Specificity of the Na+-dependent monocarboxylic acid transport pathway in rabbit renal brush border membranes

The substrate specificity of a Na+-dependent transport pathway for L-lactate was studied in rabbit renal brush border membrane vesicles. Jmax for L-lactate transport was unaffected by the presence of a fixed concentration of two different short-chain monocarboxylic acids, while the apparent Kt(Ka) for L-lactate increased, and this is compatible with competitive inhibition. The inhibitor constants ("Ki"'s) for the transport pathway for the two solutes examined closely corresponded to the respective "Ki"'s derived from a Dixon plot. A broad range of compounds were then tested as potential inhibitors of L-lactate transport, and the "Ki"'s thereby derived yielded specific information regarding optimal substrate recognition by the carrier. A single carboxyl group is an absolute requirement for recognition, and preference is given to 3 to 6 C chain molecules. Addition of ketone, hydroxyl and, particularly, amine groups at any carbon position, diminishes substrate-carrier interaction. Intramolecular forces, notably the inductive effects of halogens, may play a role in enhancing substrate-carrier interaction; however, no correlation was found between pKa and "Ki" for the substrates examined. We conclude that a separate monocarboxylic acid transport pathway, discrete from either the D-glucose, alpha or beta neutral amino-acid, or dicarboxylic acid carriers, exists in the renal brush border, and this handles a broad range of monocarboxylates.     

Carrier-mediated uptake of lactate in rat hepatocytes. Effects of pH and possible mechanisms for L-lactate transport

The rate of uptake and the distribution ratio between intra- and extracellular compartments of L- and D-lactate were studied in hepatocyte preparations from fed rats. L- and D-lactate uptake apparently depended on both passive diffusion and carrier-mediated components. The apparent Km of the high-affinity carrier for L-lactate was in the range of 1.8 mM. The reciprocal competitive inhibitions between isomers of lactate suggest that L- and D-lactate might be transported by distinct carriers. Lactate transport was inhibited by various anions; pyruvate was the most potent anion, whereas only high concentrations of ketone bodies were effective. Acidic extracellular pH enhanced lactate uptake, this effect being more pronounced for L-lactate. At low pH, L-lactate was concentrated into hepatocytes, but its affinity for the carrier appeared unchanged, suggesting the existence of a process gaining energy from the pH gradient across the cell membrane. In the hypothesis of a lactate/H+ symport, the affinity for H+ was not dependent on lactate concentration and the apparent Km for H+ corresponded to a pH of 7.34. No trans-stimulation of lactate uptake after prior loading of the cells with pyruvate or lactate was observed. The present data suggest that, at physiological concentrations, lactate uptake by the liver might be largely carrier-mediated and the rate of transport across the liver cell membrane may be of a magnitude relatively comparable to the rate of metabolism.     

The kinetics of transport of lactate and pyruvate into isolated cardiac myocytes from guinea pig. Kinetic evidence for the presence of a carrier distinct from that in erythrocytes and hepatocytes.

1. Time courses for the uptake of L-lactate, D-lactate and pyruvate into isolated cardiac ventricular myocytes from guinea pig were determined at 11 degrees C or 0 degrees C (for pyruvate) in a citrate-based buffer by using a silicone-oil-filtration technique. These conditions enabled initial rates of transport to be measured without interference from metabolism of the substrates. 2. At a concentration of 0.5 mM, transport of all these substrates was inhibited by approx. 90% by 5 mM-alpha-cyano-4-hydroxycinnamate; at 10 mM-L-lactate a considerable portion of transport could not be inhibited. 3. Initial rates of L-lactate and pyruvate uptake in the presence of 5 mM-alpha-cyano-4-hydroxycinnamate were linearly related to the concentration of the monocarboxylate and probably represented diffusion of the free acid. The inhibitor-sensitive component of uptake obeyed Michaelis-Menten kinetics, with Km values for L-lactate and pyruvate of 2.3 and 0.066 mM respectively. 4. Pyruvate and D-lactate inhibited the transport of L-lactate, with Ki values (competitive) of 0.077 and 6.6 mM respectively; the Ki for pyruvate was very similar to its Km for transport. The Ki for alpha-cyano-4-hydroxycinnamate as a non-competitive inhibitor was 0.042 mM. 5. These results indicate that L-lactate, D-lactate and pyruvate share a common carrier in guinea-pig cardiac myocytes; the low stereoselectivity for L-lactate over D-lactate and the high affinity for pyruvate distinguish it from the carrier in erythrocytes and hepatocytes. The metabolic roles for this novel carrier in heart are discussed.     

