Stimulation of toad lens epithelial Na+/H+ exchange activity by hypertonicity

Incubation of toad lenses with the acetoxymethyl ester of 2',7'-biscarboxyethyl-5(6)-carboxy-fluorescein led to a highly selective accumulation of the de-esterified, pH-sensitive form of the dye in the epithelial cells, enabling the continuous fluorometric monitoring of epithelial intracellular pH (pHi) in intact lenses. The effects of changes in extralenticular Na+] and of amiloride-addition indicated that the epithelium contains an amiloride-sensitive Na+/H+ antiport. Exposure of lenses to hypertonic conditions (by the addition of sucrose to the medium) resulted in a biphasic change in pHi; a rapid initial, 'spike-like' decrease was immediately followed by a persistent reversal that raised pHi in CO2/HCO3- -rich and -free media by 0.13 and 0.18 units, respectively. Under CO2/HCO3- -free conditions, the hypertonic exposure raised pHi to a value near the calculated equilibrium position for a lens Na+/H+ exchanger. At this point, monensin addition did not affect pHi, suggesting that the tonicity shift had induced a rapid endogenous Na+/H+ exchange activity. In contrast, in the presence of 1 mM amiloride or in the absence of extralenticular Na+, sucrose addition induced only a persistent pHi decrease, which could be reversed (in the 'amiloride' case) by monensin addition. These results demonstrate that the hypertonic exposure induced an epithelial cell acidification as well as a stimulation of the Na+/H+ exchange activity which reverted the acidification. The hypertonic exposure also elicited pHi increases in lenses that had been preacidified by the 'NH4+ loading' or 'pCO2 raise' methods, indicating that the onset of the stimulation could not be attributed to a pHi decrease.(ABSTRACT TRUNCATED AT 250 WORDS)