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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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Citing This Article：

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).