我们以前报道过,大鼠尾动脉平滑肌中高浓度钾的去极化刺激引起的持续收缩成分涉及Ca2诱导的Ca2敏化机制,由此Ca2通过电压门控Ca2通道进入激活富含脯氨酸的酪氨酸激酶2(Pyk2),导致RhoA/Rho相关激酶(ROCK)的激活。在本研究中,我们研究了Pyk2介导的RhoA/ROCK激活在由Ca2离子载体诱导的胞浆游离Ca2浓度([Ca2]i)升高介导的收缩中的潜在作用,离子霉素,而不是去极化刺激。由于Ca2的流入,离子霉素(60µM)引起大鼠尾动脉平滑肌的缓慢和持续收缩。用肌球蛋白轻链激酶(MLCK)抑制剂进行预处理,ML-9(30µM),抑制离子霉素诱导的收缩的早期阶段(4分钟)和持续阶段(30分钟)。另一方面,岩石抑制剂,HA-1077(3µM),和Pyk2抑制剂,水杨酸钠(10mM)和PF-431396(3µM),仅抑制离子霉素诱导的收缩的持续阶段。钙调蛋白(CaM)抑制剂,W-7(150µM),但不是W-5(150µM),抑制了收缩的早期阶段。离子霉素诱导的20kDa肌球蛋白轻链(LC20)磷酸化的早期或持续增加被每种抑制剂以类似于收缩减弱的方式抑制。这些结果表明,离子霉素诱导的收缩的早期阶段是由MLCK激活介导的[Ca2]i升高,而离子霉素诱导的收缩的持续阶段涉及RhoA/ROCK激活和通过[Ca2]i升高通过CaM非依赖性Pyk2激活抑制肌球蛋白轻链磷酸酶(MLCP)。
We previously reported that the sustained component of contraction induced by depolarizing stimulation by high K+ concentration in rat caudal arterial smooth muscle involves a Ca2+-induced Ca2+ sensitization mechanism whereby Ca2+ entry through voltage-gated Ca2+ channels activates proline-rich tyrosine kinase 2 (Pyk2), leading to activation of RhoA/Rho-associated kinase (ROCK). In the present study, we investigated a potential role for Pyk2-mediated RhoA/ROCK activation in contraction mediated by elevation of cytosolic free Ca2+ concentration ([Ca2+]i) induced by a Ca2+ ionophore,
ionomycin, rather than by depolarizing stimulation.
Ionomycin (60 µM) induced slow and sustained contraction of rat caudal arterial smooth muscle due to influx of Ca2+. Pre-treatment with a myosin light chain kinase (MLCK) inhibitor, ML-9 (30 µM), inhibited both the early phase (4 min) and the sustained phase (30 min) of ionomycin-induced contraction. On the other hand, a ROCK inhibitor, HA-1077 (3 µM), and Pyk2 inhibitors, sodium salicylate (10 mM) and PF-431396 (3 µM), suppressed only the sustained phase of ionomycin-induced contraction. A calmodulin (CaM) inhibitor, W-7 (150 µM), but not W-5 (150 µM), suppressed the early phase of contraction. Early or sustained increase of
ionomycin-induced 20 kDa light chain of myosin (LC20) phosphorylation was inhibited by each inhibitor in a manner similar to the attenuation of contraction. These results indicate that the early phase of
ionomycin-induced contraction is mediated by MLCK activation by [Ca2+]i elevation, whereas the sustained phase of
ionomycin-induced contraction involves RhoA/ROCK activation and inhibition of myosin light chain phosphatase (MLCP) through CaM-independent Pyk2 activation by [Ca2+]i elevation.