L-lactate Transport Research Articles

Lactate transport in rat adipocytes: identification of monocarboxylate transporter 1 (MCT1) and its modulation during streptozotocin-induced diabetes

We have characterised L-lactate transport in rat adipocytes and determined whether these cells express a carrier belonging to the monocarboxylate transporter family. L-Lactate was taken up by adipocytes in a time-dependent, non-saturable manner and was inhibited (by ∼90%) by α-cyano-4-hydroxycinnamate. Lactate transport was stimulated by 3.7-fold upon lowering extracellular pH from 7.5 to 6.5 suggesting the presence of a lactate/proton-cotransporter. Antibodies against m ono c arboxylate t ransporter 1 (MCT1) reacted positively with plasma membranes (PM), but not with intracellular membranes, prepared from adipocytes. MCT1 expression was down-regulated in PM of adipocytes from diabetic rats, which also displayed a corresponding loss (∼64%) in their capacity to transport lactate. The data support a role for MCT1 in lactate transport and suggest that changes in MCT1 expression are likely to have important implications for adipocyte lactate metabolism.

A Monocarboxylate Transporter MCT1 is Located at the Basolateral Pole of Rat Jejunum

We have functionally expressed and identified a monocarboxylate transporter (MCT1) from rat jejunal enterocyte and we provide evidence for its basolateral localization. Poly(A)+ RNA isolated from rat jejunum was injected into Xenopus laevis oocytes and expression of a proton-lactate symporter was investigated by means of L-[14C]lactate uptake. The existence of an endogenous capacity for L-lactate transport was demonstrated; when, however, oocytes were injected with jejunal mRNA, an expressed L-lactate uptake was seen which differed from the endogenous transporter since it was significantly pH dependent. After sucrose density gradient fractionation, the highest expression of the pH-dependent lactate uptake was detected with the mRNA size fraction of about 2-3 kb in length. The substrate specificity, stereoselectivity and sensitivity to pCMBS (an organomercurial thiol reagent that modifies cysteine residues) of the expressed transport were in good agreement with results previously obtained using isolated jejunal basolateral membranes. Using the reverse transcriptase-polymerase chain reaction, the presence of mRNA coding for the MCT1 isoform was demonstrated in jejunal enterocytes. These data, together with previous results, suggest that MCT1 is a major route for lactate efflux across the basolateral membrane of rat jejunum; this is in contrast to current opinion which restricts the presence of MCT1 to the apical membrane of the whole small intestine.

Identification and characterization of a monocarboxylate transporter (MCT1) in pig and human colon: its potential to transport L-lactate as well as butyrate.

1. Oligonucleotide primers based on the human heart monocarboxylate transporter (MCT1) cDNA sequence were used to isolate a 544 bp cDNA product from human colonic RNA by reverse transcription-polymerase chain reaction (RT-PCR). The sequence of the RT-PCR product was identical to that of human heart MCT1. Northern blot analysis using the RT-PCR product indicated the presence of a single transcript of 3.3 kb in mRNA isolated from both human and pig colonic tissues. Western blot analysis using an antibody to human MCT1 identified a specific protein with an apparent molecular mass of 40 kDa in purified and well-characterized human and pig colonic lumenal membrane vesicles (LMV). 2. Properties of the colonic lumenal membrane L-lactate transporter were studied by the uptake of L-[U-14C]lactate into human and pig colonic LMV. L-Lactate uptake was stimulated in the presence of an outward-directed anion gradient at an extravesicular pH of 5.5. Transport of L-lactate into anion-loaded colonic LMV appeared to be via a proton-activated, anion exchange mechanism. 3. L-Lactate uptake was inhibited by pyruvate, butyrate, propionate and acetate, but not by Cl- and SO4(2-). The uptake of L-lactate was inhibited by phloretin, mercurials and alpha-cyano-4-hydroxycinnamic acid (4-CHC), but not by the stilbene anion exchange inhibitors, 4,4'-diisothiocyanostilbene-2, 2'-disulphonic acid (DIDS) and 4-acetamido-4'-isothiocyanostilbene-2, 2'-disulphonic acid (SITS). 4. The results indicate the presence of a MCT1 protein on the lumenal membrane of the colon that is involved in the transport of L-lactate as well as butyrate across the colonic lumenal membrane. Western blot analysis showed that the abundance of this protein decreases in lumenal membrane fractions isolated from colonic carcinomas compared with that detected in the normal healthy colonic tissue.