A mitochondrial monocarboxylate transporter in rat liver and heart and its possible function in cell control.

Several hydroxy- and keto-substituted monocarboxylates were found to undergo co- as well as counter-exchange across the mitochondrial membrane. The results argue against a simple Donnan system and may be explained by the existence of a transporter for monocarboxylates. In support of this explanation it was apparently possible to 'pump' pyruvate to the sucrose-inaccessible space by using the dicarboxylate transporter. Further, several aromatic and aliphatic analogues of pyruvate, but not of di- or tri-carboxylate transport inhibitors, have been shown to prevent pyruvate-exchange reactions. Palmitoylcarnitine was found to have a much stronger affinity for the carrier than either carnitine or pyruvate and the possible consequences of this for carnitine-palmitoylcarnitine exchange and on the control of the pyruvate dehydrogenase complex are explored. In view of the range of transport inhibitors and substrates it is suggested that the carrier has a fairly broad specificity. 'Inhibitor-stop' kinetic studies show that the speed of translocation of pyruvate at 1 degrees C is of the same order as malate. The possible correlation between the role of a hydroxy-keto acid transporter in substrate exchange and some whole animal experiments is briefly discussed. It is proposed that for reasons of control the cell will require membrane monocarboxylate transporters no less than di- or tri-carboxylate carriers.     

Transport of organic anions by the lysosomal sialic acid transporter: a functional approach towards the gene for sialic acid storage disease

Transport of sialic acid through the lysosomal membrane is defective in the human sialic acid storage disease. The mammalian sialic acid carrier has a wide substrate specificity for acidic monosaccharides. Recently, we showed that also non-sugar monocarboxylates like L-lactate are substrates for the carrier. Here we report that other organic anions, which are substrates for carriers belonging to several anion transporter families, are recognized by the sialic acid transporter. Hence, the mammalian system reveals once more novel aspects of solute transport, including sugars and a wide array of non-sugar compounds, apparently unique to this system. These data suggest that the search for the sialic acid storage disease gene can be initiated by a functional selection of genes from a limited number of anion transporter families. Among these, candidates will be identified by mapping to the known sialic acid storage disease locus.     

An endogenous monocarboxylate transport in Xenopus laevis oocytes

We investigated the existence of an endogenous system for lactate transport in Xenopus laevis oocytes. (36)Cl-uptake studies excluded the involvement of a DIDS-sensitive anion antiporter as a possible pathway for lactate movement. L-[(14)C]lactate uptake was unaffected by superimposed pH gradients, stimulated by the presence of Na(+) in the incubating solution, and severely reduced by the monocarboxylate transporter inhibitor p-chloromercuribenzenesulphonate (pCMBS). Transport exhibited a broad cation specificity and was cis inhibited by other monocarboxylates, mostly by pyruvate. These results suggest that lactate uptake is mediated mainly by a transporter and that the preferred anion is pyruvate. [(14)C]pyruvate uptake exhibited the same pattern of functional properties evidenced for L-lactate. Kinetic parameters were calculated for both monocarboxylates, and a higher affinity for pyruvate was revealed. Various inhibitors of monocarboxylate transporters reduced significantly pyruvate uptake. These studies demonstrate that Xenopus laevis oocytes possess a monocarboxylate transport system that shares some functional features with the members of the mammalian monocarboxylate cotransporters family, but, in the meanwhile, exhibits some particular properties, mainly concerning cation specificity.     

