title: Evidence for Na-K-Cl cotransport in alveolar epithelial cells: effect of phorbol ester and osmotic stress.
abstract: We have investigated the presence of Na-K-Cl cotransport in alveolar type II cells using uptake of 86Rb. Several data support the presence of a Na-K-Cl cotransport in these cells. First, a large fraction of ouabain-resistant 86Rb uptake was inhibited by bumetanide and furosemide. Second, bumetanide-sensitive 86Rb uptake required the presence of Na+ and Cl- in the incubation medium; dependency on extracellular Na+ and K+ was hyperbolic, with a Km of 14.6 mM and 8.3 mM, respectively, while dependency on extracellular Cl- was sigmoidal, which suggests a 1:1:2 stoichiometry. Third, a fraction of amiloride-insensitive 22Na influx was deeply inhibited by bumetanide. 22Na influx was dependent on the presence of extracellular K+ and Cl-. Since Na-K-Cl activity dramatically decreased with time in culture, further characterization of the cotransport on polarized cells could not be performed. The phorbol ester PMA inhibited Na-K-Cl cotransport in a time- and concentration-dependent manner. This inhibition was mimicked by oleoylacetylglycerol, dioctanoylglycerol, and the diacylglycerol kinase inhibitor R59022, and was reversed by an antagonist of PKC, staurosporine. Since the Na-K-Cl cotransport has been reported to be involved in cell volume regulation, we investigated its modulation by changes in extracellular osmolarity. Na-K-Cl activity was increased after a two-step procedure: swelling in hypotonic medium followed by shrinking in hypertonic medium. Under these conditions, cotransport activity increased whenever PKC activity was up- or downregulated, which suggests that the cell volume-induced modulation of the cotransport is independent from the PKC activity. Though we were not able to determine the polarity of the cotransport, it may also be involved in the absorptive function of alveolar type II cells, and would provide an alternate pathway for sodium entry.
authors.affiliation: Department of Physiology, Faculté de Médecine Xavier Bichat, Paris, France.
authors.forename: C
authors.initials: C
authors.lastname: Clerici
authors.forename: S
authors.initials: S
authors.lastname: Couette
authors.forename: A
authors.initials: A
authors.lastname: Loiseau
authors.forename: P
authors.initials: P
authors.lastname: Herman
authors.forename: C
authors.initials: C
authors.lastname: Amiel
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D004848:Epithelium / Q000378:metabolism; D017136:Ion Transport / Q000187:drug effects; D008168:Lung / Q000378:metabolism*; D009997:Osmotic Pressure; D010703:Phorbol Esters / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012413:Rubidium; D012964:Sodium / Q000378:metabolism*
chemical_list: D002712:Chlorides; D010703:Phorbol Esters; D012964:Sodium; D012413:Rubidium; D011188:Potassium