Purification of the Lysosomal Sialic Acid Transporter: FUNCTIONAL CHARACTERISTICS OF A MONOCARBOXYLATE TRANSPORTER

Sialic acid and glucuronic acid are monocarboxylated monosaccharides, which are normally present in sugar side chains of glycoproteins, glycolipids, and glycosaminoglycans. After degradation of these compounds in lysosomes, the free monosaccharides are released from the lysosome by a specific membrane transport system. This transport system is deficient in the human hereditary lysosomal sialic acid storage diseases (Salla disease and infantile sialic acid storage disease, OMIM 269920). The lysosomal sialic acid transporter from rat liver has now been purified to apparent homogeneity in a reconstitutively active form by a combination of hydroxyapatite, lectin, and ion exchange chromatography. A 57-kDa protein correlated with transport activity. The transporter recognized structurally different types of acidic monosaccharides, like sialic acid, glucuronic acid, and iduronic acid. Transport of glucuronic acid was inhibited by a number of aliphatic monocarboxylates (i.e. lactate, pyruvate, and valproate), substituted monocarboxylates, and several dicarboxylates. cis-Inhibition, trans-stimulation, and competitive inhibition experiments with radiolabeled glucuronic acid as well as radiolabeled L-lactate demonstrated that L-lactate is transported by the lysosomal sialic acid transporter. L-Lactate transport was proton gradient-dependent, saturable with a Km of 0.4 mM, and mediated by a single mechanism. These data show striking biochemical and structural similarities of the lysosomal sialic acid transporter with the known monocarboxylate transporters of the plasma membrane (MCT1, MCT2, MCT3, and Mev).

Facilitated transport of lactate by rat jejunal enterocyte.

L-lactate transport mechanism across rat jejunal enterocyte was investigated using isolated membrane vesicles. In basolateral membrane vesicles L-lactate uptake is stimulated by an inwardly directed H+ gradient; the effect of the pH difference is drastically reduced by FCCP, pCMBS and phloretin, while furosemide is ineffective. The pH gradient effect is strongly temperature dependent. The initial rate of the proton gradient-induced lactate uptake is saturable with respect to external lactate with a K(m) of 39.2 +/- 4.8 mM and a Jmax of 8.9 +/- 0.7 nmoles mg protein-1 sec-1. A very small conductive pathway for L-lactate is present in basolateral membranes. In brush border membrane vesicles both Na+ and H+ gradients exert a small stimulatory effect on lactate uptake. We conclude that rat jejunal basolateral membrane contains a H(+)-lactate cotransporter, whereas in the apical membrane both H(+)-lactate and Na(+)-lactate cotransporters are present, even if they exhibit a low transport rate.

Lactate transport across the bovine rumen epithelium in vitro.

The in vitro transport of L-lactate across rumen epithelium was found to be a passive, mass-action regulated diffusion, and no saturable transport mechanisms were involved at concentrations from 2-20 mmol/L. During this increase in L-lactate concentrations the epithelial resistance and potential difference increased significantly, indicating an epithelial change towards a more tight epithelium. The net sodium transport was significantly reduced by 45% at 20 mmol/L lactate as compared to lactate free conditions.

1H- and 31P-nuclear magnetic resonance studies of l-lactate transport in isolated muscle fibers from the spiny lobster Panulirus argus

Proton (1H) and phosphorus (31P) nuclear magnetic resonance (NMR) spectroscopy were used to investigate the mode of transport of l-lactate across the plasma membranes of the abdominal extensor muscles of the spiny lobster Panulirus argus. Individual fibers or bundles of 2­3 fibers were superfused in a dual-tuned (1H, 31P) microsolenoid NMR probe. 1H-NMR spectra were diffusion-weighted, which eliminated the signal contribution of the fast-flowing extracellular lactate but retained that of intracellular lactate. Well-resolved intracellular lactate signals could be acquired at 15 s intervals, permitting estimation of initial velocities (Vi) of influx and efflux during loading/unloading of muscle fibers. 31P-NMR spectra were acquired to assess cellular energy status and intracellular pH. Transport results showed that Vi values for influx and efflux were a linear function of total lactate concentration, displaying no saturation effects. The rate of lactate influx was enhanced by increasing the concentration of the free acid by altering the superfusate pH. Vi values for influx and efflux of d- and l-lactate were identical. Finally, traditional inhibitors of monocarboxylate and/or anion transport had no effect on influx/efflux of lactate from these cells. The above results strongly suggest that the primary mode of lactate transport is by passive diffusion. These cells appear to lack a monocarboxylate transporter, which may be related to the apparent absence of organ-specific compartmentation of lactate metabolism in crustaceans.

