ION SELECTIVITY OF VOLUME REGULATORY MECHANISMS PRESENT DURING A HYPOOSMOTIC CHALLENGE IN VESTIBULAR DARK CELLS

Volume regulation during a hypoosmotic challenge (RVD) in vestibular d ark cells from the gerbilline inner ear has previously been shown to d epend on the presence of cytosolic K+ and Cl-, suggesting that it invo lves KCl efflux. The aim of the present study was to characterize hypo osmotically-induced KCI transport under conditions where a hypoosmotic challenge causes KCI influx via the pathways normally used for efflux . Net osmolyte movements were monitored as relative changes in cell vo lume 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-diuretic s (piretanide or bumetanide) caused an increase in CH by about a facto r 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 hyp oosmotic challenge in the presence of 79 mM K+ and loop-diuretics, how ever, 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 swell ing. Secondary swelling depended on the presence of extracellular K+ a nd Cl- suggesting that it involved KCl influx followed by water. The i on selectivity of secondary swelling was K+ = Rb+ > Cs+ much greater t han Na+ = NMDG(+) and Cl- = NO3- = SCN- much greater than gluconate(-) . Secondary swelling was not inhibited by Ba2+, tetraethylammonium, qu inidine, lidocaine, amiloride, 4,4'-diisothiocyanatostilbene-2,2'-disu lfonic acid, 4-acetamido-4'-diisothiocyanatostilbene- acid, 4,4'-dinit rostilbene-2,2'-disulfonic acid, 5-nitro-2(3-phenylpropylamino)benzoic acid, acetazolamide, or ethoxyzolamide. These data define a profile o f the hypoosmotically-induced KCl transport pathways. The ion selectiv ity and the blocker insensitivity are consistent with the involvement of the apical slowly activating K+ channel (I-sk or minK channel) and the basolateral 360 pS Cl- channel. The involvement of these channels, however, remains to be demonstrated.