Miao Liyan, Dai Yun, Zhang John
Department of Neurosurgery, University of Mississippi Medical Center, Jackson, Mississippi 39216, USA.
Am J Physiol Heart Circ Physiol. 2002 Sep;283(3):H983-9. doi: 10.1152/ajpheart.00141.2002.
This study was undertaken to demonstrate the role of the RhoA/Rho kinase pathway in endothelin-1 (ET-1)-induced contraction of the rabbit basilar artery. Isometric tension and Western blot were used to examine ET-1-induced contraction and RhoA activation. The upstream effect on ET-1-induced RhoA activity was determined by using ET(A) and ET(B) receptor antagonists, protein kinase C (PKC), tyrosine kinase, and phosphatidylinositol-3 kinase inhibitors. The downstream effect of ET-1-induced contraction and RhoA activity was studied in the presence of the Rho kinase inhibitor Y-27632. The effect of Rho kinase inhibitor on ET-1-induced myosin light chain (MLC) phosphorylation was investigated by using urea-glycerol-PAGE immunoblotting. We found 1) ET-1 increased RhoA activity (membrane binding RhoA) in a concentration-dependent manner; 2) ET(A), but not ET(B), receptor antagonist abolished the effect of ET-1 on RhoA activation; 3) phosphodylinositol-3 kinase inhibitor, but not PKC and tyrosine kinase inhibitors, reduced ET-1-induced RhoA activation; 4) Rho kinase inhibitor Y-27632 (10 microM) inhibited ET-1-induced contraction; and 5) ET-1 increased the level of MLC phosphorylation. Rho kinase inhibitor Y-27632 reduced the effect of ET-1 on MLC phosphorylation. This study demonstrated that RhoA/Rho kinase activation is involved in ET-1-induced contraction in the rabbit basilar artery. Phosphodylinositol-3 kinase and MLC might be the upstream and downstream factors of RhoA activation.
本研究旨在证明RhoA/Rho激酶通路在内皮素-1（ET-1）诱导的兔基底动脉收缩中的作用。采用等长张力和蛋白质免疫印迹法检测ET-1诱导的收缩及RhoA激活情况。通过使用ET(A)和ET(B)受体拮抗剂、蛋白激酶C（PKC）、酪氨酸激酶和磷脂酰肌醇-3激酶抑制剂来确定ET-1诱导的RhoA活性的上游效应。在存在Rho激酶抑制剂Y-27632的情况下研究ET-1诱导的收缩和RhoA活性的下游效应。通过尿素-甘油-聚丙烯酰胺凝胶电泳免疫印迹法研究Rho激酶抑制剂对ET-1诱导的肌球蛋白轻链（MLC）磷酸化的影响。我们发现：1）ET-1以浓度依赖性方式增加RhoA活性（膜结合RhoA）；2）ET(A)受体拮抗剂而非ET(B)受体拮抗剂消除了ET-1对RhoA激活的作用；3）磷脂酰肌醇-3激酶抑制剂而非PKC和酪氨酸激酶抑制剂降低了ET-1诱导的RhoA激活；4）Rho激酶抑制剂Y-27632（10 microM）抑制了ET-1诱导的收缩；5）ET-1增加了MLC磷酸化水平。Rho激酶抑制剂Y-27632降低了ET-1对MLC磷酸化的作用。本研究表明，RhoA/Rho激酶激活参与了ET-1诱导的兔基底动脉收缩。磷脂酰肌醇-3激酶和MLC可能分别是RhoA激活的上游和下游因素。