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 other 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 associated proton influx. Transport kinetics were similar, but not identical, to those determined using the same technique for the monocarboxylate transporter (MCT) of Ehrlich Lettré tumor cells (MCT1) (Carpenter, L., and Halestrap, A. P. (1994) Biochem. J. 304, 751-760). Km 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 similar in both cell types, whereas in hepatocytes the Km values were higher than MCT1 for pyruvate (1.3 mM, cf. 0.72 mM), D-3-hydroxybutyrate (24.7 mM, cf. 10.1 mM), D-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 Km values than MCT1 for glycolate, chloroacetate, dichloroacetate, and 2-hydroxy-2-methylpropionate. Differences in stereoselectivity were also detected; both carriers showed a lower Km for L-lactate than D-lactate, while hepatocyte MCT exhibited a lower Km 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, phloretin, and niflumic acid, inhibited hepatocyte MCT with K0.5 values significantly higher than for tumor cell MCT1, while stilbene disulfonate derivatives and p-chloromercuribenzene sulfonate had similar K0.5 values in both cell types. The branched chain ketoacids alpha-ketoisocaproate and alpha-ketoisovalerate were also potent inhibitors of hepatocyte MCT with K0.5 values of 270 and 340 microM, respectively. The activation energy of L-lactate transport into hepatocytes was 58 kJ mol-1, and measured rates of transport at 37 degrees C were considerably greater than those required for maximal rates of gluconeogenesis. The properties of the hepatocyte monocarboxylate transporter are consistent with the presence of a distinct isoform of MCT in liver cells as suggested 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. Biol. Chem. 270, 1843-1849).

Characterization of the Enhanced Transport of L- and D-Lactate into Human Red Blood Cells Infected with Plasmodium falciparum Suggests the Presence of a Novel Saturable Lactate Proton Cotransporter

Human erythrocytes parasitized with the malarial protozoan Plasmodium falciparum showed rates of L-lactate, D-lactate, and pyruvate uptake many fold greater than control cells. Thus it was necessary to work at 0 degrees C to resolve true initial rates of transport. Studies on the dependence of the rate of transport on substrate concentration implied the presence in parasitized cells of both a saturable mechanism blocked by alpha-cyano-4-hydroxycinnamate (CHC) and a nonsaturable mechanism insensitive to CHC. The former was dominant at physiological substrate concentrations with Km values for pyruvate and D-lactate of 2.3 and 5.2 mM, respectively, with no stereoselectivity for L- over D-lactate. CHC was significantly less effective as an inhibitor of lactate transport in parasitized erythrocytes than in uninfected cells, whereas p-chloromercuribenzenesulfonate, a potent inhibitor in control cells, gave little or no inhibition of lactate transport into parasitized erythrocytes. Inhibition of transport into infected cells was also observed with phloretin, furosemide, niflumic acid, stilbenedisulfonate derivatives, and 5-nitro-2-(3-phenylpropylamino)benzoic acid at concentrations similar to those that inhibit the lactate carrier of control erythrocytes. These compounds were more effective inhibitors of the rapid transport of chloride into infected cells than of lactate transport, whereas CHC was more effective against lactate transport. This implies that different pathways are involved in the parasite-induced transport pathways for lactate and chloride. The transport of L-lactate into infected erythrocytes was also inhibited by D-lactate, pyruvate, 2-oxobutyrate, and 2-hydroxybutyrate. The intracellular accumulation of L-lactate at equilibrium was dependent on the transmembrane pH gradient, suggesting a protogenic transport mechanism. Our data are consistent with lactate and pyruvate having direct access to the malarial parasite, perhaps via the proposed parasitophorous duct or some close contact between the host cell and parasite plasma membranes, with transport across the latter by both a proton-linked carrier (CHC-sensitive, saturable, and the major route) and free diffusion of the undissociated acid (CHC-insensitive, unsaturable, and a minor route).

