine,while PMA reduced the effect of nifedipine on blood vessels(P<0.05).After the incubation of the ROCK inhibitor fasudil(10~(-6)mol/L)and ROCK agonist angiotensin Ⅱ(Ang-Ⅱ,10~(-9)mol/L),fasudil increased the relaxation induced by nifedipine,while Ang-Ⅱ reduced the effect of nifedipine on blood vessels(P<0.05).The relaxation induced by nifedipine was not statistically inhibited by BaCl_2(10~(-4)mol/L),tetraethylammonium(10~(-3)mol/L),glibenclamide(10~(-5)mol/L)and 4-aminopyridine(10~(-3)mol/L).In calcium-free and high-potassium solution,pre-treatment with nifedipine(10~(-9)mol/L,5×10~(-8)mol/L and 10~(-6)mol/L)inhibited calcium-induced contraction of the aortic rings(P<0.05).However,nifedipine pre-treatment did not affect the contraction induced by NE in Ca~(2+)-free medium.CONCLUSION:Nifedipine exhibits vasodilatation effect in a dose-dependent manner and the vasodilatation activity is endothelium-independent.The vasodilatation effect of nifedipine may be related to the inhibition of extracellular calcium influx,and inhibition of PKC and ROCK enhances the vasodilatation effect of nifedipine.
AIM:To investigate the effects of Rho-associated kinase(ROCK)and protein kinase C(PKC)on the relaxation of isolated rat aortic rings induced by nifedipine and the mechanisms.METHODS:The changes of tension in vascular rings induced by nifedipine under the basic condition and pre-contracted by norepinephrine(NE,10~(-6)mol/L)or KCl(60 mmol/L)were observed.The effects of ROCK and PKC on the vasodilation induced by nifedipine were studied using the vascular ring perfusion device.RESULTS:Nifedipine(10~(-10)mol/L,10~(-9)mol/L,10~(-8)mol/L,10~(-7)mol/L,10~(-6)mol/L and 10~(-5)mol/L)had no significant relaxation effect on isolated aortic rings under basic condition.Nifedipine induced dose-dependent relaxation in both endothelium-intact and endothelium-denuded aortic rings pre-contracted by 10~(-6)mol/L NE and 60 mmol/L KCl(P<0.05).No obvious difference between endothelium-intact group and endothelium-denuded group was observed.After incubation of the PKC inhibitor staurosporine(STA,10~(-8)mol/L)and PKC agonist phorbol 12-myristate 13-acetate(PMA,10~(-7)mol/L),STA increased the relaxation induced by nifedipine,while PMA reduced the effect of nifedipine on blood vessels(P<0.05).After the incubation of the ROCK inhibitor fasudil(10~(-6)mol/L)and ROCK agonist angiotensin Ⅱ(Ang-Ⅱ,10~(-9)mol/L),fasudil increased the relaxation induced by nifedipine,while Ang-Ⅱ reduced the effect of nifedipine on blood vessels(P<0.05).The relaxation induced by nifedipine was not statistically inhibited by BaCl_2(10~(-4)mol/L),tetraethylammonium(10~(-3)mol/L),glibenclamide(10~(-5)mol/L)and 4-aminopyridine(10~(-3)mol/L).In calcium-free and high-potassium solution,pre-treatment with nifedipine(10~(-9)mol/L,5×10~(-8)mol/L and 10~(-6)mol/L)inhibited calcium-induced contraction of the aortic rings(P<0.05).However,nifedipine pre-treatment did not affect the contraction induced by NE in Ca~(2+)-free medium.CONCLUSION:Nifedipine exhibits vasodilatation effect in a dose-dependent manner and the vasodilatation activity is endothelium-independent.The vasodilatation effect of nifedipine may be related to the inhibition of extracellular calcium influx,and inhibition of PKC and ROCK enhances the vasodilatation effect of nifedipine.
引文
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[9]Kandabashi T,Shimokawa H,Miyata K,et al.Evidence for protein kinase C-mediated activation of Rho-kinase in a porcine model of coronary artery spasm[J].Arterioscler Thromb Vasc Biol,2003,23(12):2209-2214.
