HORMONAL AND IONIC CONTROL OF METABOLISM
新陈代谢的激素和离子控制
基本信息
- 批准号:2733977
- 负责人:
- 金额:$ 22.83万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:1993
- 资助国家:美国
- 起止时间:1993-07-15 至 2000-06-30
- 项目状态:已结题
- 来源:
- 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
项目摘要
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):
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
这些亚型中的任何一个是否被PKC或Ca~(2+)激活
要么是K+,要么是全部。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。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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HOWARD RASMUSSEN其他文献
HOWARD RASMUSSEN的其他文献
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{{ truncateString('HOWARD RASMUSSEN', 18)}}的其他基金
CORE--CELL BIOLOGY AND IMMUNOBIOLOGY FACILITY
核心--细胞生物学和免疫生物学设施
- 批准号:
6239092 - 财政年份:1997
- 资助金额:
$ 22.83万 - 项目类别:
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