The kinetics, substrate and inhibitor specificity of the lactate transporter of Ehrlich-Lettre tumour cells studied with the intracellular pH indicator BCECF

1. Suspensions of cultured Ehrlich-Lettre tumour cells were loaded with the pH-sensitive fluorescent indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and changes in intracellular pH upon addition of L-lactate and other monocarboxylates were continuously monitored by fluorimetry using dual-wavelength excitation (450/500 nm) and single-wavelength emission (> 520 nm). 2. The rapid fluorescence changes were analysed by first-order regression analysis, and with suitable calibration procedures this enabled calculation of initial rates of proton uptake associated with monocarboxylate transport. 3. The stoichiometry was shown to be one proton per lactate molecule transported. 4. The kinetics of carrier-mediated transport of a wide range of monocarboxylates were determined at 25 degrees C. The Km values for L-lactate, pyruvate and D-lactate were found to be 4.54, 0.72 and 27.5 mM respectively, similar to values found previously for rat erythrocytes. This similarity was shared with a wide range of variously substituted C2, C3 and C4 monocarboxylates, all of which were transported with similar Vmax. No stereoselectivity was found in the Km values for D- and L-2-chloropropionate (0.75 mM) or D- and L-3-hydroxybutyrate (11 mM), but in the latter case the Vmax. of the D-isomer was twice that of the L-isomer. 5. The temperature-dependence of L-lactate transport demonstrated a transition point, with activation energies of 60 and 109 kJ.mol-1 above and below 19 degrees C respectively The Km for L-lactate below the transition temperature was about half that above it. 6. Inhibition of lactate transport into tumour cells by a wide range of compounds known to inhibit the erythrocyte monocarboxylate carrier was analysed. Patterns of inhibition were similar to those seen in the erythrocyte, but the Ki values were 2-4-fold higher in the tumour cells. 7. It is concluded that tumour cells contain an isoform of the monocarboxylate carrier with functional properties almost identical with that found in erythrocytes. This is probably identical with MCT1, which was recently cloned and sequenced from Chinese Hamster Ovary cells [Kim Garcia, Goldstein, Pathak, Anderson and Brown (1994) Cell 76, 865-873].

Demonstration of organic anion transport in T lymphocytes. L-lactate and fluo-3 are target molecules.

In this paper, we describe for the first time the existence of organic anion transport in T lymphocytes, exemplified by the transmembrane transport of the anions L-lactate and the Ca2+ indicator fluo-3. The transport of either anion was found to be inhibitable by probenecid, a common blocker of organic anion transport. Transport of L-lactate was observed in long-term cultured T cell lines, as well as in freshly ex vivo isolated T cells, and occurred via a saturable, pH-dependent, and stereospecific process. L-Lactate uptake was dependent on the activation state of the T cells, because activation of T cells by Con A strongly enhanced accumulation of L-lactate from the medium. Because L-lactate may be transported bidirectionally through the T cell membrane in vivo, different physiologic roles of L-lactate transport are discussed. L-Lactate uptake may serve as an alternative source of energy in an inflamed, glucose-deficient tissue or may represent a prerequisite for the earlier-published immunoregulatory function of this molecule on T cells. On the other hand, release of L-lactate emerging from glycolysis could be necessary to avoid acidification of the cell. The fact that the Ca2+ indicator fluo-3 is also transported through the cellular membranes of long-term cultured T cells via organic anion transport has important implications for the determination of Ca2+ influx into T cells. Even though the transport of both molecules, L-lactate and fluo-3, represents organic anion transport, evidence is presented that confirms that the respective transport systems are different.

Partial purification and reconstitution of the sarcolemmal L-lactate carrier from rat skeletal muscle.

Purified sarcolemmal membranes from mixed rat hindlimb muscle were solubilized with octylglucoside and the extract subjected to hydroxylapatite (HA) chromatography. Following protein elution with a sodium phosphate gradient and detergent removal by dialysis, the HA eluate was reconstituted into asolectin liposomes using a freeze-thaw procedure. Specific L-[14C]lactate transport activity eluting from the 0.2 M sodium phosphate fraction was 30-fold higher compared with native sarcolemmal vesicles (31.64 versus 1.06 nmol/min per mg). The reconstituted carrier exhibited Michaelis-Menten saturation kinetics with Km and Vmax. values of 46.2 +/- 6.6 mM and 498.7 +/- 17.2 nmol/15 s per mg respectively. L-Lactate transport activity was inhibited 57% by preincubation of proteoliposomes with 10 mM alpha-cyano-4-hydroxycinnamate, a known inhibitor of lactate transport. Analysis of the HA eluates by SDS/PAGE showed the presence of a 34 kDa band corresponding to lactate transport activity. Reconstitution of lactate transport activity eluting from the HA column, together with SDS/PAGE analysis suggests the presence of a 34 kDa polypeptide mediating sarcolemmal lactate exchange in rat skeletal muscle.

