HORMONAL AND IONIC CONTROL OF METABOLISM

新陈代谢的激素和离子控制

• 批准号: 2443924
• 负责人: HOWARD RASMUSSEN
• 金额: $ 22.31万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-15 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2443924
• 关键词: abstracting; aldosterone; angiotensin II; cAMP response element binding protein; calcium flux; calcium indicator; confocal scanning microscopy; cytochalasins; enzyme activity; gene expression; hormone binding protein; hormone regulation /control mechanism; immunocytochemistry; isozymes; laboratory rabbit; membrane activity; membrane potentials; potassium; protein kinase A; protein kinase C; protein structure function; tissue /cell culture; western blottings

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.

描述（改编自申请人的摘要）： 提议的工作是定义酶、蛋白激酶 C 的作用 (PKC) 和蛋白激酶 A (PKA)，在持续相的调节中 血管紧张素 II- 和 K+ 诱导牛肾上腺醛固酮分泌的影响 肾小球细胞，以及 PKC 在这一现象中所起的作用 对血管紧张素 II (AII) 的时间依赖性增强 (TDP) 反应。 一个 将确定这些中存在的 PKC 的不同亚型 使用同种型特异性多克隆抗体进行蛋白质印迹分析。 一旦同种型被确定，然后重新分布， 易位，并激活不同亚型以响应 不同的激动剂将分别通过西方分析来测量 具有异构体特异性多克隆抗体的细胞级分， 使用亚型特异性的共聚焦激光显微镜进行免疫细胞化学 单克隆抗体和特定 PKC 底物的磷酸化， MARCKS，或用[32P]-磷酸盐预先标记的细胞中的底物肽。 这 钙进入途径和 PKC 激活之间的联系将是 使用 Ca2+ 流入量、fura 2 荧光和变化测量进行的研究 通过免疫细胞化学观察小窝分布。 PKC的相关角色 和PKA在醛固酮分泌调节中的作用将被研究 鉴定这些细胞中腺苷酸环化酶的异构体并确定 这些亚型中的任何一个是否被 PKC 或 Ca2+ 激活以响应 K+ 或 AII。 PKA 的激活将使用以下方法确定 8-叠氮基-cAMP 结合并通过测量结合的程度和持续阶段 通过比较醛固酮的激活情况来检查醛固酮的分泌。 具有类固醇急性调节mRNA表达的同工酶 蛋白质（StARP）。 cAMP 反应元件结合的磷酸化 蛋白质（CREB）也将被研究对几种激动剂的反应 确定这些事件在时间和幅度上是否相关 与醛固酮分泌和 TDP 的时间进程和幅度有关。 这 细胞松弛素 D 破坏肌动蛋白结构的效果 还将审查这些事件的情况。 申请人认为新 这项研究提供了对醛固酮的机制的见解 分泌受到调节可能与以下领域相关 高血压。