REGULATION OF CARDIAC CROSS-BRIDGE KINETICS BY SECOND MESSENGER CASCADES

这项研究的长期目标是阐明 激动剂与磷酸肌醇水解偶联的机制 影响哺乳动物心脏的跨桥功能。 两个一般 提出了以下方法：1）解剖完整的信号通路， 确定不同的第二信使级联激活的差异 激动剂，和2）评估蛋白激酶对交叉- 皮肤肌细胞中的桥动力学。 最初的实验旨在 表征暴露时发生的细胞内生化变化 心肌细胞对受体激动剂，特别是那些刺激 磷酸肌醇水解 特定脂质升高，易位 蛋白激酶C亚型和收缩蛋白磷酸化 将评估对激动剂的反应，所述激动剂引起细胞内的独特变化， 电起搏心室肌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的影响 还将测定对激动剂的反应。 然后蛋白激酶C将 在活的肌细胞内以受控的方式通过光释放 二酰基甘油。 甘油二酯的时间进程和剂量依赖性 将建立变力性和肌丝蛋白磷酸化。 重点将放在某些物种的潜力， 二酰基甘油选择性激活蛋白激酶C亚型， 直接或作为二酰基甘油代谢的结果。 皮肤肌细胞 将接受蛋白激酶A、c-α、C-α或肌球蛋白光治疗 链激酶，然后几个活跃的跨桥过程将被测量 包括活化和弛豫速率，以及解离的动力学 π和ADP的含量 这些实验的结果应该 提供了对假定的第二信使的作用的独特见解， 蛋白激酶及其靶蛋白在介导 表面受体刺激对跨桥动力学的影响。 因此，该项目 直接涉及总体方案的具体目标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.