Lactate transport in rat sarcolemmal vesicles and intact skeletal muscle, and after muscle contraction

To determine whether it was possible to measure lactate transport rates into intact skeletal muscles, the transport of lactate (zero-trans) was determined in soleus muscle strips incubated in vitro and compared with lactate transport in sarcolemmal vesicles. In addition, the effects of muscle contractility on lactate transport were investigated in electrically-stimulated soleus muscle strips. In both the intact muscle and the sarcolemmal preparations the rates of transport were saturable, stereospecific, and inhibitable by monocarboxylates (pyruvate, alpha-cyano-4-hydroxycinnamate) and a protein modifier (N-ethylmaleimide; P < 0.05). The anion exchange inhibitor SITS had no effect on lactate uptake (P > 0.05). In both preparations lactate transport followed an inwardly directed proton gradient. Relative comparisons (%) between the preparations indicated a similar slope of increasing transport rates with increasing lactate concentrations and similar responses to a changing pH environment. These characterizations of L-lactate transport into isolated sarcolemmal vesicles and muscle strips revealed that both preparations yielded similar conclusions regarding the transmembrane movement of L-lactate. By using this more physiological muscle preparation, contractile activity, induced by electrical stimulation, did not increase lactate uptake in skeletal muscle in the post-exercise period whereas under similar conditions a marked increase in 2-deoxy-D-glucose uptake occurred (+ 47%; P < 0.05). These data suggest that the transport of glucose and lactate in contracting muscle is regulated differently. These studies also show that the incubated muscle strip preparation may be useful for studying lactate transport in an intact cell system during physiological experiments.

Reconstitution of the lactate carrier from rat skeletal-muscle sarcolemma.

The lactate carrier was solubilized from purified rat skeletal-muscle sarcolemma with the detergent decanoyl-N-methyl-glucamide and the solubilized carrier was reconstituted into phospholipid vesicles. Reconstituted proteoliposomes showed a faster time course of L-lactate uptake than did protein-free liposomes. The rate of L-lactate uptake into the proteoliposomes was inhibited by the lactate-transport inhibitors p-chloromercuribenzenesulphonate, diethyl pyrocarbonate, alpha-cyano-4-hydroxycinnamate and quercetin. In contrast, the anion-exchange inhibitor DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulphonate) had almost no effect on the uptake. The extent of L-lactate uptake at equilibrium was not affected by the presence of the transport inhibitors, but was sensitive to osmotic strength. L-Lactate and pyruvate, but not D-lactate, inhibited L-lactate uptake when present at 10-fold excess. The properties of L-lactate transport in reconstituted proteoliposomes were similar to those observed in native sarcolemmal vesicles, i.e. the lactate carrier seems to retain its transport characteristics during the solubilization and reconstitution steps.

Effects of an Aspergillus oryzae fermentation extract and other factors on lactate utilization by the ruminal bacterium Megasphaera elsdenii.

The objective of this study was to determine the effects of an Aspergillus oryzae fermentation extract (Amaferm) as well as other factors on lactate utilization by the ruminal bacterium Megasphaera elsdenii B159. Addition of Amaferm or a filter-sterilized Amaferm filtrate stimulated L-lactate uptake by both M. elsdenii and the ruminal selenomonad strain H18. Growth of M. elsdenii in medium that contained DL-lactate (2 g/L), Trypticase, and yeast extract was only slightly stimulated by the addition of 5% (vol/vol) Amaferm filtrate after 24 h. However, growth of M. elsdenii in a similar medium lacking Trypticase and yeast extract was increased over twofold by the addition of either 2 or 5% (vol/vol) Amaferm filtrate. These results suggest that Amaferm provides growth factors (i.e., amino acids, B vitamins) to support growth of M. elsdenii on lactate. There was no inhibition of L-lactate uptake when lactate-grown cells of M. elsdenii were incubated with excess (10 mM) glucose, sucrose, or maltose. In addition, when cells were grown on glucose, sucrose, or maltose rather than lactate there was little difference in L-lactate uptake, suggesting that L-lactate transport in M. elsdenii is not subject to catabolite repression by these soluble sugars. Both K+ and Na+ had little effect on L-lactate uptake. Uptake was unaffected at extracellular pH values between 6.0 and 8.0, whereas pH values of 5.0 and 4.0 increased uptake. In addition, L-lactate uptake was inhibited between 34 and 61% by protonophores. These results suggest that protons may be involved in the uptake of L-lactate by M. elsdenii B159.