Transport and metabolism of L-lactate occur in mitochondria from cerebellar granule cells and are modified in cells undergoing low potassium dependent apoptosis

Having confirmed that externally added L-lactate can enter cerebellar granule cells, we investigated whether and how L-lactate is metabolized by mitochondria from these cells under normal or apoptotic conditions. (1) L-lactate enters mitochondria, perhaps via an L-lactate/H+ symporter, and is oxidized in a manner stimulated by ADP. The existence of an L-lactate dehydrogenase, located in the inner mitochondrial compartment, was shown by immunological analysis. Neither the protein level nor the Km and Vmax values changed en route to apoptosis. (2) In both normal and apoptotic cell homogenates, externally added L-lactate caused reduction of the intramitochondrial pyridine cofactors, inhibited by phenylsuccinate. This process mirrored L-lactate uptake by mitochondria and occurred with a hyperbolic dependence on L-lactate concentrations. Pyruvate appeared outside mitochondria as a result of external addition of L-lactate. The rate of the process depended on L-lactate concentration and showed saturation characteristics. This shows the occurrence of an intracellular L-lactate/pyruvate shuttle, whose activity was limited by the putative L-lactate/pyruvate antiporter. Both the carriers were different from the monocarboxylate carrier. (3) L-lactate transport changed en route to apoptosis. Uptake increased in the early phase of apoptosis, but decreased in the late phase with characteristics of a non-competitive like inhibition. In contrast, the putative L-lactate/pyruvate antiport decreased en route to apoptosis with characteristics of a competitive like inhibition in early apoptosis, and a mixed non-competitive like inhibition in late apoptosis.     

Pyruvate transport across the plasma membrane of the bloodstream form of Trypanosoma brucei is mediated by a facilitated diffusion carrier.

The characteristics of pyruvate transport across the plasma membrane in the bloodstream form of Trypanosoma brucei were studied using [14C]pyruvate in combination with the silicone-oil centrifugation technique. We present evidence for the existence of a facilitated diffusion carrier in the plasma membrane of T. brucei which specifically mediates the translocation of pyruvate. The uptake of pyruvate followed saturation kinetics (Km 1.96 +/- 0.28 mM; Cmax 36.61 +/- 1.15 nmol pyruvate/30 sec.mg protein), after correction of the data for a nonsaturable diffusion component. The uptake of pyruvate was competitively inhibited by a number of (oxo)monocarboxylic acids, including pyruvate analogs and metabolically related substances, but not by L-lactate. The transport exhibited the phenomenon of transacceleration, indicative for the involvement of a facilitated diffusion carrier. The carrier is highly specific for pyruvate and differs from other known monocarboxylate carriers present in the mitochondrial and/or plasma membrane of other eukaryotic cells in that it does not transport L-lactate.     

Defective cation-coupling mutants of Escherichia coli Na+/proline symport carrier. Characterization and localization of mutations

A major proline carrier in Escherichia coli encoded by the putP gene mediates proline/Na+ or Li+ symport. Proline carrier mutants with altered cation specificity were obtained by mutagenesis with nitrous acid in vitro of a plasmid carrying the wild-type putP gene. Two mutant strains harboring plasmid pMOP4135 and pMOP4141 could transport proline efficiently only in the presence of an increased concentration of sodium ion. Mutations of these plasmids, putP4135 and putP4141, caused reduction of affinity for Na+ of proline transport and binding, without remarkable change in the affinity for proline or in production of the carriers. Consistent with the lower affinity of the putP4141 carrier for Na+, the mutant carrier was supersensitive to N-ethylmaleimide inhibition. The pH dependence of proline binding was also changed in these mutant carriers. The lesions of putP4135 and putP4141 were located in the N-terminal part of the putP gene (ClaI-PvuII fragment) by in vitro recombination and subsequent examination of the phenotype of the transformants. DNA sequencing of these fragments revealed one base alteration of G to A at nucleotides 299 and 656 in pMOP4141 and pMOP4135, respectively, which corresponded to amino acid changes from Gly22 to glutamic acid and Cys141 to tyrosine, respectively.     

