EFFECTS OF LOW-MOLECULAR GTP BINDING PROTEINS AND TYROSINE KINASE ACTIVATION ON ION CHANNELS OF MYOCARDIAL CELLS.

本课题旨在确定低分子GTP结合蛋白（小G蛋白）和胞内酪氨酸激酶的激活是否在细胞表面受体的信号转导机制中起关键作用。采用膜片钳法观察了肉毒杆菌C_3毒素修饰的小G蛋白和胰岛素对兔心脏单个心肌细胞膜电流系统（I_<Na>、I_<Ca>、I_ k、I_<K1>和I_<to>）的影响。在一些ex13EA实验中，用玻璃微电极记录了兔和豚鼠乳头肌心脏的动作电位，用氢离子选择性微电极监测了细胞内ph。得到了以下结果：1)肉毒杆菌C_3毒素对小G蛋白（rho蛋白）adp核糖基化不影响I_<Na>、I_<Ca>、I_ k、I_<K1>和I_<to>，提示rho蛋白不是控制受体介导的离子通道变化的信号转导通路的活跃成员。2)胰岛素对家兔单心室细胞主要膜电流无明显影响，对豚鼠乳头肌Na^+/H^+交换体和阴离子转运体无明显影响。这一发现表明酪氨酸激酶在胰岛素受体胞内区域存在13ea \: nt并不调节离子通道功能。然而，胰岛素确实抑制Na^+泵电流。这一出人意料的结果值得进一步研究。3)百日咳毒素诱导的高分子G蛋白（可能是G_i）功能失活增强了alpha_1-肾上腺素能受体介导的细胞反应，如动作电位持续时间延长、正性肌力效应和ph13EA\：磷脂酰肌苷水解。这一发现否定了G_i作为高分子G蛋白偶联α _1-肾上腺素能受体的候选者，并提示G_i对细胞内信号转导系统的动态抑制。

The aim of this research project is to determine whether low-molecular GTP binding proteins (small G protein) and activation of intracellular tyrosine kinase play crucial roles in signal transduction mechanism of cell-surface receptors.Using a patch clamp method, we observed influences of modified small G protein by botulinum C_3 toxin and of insulin on membrane current system (I_<Na>, I_<Ca>, I_K, I_<K1> and I_<to>) of single myocardial cells obtained from rabbit hearts. In some ex13EA\ : periments, action potentials of rabbit and guinea-pig papillary muscles hearts were recorded by means of glass microelectrodes, and intracellular _pH was monitored with hydrogen-ion selective microelectrodes. The following results were obtained.1) ADP-ribosylation of small G protein (rho protein) by botulinum C_3 toxin did not affect I_<Na>, I_<Ca>, I_K, I_<K1> and I_<to>, suggesting that rho ptotein is not an active member of signal transduction pathway controlling receptor-mediated alterat13EA\ : ions of ion-channels.2) Insulin did not alter in any rate major membrane currents of single ventricular cells of rabbits and also did not affect Na^+/H^+ exchangers and anion transporters in guinea-pig papillary muscles. This finding implies that tyrosine kinase prese13EA\ : nt in intracellular domain of insulin receptor does not modulate ion channel functions. However, insulin did inhibit Na^+ pump current. This unexpected results warrant further study.3) Functional inactivation of high-molecular G protein (probably G_i) induced by pertussis toxin potentiated alpha_1-adrenoceptor mediated cellular responses such as prolonged action potential duration, positive inotropic effect and hydrosis of ph13EA\ : osphatidyl inositides. This finding denies G_i as a candidate of high-molecular G protein coupling to alpha_1-adrenoceptor and suggests dynamic inhibition of G_i on intracellular signal transduction system.