REGULATION OF CARDIAC CROSS-BRIDGE KINETICS BY SECOND MESSENGER CASCADES

这项拟议研究的长期目标是阐明分子 激动剂与肌醇磷脂水解物偶联的机制 影响哺乳动物心脏的跨桥功能。两位将军 建议的方法是：1)解剖完整的信号通路以 确定不同组织激活的第二信使级联的差异 激动剂，以及2)评估蛋白激酶对交叉反应的影响。 去皮心肌细胞的桥接动力学。最初的实验旨在 描述暴露于放射性物质时发生的细胞内生化变化 心肌细胞对受体激动剂的作用，特别是那些刺激 磷脂酰肌醇水解法。特定脂类升高，移位 蛋白激酶C亚型与收缩蛋白的磷酸化 将在对激动剂的反应中进行评估，这些激动剂会导致 电起搏心脏的收缩幅度和收缩时程 肌细胞。选择性蛋白激酶抑制剂对生理学的影响 对激动剂的反应也将决定。然后蛋白激酶C将被 通过光释放在活的心肌细胞内以受控的方式激活 甘油二酯。甘油二酯的时间进程和剂量依赖关系 肌力和肌丝蛋白的磷酸化将被建立。 重点将放在某些物种的潜力上 二酰甘油醇选择性激活蛋白激酶C亚型 直接或由于甘油二酯代谢的结果。去皮心肌细胞 将用蛋白激酶A、C-α、C-α或肌球蛋白光治疗 链激酶，然后将测量几个活跃的跨桥过程 包括活化速率和松弛速率，以及离解动力学 跨桥的PI和ADP。这些实验的结果应该是 对假定的第二信使的角色提供独特的见解， 蛋白激酶及其靶蛋白在介导血管紧张素转换酶的作用 表面受体刺激对跨桥动力学的影响。因此，这个项目 直接针对总体计划的具体目标2、3和4。

The long range goals of the proposed research are to elucidate molecular mechanisms by which agonists coupled to phosphoinositide hydrolysis influence cross-bridge function in the mammalian heart. Two general approaches are proposed: 1) dissection of intact signalling pathways to identify differences in second messenger cascades activated by different agonists, and 2) evaluation of the effects of protein kinases on cross- bridge kinetics in skinned myocytes. Initial experiments are designed to characterize intracellular biochemical changes that occur on exposure of cardiac myocytes to receptor agonists, particularly those that stimulate phosphoinositide hydrolysis. Elevation of specific lipids, translocation of protein kinase C isoforms and phosphorylation of contractile proteins will be assessed in response to agonists that cause unique changes in the twitch amplitude and twitch time course of electrically paced ventricular myocytes. Effects of selective protein kinase inhibitors on physiological responses to agonists will also determined. Protein kinase C will then be activated within living myocytes in a controlled way by photorelease of diacylglycerols. The time course and diacylglycerol dose dependence of inotropy and myofilament protein phosphorylation will be established. Emphasis will be placed on the potential for certain species of diacyglcyerol to selectively activate protein kinase C isoforms either directly or as a result of diacylglycerol metabolism. Skinned myocytes will be treated with protein kinases A, c-alpha, C-alpha or myosin light chain kinase, then several active cross-bridge processes will be measured including activation and relaxation rates, and the kinetics of dissociation of Pi and ADP from the cross-bridge. Results from these experiments should provide unique insights into the roles of putative second messengers, protein kinases, and their target proteins in mediating the effects of surface receptor stimulation on cross-bridge kinetics. Thus, this project directly addresses specific objective 2, 3, and 4 of the overall program.