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

为了研究静脉麻醉药对猪脑动脉平滑肌收缩时钙致敏的影响，1)在恒定（夹紧）细胞内钙浓度下，测定了渗透平滑肌的等长力。用PGF2α、内皮素、U46619或GTPγS刺激平滑肌。2)为了观察GTP小结合蛋白之一RhoA及其下游效应物Rho-kinase （Rock II）参与钙致敏作用，我们使用了RhoA抑制剂Rho-kinase的外泌酶C3 Y-27632。静脉麻醉药具有血管舒张作用，观察其对脑血管痉挛的潜在治疗效果。我们发现1)RhoA/ rho激酶可能与PGF2α、内皮素、U-46619或GTPγS诱导脑动脉收缩的钙致敏机制有关。2)静脉麻醉药、氯胺酮、硫胺醛、异丙酚对PGF2α、内皮素、U-46619、GTPγS诱导的脑动脉收缩的钙致敏作用影响不大。3)高浓度氯胺酮在pCa 4.5诱导松弛时，增强了肌球蛋白磷酸酶的活性，使用肌球蛋白激酶抑制剂ML-9.4) Western blotting分析显示脑动脉中存在RhoA、rho激酶（Rock II）、RhoGDI。氯胺酮增强肌球蛋白磷酸酶活性的机制尚不清楚。另一种钙致敏收缩的机制，通过PKC与同工酶的规范，有待进一步的研究。综上所述，通过脑动脉RhoA/ rho激酶抑制收缩的钙致敏可能是人类蛛网膜下腔出血后血管痉挛的潜在药物干预之一。

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.