Ion selectivity of volume regulatory mechanisms present during a hypoosmotic challenge in vestibular dark cells

Abstract

Volume regulation during a hypoosmotic challenge (RVD) in vestibular dark cells from the gerbilline inner ear has previously been shown to depend on the presence of cytosolic K+ and Cl-, suggesting that it involves KCl efflux. The aim of the present study was to characterize hypoosmotically-induced KCl transport under conditions where a hypoosmotic challenge causes KCl influx via the pathways normally used for efflux. Net osmolyte movements were monitored as relative changes in cell volume measured as epithelial cell height (CH). A hypoosmotic challenge (298 to 154 mosM) in the presence of 3.6 or 25 mM K+ and loop-diuretics (piretanide or bumetanide) caused an increase in CH by about a factor of 1.2 presumably due to the net effect of primary swelling defined as osmotic dilution of the cytosol and RVD involving KCl efflux. A hypoosmotic challenge in the presence of 79 mM K+ and loop-diuretics, however, caused CH to increase by a factor of over 2.4. Presumably, this large increase in CH was due to the sum of primary and secondary swelling. Secondary swelling depended on the presence of extracellular K+ and Cl- suggesting that it involved KCl influx followed by water. The ion selectivity of secondary swelling was K+ = Rb+ > Cs+ >> Na+ = NMDG+ and Cl- = NO3- = SCN- >> gluconate-. Secondary swelling was not inhibited by Ba2+, tetraethylammonium, quinidine, lidocaine, amiloride, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, 4-acetamido-4'-diisothiocyanatostilbene-2,2'-disulfonic acid, 4,4'-dinitrostilbene-2,2'-disulfonic acid, 5-nitro-2(3-phenylpropylamino)benzoic acid, acetazolamide, or ethoxyzolamide. These data define a profile of the hypoosmotically-induced KCl transport pathways. The ion selectivity and the blocker insensitivity are consistent with the involvement of the apical slowly activating K+ channel (IsK or minK channel) and the basolateral 360 pS Cl- channel. The involvement of these channels, however, remains to be demonstrated.