The kinetics of transport of lactate and pyruvate into rat hepatocytes. Evidence for the presence of a specific carrier similar to that in erythrocytes.
Time courses of L-lactate and pyruvate uptake into isolated rat hepatocytes were measured in a citrate-based medium to generate a pH gradient (alkaline inside), by using the silicone-oil-filtration technique at 0 degrees C to minimize metabolism. At low concentrations of lactate and pyruvate (0.5 mM), transport was inhibited by over 95% by 5 mM-alpha-cyano-4-hydroxycinnamate, whereas at higher concentrations (greater than 10 mM) a significant proportion of transport could not be inhibited. The rate of this non-inhibitable transport was linearly related to the substrate concentration, was less with pyruvate than with L-lactate, and appeared to be due to diffusion of undissociated acid. Uptake of D-lactate was not inhibited by alpha-cyano-4-hydroxycinnamate and occurred only by diffusion. Kinetic parameters for the carrier-mediated transport process were obtained after correction of the initial rates of uptake of lactate and pyruvate in the absence of 5 mM-alpha-cyano-4-hydroxycinnamate by that in the presence of inhibitor. Under the conditions used, the Km values for L-lactate and pyruvate were 2.4 and 0.6 mM respectively and the Ki for alpha-cyano-4-hydroxycinnamate as a competitive inhibitor was 0.11 mM. Km values for the transport of L-lactate and pyruvate into rat erythrocytes under similar conditions were 3.0 and 0.96 mM. The Vmax. of lactate and pyruvate transport into hepatocytes at 0 degrees C was 3 nmol/min per mg of protein. Carrier-mediated transport of 0.5 mM-L-lactate was inhibited by 0.2 mM-p-chloromercuribenzenesulphonate (greater than 90%), 0.5 mM-quercetin (80%), 0.6 mM-isobutylcarbonyl-lactyl anhydride (70%) and 0.5 mM-4,4'-di-isothiocyanostilbene-2,2'-disulphonate (50%). A similar pattern of inhibition of lactate transport is seen in erythrocytes. It is suggested that the same or a similar carrier protein exists in both tissues. The results also show that L-lactate transport into rat hepatocytes is very rapid at physiological temperatures and is unlikely to restrict the rate of its metabolism. Differences between our results and those of Fafournoux, Demigne & Remesy [(1985) J. Biol. Chem. 260, 292-299] are discussed.
Edlund
G L
GL
Department of Biochemistry, University of Bristol, U.K.
Halestrap
A P
AP
D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002469:Cell Separation; D003373:Coumaric Acids / Q000494:pharmacology; D004912:Erythrocytes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007424:Intracellular Fluid / Q000378:metabolism; D007773:Lactates / Q000493:pharmacokinetics*; D019344:Lactic Acid; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D011773:Pyruvates / Q000493:pharmacokinetics*; D019289:Pyruvic Acid; D051381:Rats; D011919:Rats, Inbred Strains
D000963:Antimetabolites; D003373:Coumaric Acids; D007773:Lactates; D011773:Pyruvates; C007175:alpha-cyano-4-hydroxycinnamate; D019344:Lactic Acid; D019289:Pyruvic Acid