Regulation of Epithelial Na+ Transport by Soluble Adenylyl Cyclase in Kidney Collecting Duct Cells
| Autor: | Huamin Wang, Núria M. Pastor-Soler, Robert S. Edinger, Nicholas M. Oyster, John P. Johnson, Jochen Buck, Kenneth R. Hallows, Michael B. Butterworth, Lonny R. Levin, Hui Li |
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| Rok vydání: | 2009 |
| Předmět: |
Male
Epithelial sodium channel medicine.medical_specialty Adenosine ATPase Immunoblotting Fluorescent Antibody Technique Biochemistry Epithelium Ouabain Rats Sprague-Dawley Xenopus laevis chemistry.chemical_compound Internal medicine medicine Animals Biotinylation Kidney Tubules Collecting RNA Small Interfering Epithelial Sodium Channels education Aldosterone Molecular Biology Cells Cultured Ion transporter education.field_of_study Ion Transport Estradiol biology Reabsorption Sodium Cell Biology Apical membrane Soluble adenylyl cyclase Rats Cell biology Membrane Transport Structure Function and Biogenesis Endocrinology chemistry Adenylyl Cyclase Inhibitors Oocytes biology.protein Sodium-Potassium-Exchanging ATPase Adenylyl Cyclases Sodium Channel Blockers medicine.drug |
| Zdroj: | Journal of Biological Chemistry. 284:5774-5783 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m805501200 |
| Popis: | 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 I(sc) over 4 h, an effect that was abolished in the presence of KH7. The sAC contribution to I(sc) 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 mpkCCD(c14) 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. |
| Databáze: | OpenAIRE |
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