Mechanism of sgk action in the collecting duct
SGK在集合管中的作用机制
基本信息
- 批准号:6727493
- 负责人:
- 金额:$ 29.23万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2002
- 资助国家:美国
- 起止时间:2002-04-01 至 2006-01-31
- 项目状态:已结题
- 来源:
- 关键词:Xenopus oocytealdosteroneamilorideapical membranearginine vasopressincell lineimmunologic assay /testimmunoprecipitationlaboratory rabbitmass spectrometrymembrane activityphosphatidylinositol 3 kinasephosphopeptidesphosphorylationprotein kinaseprotein localizationprotein purificationprotein transportregulatory generenal tubular transportrenal tubulesaluresissodium channeltranscription factor
项目摘要
The long-term objective of these studies is to understand the molecular mechanisms through which aldosterone stimulates Na transport and thereby regulates blood pressure. The foundation for the proposed studies is the recent identification of serum- and glucocorticoid-induced kinase (sgk) as an aldosterone-induced early response gene in the cortical collecting duct (CCD). We also demonstrated that in Xenopus oocytes expressing the epithelial Na channel (ENaC) sgk stimulates amiloride-sensitive Na current. These observations make sgk a strong candidate for mediating the early actions of aldosterone on Na transport. We established that in oocytes sgk increases the number of ENaC subunits in the cell membrane and has no effect on open the probability of ENaCs or their rate of their endocytosis, suggesting that in this system sgk increases ENaC exocytosis or recycling. We propose the following specific aims. Aim 1 will test the hypothesis that sgk increases Na absorption in mammalian collecting duct cells by increasing exocytosis or recycling of ENaC, similarly as it does in Xenopus oocytes. The effects of up- or down-regulating sgk in CCD cells on ENaC will be determined by measuring amiloride-sensitive short-circuit current, and analyzing its fluctuations by noise analysis, by determining the amount of ENaC in the apical membrane using biochemical approaches, and by determining the rate of removal of ENaC from the apical membrane. Aim 2 is to elucidate the mechanism by which sgk increases surface expression of ENaC. We will determine if the effect of sgk is mediated by post-translational or transcriptional events by monitoring ENaC current in oocytes following injection of sgk protein and/or RNA synthesis inhibitors. The subcellular localization of sgk will be determined using GFP-sgk chimeras or antibodies against sgk. Finally, in this aim we will identify the downstream targets of sgk action by testing the effects of candidate proteins and by phosphopeptide mapping. Aim 3 is to determine the role of sgk in AVP-stimulated Na transport in CCD cells. The effects of AVP and PI3K inhibitors on the activity and phosphorylation of sgk in mammalian CCD cells will be determined, and these effects will be correlated with changes in amiloride-sensitive current, in normal CCD cells and cells in which endogenous sgk levels are down- regulated. Since the ultimate target of sgk action seems to be a step in ENaC trafficking, identification of the downstream effectors of sgk could lead to insights into the mechanism of ENaC trafficking, which is poorly understood. Unraveling the molecular steps leading to hormone-stimulated Na transport in the CCD could also lead to the identification of genes that might be mutated in some forms of human hypertension, and may eventually lead to therapeutic measures that interrupt this pathway.
这些研究的长期目标是了解醛固酮刺激钠转运从而调节血压的分子机制。提出的研究的基础是最近发现血清和糖皮质激素诱导的激酶(sgk)是皮质集管(CCD)中醛固酮诱导的早期反应基因。我们还证明,在非洲爪蟾卵母细胞中,表达上皮Na通道(ENaC)的ssk刺激了阿米洛利敏感的Na电流。这些观察结果使ssk成为介导醛固酮对钠转运的早期作用的强有力的候选者。我们发现,在卵母细胞中,sgk增加了细胞膜中ENaC亚基的数量,但对ENaC的打开概率或内吞率没有影响,这表明在该系统中,sgk增加了ENaC的胞吐或再循环。我们提出以下具体目标。目的1将验证sgk通过增加ENaC的胞吐或再循环来增加哺乳动物收集管细胞对Na的吸收的假设,类似于在非洲爪蟾卵母细胞中的作用。CCD细胞中上调或下调sgk对ENaC的影响将通过测量amiloride敏感短路电流,并通过噪声分析分析其波动,通过生化方法确定ENaC在根尖膜中的含量,并通过确定根尖膜上ENaC的去除速率来确定。目的2是阐明sgk增加ENaC表面表达的机制。我们将通过注射sgk蛋白和/或RNA合成抑制剂后监测卵母细胞中的ENaC电流来确定sgk的作用是否由翻译后或转录事件介导。sgk的亚细胞定位将使用GFP-sgk嵌合体或针对sgk的抗体来确定。最后,在这个目标中,我们将通过测试候选蛋白的作用和磷酸肽定位来确定sgk作用的下游靶点。目的3是确定ssk在avp刺激的CCD细胞钠转运中的作用。AVP和PI3K抑制剂对哺乳动物CCD细胞中sgk活性和磷酸化的影响将被确定,这些影响将与正常CCD细胞和内源性sgk水平下调的细胞中阿米洛胺敏感电流的变化相关。由于sgk作用的最终目标似乎是ENaC贩运的一个步骤,因此确定sgk的下游效应物可能有助于深入了解ENaC贩运的机制,这一机制尚不清楚。揭示导致CCD中激素刺激的钠转运的分子步骤也可能导致在某些形式的人类高血压中可能发生突变的基因的鉴定,并可能最终导致阻断这一途径的治疗措施。
项目成果
期刊论文数量(0)
专著数量(0)
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会议论文数量(0)
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GEZA FEJES-TOTH其他文献
GEZA FEJES-TOTH的其他文献
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{{ truncateString('GEZA FEJES-TOTH', 18)}}的其他基金
The Role of Cardiovascular Mineralocorticoid Receptors in Health and Disease
心血管盐皮质激素受体在健康和疾病中的作用
- 批准号:
7575668 - 财政年份:2008
- 资助金额:
$ 29.23万 - 项目类别:
The Role of Cardiovascular Mineralocorticoid Receptors in Health and Disease
心血管盐皮质激素受体在健康和疾病中的作用
- 批准号:
7780010 - 财政年份:2008
- 资助金额:
$ 29.23万 - 项目类别:
The Role of Cardiovascular Mineralocorticoid Receptors in Health and Disease
心血管盐皮质激素受体在健康和疾病中的作用
- 批准号:
7369910 - 财政年份:2008
- 资助金额:
$ 29.23万 - 项目类别:
MOLECULAR MECHANISM OF MINERALOCORTICOID RECEPTOR ACTION
盐皮质激素受体作用的分子机制
- 批准号:
6350734 - 财政年份:2000
- 资助金额:
$ 29.23万 - 项目类别:
MOLECULAR MECHANISM OF MINERALOCORTICOID RECEPTOR ACTION
盐皮质激素受体作用的分子机制
- 批准号:
6498156 - 财政年份:2000
- 资助金额:
$ 29.23万 - 项目类别:
MOLECULAR MECHANISM OF MINERALOCORTICOID RECEPTOR ACTION
盐皮质激素受体作用的分子机制
- 批准号:
6044901 - 财政年份:2000
- 资助金额:
$ 29.23万 - 项目类别:
MOLECULAR MECHANISM OF MINERALOCORTICOID RECEPTOR ACTION
盐皮质激素受体作用的分子机制
- 批准号:
6628558 - 财政年份:2000
- 资助金额:
$ 29.23万 - 项目类别:
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