PKC及蛋白酪氨酸磷酸化信号通路对血管平滑肌细胞表型转化过程中容积调节性氯通道的作用

Regulation of PKC and Protein Tyrosine Phosphorylation on VRCC During the Phenotype Modulation of Femoral Artery and Vein Smooth Muscle cells

【目的】比较蛋白激酶C(PKC)及蛋白酪氨酸磷酸化信号通路对大鼠股动脉平滑肌细胞(femoral artery smoothmuscle cells,FASMC)和股静脉平滑肌细胞(FVSMC)上容积调节性氯通道的调节作用的差异,同时观察这一调节作用在动静脉平滑肌细胞表型转化过程中的变化。【方法】分别选取FASMC和FVSMC的原代,四代和八代作为观察血管平滑肌细胞表型转化的模型。采用全细胞膜片钳技术记录血管平滑肌细胞表型转化过程中容积调节性氯通道(VRCC)的变化以及蛋白酪氨酸激酶(PTK)抑制剂genistein、蛋白酪氨酸磷酸酶(PTP)抑制剂sodium orthevanadate和蛋白激酶C激动剂PDBu对这一变化的调节作用。【结果】低渗溶液在动脉和静脉平滑肌细胞均可激活VRCC,且动脉平滑肌细胞的VRCC活性大于静脉平滑肌细胞。PTK抑制剂genistein对原代、4代、8代大鼠FASMC和FVSMC的VRCC活性均具有明显抑制作用且随着培养代数增加而逐渐增强;PTP抑制剂sodium orthevanadate对原代、4代、8代大鼠FASMC和FVSMC的VRCC活性均具有增强作用但逐渐减弱;PKC激动剂PDBu对原代、4代、8代大鼠FASMC和FVSMC的VRCC活性均具有抑制作用且逐渐增强。与同代的FASMC相比,genistein,sodium orthevanadate和PDBu对FVSMC的VRCC活性的调节作用更强。【结论】蛋白酪氨酸激酶、酪氨酸磷酸酶和蛋白激酶C对股静脉平滑肌细胞VRCC的调节作用强于股动脉平滑肌细胞。且随着血管平滑肌细胞由收缩型向合成型转化,VRCC的活性及酪氨酸蛋白的磷酸化调节作用均有逐渐增强的趋势,而蛋白激酶C对VRCC的抑制作用增强。

[Objective] To compare the differences between the effects of protein kinase C （PKC） and protein tyrosine phosphorylation on volume-regulated chloride channel （VRCC） during the phenotype modulation of smooth muscle cells in artery and vein.[Methods] We used the whole cell patch clamp to evaluate swelling induced Cl-currents （ICl.vol） in primay,Ⅳ and Ⅷ passage rat femoral artery （FASMC） and vein smooth muscle cells （FVSMC） ; and the regulative the effects of protein tyrosine kinase （PTK） inhibitor genistein,protein tyrosine phosphatase （PTP） inhibitor sodium orthovanadate,and PKC activator PDBu on VRCC activity in these cells were examined.[Results] Hypotonic solution induced larger ICl.vol in FASMC than in FVSMC.PTK inhibitor genistein and PKC activator PDBu inhibited VRCC activity in both FASMC and FVSMC,and this inhibitory effect was gradually enhanced in the cells from primary to passage Ⅷ.Whereas PTP inhibitor sodium orthovanadate increased VRCC activity in both FASMC and FVSMC,and this increase was gradually decreased as cell passages increased.Moreover,at the same cell passage,the regulatory effects of genistein,PDBu,and sodium orthovanadate on VRCC activity were more prominent in FVSMC than in FASMC.[Conclusion] PTK,PTP,and PKC could regulate VRCC activity,which were more prominent in FVSMC than in FASMC.Moreover,VRCC activity and the regulatory effects of PKC and protein tyrosine phosphorylation on VRCC were gradually increased as vascular cells switch from contractile to proliferative phenotypes.