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UTP经PLC--IP_(3)信号通路调控猪冠状动脉平滑肌细胞自发性瞬时外向电流

李鹏云, 曾晓荣, 杨艳, 蔡芳, 李妙龄, 刘智飞, 裴洁, 周文

泸州医学院心肌电生理学研究室.四川,泸州 646000

摘要

该文采用全细胞穿孔膜片钳技术研究UTP对急性酶分离的猪冠状动脉平滑肌细胞（coronary artery smooth muscle cells,CASMCs）自发性瞬时外向电流（spontaneous transient outward currents，STOCs）的作用，探讨细胞内Ca~(2+)释放在UTP产物三磷酸肌醇（inositol 1,4,5--trisphosphate，IP_(3)）调控STOCs过程中的作用机制。结果显示：（1）UTP（40#mu#mol／L）可明显激活CASMCs的STOCs，使其幅度和频率分别增加（57.54±5.34）％和（77.46±8.42）％（{sl P}<0.01，{sl n}=38）。（2）磷脂酶C（phospholipase C，PLC）阻断剂U73122（5#mu#mol／L）可明显抑制STOCs的活性，使其幅度和频率分别降低（31.04±7.46）％和（41.65±16.59）％（{sl P}=0,05，{sl n}=10）；细胞外再加入UTP不能再次激活STOCs（{sl n}=7）。（3）L型电压依赖性钙通道（L--type voltage--dependent Ca~(2+) channels，L--VDCCs）阻断剂verapamil（20#mu#mol／L）和CdCl_(2)（200#mu#mol／L）几乎不影响UTP对STOCs活性的调节（{sl n}=8）。（4）1#mu#mol／L的bisindolylmaleimide I[BisI，蛋白激酶C（protein kinase C，PKC）的特异性阻断剂]可明显激活STOCs，使其幅度和频率分别增加（65.44±24.66）％和（61.35±21.47）％（{sl P}<0.01，{sl n}=12），细胞外再加入UTP（40#mu#mol／L）可使STOCs的幅度及频率进一步明显增加（{sl P}<0.05，{sl P}<0.01，{sl n}=12），细胞外继续加入ryanodine（50#mu#mol/L）则可完全阻断STOCs。（5）UTP（40#mu#mol／L）预处理细胞后，IP_(3)受体（IP_(3) receptors，IP_(3)Rs）阻断剂2--arninocthoxydiphenyl borate（2--APB，40#mu#mol／L）可使STOCs的幅度降低（24.08±3.97）％（{sl P}<0.05，{sl n}=8），对其频率的影响较小（{sl n}=8）；而80#mu#mol／L的2--APB则可明显抑制STOCs的活性，使其幅度和频率分别降低（31.43±6.34）％和（40.59±19.01）％（{sl P}<0.05，{sl P}<0.01，{sl n}=6），细胞外继续加入高浓度的ryanodine（50#mu#mol／L）可完全抑制STOCS（{sl n}=6）。用2--APB（40#mu#mol／L）或ryanodine（50#mu#mol／L）预处理细胞后，UTP（40#mu#mol／L）不能再次激活STOCs。以上结果提示：UTP主要通过PLC--IP_(3)信号通路激活急性酶分离的猪CASMCs的STOCs，IP_(3)Rs和ryanodine受体（ryanodine receptors，RyRs）介导的细胞内Ca~(2+)释放在此过程中发挥重要作用。

关键词： 大电导钙激活钾通道; 三磷酸肌醇; 自发性瞬时外向电流; 猪冠状动脉平滑肌细胞; 膜片钳技术

UTP regulates spontaneous transient outward currents in porcine coronary artery smooth muscle cells through PLC--IP_(3) signaling pathway

Li Pengyun, Zeng Xiaorong, Yang Yan, Cai Fang, Li Miaoling, Liu Zhifei, Pei Jie, Zhou Wen

Department of Myocardial Electrophysiology, Luzhou Medical College.Luzhou 646000,Sichuan

