MECHANISMS OF RHO KINASE REGULATION OF VASCULAR REACTIVITY FOLLOWING HEMORRHAGIC SHOCK IN RATS
MECHANISMS OF RHO KINASE REGULATION OF VASCULAR REACTIVITY FOLLOWING HEMORRHAGIC SHOCK IN RATS
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DOI：
10.1097/shk.0b013e318063e477
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发表时间：
2007-07
期刊：
影响因子：
3.1
通讯作者：
Tao Li;Liangming Liu;Jian-cang Liu;Jia Ming;Jing Xu;Guangming Yang;Yuan Zhang
中科院分区：
文献类型：
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作者：
Tao Li;Liangming Liu;Jian-cang Liu;Jia Ming;Jing Xu;Guangming Yang;Yuan Zhang
我们前期的研究表明Rho激酶参与了休克后血管反应性降低的调控。本研究的目的是探讨其机制。用离体失血性休克大鼠肠系膜上动脉（SMA），研究了Rho激酶调节血管对钙敏感性的反应性与肌球蛋白轻链磷酸酶（MLCP）和肌球蛋白轻链激酶（MLCK）的关系。在离体器官灌注系统下，在去极化条件下（120 mM K+），通过测量去甲肾上腺素（NE）和钙的累积引起的收缩，观察SMA的血管反应性和钙敏感性。采用缺氧处理血管平滑肌细胞（VSMCs），研究Rho激酶对血管平滑肌细胞MLCP和MLCK活性及20-kDa肌球蛋白轻链（MLC20）磷酸化的影响。采用免疫沉淀法和Western blotting检测肠系膜动脉VSMC轻链（20kda）磷酸化水平。采用酶催化法测定了MLCP和MLCK的活性。采用荧光异硫氰酸盐偶联牛血清白蛋白经transwell累计浸润率测定VSMC的收缩反应。结果表明，失血性休克后，SMA对NE和钙的血管反应性和钙敏感性以及缺氧后VSMC对NE的收缩反应均显著降低。血管紧张素II (Ang-II)， Rho激酶刺激剂，显著改善缺氧或失血性休克引起的血管反应性和钙敏感性下降。特异性Rho激酶抑制剂Y-27632可消除Ang-II对血管反应性的影响。MLCP抑制剂Calyculin A进一步增强了ang - ii诱导的钙敏感性升高，而MLCK抑制剂ML-9则没有作用。进一步研究表明，Ang-II逆转了缺氧诱导的VSMC中MLCP活性的升高，并增加了缺氧诱导的VSMC中MLC20磷酸化的降低。提示Rho激酶在失血性休克后血管反应性的调节中起重要作用。其机制可能与其钙敏感性调节有关。Rho激酶可能通过抑制MLCP活性和增加MLC20的磷酸化来上调VSMC的钙敏感性。
Our previous research showed that Rho kinase took part in the regulation of vascular hyporeactivity after shock. The objective of the present study was to investigate its mechanism. With isolated superior mesenteric artery (SMA) from hemorrhagic shock rats, we studied the relationship of Rho kinase regulating vascular reactivity to calcium sensitivity and myosin light chain phosphatase (MLCP) and myosin light chain kinase (MLCK). The vascular reactivity and calcium sensitivity of SMA were observed by measuring the contraction initiated by accumulative norepinephrine (NE) and calcium under depolarizing condition (120 mM K+) with an isolated organ perfusion system. Hypoxia-treated vascular smooth muscle cells (VSMCs) were used to study the effects of Rho kinase on the activity of MLCP and MLCK and the phosphorylation of 20-kDa myosin light chain (MLC20). Myosin light chain (20 kDa) phosphorylation of VSMC in mesenteric artery was detected by immunoprecipitation and Western blotting. The activity of MLCP and MLCK was assayed by enzymatic catalysis. The contractile response of VSMC was measured by the ratio of accumulative infiltration of fluorescent isothiocyanate-conjugated bovine serum albumin through transwell. The results indicated that the vascular reactivity and calcium sensitivity of SMA to NE and calcium following hemorrhagic shock and the contractile response of VSMC to NE following hypoxia were significantly decreased. Angiotensin II (Ang-II), the Rho kinase stimulator, significantly improved hypoxia or hemorrhagic shock-induced decrease of vascular reactivity and calcium sensitivity. These effects of Ang-II on vascular reactivity were abolished by Y-27632, the specific Rho kinase inhibitor. Calyculin A, the MLCP inhibitor, further enhanced Ang-II-induced increase of calcium sensitivity, but ML-9, the MLCK inhibitor, had no effect. Further studies showed Ang-II reversed the hypoxia-induced increase of MLCP activity and increased the hypoxia-induced decrease of MLC20 phosphorylation in VSMC. It was suggested that Rho kinase played an important role in the regulation of vascular reactivity after hemorrhagic shock. The mechanisms may be related to its calcium sensitivity regulation. Rho kinase up-regulates calcium sensitivity of VSMC possibly through inhibiting the activity of MLCP and increasing the phosphorylation of MLC20.