Intracellular pH regulation in basal corneal epithelial cells measured in corneal explants: Characterization of [formula omitted] exchange

Abstract

Intracellular pH (pH i) was measured in basal corneal epithelial cells from fresh corneal explants using the pH sensitive fluorescent dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The overlying superficial and wing cells were removed by mechanical scraping to expose basal cells attached to their basal lamina. Tissue pieces with attached, dye-loaded basal cells were mounted in a microscope-stage-perfusion chamber which allowed rapid changes of Ringer's bathing solutions while measuring BCECF fluorescence. In NaCl-Ringer's (bicarbonate free), pH o 7·40, resting cell pH 1 was 7·34±0·03 (± s.e.m., n = 31). Buffering capacity measured by NH 4Cl treatment was 31 m m pH at pH i 7·34 and increased with decreasing pH i. Recovery from 20 m m NH 4Cl-induced acid loads was dependent on the presence of Na and inhibited by 1 m m amiloride. Adding amiloride to resting cells caused a slow, reversible acidification (0·04 pH units min −1). These results indicate the presence of Na : H exchange, its role in responding to acid loads and in maintaining resting cell pH i. Activation of Na : H by Na o showed simple saturation kinetics, with K m = 44 m m. Net proton efflux via Na : H exchange increased with decreasing pH i and was enhanced by depleting cells of Na i suggesting roles for both pH i and Na i in control of Na : H activation.