[10]Shimokawa H,Sunamura S,Satoh K.Rho A/Rho-kinase in the cardiovascular system[J].Circ Res,2016,118(2):352-366.
[11]Sanderson MJ,Delmotte P,Bai Y,et al.Regulation of airway smooth muscle cell contractility by Ca2+signaling and sensitivity[J].Proc Am Thorac Soc,2008,5(1):23-31.
[12]Robles JC,Sturek M,Parker JL,et al.Ca2+sensitization and PKC contribute to exercise training-enhanced contractility in porcine collateral-dependent coronary arteries[J].Am J Physiol Heart Circ Physiol,2011,300(4):H1201-H1209.
[13]Kizub IV,Pavlova OO,Johnson CD,et al.Rho kinase and protein kinase C involvement in vascular smooth muscle myofilament calcium sensitization in arteries from diabetic rats[J].Br J Pharmacol,2010,159(8):1724-1731.
[14]Ko EA,Han J,Jung ID,et al.Physiological roles of K+channels in vascular smooth muscle cells[J].J Smooth Muscle Res,2002,44(2):65-81.
[15]Silva MT,Ribeiro FP,Medeiros MA,et al.The vasorelaxant effect of p-cymene in rat aorta involves potassium channels[J].Sci World J,2015,2015:458080.
[2]Guan RG,Xu XY,Chen MH,et al.Advances in the studies of roles of Rho/Rho-kinase in diseases and the development of its inhibitors[J].Eur J Med Chem,2013,70:613-622.
[3]Steinberg SF.Cardiac actions of protein kinase C isoforms[J].Physiology(Bethesda),2012,27(3):130-139.
[4]田晴晴,罗乐,张夏丽,等.白杨素对大鼠主动脉舒张作用的影响[J].中国病理生理杂志,2016,32(4):618-622.
[5]张洪平,李亚娟,章丹丹,等.辛夷二氯甲烷提取物对离体大鼠胸主动脉环的舒张作用及其机制[J].中国病理生理杂志,2010,26(9):1689-1694.
[6]Loirand G,Guerin P,Pacaud P.Rho kinases in cardiovascular physiology and pathophysiology[J].Circ Res,2006,98(3):322-334.
[7]闫美灵,李京波.PKC在心血管疾病中的研究进展[J].医学研究杂志,2014,43(9):7-9.
[8]乔树斌,李险峰.硝苯地平的临床应用进展[J].中国基层药,2006,13(8):1379-1380.
[9]Kandabashi T,Shimokawa H,Miyata K,et al.Evidence for protein kinase C-mediated activation of Rho-kinase in a porcine model of coronary artery spasm[J].Arterioscler Thromb Vasc Biol,2003,23(12):2209-2214.
[10]Shimokawa H,Sunamura S,Satoh K.Rho A/Rho-kinase in the cardiovascular system[J].Circ Res,2016,118(2):352-366.
[11]Sanderson MJ,Delmotte P,Bai Y,et al.Regulation of airway smooth muscle cell contractility by Ca2+signaling and sensitivity[J].Proc Am Thorac Soc,2008,5(1):23-31.
[12]Robles JC,Sturek M,Parker JL,et al.Ca2+sensitization and PKC contribute to exercise training-enhanced contractility in porcine collateral-dependent coronary arteries[J].Am J Physiol Heart Circ Physiol,2011,300(4):H1201-H1209.
[13]Kizub IV,Pavlova OO,Johnson CD,et al.Rho kinase and protein kinase C involvement in vascular smooth muscle myofilament calcium sensitization in arteries from diabetic rats[J].Br J Pharmacol,2010,159(8):1724-1731.
[14]Ko EA,Han J,Jung ID,et al.Physiological roles of K+channels in vascular smooth muscle cells[J].J Smooth Muscle Res,2002,44(2):65-81.
[15]Silva MT,Ribeiro FP,Medeiros MA,et al.The vasorelaxant effect of p-cymene in rat aorta involves potassium channels[J].Sci World J,2015,2015:458080.