HORMONAL AND IONIC CONTROL OF METABOLISM

描述(改编自申请人的摘要)： 建议的工作是确定这些酶的作用，即蛋白激酶C 蛋白激酶C(PKC)和蛋白激酶A(PKA)在持续相调节中的作用 血管紧张素II和K诱导牛肾上腺分泌醛固酮的研究 肾小球细胞，以及PKC在肾小球疾病中的作用。 血管紧张素II(AII)的时间依赖增强效应(TDP)。一个 将对这些细胞中存在的不同PKC亚型进行测定 用异构体特异性多克隆抗体进行Western分析。 一旦确定了异构体，然后重新分配， 不同异构体的移位和激活 不同的激动剂将分别通过Western分析进行测量 含有异构体特异性多克隆抗体的细胞组分， 异构体特异性激光共聚焦显微镜免疫细胞化学 单抗和特定PKC底物的磷酸化， Marcks，或细胞中用[32P]-磷酸预先标记的底物多肽。这个 钙进入途径和蛋白激酶C激活之间的联系将是 应用钙离子内流、Fura-2荧光及其变化的研究 免疫细胞化学检测小窝分布。PKC的相关作用 和PKA在调节醛固酮分泌中的作用将通过 鉴定腺酰环化酶在这些细胞中的异构体并测定 这些异构体中的任何一种是否被PKC或钙激活以响应 不是K就是AII。将使用以下方法确定PKA的激活 通过测量8-叠氮基-cAMP结合的程度和持续时相 醛固酮的分泌将通过比较 类固醇急性调节基因表达的同工酶 蛋白质(Starp)。CAMP反应元件结合的磷酸化 还将研究蛋白质(CREB)对几种激动剂的反应 确定这些事件中是否有任何事件在时间和强度上相关 与醛固酮分泌和TDP的时间进程和大小有关。这个 细胞松弛素D对肌动蛋白结构的破坏作用 也将对这些事件进行审查。申请人认为新的 这项研究为醛固酮的作用机制提供了见解 分泌是受调控的，可能与 高血压。

DESCRIPTION (Adapted from the Applicant's Abstract): The aim of the proposed work is that of defining the role of the enzymes, protein kinase C (PKC) and protein kinase A (PKA), in the regulation of the sustained phase of angiotensin II- and K+-induced aldosterone secretion from bovine adrenal glomerulosa cells, and the role that PKC plays in the phenomenon of time-dependent potentiation (TDP) in response to angiotensin II (AII). A determination will be made of the different isoforms of PKC present in these cells using Western analysis with isoform-specific polyclonal antibodies. Once the isoforms have been determined, then the redistribution, translocation, and activation of the different isoforms in response to different agonists will be measured by, respectively, Western analysis of cell fractions with isoform-specific polyclonal antibodies, immunocytochemistry with confocal laser microscopy using isoform specific monoclonal antibodies, and the phosphorylation of a specific PKC substrate, MARCKS, or substrate peptides in cells prelabeled with [32P]-phosphate. The link between the pathways of calcium entry and the activation of PKC will be studies using measurements of Ca2+ influx, fura 2 fluorescence and changes in caveolae distribution by immunocytochemistry. The related roles of PKC and PKA in the regulation of aldosterone secretion will be studied by identifying the isoforms of adenyl cyclase in these cells and determining whether any of these isoforms are activated by PKC or Ca2+ in response to either K+ or AII. The activation of PKA will be determined using 8-azido-cAMP binding and by measuring the extent and the sustained phase of aldosterone secretion will be examined by comparing the activation of the isoenzymes with the expression of the mRNA for the steroid acute regulatory protein (StARP). The phosphorylation of the cAMP response element binding protein (CREB) will also be studied in response to several agonists to determine whether or not any of these events correlate in time and magnitude with the time course and magnitude of aldosterone secretion and TDP. The effect that the disruption of the actin architecture, by cytochalasin D, has on these events will also be examined. The applicant believes the new insight provided by this research into the mechanisms by which aldosterone secretion is regulated are likely to be relevant to the field of hypertension.