CONTROL OF METABOLISM IN NORMAL AND DISEASE STATES

该项目的长期目标是阐明机制， 神经激活的细胞内信号传导的生理意义 肝细胞中的系统。 现在已经知道，各种各样的 Ca 2+动员激素由磷脂酶C的活化介导， 这导致磷脂酰肌醇多磷酸迅速分解， 质膜与同时生产的Ca 2+动员 第二信使，肌醇1，4，5-三磷酸，和蛋白激酶C 活化剂，1，2-二酰基甘油。 本提案的主要目的 将是a）调查耦合机制的性质， 受体占据引起磷脂酶C的活化，和B） 表征靶磷酸化的功能效应和性质 二酰基甘油和佛波酯介导的激活诱导的蛋白质 蛋白激酶C 要研究的假设之一是 细胞膜受体与肌醇的分子偶联 磷酸盐代谢是由一种GTP结合蛋白介导的， 从已知的刺激性（Gs）和抑制性（Gi）GPT结合蛋白， 与腺苷酸环化酶相互作用。 这种推定的GTP结合蛋白将是 分离、纯化及其对磷脂酶C的作用， 重组实验使用纯化的磷脂酶C插入到 磷脂囊泡。 其他可能的相互作用的靶位点 将研究GTP结合蛋白以及可能的调节 GTP结合的α和β亚基所产生的影响 cAMP介导的蛋白激酶和蛋白激酶C。 的 激动剂刺激的肌醇脂质周转的动力学将是 研究使用同位素技术和可能的意义 评价了蛋白激酶C对肌醇脂质激酶的激活作用。 的 负责钙离子激活流入细胞的机制 将进行研究，并评估Ca 2+内流发生的假设 通过由蛋白激酶产生的能量有利的Ca 2 +/Na+交换 C介导的Na+/H+交换活化。 进一步的研究涉及 调查的本地化和可能的异质性 Ins-1，4，5-P3介导的完整细胞中的钙池 显微荧光分光光度法;胰高血糖素介导钙离子的机制 在肝细胞中的动员;和胰岛素介导的作用。 被提议者 研究与激素作用机制直接相关， 糖尿病相关研究领域内的刺激-分泌偶联。

The long term objective of this project is to elucidate the mechanisms and physiological significance of hormonally-activated intracellular signalling systems in hepatocytes. It is now known that the effects of a variety of Ca2+-mobilizing hormones are mediated by activation of phospholipase C, which causes a rapid breakdown of phosphatidylinositol polyphosphates in the plasma membrane with a simultaneous production of the Ca2+ -mobilizing second messenger, inositol 1, 4,5-trisphosphate, and the protein kinase C activator, 1,2-diacylglycerol. The major purpose of the present proposal will be a) to investigate the nature of the coupling mechanism whereby receptor occupancy causes an activation of phospholipase C, and b) to characterize the functional effects and nature of target phosphorylated proteins induced by diacylglycerol and phorbol ester-mediated activation of protein kinase C. One of the hypotheses to be investigated is that the molecular coupling between the plasma membrane receptor and inositol phosphate metabolism is mediated by a GTP-binding protein that is distinct from the stimulatory (Gs) and inhibitory (Gi) GPT-binding proteins known to interact with adenylate cyclase. This putative GTP-binding protein will be isolated, purified and its effects on phospholipase C characterized by reconstitution experiments using purified phospholipase C inserted into phospholipid vesicles. Other possible target sites for interaction of the GTP-binding protein will be investigated together with possible modulation of the effects exerted by the alpha and beta subunits of GTP-binding proteins by cAMP-mediated protein kinase and by protein kinase C. The kinetics of the agonist-stimuated turnover of the inositol lipids will be investigated using isotopic techniques and the significance of possible activation of inositol lipid kinases by protein kinase C evaluated. The mechanism responsible for hormone-activated influx of Ca2+ into the cell will be investigated and the hypothesis evaluated that Ca2+ influx occurs by a Ca2+/Na+ exchange made energetically favorable by a protein kinase C-mediated activation of Na+/H+ exchange. Further studies relate to investigations of the localization and possible heterogeneity of Ins-1,4,5-P3-mediated calcium pools in intact cells using microspectrofluorometry techniques; the mechanism of glucagon-mediated Ca2+ mobilization in hepatocytes; and insulin-mediated effects. The propoed studies are directly relevant to the mechanisms of hormone action and stimulus-secretion coupling within the area of diabetes related research.