Clinical assay of the human erythrocyte lactate transporter. I. Principles, procedure, and validation

A clinically applicable method for the assay of lactate efflux from human red cells has been developed and described in detail. It requires only small volumes of blood and routine chemicals, and evaluates the process under physiological conditions and direction of lactate loading and transport. The decline of red cell lactate level fit a first order decay curve reasonably well, and better than the fit to zero order or second order plots. Bias is controlled by the use of least-squares curve fitting for all assays, and constraints on the elimination of outlier points. The assay shows a variety of inhibitor effects that may be considered typical for this transporter: potent inhibition by p-hydroxymercuribenzoate, but not by other types of sulfhydryl reagents; marked inhibition by phloretin, quercitin, and 1-fluoro-2,4-dinitrobenzene; lack of inhibition by the amine-reactive agents that block the chloride/carbonate exchanger, DIDS and SITS; and reversible competitive inhibition by alpha-cyano-4-OH-cinnamic acid. Harmaline and N-I-succinimide also produced effective inhibition. The assay also demonstrated transacceleration of L-lactate efflux in the presence of external additions of D-lactate, glycollate, iodoacetate, fluoropyruvate, and bromopyruvate, which are substituted monocarboxylates like lactate, but not by iodoacetamide or L-alanine. Such activation is a manifestation of a macromolecular carrier in operation, and cannot be explained by a pore or channel. These findings satisfy all reasonable criteria for a satisfactory and sensitive lactate transporter assay, which should be adequate to evaluate volunteers and patients for the normal range of this carrier, and to seek possible deficient states.     

Rate Equations and Kinetics of Uptake of α-Aminoisobutyric Acid and γ-Aminobutyric Acid by Mouse Cerebrum Slices Incubated in Media Containing L(+)-Lactate or a Mixture of Succinate, L-Malate, and Pyruvate as the Energy Source

Influx of alpha-aminoisobutyric acid (AIB) and gamma-aminobutyric acid (GABA) by mouse cerebrum slices incubated with L-lactate or a mixture of succinate, L-malate, and pyruvate (SMP) as the energy source follows the phenomenological rate equation for influx from pyruvate and glucose media: v = Vmax/(1 + Kt/S) + kuS, where v is rate and S is concentration of amino acid. There are two kinetically distinct, parallel components for concentrative uptake, one saturable, and one unsaturable. Rates are less with lactate than with pyruvate and still less with SMP (only GABA was studied), disproving the hypotheses that lower rates with pyruvate compared to glucose are due to an abnormal redox state in the tissue or to a Krebs cycle unbalanced by input at only one point. The carriers for AIB and GABA are qualitatively different. In lactate medium the capacity of each AIB carrier is unchanged but its affinity is reduced to one-third. In lactate and SMP media, the capacity of the saturable GABA carrier is diminished although its affinity is increased. Rates from these media with added glucose or a glucose analog confirm that amino acid and glucose fluxes are not coupled.     

Polarized expression of different monocarboxylate transporters in rat medullary thick limbs of Henle.

Extracellular lactic acid is a major fuel for the mammalian medullary thick ascending limb (MTAL), whereas under anoxic conditions, this nephron segment generates a large amount of lactic acid, which needs to be excreted. We therefore evaluated, at both the functional and molecular levels, the possible presence of monocarboxylate transporters in basolateral (BLMVs) and luminal (LMVs) membrane vesicles isolated from rat MTALs. Imposing an inward H(+) gradient induced the transient uphill accumulation of L-[(14)C]lactate in both types of vesicles. However, whereas the pH gradient-stimulated uptake of L-[(14)C]lactate in BLMVs was inhibited by anion transport blockers such as alpha-cyano-4-hydroxycinnamate, 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS), and furosemide, it was unaffected by these agents in LMVs, indicating the presence of a L-lactate/H(+) cotransporter in BLMVs, but not in LMVs. Under non-pH gradient conditions, however, the uptake of L-[(14)C]lactate in LMVs was transstimulated 100% by L-lactate, but by only 30% by D-lactate. Furthermore, this L-lactate self-exchange was markedly inhibited by alpha-cyano-4-hydroxycinnamate and DIDS and almost completely by 1 mM furosemide, findings consistent with the existence of a stereospecific carrier-mediated lactate transport system in LMVs. Using immunofluorescence confocal microscopy and immunoblotting, the monocarboxylate transporter (MCT)-2 isoform was shown to be specifically expressed on the basolateral domain of the rat MTAL, whereas the MCT1 isoform could not be detected in this nephron segment. This study thus demonstrates the presence of different monocarboxylate transporters in rat MTALs; the basolateral H(+)/L-lactate cotransporter (MCT2) and the luminal H(+)-independent organic anion exchanger are adapted to play distinct roles in the transport of monocarboxylates in MTALs.     

