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目的观察肌球蛋白轻链(MLC20)磷酸化在蛋白激酶Cα(protein kinase Cα,PKCα)、蛋白激酶Cε(protein kinase Cε,PKCε)调节大鼠失血性休克后血管钙敏感性中的作用,进一步探讨休克血管钙失敏的发生机制。方法采用缺氧大鼠血管平滑肌细胞(vascular smooth muscle cell,VSMC)和失血性休克后大鼠肠系膜上动脉(superior mesenteric artery,SMA),运用Western blot、底物磷酸化等技术方法,观察PKCα、PKCε激动剂作用下,以及抑制CPI-17、ILK、ZIPK后再给予PKCα、PKCε激动剂时,肌球蛋白轻链磷酸酶(myosin light chain phosphatase,MLCP)活性和MLC20磷酸化的变化。结果①缺氧2h后VSMC中MLCP活性增高至正常对照的150.1%(P<0.01),PKCα、PKCε激动剂可显著抑制其增高,使其分别降低至正常对照的103.0%和96.3%(P<0.01),抑制CPI-17、ILK、ZIPK,可显著抑制PKCα激动剂降低MLCP活性的作用,使其分别增高至正常对照的136.7%、127.6%和116.7%(P<0.01);抑制CPI-17、ILK、ZIPK,也可显著抑制PKCε激动剂降低MLCP活性的作用,使其分别增高至134.0%、128.5%和126.0%(P<0.01)。②失血性休克2h后SMA中MLC20磷酸化水平由32.4%显著降低至10.4%(P<0.01),PKCα、PKCε激动剂可显著抑制其降低,使其分别增高至26.4%和25.6%(P<0.01),抑制CPI-17、ILK、ZIPK,可显著削弱PKCα激动剂的增高MLC20磷酸化的作用,使其分别降低至12.7%、10.0%和15.7%(P<0.01);也可显著削弱PKCε激动剂增高MLC20磷酸化的作用,使其分别降低至10.1%、12.5%和10.3%(P<0.01)。结论PKCα、PKCε可能通过CPI-17、ILK、ZIPK调节MLCP活性和MLC20磷酸化,调节失血性休克血管平滑肌的钙敏感性。
Objective To investigate the role of phosphorylation of myosin light chain (MLC20) in the regulation of vascular calcium sensitivity after hemorrhagic shock in rats with protein kinase Cα (PKCα) and protein kinase Cε (PKCε) To explore the mechanism of shock vascular calcium desensitization. Methods Hypoxic rat vascular smooth muscle cells (VSMCs) and hemorrhagic shock rat superior mesenteric artery (SMA) were treated with Western blot and substrate phosphorylation to observe the effects of PKCα, PKC|Ã agonist, as well as the changes of myosin light chain phosphatase (MLCP) activity and phosphorylation of MLC20 when PKC|Á and PKC|Ã agonist were inhibited after CPI-17, ILK and ZIPK. Results ① After 2 hours of hypoxia, MLCP activity in VSMCs increased to 150.1% (P <0.01) of normal controls. PKCα and PKCε agonists significantly inhibited them to 103.0% and 96.3% of normal controls (P < 0.01). Inhibition of CPI-17, ILK and ZIPK significantly inhibited the decrease of MLCP activity by PKCα agonists, which were increased to 136.7%, 127.6% and 116.7% (P <0.01), respectively. , ILK and ZIPK also significantly inhibited the decrease of MLCP activity by PKCε agonists, which were increased to 134.0%, 128.5% and 126.0%, respectively (P <0.01). ② The phosphorylation level of MLC20 in SMA decreased significantly from 32.4% to 10.4% (P <0.01) at 2h after hemorrhagic shock. PKCα and PKCε agonists significantly decreased MLC20 levels to 26.4% and 25.6%, respectively (P < 0.01). Inhibition of CPI-17, ILK and ZIPK significantly reduced the phosphorylation of MLC20 by PKCα agonist and decreased them to 12.7%, 10.0% and 15.7%, respectively (P <0.01) Agonists increased the phosphorylation of MLC20 and decreased them to 10.1%, 12.5% and 10.3%, respectively (P <0.01). Conclusion PKCα and PKCε may regulate MLCP activity and phosphorylation of MLC20 through CPI-17, ILK and ZIPK and regulate calcium sensitivity of vascular smooth muscle in hemorrhagic shock.