ERK和P38MAPK升高MLC20磷酸化调节大鼠平滑肌低氧反应性

摘要

目的 研究丝裂原活化蛋白激酶(MAPK)亚类ERK、P38 MAPK和JNK在低氧血管平滑肌收缩反应性调节中的作用,并从肌球蛋白轻链(MLC20)磷酸化的钙敏感性调节途径上初步探讨其机制.方法 用低氧培养血管平滑肌细胞(VSMC)模型和大鼠失血性休克模型,用荧光法检测VSMC收缩反应,用Western blot检测血管平滑肌MLC20磷酸化水平,用离体血管张力测定技术观察休克血管钙敏感性.结果 低氧损伤使VSMC对去甲肾上腺素(NE)诱导的收缩反应性明显降低,同时休克血管平滑肌MLC20磷酸化水平和血管钙敏感性降低;给予具有MAPK激动作用的AngⅡ处理可恢复低氧VSMC的收缩反应性、升高休克血管MLC20磷酸化水平和钙敏感性.PD98059(ERK抑制剂)、SB203580(P38 MAPK抑制剂)和SP600125(JNK抑制剂)均可拮抗AngⅡ诱导低氧VSMC反应性升高的作用;同时ERK和P38 MAPK的抑制剂也拮抗了AngⅡ诱导的休克血管MLC20磷酸化水平和钙敏感性升高,而JNK抑制剂无明显作用.结论 ERK、P38 MAPK和JNK都参与了低氧VSMC收缩反应性的调节,其中ERK和P38MAPK的作用机制可能与MLC20磷酸化的钙敏感性调节途径有关,而JNK可能通过其他途径发挥调节低氧VSMC反应性的作用.%Objective To observe the effect of ERK, P38 MAPK and JNK on the contractile response of vascular smooth muscle to norepinephrine (NE) after hypoxia, and explore its calcium sensitivity regulation mechanism of myosin light chain (MLC20) phosphorylation.Methods With hypoxia-treated vascular smooth muscle cell (VSMC) and hemorrhagic shock model of rats, the contractile response of VSMC to NE after hypoxia was observed by fluorometric method.At the same time, the MLC20 phosphorylation of superior mesenteric artery (SMA) in hemorrhagic shock rats was examined by Western blot, and the calcium sensitivityof SMA was assayed with isolated organ perfusion system.Results After hypoxia, the contractile response of VSMC to NE was significantly decreased.Meanwhile the level of MLC20 phosphorylation and calcium sensitivity of SMA after shock were decreased.Angiotensin Ⅱ ( Ang Ⅱ ), with the effect of MAPK stimulation, increased the contractile response of VSMC to NE after hypoxia, and increased the level of MLC20 phosphorylation and calcium sensitivity of SMA after shock.PD98059 ( ERK inhibitor), SB203580 ( P38 MAPK inhibitor) and SP600125 ( JNK inhibitor) antagonized the effects of Ang Ⅱ -induced increase of the contractile response of VSMC after hypoxia.ERK and P38 MAPK inhibitors also antagonized the effects of Ang Ⅱ -induced increased the level of MLC20 phosphorylation and calcium sensitivity of SMA after shock, while JNK inhibitor had no effect.Conclusion ERK, P38 MAPK and JNK are involved in the regulation of the contractile response of VSMC after hypoxia, and the mechanism of ERK and P38 MAPK is potentially mediated through MLC20 phosphorylation dependent mechanism (calcium sensitivity regulation pathway), while JNK may be partially involved in regulating of the contractile response of VSMC by other mechanism.