VESTIBULAR DARK CELLS CONTAIN THE NA+ H+ EXCHANGER NHE-1 IN THE BASOLATERAL MEMBRANE/

Vestibular dark cells and strial marginal cells transport K+ by simila r mechanisms. We have shown that K+ transport in vestibular dark cells is sensitive to the cytosolic pH (pH(i)) (Wangemann et al. (1995a): J . Membrane Biol. 147: 255-262). The present study addresses pharmacolo gically the questions whether vestibular dark cells and strial margina l cells from the gerbil contain a Na+/H+ exchanger (NHE) and in which membrane, apical or basolateral, NHE is located. Further, the study ad dresses the question which NHE subtype is present in vestibular dark c ells. pH(i) was measured micro-fluorometrically with the pH-sensitive dye 2',7'-bicarboxyethyl-5(6)-carboxyfluorescein (BCECF), cell volume which is a measure of the net balance between ion influx and efflux wa s monitored as cell height (CH) and the equivalent short circuit curre nt (I-sc) which is a measure of transepithelial K+ secretion was calcu lated from measurements of the transepithelial voltage (V-t) and the t ransepithelial resistance (R(t)). Changes in pH(i) were induced by 20 or 40 mM propionate. In the presence of propionate a transient acidifi cation of pH(i) was observed in vestibular dark cells as well as a sub sequent alkalization to pH(i) values exceeding those under control con ditions. The alkalization of pH(i) in the presence of propionate was i nhibited by the NHE blockers amiloride and EIPA. Proionate-induced swe lling of vestibular dark cells was inhibited by amiloride. The NHE blo cker amiloride cased in vestibular dark cells an acidification of pH(i ) and a decrease in CH. Amiloride caused in both vestibular dark cells and strial marginal cells a transient stimulation of I-sc when added to the basolateral side but not when added to the apical side. Similar effects on pH(i) were observed in vestibular dark cells with the amil oride analog ethyl-isopropyl-amiloride (EIPA) and similar effects on I -sc were observed with EIPA and the NHE blocker HOE694 when applied to the basolateral side of vestibular dark cells epithelium. The IC50 fo r these basolaterial effects of EIPA, HOE694 and amiloride on I-sc in vestibular dark cells were 2 X 10(-7) M, 8 X 10(-7) M and 4 X 10(-5) M . These observations suggest that vestibular dark cells and strial mar ginal cells contain NHE in their basolateral membrane, that K+ transpo rt in strial marginal cells is pH(i) sensitive similar to K+ transport in vestibular dark cells and that NHE in vestibular dark cells consis ts of the subtype NHE-1.