Abstract

The aim of the present study was to investigate the effects of inositol 1,4,5-trisphosphate （IP_(3)）-generating agonist UTP on spontaneous transient outward currents （STOCs）, and explore the role of intracellular Ca~(2+) release in the current response mediated by IP3 in porcine coronary artery smooth muscle cells （CASMCs）. The coronary artery was excised from the fresh porcine heart and cut into small segments （2 mm × 5 mm） and then transferred to enzymatic dissociation solution for incubation. Single CASMCs were obtained by two-step enzyme digestion at 37℃. STOCs were recorded and characterized using the perforated whole-cell patch-clamp configuration in freshly isolated porcine CASMCs. The currents were amplified and filtered by patch-clamp amplifier （Axopatch 200B）, and then the digitized data were recorded by pClamp 9.0 software and further analyzed by MiniAnalysis 6.0 program. The results were as follows： （1） UTP led to conspicuous increases in STOC amplitude by （57.54±5.34）% and in frequency by （77.46±8.42）% （P〈0.01,n=38）. （2） The specific blocker of phospholipase C （PLC）-U73122 （5 #mu#mol/L） remarkably reduced STOC amplitude by （31.04±7.46）% and frequency by （41.65±16.59）%, respectively （P〈0.05, n=10）. In the presence of U73122, UTP failed to reactivate STOCs （n=7）. （3） Verapamil （20 #mu#mol/L） and CdCl_(2) （200 #mu#mol/L）, two blockers of L-type voltage-dependent Ca~(2+) channels, had little effects on STOCs initiated by UTP （n=8）. （4） 1#mu#mol/L bisindolylmaleimide I （BisI）, a potent blocker of protein kinase C （PKC）, significantly increased STOC amplitude by （65.44±24.66）% and frequency by （61.35±21.47）% （P〈0.01, n=12）; UTP （40 #mu#mol/L）, applied in the presence of 1 #mu#mol/L BisI, could further increase STOC activity （P〈0.05, P〈0.01, n=12）. Subsequent application of ryanodine （50 #mu#mol/L） abolished STOC activity. （5） In the presence of UTP （40 #mu#mol/L）, inhibition of IP_(3) receptors （IP_(3)Rs） by 2-aminoethoxydiphenyl borate （2-APB, 40 #mu#mol/L） reduced STOC amplitude by （24.08±3.97）% （P〈0.05, n=8）, but had little effect on STOC frequency （n=8）. While application of 2-APB （80 #mu#mol/L） significantly reduced STOC amplitude by （31.43±6.34）% and frequency by （40.59±19.01）%, respectively （P〈0.05, P〈0.01, n=6）. Subsequent application of ryanodine （50 #mu#mol/L） completely blocked STOC activity. Pretreatment of cells with 2-APB （40 #mu#mol/L） or ryanodine （50 #mu#mol/L）, UTP （40 #mu#mol/L） failed to reactivate STOCs. The results suggest that UTP activates STOCs mainly via PLC and IP_(3)-dependent mechanisms. Complex Ca~(2+)-mobilization pathways are involved in UTP-mediated STOC activation in porcine CASMCs.

Key words: large-conductance Ca~(2+)-activated K~(+) channels;Inositol 1,4,5-trisphosphate;spontaneous transient outward currents;porcine coronary artery smooth muscle cells;patch-clamp technique

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引用本文：

李鹏云, 曾晓荣, 杨艳, 蔡芳, 李妙龄, 刘智飞, 裴洁, 周文. UTP经PLC--IP_(3)信号通路调控猪冠状动脉平滑肌细胞自发性瞬时外向电流[J]. 生理学报 2008; 60 (1): 65-73.

Li Pengyun, Zeng Xiaorong, Yang Yan, Cai Fang, Li Miaoling, Liu Zhifei, Pei Jie, Zhou Wen. UTP regulates spontaneous transient outward currents in porcine coronary artery smooth muscle cells through PLC--IP_(3) signaling pathway. Acta Physiol Sin 2008; 60 (1): 65-73 (in Chinese with English abstract).