PROTON LACTATE COTRANSPORT IN THE APICAL MEMBRANE OF FROG RETINAL-PIGMENT EPITHELIUM

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 intrace llular microelectrodes or a pH-sensitive dye, 2',7'-bis(2-carboxyethyl )-5,6-carboxyfluorescein (BCECF). To estimate the rate of lactate infl ux from the change in intracellular pH, we used the NH4 pulse techniqu e to measure intracellular buffering capacity and its dependence on in tracellular pH. We found that the buffering capacity was 16 mM at pH(i ) = 7.28, and that it increased as intracellular pH decreased. Intrace llular pH was monitored with the tissue bathed in nominally HCO3-free (Hepes buffered) Ringer. The perfusate on the apical side of the epith elium 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 r ate of intracellular acidification. These influxes were plotted as fun ctions of the concentrations of lactate or pyruvate and this relations hip 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 di fferences in the rates of acid influx caused by L- or D-lactate. The r ates 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-rea gent mersalyl acid (2 nM). These results indicate the presence of a pr oton-lactate cotransport system in the apical membrane of the frog RPE .