Regulation of Epithelial Na+ Transport by Soluble Adenylyl Cyclase in Kidney Collecting Duct Cells
Regulation of Epithelial Na+ Transport by Soluble Adenylyl Cyclase in Kidney Collecting Duct Cells
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DOI：
10.1074/jbc.m805501200
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发表时间：
2009-02-27
影响因子：
4.8
通讯作者：
Pastor-Soler, Nuria M.
中科院分区：
文献类型：
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作者：
Hallows, Kenneth R.;Wang, Huamin;Pastor-Soler, Nuria M.
碱中毒损害利尿剂的利钠反应，但其潜在机制尚不清楚。可溶性腺苷酸环化酶（sAC）是一种化学传感器，介导细胞内微域中碳酸氢盐依赖性cAMP的升高。我们推测sAC可能是肾内Na+转运的重要调节因子。大鼠肾脏共聚焦图像显示sAC在收集管细胞中特异性免疫定位，免疫印迹证实sAC在小鼠皮质收集管（mpkCCD(c14)）细胞中表达。这些细胞表现出醛固酮刺激的跨上皮Na+电流，这种电流依赖于顶端上皮Na+通道（ENaC）和基底外侧Na+， K+- atp酶。RNA干扰介导的60-70%的sAC表达下调可明显抑制mpkCCD（c14）细胞的基底上皮短路电流（I-sc）。此外，sAC抑制剂KH7和2-羟基雌二醇在30分钟内降低了这些细胞中50-60%的I-sc。8-溴腺苷-3',5'-环单磷酸基本上恢复了KH7对经上皮Na+电流的抑制。醛固酮在4小时内使enact依赖的Isc增加一倍，在KH7存在时这种效应被消除。sAC对Isc的贡献不受制氨抑素介导的根尖膜通透性的影响，而sAC依赖的Na+电流被基底外侧乌巴因完全抑制，这表明Na+， K+- atp酶，而不是ENaC，是sAC的相关转运蛋白靶点。事实上，sAC过表达和KH7处理都不能调节非洲爪蟾卵母细胞中的ENaC电流。mpkCCDc14细胞的atp酶和生物素化实验表明，sAC抑制降低了Na+， K+- atp酶的催化活性，而不是表面表达。综上所述，这些结果表明，sAC调节基础和激动剂刺激的肾收集管Na+重吸收，从而增强Na+， K+- atp酶活性。
Alkalosis impairs the natriuretic response to diuretics, but the underlying mechanisms are unclear. The soluble adenylyl cyclase (sAC) is a chemosensor that mediates bicarbonate-dependent elevation of cAMP in intracellular microdomains. We hypothesized that sAC may be an important regulator of Na+ transport in the kidney. Confocal images of rat kidney revealed specific immunolocalization of sAC in collecting duct cells, and immunoblots confirmed sAC expression in mouse cortical collecting duct (mpkCCD(c14)) cells. These cells exhibit aldosterone-stimulated transepithelial Na+ currents that depend on both the apical epithelial Na+ channel (ENaC) and basolateral Na+, K+-ATPase. RNA interference-mediated 60-70% knockdown of sAC expression comparably inhibited basal transepithelial short circuit currents (I-sc) in mpkCCD(c14) cells. Moreover, the sAC inhibitors KH7 and 2-hydroxyestradiol reduced I-sc in these cells by 50-60% within 30 min. 8-Bromoadenosine-3',5'-cyclic-monophosphate substantially rescued the KH7 inhibition of transepithelial Na+ current. Aldosterone doubled ENaC-dependent Isc over 4 h, an effect that was abolished in the presence of KH7. The sAC contribution to Isc was unaffected with apical membrane nystatin-mediated permeabilization, whereas the sAC-dependent Na+ current was fully inhibited by basolateral ouabain treatment, suggesting that the Na+, K+-ATPase, rather than ENaC, is the relevant transporter target of sAC. Indeed, neither overexpression of sAC nor treatment with KH7 modulated ENaC currents in Xenopus oocytes. ATPase and biotinylation assays in mpkCCDc14 cells demonstrated that sAC inhibition decreases catalytic activity rather than surface expression of the Na+, K+-ATPase. In summary, these results suggest that sAC regulates both basal and agonist-stimulated Na+ reabsorption in the kidney collecting duct, acting to enhance Na+, K+-ATPase activity.