THE KINETICS, SUBSTRATE, AND INHIBITOR SPECIFICITY OF THE MONOCARBOXYLATE (LACTATE) TRANSPORTER OF RAT-LIVER CELLS DETERMINED USING THE FLUORESCENT INTRACELLULAR PH INDICATOR, 2',7'-BIS(CARBOXYETHYL)-5(6)-CARBOXYFLUORESCEIN

The kinetics of transport of L lactate, pyruvate, ketone bodies, and o ther monocarboxylates into isolated hepatocytes from starved rats were measured at 25 degrees C using the intracellular pH-sensitive dye, 2' ,7'-bis(carboxyethyl) 5(6)-carboxyfluorescein, to detect the associate d proton influx. Transport kinetics were similar, but not identical, t o those determined using the same technique for the monocarboxylate tr ansporter (MCT) of Ehrlich Lettre tumor cells (MCT1) (Carpenter, L., a nd Halestrap, A. P. (1994) Biochem. J. 304, 751-760). K-m values for L -lactate (4.7 mM), D-lactate (27 mM), D,L-2-hydroxybutyrate (3.3 mM), L-3-hydroxybutyrate (12.7 mM), and acetoacetate (6.1 mM) were very sim ilar in both cell types, whereas in hepatocytes the K-m values were hi gher than MCT1 for pyruvate (1.3 mM, cf. 0.72 mM), D-3-hydroxybutyrate (24.7 mM, cf. 10.1 mM), L-2-chloropropionate (1.3 mM, cf. 0.8 mM), 4- hydroxybutyrate (18.1 mM, cf. 7.7 mM), and acetate (5.4 mM, cf. 3.7 mM ). In contrast, the hepatocyte carrier had lower K-m values than MCT1 for glycolate, chloroacetate, dichloroacetate, and 2-hydroxy-2-methylp ropionate. Differences in stereoselectivity were also detected; both c arriers showed a lower K-m for L-actate than D-lactate, while hepatocy te MCT exhibited a lower K-m for D- than L-2-chloropropionate and for L- than D-3-hydroxybutyrate; this is not the case for MCT1. A range of inhibitors of MCT1, including alpha-cyanocinnamate derivatives, phlor etin, and niflumic acid, inhibited hepatocyte MCT with K-0.5 values si gnificantly higher than for tumor cell MCT1, while stilbene disulfonat e derivatives and p-chloromercuribenzene sulfonate had similar K-0.5 v alues in both cell types. The branched chain ketoacids alpha-ketoisoca proate and alpha-ketoisovalerate were also potent inhibitors of hepato cyte MCT with K-0.5 values of 270 and 340 mu M, respectively. The acti vation energy of L-lactate transport into hepatocytes was 58 kJ mol(-1 ), and measured rates of transport at 37 degrees C were considerably g reater than those required for maximal rates of gluconeogenesis. The p roperties of the hepatocyte monocarboxylate transporter are consistent with the presence of a distinct isoform of MCT in liver cells as sugg ested by the cloning and sequencing of MCT2 from hamster liver (Garcia , C. K., Brown, M. S., Pathak, R. K., and Goldstein, J. L. (1995) J. B iol. Chem. 270, 1843-1849).