静脉麻醉药抑制心肌细胞收缩反应与蛋白激酶A信号调节机制

The inhibitory effects of anesthetics on circulatory system causes reduction of blood perfusion, leading to the damage of organic function such as heart and brain. The suppression of anesthetics on vascular and myocardial contraction are the primary causes of inhibiting circulatory function. We previously studied the effects of anesthetics on vascular smooth muscle contraction, demonstrating that the inhibition on the calcium-sensitive mechanism (such as protein kinase C, Rho kinase,p44/42 MAPKs) is one of the important mechanisms of anesthetic attenuating vascular smooth muscle constriction. Differing from the regulatory mechanism of the vascular smooth muscle contraction, cAMP-dependent protein kinase A (PKA) pathway plays an important role in the regulation of myocardial contraction. PKA is able to phosphorylate L-type calcium channel protein and the ryanodine receptor, promoting the extracelluar Ca2+ influx and Ca2+ release from sarcoplasmic reticulum (SR) and increasing intracellular Ca2+ concentration. PKA is also capable of phosphorylating phospholamban and myosin light chain, and finally of strengthening myocardial contractility. It has not been confirmed that whether the inhibition of anesthetics on cardiac contraction is related to the PKA signaling pathway. The purpose of the study is expected to demonstrate that intravenous anesthetics (propofol, midazolam and etomidate) weaken myocardial contraction by, at least in part, directly inhibiting PKA pathway-mediated myocardial intracellular calcium concentration and other intracellular episodes, by detecting or measureing myocardial cell membrane calcium channel current(using patch clamp technique), intracellular calcium concentration(using laser scanning confocal microscope), PKA and related protease activity(using western blot, Elisa and isotope technique) and myocardial cell morphology(using inverted microscope and phase contrast microscope) with PKA agonist and antagonist as tool agents.

麻醉药的循环功能抑制作用可引起心脑等重要脏器的供血减少而致其功能受损，影响手术病人安全。这一负性影响主要起因于麻醉药抑制血管和心肌收缩功能。研究麻醉药对血管和心肌收缩功能的影响机制对探寻防治对策具有重要的理论意义。申请者等前期对麻醉药抑制血管平滑肌收缩功能及其影响机制进行了较为系统的研究，结果表明对以蛋白激酶C为主的钙敏感调节机制的抑制是麻醉药减弱血管平滑肌收缩功能的重要机制之一。与血管平滑肌收缩调节机制不同，蛋白激酶A（PKA）通路（通过开放钙通道、磷酸化相关蛋白等）在心肌收缩功能的调节过程中起到重要作用。本课题拟借用PKA激动剂和抑制剂等工具，采用生物化学、电生理学和形态学等方法，创新性地研究静脉麻醉药对PKA活性以及其所调节的心肌细胞膜钙通道电流、细胞内钙离子浓度、相关蛋白酶的活性和心肌细胞形态等靶目标的影响，以阐明静脉麻醉药至少部分通过直接抑制PKA通路来减弱心肌细胞收缩功能。

麻醉药对循环功能的负性影响可引起心脑等重要脏器的供血减少而致其功能受损，主要起因于麻醉药抑制血管和心肌收缩功能。众所周知，儿茶酚胺及肾上腺素受体主要通过调节心肌收缩来影响心脏功能，cAMP/PKA通路激活多各信号途径参与心脏收缩的调节。本研究主要探讨了麻醉药对血管和心肌收缩功能的影响机制，旨在明确异丙酚是否通过调节PKA磷酸化状态降低心肌收缩功能，并证明无β受体激动剂存在时，异丙酚能否直接抑制PKA从而影响其下游蛋白抑制心肌收缩。本研究采用离体器官灌注及分子水平实验技术进行验证，发现异丙酚能够部分通过调节cAMP依赖性蛋白激酶的磷酸化状态调节心脏功能，无β受体激动剂存在时，异丙酚直接抑制PKA从而影响其下游蛋白抑制心肌收缩。本研究为临床麻醉药对心脏抑制作用的机制研究提供了新思路，对探寻防治麻醉药循环抑制作用的措施具有重要意义。