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Abstract
PURPOSE: To study transport mechanisms for small monocarboxylic acids in the apical and basolateral membranes of freshly isolated, human fetal retinal pigment epithelium. METHODS: The epithelium was mounted in a small Ussing chamber that allowed separate perfusion of both the apical and basal compartments and simultaneous measurements of intracellular pH, transepithelial potential, and tissue resistance. Intracellular pH was measured using a pH-sensitive dye, 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein. RESULTS: When 10-100 mM lactate or pyruvate was added to the apical bath the cells acidified by 0.10-0.25 pH units. There were no differences between the initial rates of intracellular acidification produced by L-lactate and D-lactate. These rates could be described as Michaelis-Menten functions of the concentrations of lactate and pyruvate. The Km values were 42 +/- 12 mM for L-lactate and 34 +/- 8 mM for pyruvate. The rates of acidification caused by 50 mM L-lactate were reversibly reduced by 44% or 35% after apical administration of probenecid (2 mM) or alpha-cyano-4-hydroxycinnamate (2 mM), and irreversibly reduced by 78% after apical administration of the sulfhydryl-reagent mersalyl acid (2 mM). The intracellular acidifications caused by apical pyruvate (50 mM) were completely and reversibly inhibited by 50 mM apical L-lactate. Addition of 50 to 100 mM lactate to the basal bath caused intracellular alkalinizations, which could be inhibited by Na+ removal in the basal bath or by 2 mM alpha-cyano-4-hydroxycinnamate in the apical bath.