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Acute inhibition of the epithelial sodium channel.

机译：急性抑制上皮钠通道。

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摘要

The epithelial sodium channel (ENaC) is expressed in a variety of tissues, including the renal collecting duct, where it constitutes the rate-limiting step for sodium reabsorption. First, the ERK1/2-mediated down-regulation of ENaC was characterized in primary and immortalized renal collecting duct cells (mCT12). Addition of epidermal growth factor (EGF) to polarized monolayers reduced amiloride-sensitive short-circuit current (ISC) by 15-25%. Exposure of mCT12 cells to EGF caused an increase in phosphorylation of ERK1/2; pretreatment of monolayers with a MEK inhibitor prevented this phosphorylation and significantly reduced EGF-induced inhibition of amiloride-sensitive I SC. The results of these studies demonstrate that acute inhibition of ISC by EGF involves ERK1/2 activation.; Liddle's syndrome is caused by gain-of-function mutations in the beta- and gamma-subunits of ENaC, resulting in enhanced Na+ reabsorption and hypertension. In the second part of this study the effect of EGF on sodium absorption in primary renal collecting duct cells derived from a Liddle's mouse model was evaluated. It was found that EGF inhibited ISC by 24+/-5% in wild-type cells but only by 6+/-3% in homozygous mutant cells. EGF-induced ERK1/2 phosphorylation was similar in +/+ and L/L cells, and prolonged exposure to EGF decreased ENaC expression by ∼50% in both. Acute inhibition of ENaC activity by EGF requires an intact beta-ENaC C-terminus, whereas EGF-induced down-regulation of ENaC expression does not.; Finally, in order to elucidate the role of specific regions of the beta-ENaC C-terminus, MDCK cell lines expressing beta-ENaC with mutations of the PY motif, ERK phosphorylation site, and C-terminus truncation were created. All mutants exhibited significant attenuation of EGF-induced inhibition of sodium current. In MDCK cells with wild-type beta-ENaC, EGF-induced inhibition of ISC (30min) was fully reversed by exposure to an ERK kinase inhibitor and occurred with no change in ENaC surface expression, indicative of an effect on channel open probability (Po). At later times (>30min), EGF-induced inhibition of ISC was not reversed by an ERK kinase inhibitor and was accompanied by a decrease in ENaC surface expression. These results are consistent with a reversible ERK-mediated decrease in ENaC P o followed by an irreversible retrieval of sodium channels from the apical membrane.
机译：上皮钠通道（ENaC）在包括肾脏收集管在内的多种组织中表达，在其中构成钠再吸收的限速步骤。首先，ERK1 / 2介导的ENaC下调的特征在于原代和永生化的肾收集导管细胞（mCT12）。在极化的单层膜上添加表皮生长因子（EGF）可将阿米洛利敏感的短路电流（ISC）降低15-25％。 mCT12细胞暴露于EGF导致ERK1 / 2磷酸化增加；用MEK抑制剂预处理单层可防止这种磷酸化作用，并显着降低EGF诱导的对阿米洛利敏感性I SC的抑制。这些研究的结果表明，EGF对ISC的急性抑制作用涉及ERK1 / 2激活。 Liddle综合征是由ENaC的β和γ亚基的功能获得性突变引起的，从而导致Na +重吸收增强和高血压。在这项研究的第二部分中，评估了EGF对源自Liddle小鼠模型的原代肾收集管细胞中钠吸收的影响。发现EGF在野生型细胞中抑制ISC 24 +/- 5％，而在纯合突变细胞中仅抑制6 +/- 3％。 EGF诱导的ERK1 / 2磷酸化在+ / +和L / L细胞中相似，并且长时间暴露于EGF会使两者的ENaC表达降低约50％。 EGF对ENaC活性的急性抑制需要完整的β-ENaCC末端，而EGF诱导的ENaC表达下调则不需要。最后，为了阐明β-ENaCC末端特定区域的作用，创建了表达β-ENaC的PCK基序，ERK磷酸化位点和C末端截短突变的MDCK细胞系。所有突变体均表现出EGF诱导的钠电流抑制作用的显着减弱。在具有野生型β-ENaC的MDCK细胞中，暴露于ERK激酶抑制剂可完全逆转EGF诱导的对ISC的抑制作用（<30min），并且在ENaC表面表达无变化的情况下发生，表明对通道打开概率的影响（宝）。在随后的时间（> 30分钟），ERK激酶抑制剂未逆转EGF诱导的ISC抑制作用，并伴有ENaC表面表达的降低。这些结果与可逆性ERK介导的ENaC P o降低，随后不可逆地从根尖膜吸收钠通道相一致。

著录项

  • 作者

    Falin, Rebecca Anne.;

  • 作者单位

    Case Western Reserve University.;

  • 授予单位 Case Western Reserve University.;
  • 学科 Biology Cell.; Biophysics General.
  • 学位 Ph.D.
  • 年度 2008
  • 页码 172 p.
  • 总页数 172
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 细胞生物学;生物物理学;
  • 关键词

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