Transport of L-Lactate, D-Lactate, and glycolate by the LldP and GlcA membrane carriers of Escherichia coli.

To examine the substrate specificity of the membrane transport carriers LldP (L-lactate permease) and GlcA (glycolate permease) of Escherichia coli, a mutant strain lacking their structural genes and blocked in the metabolism of the tested substrates was constructed and transformed with a plasmid bearing either the lldP or the glcA gene. Each transformant acquired the ability to accumulate L-lactate, D-lactate, and glycolate against a high concentration gradient. Substrate accumulation was inhibited by carbonyl cyanide m-chlorophenylhydrazone, a hydrophobic proton conductor that dissipates proton motive force. Competition of (14)C-L-lactate transport by nonradioactive L-lactate, D-lactate, and glycolate in LldP synthesizing cells and competition of (14)C-glycolate transport by the same three substrates in GlcA synthesizing cells showed that both carriers effectively transported all three substrates with a K(i) value ranging from 10 to 20 microM. D-Lactate does not appear to have a permease of its own. Utilization of the compound depends mainly on LldP.     

The separation of partially modified lactate dehydrogenase by affinity chromatography. The specific activity of protomers?

Pig heart lactate dehydrogenase (L-lactate: NAD+ oxidoreductase, EC 1.1.1.27) was partially inactivated by reaction with phenylmaleimide. The resulting protein mixture was separated by affinity chromatography on a Sepharose-linked oxamate column yielding three distinct enzyme fractions with one, two and three out of four subunits being modified. The specific activities of these samples were lower than expected from the degree of modification, while the maximum binding capacity of NADH-oxamate related well to the number of catalytic centers modified. A possible cooperative effect in the native enzyme is discussed.     

Evidence for a lactate transport system in the sarcolemmal membrane of the perfused rabbit heart: kinetics of unidirectional influx, carrier specificity and effects of glucagon

The kinetics and specificity of L-lactate transport into cardiac muscle were studied during a single transit through the isolated perfused rabbit heart using a rapid (15 s) paired-tracer dilution technique. Kinetic experiments revealed that lactate influx was highly stereospecific and saturable with an apparent Kt = 19 +/- 6 mM and a Vmax = 8.4 +/- 1.5 mumol/min per g (mean +/- S.E., n = 14 hearts). At high perfusate concentrations (10 mM), the inhibitors alpha-cyano-4-hydroxycinnamate (Ki = 7.3 mM), pyruvate (Ki = 6.5 mM), acetate (Ki = 19.4 mM) and chloroacetate (Ki = 28 mM) reduced L-lactate influx, and Ki values were estimated assuming a purely competitive interaction of the inhibitors with the monocarboxylate carrier. The monocarboxylic acids [14C]pyruvate and [3H]acetate were themselves transported, and sarcolemmal uptakes of respectively 38 +/- 1% and 70 +/- 8% were measured relative to D-mannitol. Perfusion of hearts for 10-30 min with 0.15 or 1.5 microM glucagon increased myocardial lactate production and simultaneously inhibited tracer uptake of lactate, pyruvate and acetate. It is concluded that a stereospecific lactate transporter exhibiting an affinity for other substituted monocarboxylic acids is operative in the sarcolemmal plasma membrane of the rabbit myocardium.     

The kinetics of glutamine transport in bovine lymphocytes.

The transport of glutamine was examined in bovine peripheral lymphocytes which had been cultured in the presence or absence of Concanavalin A (Con A). Glutamine transport was mediated by a triphasic transport system in both cell populations. The calculated kinetic parameters were: Km 1.0, 4.7 and 12.7 mM and Vmax 4.5, 6.0 and 9.0 nmol/min per mg protein respectively. Con A augmented the capacity rather than the affinity of the glutamine transport systems (Vmax rates being 8.0, 12.2 and 38.0 nmol/min per mg protein respectively). Transporter I displayed Michaelis-Menton kinetics, while transporters II and III were co-operative carriers possessing Hill coefficients of 2.3 and 9.5 respectively. Preliminary studies using amino acid and ion inhibition studies suggested that transporter I was a system ASC-type carrier, transporter III a system L carrier, while the nature of transporter II was unclear.     

