CONTROL OF METABOLISM IN NORMAL AND DISEASE STATES

这个项目的长期目标是阐明这些机制和 激素激活的细胞内信号转导的生理学意义 肝细胞中的系统。现在已经知道，各种不同的 钙动员激素是通过激活磷脂酶C来调节的， 这会导致磷脂酰肌醇多磷酸盐在 同时产生钙离子动员的质膜 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。本提案的主要目的是 将是a)调查耦合机制的性质，由此 受体占位导致磷脂酶C的激活，以及b) 表征磷酸化靶标的功能效应和性质 甘油二酯和佛波酯诱导的蛋白质激活 蛋白激酶C。有待研究的假说之一是 质膜受体与肌醇的分子偶联 磷酸盐代谢是由一种不同的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 与腺苷环化酶相互作用。这个假定的GTP结合蛋白将是 分离纯化及其对磷脂酶C的影响 纯化的磷脂酶C插入重组载体的实验研究 磷脂小泡。其他可能的目标站点，用于 将一起研究GTP结合蛋白及其可能的调节 GTP结合的α和β亚基所起的作用 通过cAMP介导的蛋白激酶和蛋白激酶C。 激动剂引发的肌醇脂类周转的动力学将是 利用同位素技术进行的研究及其可能的意义 评价蛋白激酶C对肌醇类脂蛋白的激活作用。这个 激素激活的钙离子流入细胞的机制 将被调查并评估发生钙内流的假说 通过一种蛋白激酶的能量有利的钙/钠交换 C介导的Na/H交换的激活。进一步的研究涉及到 的局部性和可能的异质性的研究 INS-1，4，5-P3介导的完整细胞内钙池 显微荧光技术；胰高血糖素介导的钙机制 肝细胞的动员；以及胰岛素介导的效应。最合适的 研究直接与激素的作用机制和 糖尿病相关研究领域内的刺激-分泌耦合。

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.