Transport of lactate and other monocarboxylates across mammalian plasma membranes

Transport of L-lactate across the plasma membrane is of considerable importance to almost all mammalian cells. In most cells a specific H(+)-monocarboxylate cotransporter is largely responsible for this process; the capacity of this carrier is usually very high, to support the high rates of production or utilization of L-lactate. The best characterized H(+)-monocarboxylate transporter is that of the erythrocyte membrane, which transports L-lactate and a wide range of other aliphatic monocarboxylates, including pyruvate and the ketone bodies acetoacetate and beta-hydroxybutyrate. This carrier is inhibited by alpha-cyanocinnamate derivatives and some stilbene disulfonates and has been identified as a protein of 35-50 kDa on the basis of purification and specific labeling experiments. Other cells possess similar alpha-cyanocinnamate-sensitive H(+)-linked monocarboxylate transporters, but in some cases there are significant differences in the properties of these systems, sufficient to suggest the existence of a family of such carriers. In particular, cardiac muscle and tumor cells have transporters that differ in their Km values for certain substrates (including stereoselectivity for L- over D-lactate) and in their sensitivity to inhibitors. Mitochondria, bacteria, and yeast also possess H(+)-monocarboxylate transporters that share some properties in common with those in the mammalian plasma membrane but are adapted to their specific roles. However, there are distinct Na(+)-monocarboxylate cotransporters on the luminal surface of intestinal and kidney epithelia, which enable active uptake of lactate, pyruvate, and ketone bodies in these tissues. This article reviews the properties of these transport systems and their role in mammalian metabolism.

Characterization of the inhibition by stilbene disulphonates and phloretin of lactate and pyruvate transport into rat and guinea-pig cardiac myocytes suggests the presence of two kinetically distinct carriers in heart cells.

1. The kinetics of transport of pyruvate (Km 0.20 mM), L-lactate (Km 2.2 mM) and D-lactate (Ki 10.2 mM) into rat cardiac myocytes were studied and compared with those for guinea-pig heart cells [Poole, Halestrap, Price and Levi (1989) Biochem. J. 264, 409-418] whose equivalent values were 0.07, 2.3 and 6.6 mM respectively. Maximal rates of transport were about 5-fold higher in the rat heart cells. 2. 4,4'-Dibenzamidostilbene-2,2'-disulphonate (DBDS), a powerful inhibitor of monocarboxylate transport into erythrocytes [Poole & Halestrap (1991) Biochem. J. 275, 307-312], was found to be a potent but apparently partial inhibitor of lactate and pyruvate transport, with an apparent Ki value at 0.5 mM L-lactate of about 16 microM in both species. Maximal inhibition was 50% and 80% in rat and guinea-pig cells respectively. 3. The maximal extent of inhibition and apparent Ki values were dependent on both the substrate transported and its concentration. Maximum inhibition was less and the Ki was greater at higher substrate concentrations. 4. A variety of other stilbene disulphonates were studied which showed different Ki values and maximal extents of inhibition. 5. Phloretin was a significantly less potent inhibitor of transport into both rat (Ki 25 microM) and guinea-pig (Ki 16 microM) heart cells than into rat erythrocytes (Ki 1.4 microM). In the rat but not the guinea-pig heart cells, inhibition appeared partial (maximal inhibition 84%). 6. We demonstrate that our results can be explained by the presence of two monocarboxylate carriers in heart cells, both with Km values for L-lactate of about 2 mM and inhibited by alpha-cyano-4-hydroxycinnamate, but with different affinities for other substrates and inhibitors. One carrier is sensitive to inhibition by stilbene disulphonates and has lower Km values for pyruvate (0.05-0.10 mM) and D-lactate (5 mM), whereas the other has higher Km values for pyruvate (0.30 mM) and D-lactate (25 mM), and is relatively insensitive to stilbene disulphonates. Rat heart cells possess more of the latter carrier and guinea-pig heart cells more of the former. 7. The significance of these results for the study of lactate transport in the perfused heart is discussed.

