8-pCPT-cGMP stimulates αβγ-ENaC activity in oocytes as an external ligand requiring specific nucleotide moieties
8-pCPT-cGMP stimulates αβγ-ENaC activity in oocytes as an external ligand requiring specific nucleotide moieties
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DOI：
10.1152/ajprenal.00307.2009
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发表时间：
2010-02-01
影响因子：
4.2
通讯作者：
Ji, Hong-Long
中科院分区：
文献类型：
--
作者：
Nie, Hong-Guang;Zhang, Wei;Ji, Hong-Long
聂华光，张伟，韩德英，李庆宁，李健，赵仁忠，苏晓芳，彭建波，季洪华。8-pCPT-cGMP作为需要特定核苷酸部分的外部配体刺激卵母细胞中的α β γ-ENaC活性。美国肾脏生理学杂志298：F323-F334，2010年。首次发表于2009年12月9日; doi：10.1152/ajprenal.00307.2009.-上皮钠通道（ENaC）除了由广谱的其他蛋白激酶调节之外，还由蛋白激酶A调节。目前尚不清楚cGMP/PKG信号是否可能调节ENaC活性。我们检测了非洲爪蟾卵母细胞中表达的α β γ-ENaC通道对8-（4-氯苯硫基）-cGMP（8-pCPT-cGMP）（一种细胞渗透性cGMP类似物）的反应。该化合物以剂量依赖性方式刺激人α β γ-ENaC活性，但不渗透细胞的cGMP没有影响。在表达小鼠或大鼠α β γ-ENaC通道的卵母细胞中观察到cGMP的类似刺激作用。8-pCPT-cGMP激活电流与α-β-γ-ENaC通道具有相同的离子选择性和阿米洛利敏感性，表明cGMP激活电流与表达的ENaC相关。β-苯基-1，N-2-乙烯基-8-溴-cGMP的PKGI激活剂Sp异构体未引起ENaC电流的升高，8-pCPT-cGMP诱导的ENaC通道激活可通过将卵母细胞与PKG抑制剂孵育而被阻断，但与PKA、MEK、MAP和PKC的其他cGMP敏感性激酶灭活剂孵育则无此作用。令人惊讶的是，这两个定点突变的推定共识PKG磷酸化位点和整个胞质NH 2-和COOH-末端尾部的截短没有改变8-pCPT-cGMP的响应。在使用小干扰RNA敲低PKGII表达的细胞中，8-pCPT-cGMP以相同程度激活ENaC活性。与8-CPT-cAMP类似，8-pCPT-cGMP能够以相同的方式在无细胞外向贴片中激活ENaC。我们的结论是，快速上调人α β γ ENaC活性的卵母细胞由外部8-pCPT-cGMP和4-chlorothiolphenol-cAMP依赖于parachlorophenylthiol和羟基基团，和8-pCPT-cGMP可能作为一个新的ENaC配体除了激活PKG信号。
Nie HG, Zhang W, Han DY, Li QN, Li J, Zhao RZ, Su XF, Peng JB, Ji HL. 8-pCPT-cGMP stimulates alpha beta gamma-ENaC activity in oocytes as an external ligand requiring specific nucleotide moieties. Am J Physiol Renal Physiol 298: F323-F334, 2010. First published December 9, 2009; doi: 10.1152/ajprenal.00307.2009.-Epithelial sodium channels (ENaC) are regulated by protein kinase A, in addition to a broad spectrum of other protein kinases. It is not clear whether cGMP/PKG signaling might regulate ENaC activity. We examined the responses of alpha beta gamma-ENaC channels expressed in Xenopus oocytes to 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP), a cell-permeable cGMP analog. This compound stimulated human alpha beta gamma-ENaC activity in a dose-dependent fashion, but cell-impermeable cGMP had no effect. Similar stimulatory effects of cGMP were observed in oocytes expressing either mouse or rat alpha beta gamma-ENaC channels. The identical ion selectivity and amiloride sensitivity of the 8-pCPT-cGMP-activated currents to those of alpha beta gamma-ENaC channels suggest that the cGMP-activated currents are associated with expressed ENaC. The PKGI activator Sp isomer of beta-phenyl-1,N-2-etheno-8-bromo-cGMP did not elicit a rise in ENaC current and that the 8-pCPT-cGMP-induced activation of ENaC channels was blocked by incubating oocytes with a PKG inhibitor, but not with other cGMP-sensitive kinase inactivators for PKA, MEK, MAP, and PKC. Surprisingly, both site-directed mutation of putative consensus PKG phosphorylation sites and truncation of entire cytosolic NH2- and COOH-terminal tails did not alter the response to 8-pCPT-cGMP. The ENaC activity was activated to the same extent by 8-pCPT-cGMP in cells in which PKGII expression was knocked down using small interfering RNA. Analog to 8-CPT-cAMP, 8-pCPT-cGMP was capable of activating ENaC in the identical manner in cell-free outside-out patches. We conclude that the rapid upregulation of human alpha beta gamma-ENaC activity in oocytes by external 8-pCPT-cGMP and 4-chlorothiolphenol-cAMP depends on the parachlorophenylthiol and the hydroxy groups, and 8-pCPT-cGMP may serve as a novel ENaC ligand in addition to activating PKG signal.