Effect of hypoxia and intravenous anesthetics on calcium sensitization of contraction in cerebral vascular smooth muscle

为研究静脉麻醉药对猪大脑动脉平滑肌收缩的钙增敏作用，1)在恒定(钳制)细胞内钙浓度的条件下，测定通透性平滑肌的等长力。用PgF_2α、ET、U46619或GTPγS_2刺激平滑肌，观察其中一种小G蛋白结合蛋白RhoA及其下游效应Rho-K(ROCKⅡ)是否参与钙增敏作用，RhoA的抑制剂外酶C3用于Rho-K，Y-27632。应用具有血管扩张作用的静脉麻醉药观察其对脑血管痉挛的潜在治疗作用。我们发现：1)RhoA/Rho-Kinase可能参与了PgF_2α、ET、U-46619或GTPγS引起的脑动脉收缩的钙敏化机制。2)静脉麻醉剂氯胺酮、硫代戊醛、异丙酚对PgF_2α、ET、U-46619或GTPγS引起的脑血管收缩的钙敏化作用几乎没有影响。3)较高浓度的氯胺酮在4.5时增强肌球蛋白磷酸酶的活性，并与肌球蛋白激酶抑制剂Pca一起使用。Ml-9.4)免疫印迹分析显示RhoA、Rho-Kinase(Rock II)、RhoGDI存在于脑动脉，但氯胺酮增强肌球蛋白磷酸酶活性的机制尚不清楚。另一种具有同工酶特性的PKC钙增敏收缩机制有待进一步研究。综上所述，RhoA/Rho-Kinase抑制脑动脉钙增敏收缩可能是治疗蛛网膜下腔出血后血管痉挛的潜在药物干预措施之一。

To study the effect of intravenous anesthetics on the calcium sensitization of contraction in porcine cerebral arterial smooth muscle,1) isometric force was measured in permeabilized smooth muscle at constant (clamped) intracellular calcium concentration. Smooth muscles were stimulated with PGF2α, endothelin, U46619, or GTPγS.2) To see the participation of one of small GTP binding protein, RhoA, and its downstream effecor, Rho-kinase (Rock II) in the calcium sensitization, the inhibitor for RhoA, exoenzyme C3, for Rho-kinase, Y-27632 were used. Intravenous anesthetics, which have a vasodilating effect, were applied to see the potential therapeutic effect on the cerebral vasospasm.We found that1) RhoA/Rho-kinase may be responsible for the mechanism of calcium sensitization of contraction induced by PGF2α, endothelin, U-46619, or GTPγS in the cerebral artery.2) Intravenous anesthetics, ketamine, thiamylal, propofol had little effect on the calcium sensitization of contraction induced by PGF2α, endothelin, U-46619, or GTPγS in the cerebral artery.3) Ketamine at higher concentration, potentiated the myosin phosphatase activity when relaxation was induced at pCa 4.5, with myosin kinase inhibitor, ML-9.4) Western blotting analysis showed the presence of RhoA, Rho-kinase (Rock II), RhoGDI in the cerebral artery.The mechanism of potentiation by ketamine of the myosin phosphatase activity remained to be clarified. Another mechanism of calcium sensitization of contraction, by PKC, with specification of isoenzyme, awaits for further investigation.In summary, it is suggested that the suppression of the calcium sensitization of contraction through RhoA/Rho-kinase in cerebral artery may be useful as one of potential pharmacological intervention for the vasospasm after subarachnoid hemorrhage in human.