Proton-Lactate Cotransport in the Apical Membrane of Frog Retinal Pigment Epithelium

Abstract

We studied lactate- and pyruvate-dependent proton transport across the apical membrane of frog RPE. The epithelium was mounted in a modified Ussing-chamber that allowed measurement of transepithelial potential and resistance while intracellular pH was measured with either intracellular microelectrodes or a pH-sensitive dye, 2′,7′-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). To estimate the rate of lactate influx from the change in intracellular pH, we used the NH 4 pulse technique to measure intracellular buffering capacity and its dependence on intracellular pH. We found that the buffering capacity was 16 mm at pH i = 7·28, and that it increased as intracellular pH decreased. Intracellular pH was monitored with the tissue bathed in nominally HCO 3-free (Hepes buffered) Ringer. The perfusate on the apical side of the epithelium was then changed to a Ringer that contained between 5 and 100 mM lactate or pyruvate. When 10-100 mM lactate or pyruvate was added to the apical bath the cells acidified by 0·05-0·:50 pH units. For each of these acidifications, the initial acid influx into the RPE cells was calculated from the intracellular buffering capacity and the initial rate of intracellular acidification. These influxes were plotted as functions of the concentrations of lactate or pyruvate and this relationship was analysed using Michaelis-Menten kinetics. The K m values were: 33 ± 5 mM for lactate and 9 ± 3 mM for pyruvate. There were no differences in the rates of acid influx caused by L- or D-lactate. The rates of acidification caused by 50 mM apical L-lactate were reversibly reduced by 56% after apical administration of probenecid (2 mM), and irreversibly reduced by 63% after apical administration of the SH-reagent mersalyl acid (2 mM). These results indicate the presence of a proton-lactate cotransport system in the apical membrane of the frog RPE.