Transport of lactate and other short-chain monocarboxylates in the yeast Saccharomyces cerevisiae.

Saccharomyces cerevisiae IGC4072 grown in lactic acid medium transported lactate by an accumulative electroneutral proton-lactate symport with a proton-lactate stoichiometry of 1:1. The accumulation ratio measured with propionate increased with decreasing pH from ca. 24-fold at pH 6.0 to ca. 1,400-fold at pH 3.0. The symport accepted the following monocarboxylates (Km values at 25 degrees C and pH 5.5): D-lactate (0.13 mM), L-lactate (0.13 mM), pyruvate (0.34 mM), propionate (0.09 mM), and acetate (0.05 mM), whereas apparently a different proton symport accepted formate (0.13 mM). The lactate system was inducible and was subject to glucose repression. Undissociated lactic acid entered the cells by simple diffusion. The permeability of the plasma membrane for undissociated lactic acid increased exponentially with pH, and the diffusion constant increased 40-fold when the pH was increased from 3.0 to 6.0.     

Ribonuclease S-peptide as a carrier in fusion proteins.

S-peptide (residues 1-20) and S-protein (residues 21-124) are the enzymatically inactive products of the limited digestion of ribonuclease A by subtilisin. S-peptide binds S-protein with high affinity to form ribonuclease S, which has full enzymatic activity. Recombinant DNA technology was used to produce a fusion protein having three parts: carrier, spacer, and target. The two carriers used were the first 15 residues of S-peptide (S15) and a mutant S15 in which Asp 14 had been changed to Asn (D14N S15). The spacer consisted of three proline residues and a four-residue sequence recognized by factor Xa protease. The target was beta-galactosidase. The interaction between the S-peptide portion of the fusion protein and immobilized S-protein allowed for affinity purification of the fusion protein under denaturing (S15 as carrier) or nondenaturing (D14N S15 as carrier) conditions. A sensitive method was developed to detect the fusion protein after sodium dodecyl sulfate-polyacrylamide gel electrophoresis by its ribonuclease activity following activation with S-protein. S-peptide has distinct advantages over existing carriers in fusion proteins in that it combines a small size (> or = 15 residues), a tunable affinity for ligand (Kd > or = 10(-9) M), and a high sensitivity of detection (> or = 10(-16) mol in a gel).     

Substrate and inhibitor specificity of the lactate carrier of human neutrophils

Summary The substrate and inhibitor specificity of the lactic acid (Lac) transport system of human neutrophils was investigated. The ability of a variety of compounds to inhibit the influx of [¹⁴C]lactate, presumably reflecting competition by substrate analogues for binding at the external translocation site, was taken as an index of affinity for the Lac carrier. pH-stat techniques were utilized to assess transportability. Results indicate a relatively low order of selectivity, the neutrophil H⁺ + lactate^– cotransport system demonstrating a broad acceptance of short-chain unsubstituted and substituted alkyl monocarboxylates as well as aromatic monocarboxylates. There was a slight preference for oxo, Cl, and OH substituents over other groups at the two-position of short chain alkyl fatty acids: all were readily transported across the plasma membrane at rates approaching that ofl-lactate itself. Aromatic acids were not transported inward by the carrier although these compounds did permeate via simple nonionic diffusion. The neutrophil Lac carrier can be blocked by a number of cyanocinnamate derivatives, the classical inhibitors of monocarboxylate transport in mitochondria, and by dithiol compounds and sulfhydryl-reactive agents. This constellation of biochemical properties is similar to the features that characterize other well described H⁺ + lactate^– cotransport systems in red blood cells, Ehrlich ascites tumor cells, hepatocytes, and cardiac sarcolemmal vesicles, although significant differences exist when comparisons are made to the Na⁺-dependent lactate transporter of the kidney proximal tubule.     

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.