The Na+/H+ exchanger in transitional cells of the inner ear

The cytosolic pH (pHi) of transitional cells from the ampulla of the gerbil was measured micro-fluorometrically with the pH-sensitive dye 2',7'-bicarboxyethyl-5(6)-carboxyfluorescein (BCECF) to assess the possible contribution of a Na+/H+ exchanger to the regulation of pHi. All experiments were conducted in virtually HCO3(-)-free solutions. Under control conditions, pHi was 7.19 and addition of 10(-5) M ethylisopropylamiloride (EIPA), a blocker of Na+/H+ exchange, caused a small but significant acidification of pHi. A transient exposure to 21.4 mM NH4Cl caused a rapid cytosolic alkalinization followed by a brisk acidification and prompt recovery of pHi to its control value. The cytosolic buffer capacity (Bi) was determined in the absence of Na+ from changes in pHi which were elicited by [NH4+] steps. Bi was 4.7 mM/pH at pHi 7.19 and varied with pHi. The initial net proton flux JH, representative of Na+/H+ exchange activity, was calculated from the product of the initial rate of alkalinization after an NH4(+)-prepulse and Bi at the corresponding pHi. JH was dependent on the extracellular Na+ with an apparent Km of 64 mM, sensitive to the cytosolic [H+] with an apparent Km of 2.7 * 10(-7) M (i.e. pH 6.6), and inhibited by EIPA with an IC50 of 5 * 10(-7) M. These data suggest that transitional cells contain in the basolateral membrane a Na+/H+ exchanger of the amiloride-sensitive subtype.