Kinetics of lactate and pyruvate transport in cultured rat myotubes.
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Skeletal muscle transport of lactate and pyruvate was studied in primary cultures of rat myotubes, applying the pH-sensitive fluorescent indicator 2', 7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The initial rate of decrease in intracellular pH (pHi) upon lactate or pyruvate incubation was used to determine total transport (carrier mediated and diffusion). Both lactate and pyruvate transport could be inhibited by a combination of 0.5 mM 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid, 5 mM mersalyl and 10 mM alpha-cyano-4-hydroxycinnamate. The kinetic parameters, Km and Vmax, for carrier-mediated transport of lactate were 9.9+/-1.1 mM and 0. 69+/-0.02 mmol l-1 s-1, respectively. For pyruvate, Km and Vmax were 4.4+/-1.3 mM and 0.30+/-0.05 mmol l-1 s-1, respectively. The diffusion component of the total transport was 0.0040+/-0.0005[S] (n=4) and 0.0048+/-0.0003[S] (n=4) for lactate and pyruvate, respectively. Furthermore, it was observed that the two monocarboxylate transporter isoforms present in mature skeletal muscles, MCT1 and MCT4 (formerly called MCT3 (M.C. Wilson, V.N. Jackson, C. Heddle, N.T. Price, H. Pilegaard, C. Juel, A. Bonen, I. Montgomery, O.F. Hutter, A.P. Halestrap, Lactic acid efflux from white skeletal muscle is catalyzed by the monocarboxylate transporter isoform MCT3, J. Biol. Chem. 273 (1998) 15920-15926)), were also expressed in primary culture of myotubes. (+info)
The lactate-proton symport of Saccharomyces cerevisiae is encoded by JEN1.
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A mutant of Saccharomyces cerevisiae deficient in the lactate-proton symport was isolated. Transformation of the mutant with a yeast genomic library allowed the isolation of the gene JEN1 that restored lactate transport. Disruption of JEN1 abolished uptake of lactate. The results indicate that, under the experimental conditions tested, no other monocarboxylate permease is able to efficiently transport lactate in S. cerevisiae. (+info)
Osmotic shock: modulation of contractile function, pHi, and ischemic damage in perfused guinea pig heart.
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To determine the contribution of changes in extracellular osmolarity to ischemic injury, isolated guinea pig hearts were perfused with hyposmotic (220 mosM) or hyperosmotic (380 mosM) buffer. 31P NMR spectroscopy was used to follow changes in intracellular pH (pHi) and energetics. Hyposmotic buffer decreased myocardial developed pressure by 30 +/- 2% and pHi by 0.02 +/- 0.01 unit, whereas hyperosmotic buffer increased myocardial developed pressure by 34 +/- 1% and pHi by 0.14 +/- 0.01 unit. All hearts recovered to control values on restoration of isosmotic (300 mosM) buffer. The hyperosmolar-induced intracellular alkalosis and developed pressure increase were not prevented by inhibition of Na+/H+ exchange with use of 1 microM HOE-642 but were abolished with use of bicarbonate-free buffers. After 20 min of total global ischemia, hearts perfused with hyposmotic buffer showed significantly greater recoveries of developed pressure, phosphocreatine, and ATP than control hearts, but hearts perfused with hyperosmotic buffer did not recover after ischemia. In conclusion, buffer osmolarities between 220 and 380 mosM alter myocardial pHi and developed pressure but are not deleterious during perfusion. However, buffer osmolarity significantly alters the extent of myocardial ischemic injury. (+info)
Distribution of the lactate/H+ transporter isoforms MCT1 and MCT4 in human skeletal muscle.
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The profiles of the lactate/H+ transporter isoforms [monocarboxylate transporter isoforms (MCT)] MCT1 and MCT4 (formerly MCT3 of Price, N. T., V. N. Jackson, and A. P. Halestrap. Biochem. J. 329: 321-328, 1998) were studied in the soleus, triceps brachii, and vastus lateralis muscles of six male subjects. The fiber-type compositions of the muscles were evaluated from the occurrence of the myosin heavy chain isoforms, and the fibers were classified as type I, IIA, or IIX. The total content of MCT1 and MCT4 was determined in muscle homogenates by Western blotting, and MCT1 and MCT4 were visualized on cross-sectional muscle sections by immunofluorescence microscopy. The Western blotting revealed a positive, linear relationship between the MCT1 content and the occurrence of type I fibers in the muscle, but no significant relation was found between MCT4 content and fiber type. Moreover, the interindividual variation in MCT4 content was much larger than the interindividual variation in MCT1 content in homogenate samples. The immunofluorescence microscopy showed that within a given muscle section, the MCT4 isoform was clearly more abundant in type II fibers than in type I fibers, whereas only minor differences existed in the occurrence of the MCT1 isoform between type I and II fibers. Together the present results indicate that the content of MCT1 in a muscle varies between different muscles, whereas fiber-type differences in MCT1 content are minor within a given muscle section. In contrast, the content of MCT4 is clearly fiber-type specific but apparently quite similar in various muscles. (+info)
Lactate transport in skeletal muscle - role and regulation of the monocarboxylate transporter.
