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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.

ABSTRACT: 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.

SUBMITTER: Poole RC

PROVIDER: S-EPMC1133596 | biostudies-other | 1989 Dec

REPOSITORIES: biostudies-other

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Project description: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.

Project description:The kinetics and activation energy of entry of pyruvate and lactate into the erythrocyte were studied at concentrations below 4 and 15mM respectively. The Km and Vmax. values for both substrates are reported, and it is shown that pyruvate inhibits 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 (20mM), 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 (less than 10 muM) concentrations of the inhibitor of Cl- transport, 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid. This inhibitor titrated out the chlordie efflux induced by pyruvate, bicarbonate, formate and fluoride, in each case total inhibition becoming apparent when approximately 1.2x10(6) molecules of inhibitor were present per erythrocyte, that is, about one inhibitor molecule per molecule of the Cl- carrier. Evan when Cl- efflux was totally blocked pyruvate and lactate uptake occurred. Kinetic evidence is 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 exists. This carrier catalyses the exchange of small carboxylate anions with intracellular lactate, is competitively inhibited by alpha-cyano-4-hydroxycinnamate and non-competitively inhibited by picrate. The Cl- carrier shows a reverse pattern of inhibition. It is concluded that 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. It is concluded that its presence in other cell membranes together with a low intracellular Cl- concentration would 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.

Project description:In order to obtain a global view of energy metabolism pathways of the sulfate-reducer Desulfovibrio vulgaris Hildenborough and the proteins involved therein whole-genome microarrays were used to compare the transcriptional response of cells grown with hydrogen/sulfate, pyruvate/sulfate, lactate/thiosulfate, and pyruvate with limiting sulfate, relative to growth in standard lactate/sulfate condition. Growth with hydrogen/sulfate showed the largest number of differently expressed genes and the largest changes in expression levels. The most up-regulated energy metabolism genes were those coding for the periplasmic [NiFeSe] hydrogenase, followed by the Ech hydrogenase, and the most down-regulated were genes coding for the Coo hydrogenase. The results point to the involvement of formate cycling and the ethanol pathway during growth on hydrogen, whereas there is evidence for CO cycling during growth on lactate and pyruvate, but not on H2. Growth with thiosulfate showed the hallmarks of a reduced energy status of the cells with down regulation of the ATP synthase and the Qmo and Dsr complexes., whereas growth with pyruvate showed the smallest differences but an increased role for the Ech hydrogenase.that in this case functions in reverse from the case of growth with H2. The multiple periplasmic hydrogenases and formate dehydrogenases, do not display the same regulation pattern showing that their metabolic roles are not totally interchangeable. This result together with the observation that several genes coding for proteins that have not been biochemically characterised were considerably affected in this study, reveals a more complex energy metabolism than previously considered and provides guidance for further studies. Keywords: Growth protocol Desulfovibrio vulgaris from our laboratory culture collection was cultured at 37°C with pyruvate as the only substrate (without sulfate as the electron acceptor) to mid-log phase. Cultures were also cultivated similarly in Lactate-Sulfate medium to mid-log phase. Gene expression profiles of cultures grown under pyruvate fermentation were compared with those of the cultures grown via sulfate reduction. Total RNA was harvested from four replicate cultures for microarray analysis. RNA extraction, purification, and labeling were performed independently on each cell sample.Two samples of each total RNA preparation were labeled, one with Cy3-dUTP and another with Cy5-dUTP for microarray hybridization.

Project description: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.

Project description:1. Studies on the kinetics of pyruvate transport into mitochondria by an 'inhibitor-stop' technique were hampered by the decarboxylation of pyruvate by mitochondria even in the presence of rotenone. Decarboxylation was minimal at 6 degrees C. At this temperature the Km for pyruvate was 0.15 mM and Vmax. was 0.54nmol/min per mg of protein; alpha-cyano-4-hydroxycinnamate was found to be a non-competitive inhibitor, Ki 6.3 muM, and phenyl-pyruvate a competitive inhibitor, Ki 1.8 mM. 2. At 100 muM concentration, alpha-cyano-4-hydroxycinnamate rapidly and almost totally inhibited O2 uptake by rat heart mitochondria oxidizing pyruvate. Inhibition could be detected at concentrations of inhibitor as low as 1 muM although inhibition took time to develop at this concentration. Inhibition could be reversed by diluting out the inhibitor. 3. Various analogues of alpha-cyano-4-hydroxycinnamate were tested on rat liver and heart mitochondria. The important structural features appeared to be the alpha-cyanopropenoate group and the hydrophobic aromatic side chain. Alpha-Cyanocinnamate, alpha-cyano-5-phenyl-2,4-pentadienoate and compound UK 5099 [alpha-cyano-beta-(2-phenylindol-3-yl)acrylate] were all more powerful inhibitors than alpha-cyano-4-hydroxycinnamate showing 50% inhibition of pyruvate-dependent O2 consumption by rat heart mitochondria at concentrations of 200, 200 and 50 nM respectively. 4. The specificity of the carrier for its substrate was studied by both influx and efflux experiments. Oxamate, 2-oxobutyrate, phenylpyruvate, 2-oxo-4-methyl-pentanoate, chloroacetate, dichloroacetate, difluoroacetate, 2-chloropropionate, 3-chloropropionate and 2,2-dichloropropionate all exchanged with pyruvate, whereas acetate, lactate and trichloroacetate did not. 5. Pyruvate entry into the mitochondria was shown to be accompanied by the transport of a proton (or by exchange with an OH-ion). This proton flux was inhibited by alpha-cyano-4-hydroxycinnamate and allowed measurements of pyruvate transport at higher temperatures to be made. The activation energy of mitochondrial pyruvate transport was found to be 113 kJ (27 kcal)/mol and by extrapolation the rate of transport of pyruvate at 37 degrees C to be 42 nmol/min per mg of protein. The possibility that pyruvate transport into mitochondria may be rate limiting and involved in the regulation of gluconegenesis is discussed. 6. The transport of various monocarboxylic acids into mitochondria was studied by monitoring proton influx. The transport of dichloroacetate, difluoroacetate and oxamate appeared to be largely dependent on the pyruvate carrier and could be inhibited by pyruvate-transport inhibitors. However, many other halogenated and 2-oxo acids which could exchange with pyruvate on the carrier entered freely even in the presence of inhibitor.

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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.

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