title: Regulation of epithelial Na+ transport by soluble adenylyl cyclase in kidney collecting duct cells.
abstract: 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.
authors.affiliation: Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15621, USA. hallows@pitt.edu
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: Hallows
authors.forename: Huamin
authors.initials: H
authors.lastname: Wang
authors.forename: Robert S
authors.initials: RS
authors.lastname: Edinger
authors.forename: Michael B
authors.initials: MB
authors.lastname: Butterworth
authors.forename: Nicholas M
authors.initials: NM
authors.lastname: Oyster
authors.forename: Hui
authors.initials: H
authors.lastname: Li
authors.forename: Jochen
authors.initials: J
authors.lastname: Buck
authors.forename: Lonny R
authors.initials: LR
authors.lastname: Levin
authors.forename: John P
authors.initials: JP
authors.lastname: Johnson
authors.forename: Núria M
authors.initials: NM
authors.lastname: Pastor-Soler
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D000450:Aldosterone / Q000494:pharmacology; D000818:Animals; D019921:Biotinylation; D002478:Cells, Cultured; D053503:Epithelial Sodium Channels / Q000378:metabolism; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism*; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D017136:Ion Transport; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000378:metabolism*; D026941:Sodium Channel Blockers / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D000067956:Adenylyl Cyclase Inhibitors; D053503:Epithelial Sodium Channels; D034741:RNA, Small Interfering; D026941:Sodium Channel Blockers; C036038:8-bromoadenosine; D000450:Aldosterone; D004958:Estradiol; D012964:Sodium; C001390:2-hydroxyestradiol; D000262:Adenylyl Cyclases; D000254:Sodium-Potassium-Exchanging ATPase; D000241:Adenosine