Stimulation of toad lens epithelial [formula omitted] exchange activity by hypertonicity

Abstract

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 (pH i) 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 pH i; a rapid initial, ‘spike-like’ decrease was immediately followed by a persistent reversal that raised pH i in CO 2 HCO 3 − -rich and -free media by 0·13 and 0·18 units, respectively. Under CO 2 HCO 3 − -free conditions, the hypertonic exposure raised pH 1 to a value near the calculated equilibrium position for a lens Na + H + exchanger. At this point, monensin addition did not affect pH 1, suggesting that the tonicity shift had induced a rapid endogenous Na + H + exchange activity. In contrast, in the presence of 1 m m amiloride or in the absence of extralenticular Na +, sucrose addition induced only a persistent pH i 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 pH i increases in lenses that had been preacidified by the ‘NH 4 + loading’ or ‘pCO 2 raise’ methods, indicating that the onset of the stimulation could not be attributed to a pH i decrease. The cellular phenomena that trigger the stimulation are unknown, however; since a hypertonic exposure will initially generate a cell volume loss, the stimulation may be related to the homeostatic control of cell volume.