K+-induced swelling of vestibular dark cells is dependent on Na+ and Cl? and inhibited by piretanide

Abstract

Epithelial cell height was measured in order to estimate the cell volume of dark cells from the ampullae of the semicircular canal of the gerbil. Under control conditions, addition of 10(-4) mol/l piretanide, 10(-5) mol/l 5-nitro-2(3-phenylpropylamino)-benzoic acid (NPPB), 5 mmol/l barium or 10(-3) mol/l quinidine had no significant effect on cell height. Addition of 10(-4) mol/l NPPB or 10(-3) mol/l ouabain led to a small significant decrease in cell height which was not reversible. Substitution of Na+ by N-methyl-D-glucamine or of Cl- by gluconate led to a significant and reversible reduction in cell height. Isotonic elevation of [K+] from 3.6 to 25 mmol/l in a PO4-buffered, HCO3-free solution led to an increase in cell height from 5.8 +/- 0.1 (SEM) to 8.7 +/- 0.2 microns (n = 62) during the first 40 s. During prolonged exposure to elevated [K+] (3-5 min; n = 19), some tissue samples underwent a regulatory volume decrease. K(+)-induced swelling was absent in both isotonic Cl(-)-free and isotonic Na(+)-free solutions and was inhibited by the loop diuretic piretanide (10(-5) and 10(-4) mol/l) or by the (Na+ + K+) ATPase inhibitor ouabain (10(-3) mol/l) or by 10(-4) mol/l NPPB. After the removal of ouabain or 10(-4) mol/l NPPB, K(+)-induced swelling under control conditions was enhanced and was less reversible as compared to control conditions before the experiment. K(+)-induced swelling was not altered by NPPB (10(-5) mol/l) or barium (5 mmol/l); however, barium slowed shrinking upon return of [K+] to control level. In the presence of 10(-3) mol/l quinidine, K(+)-induced swelling was enhanced and not reversible. These data suggest that dark cells from the semicircular canal possess an Na+2Cl-K+ cotransporter as a solute uptake mechanism and a solute efflux mechanism which is sensitive to barium and inhibited by quinidine.