Sakai Hiroyasu, Hirano Tomona, Takeyama Hisao, Chiba Yoshihiko, Misawa Miwa
Department of Pharmacology, School of Pharmacy, Hoshi University, Shinagawa-ku, Tokyo, Japan.
Can J Physiol Pharmacol. 2005 Apr;83(4):375-81. doi: 10.1139/y05-022.
It has been demonstrated that CPI-17 provokes an inhibition of myosin light chain phosphatase to increase myosin light chain phosphorylaton and Ca(2+) sensitivity during contraction of vascular smooth muscle. However, expression and agonist-mediated regulation of CPI-17 in bronchial smooth muscle have not been documented. Thus, expression and phosphorylation of CPI-17 mediated by PKC and ROCK were investigated using rat bronchial preparations. Acetylcholine (ACh)-induced contraction and Ca(2+) sensitization were both attenuated by 10(-6) mol Y-27632 /L, a ROCK inhibitor, 10(-6) mol calphostin C/L, a PKC inhibitor, and their combination. A PKC activator, PDBu, induced a Ca(2+) sensitization in alpha-toxin-permeabilized bronchial smooth muscle. In this case, the Ca(2+) sensitizing effect was significantly inhibited by caphostin C but not by Y-27632. An immunoblot study demonstrated CPI-17 expression in the rat bronchial smooth muscle. Acetylcholine induced a phosphorylation of CPI-17 in a concentration-dependent manner, which was significantly inhibited by Y-27632 and calphostin C. In conclusion, these data suggest that both PKC and ROCK are involved in force development, Ca(2+) sensitization, and CPI-17 phosphorylation induced by ACh stimulation in rat bronchial smooth muscle. As such, RhoA/ROCK, PKC/CPI-17, and RhoA/ROCK/CPI pathways may play important roles in the ACh-induced Ca(2+) sensitization of bronchial smooth muscle contraction.
已证实，CPI - 17可抑制肌球蛋白轻链磷酸酶，从而在血管平滑肌收缩过程中增加肌球蛋白轻链磷酸化及钙（Ca2+）敏感性。然而，支气管平滑肌中CPI - 17的表达及激动剂介导的调控尚未见报道。因此，利用大鼠支气管标本研究了蛋白激酶C（PKC）和Rho相关卷曲螺旋蛋白激酶（ROCK）介导的CPI - 17的表达及磷酸化。10(-6) mol/L的ROCK抑制剂Y - 27632、10(-6) mol/L的PKC抑制剂钙磷蛋白C及其联合使用，均可减弱乙酰胆碱（ACh）诱导的收缩及钙（Ca2+）致敏作用。PKC激活剂佛波酯（PDBu）可在α - 毒素通透的支气管平滑肌中诱导钙（Ca2+）致敏作用。在这种情况下，钙磷蛋白C可显著抑制钙（Ca2+）致敏作用，而Y - 27632则无此作用。免疫印迹研究证实大鼠支气管平滑肌中有CPI - 17表达。乙酰胆碱以浓度依赖的方式诱导CPI - 17磷酸化，Y - 27632和钙磷蛋白C可显著抑制该作用。总之，这些数据表明，PKC和ROCK均参与大鼠支气管平滑肌中ACh刺激诱导的力的产生、钙（Ca2+）致敏及CPI - 17磷酸化。因此，RhoA/ROCK、PKC/CPI - 17及RhoA/ROCK/CPI途径可能在ACh诱导的支气管平滑肌收缩的钙（Ca2+）致敏过程中发挥重要作用。