Transport ofl-lactate by cultured rat brain astrocytes

Several reports indicate that lactate can serve as an energy substrate for the brain. The rate of oxidation of this substrate by cultured rat brain astrocytes was 3-fold higher than the rate with glucose, suggesting that lactate can serve as an energy source for these cells. Since transport into the astrocytes may play an important role in regulating nutrient use by individuals types of brain cells, we investigated the uptake of L-[U-14C]lactate by primary cultures of rat brain astrocytes. Measurement of the net uptake suggested two carrier-mediated mechanisms and an Eadie-Hofstee type plot of the data supported this conclusion revealing 2 Km values of 0.49 and 11.38 mM and Vmax values of 16.55 and 173.84 nmol/min/mg protein, respectively. The rate of uptake was temperature dependent and was 3-fold higher at pH 6.2 than at 7.4, but was 50% less at pH 8.2. Although the lactate uptake carrier systems in astrocytes appeared to be labile when incubated in phosphate buffered saline for 20 minutes, the uptake process exhibited an accelerative exchange mechanism. In addition, lactate uptake was altered by several metabolic inhibitors and effectors. Potassium cyanide and alpha-cyano-4-hydroxycinnamate inhibited lactate uptake, but mersalyl had little or no effect. Phenylpyruvate, alpha-ketoisocaproate, and 3-hydroxybutyrate at 5 and 10 mM greatly attenuated the rate of lactate uptake. These results suggest that the availability of lactate as an energy source is regulated in part by a biphasic transport system in primary astrocytes.

Transport of L-leucine hydroxy analogue and L-lactate in human small intestinal brush border membrane vesicles.

Nitrogen load can be reduced by substituting dietary protein intake with keto or hydroxy analogues of amino acids. To investigate their intestinal absorption, human jejunal brush border membrane vesicles were used to measure uptake of L-leucine hydroxy analogue (L-LHA) and L-lactate. Uptake assays were performed under voltage-clamped conditions in the presence of valinomycin and K+ inside and outside. Both inward directed H(+)- and Na(+)-gradients stimulated uptake of both substrates. The H(+)-gradient was the major driving force and led to an increased rate of L-LHA or L-lactate transport. The proton ionophore FCCP abolished the H(+)-gradient-driven but not the Na(+)-gradient-driven uptakes of both substrates. The H(+)-gradient-driven uptake of both substrates was trans-stimulated by L-LHA, D-LHA, L-valine hydroxy analogue or L-lactate, respectively. In the presence of a Na(+)-gradient the uptake of tracer L-lactate was trans-stimulated only after preloading the vesicles with L-lactate but not by L-LHA. It can be concluded that the H(+)-gradient-driven transport of L- or D-hydroxy analogues of branched chain amino acids and L-lactate across the human intestinal brush border membrane is mediated by the same carrier.

Transport of lactate in Plasmodium falciparum-infected human erythrocytes.

The intraerythrocytic human malarial parasite Plasmodium falciparum produces lactate at a rate that exceeds the maximal capacity of the normal red cell membrane to transport lactate. In order to establish how the infected cell removes this excess lactate, the transport of lactate across the host cell and the parasite membranes has been investigated. Transport of radiolabeled L-lactate across the host cell membrane was shown to increase ca. 600-fold compared to uninfected erythrocytes. It showed no saturation with [L-lactate] and was inhibited by inhibitors of the monocarboxylate carrier, cinnamic acid derivatives (CADs), but not by the SH-reagent p-chloromercuriphenyl sulfonic acid (PCMBS). These results suggest that L-lactate is translocated through CAD-inhibitable new pathways induced in the host cell membrane by parasite activity, probably by diffusion of the acid form and through a modified native monocarboxylate:H+ symporter. Continuous monitoring of extracellular pH changes occurring upon suspension of infected cells in isoosmotic Na-lactate solutions indicates that part of the lactate egress is mediated by anionic exchange through the constitutive, but modified, anion exchanger. The transport of L-lactate across the parasite membrane is rapid, nonsaturating, and insensitive to either CADs or PCMBS, or to the presence of pyruvate. L-lactate uptake increased transiently when external pH was lowered and decreased when delta pH was dissipated by the protonophore carbonylcyanide m-chlorophenyl hydrazone (CCCP). These results are compatible with L-lactate crossing the parasite membrane either as the undissociated acid or by means of a novel type of lactate-/H+ symport.