HORMONAL AND IONIC CONTROL OF METABOLISM

描述（改编自申请人的摘要）： 建议的工作是确定酶的作用，蛋白激酶C (PKC)和蛋白激酶A（PKA），在调节持续相 血管紧张素II和K+诱导的牛肾上腺醛固酮分泌 肾小球细胞，以及PKC的作用，在这一现象中发挥作用， 时间依赖性增强（TDP）对血管紧张素II（AII）的反应。 一 将确定存在于这些细胞中的PKC的不同亚型。 使用同种型特异性多克隆抗体进行Western分析。 一旦确定了亚型，然后再分布， 易位，以及不同同种型响应于 不同的激动剂将分别通过Western分析来测量， 具有同种型特异性多克隆抗体的细胞级分， 共聚焦激光显微镜免疫细胞化学 单克隆抗体，和特定PKC底物的磷酸化， MARCKS，或用[32 P]-磷酸预标记的细胞中的底物肽。 的 钙离子进入途径和PKC激活之间的联系将是 使用测量Ca 2+内流、Fura 2荧光和变化的研究 免疫细胞化学法检测细胞膜小窝的分布。 PKC的相关作用 和PKA在调节醛固酮分泌中的作用将通过 鉴定这些细胞中腺苷酸环化酶的同种型， 这些亚型中的任何一种是否被PKC或Ca 2+激活， K+或AII。 PKA的活化将使用以下测定： 8-叠氮基-cAMP结合，并通过测量8-叠氮基-cAMP结合的程度和持续阶段， 醛固酮分泌将通过比较 同工酶与类固醇急性调节基因mRNA的表达 蛋白（StARP）。 cAMP反应元件结合的磷酸化 蛋白（CREB）也将被研究，以响应几种激动剂， 确定这些事件中的任何一个在时间和强度上是否相关 与醛固酮分泌和TDP的时间进程和幅度有关。 的 细胞松弛素D破坏肌动蛋白结构的作用， 这些事件也将受到审查。 申请人认为， 这项研究提供了深入了解醛固酮 分泌调节可能与以下领域相关： 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.