TRANSPORT OF PYRUVATE NAD LACTATE INTO HUMAN ERYTHROCYTES - EVIDENCE FOR INVOLVEMENT OF CHLORIDE CARRIER AND A CHLORIDE-INDEPENDENT CARRIER

The kinetics and activation energy of entry of pyruvate and lactate into the erythrocyte were studied at concentrations below 4 and 15 mM, respectively. The Km and Vmax values for both substrates showed that pyruvate inhibited competitively with respect to lactate and vice versa. In both cases the Km for the carboxylate as a substrate was the same as its Ki as an inhibitor. .alpha.-Cyano-4-hydroxycinnamate and its analogues inhibited the uptake of both lactate and pyruvate competitively. Inhibition was also produced by treatment of cells with fluorodinitrobenzene but not with the thiol reagents or Pronase. At high concentrations of pyruvate or lactate (20 mM), uptake of the carboxylate was accompanied by an efflux of Cl- ions. This efflux of Cl- was inhibited by .alpha.-cyano-4-hydroxycinnamate and picrate and could be totally abolished by very low (< 10 .mu.M) concentrations of the inhibitor of Cl- transport, 4,4''-di-isothiocyanostilbene-2,2''-di-sulfonic acid. This inhibitor titrated out the chloride efflux induced by pyruvate, bicarbonate, formate and fluoride, in each case total inhibition becoming apparent when approximately 1.2 .times. 106 molecules of inhibitor were present per erythrocyte, that is, about one inhibitor molecule per molecule of the Cl- carrier. Even when Cl- efflux was totally blocked pyruvate and lactate uptake occurred. Kinetic evidence was presented which suggests that the Cl- carrier can transport pyruvate and lactate with a high Km and high Vmax, but that an additional carrier with a low Km and a low Vmax also existed. This carrier catalyzed the exchange of small carboxylate anions with intracellular lactate, was competitively inhibited by .alpha.-cyano-4-hydroxycinnamate and non-competitively inhibited by picrate. The Cl- carrier showed a reverse pattern of inhibition. Net efflux of lactic acid from the cell must occur on the Cl- carrier and involve exchange with HCO3- followed by loss of CO2. The low Km carrier might be used in pyruvate/lactate or acetoacetate/.beta.-hydroxybutyrate exchanges involved in transferring reducing power across the cell membrane. The possibility that the Cl- carrier exists in cells other than the erythrocyte is discussed. Its presence in other cell membranes together with a low intracellular Cl- concentration would probably explain why the pH in the cytoplasm is lower than that of the blood, and why permeable carboxylate anions do not accumulate within the cell when added from outside.