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Skeletal muscle is the major producer of lactic acid in the body, but its oxidative fibres also use lactic acid as a respiratory fuel. The stereoselective transport of L-lactic acid across the plasma membrane of muscle fibres has been shown to involve a proton-linked monocarboxylate transporter (MCT) similar to that described in erythrocytes and other cells. This transporter plays an important role in the pH regulation of skeletal muscle. A family of eight MCTs has now been cloned and sequenced, and the tissue distribution of each isoform varies. Skeletal muscle contains both MCT1 (the only isoform found in erythrocytes but also present in most other cells) and MCT4. The latter is found in all fibre types, although least in more oxidative red muscles such as soleus, whereas expression of MCT1 is highest in the more oxidative muscles and very low in white muscles that are almost entirely glycolytic. The properties of MCT1 and MCT2 have been described in some detail and the latter shown to have a higher affinity for substrates. MCT4 has been less well characterized but has a lower affinity for L-lactate (i.e. a higher Km of 20 mM) than does MCT1 (Km of 5 mM). MCT1 expression is increased in response to chronic stimulation and either endurance or explosive exercise training in rats and humans, whereas denervation decreases expression of both MCT1 and MCT4. The mechanism of regulation is not established, but does not appear to be accompanied by changes in mRNA concentrations. However, in other cells MCT1 and MCT4 are intimately associated with an ancillary protein OX-47 (also known as CD147). This protein is a member of the immunoglobulin superfamily with a single transmembrane helix, whose expression is known to be increased in a range of cells when their metabolic activity is increased. (+info)
Characterization of the high-affinity monocarboxylate transporter MCT2 in Xenopus laevis oocytes.
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Observations on lactate transport in brain cells and cardiac myocytes indicate the presence of a high-affinity monocarboxylate transporter. The rat monocarboxylate transporter isoform MCT2 was analysed by expression in Xenopus laevis oocytes and the results were compared with the known characteristics of lactate transport in heart and brain. Monocarboxylate transport via MCT2 was driven by the H(+) gradient over the plasma membrane. Uptake of lactate strongly increased with decreasing pH, showing half-maximal stimulation at pH 7.2. A wide variety of monocarboxylates and ketone bodies, including lactate, pyruvate, beta-hydroxybutyrate, acetoacetate, 2-oxoisovalerate and 2-oxoisohexanoate, were substrates of MCT2. All substrates had a high affinity for MCT2. For lactate a K(m) value of 0.74+/-0.07 mM was determined at pH 7.0. For the other substrates, K(i) values between 100 microM and 1 mM were measured for inhibition of lactate transport, which is about one-tenth of the corresponding values for the ubiquitously expressed monocarboxylate transporter isoform MCT1. Monocarboxylate transport via MCT2 could be inhibited by alpha-cyano-4-hydroxycinnamate, anion-channel inhibitors and flavonoids. It is suggested that cells which express MCT2 preferentially use lactate and ketone bodies as energy sources. (+info)
Polarized expression of different monocarboxylate transporters in rat medullary thick limbs of Henle.
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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. (+info)
The proton-linked monocarboxylate transporter (MCT) family: structure, function and regulation.
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Monocarboxylates such as lactate and pyruvate play a central role in cellular metabolism and metabolic communication between tissues. Essential to these roles is their rapid transport across the plasma membrane, which is catalysed by a recently identified family of proton-linked monocarboxylate transporters (MCTs). Nine MCT-related sequences have so far been identified in mammals, each having a different tissue distribution, whereas six related proteins can be recognized in Caenorhabditis elegans and 4 in Saccharomyces cerevisiae. Direct demonstration of proton-linked lactate and pyruvate transport has been demonstrated for mammalian MCT1-MCT4, but only for MCT1 and MCT2 have detailed analyses of substrate and inhibitor kinetics been described following heterologous expression in Xenopus oocytes. MCT1 is ubiquitously expressed, but is especially prominent in heart and red muscle, where it is up-regulated in response to increased work, suggesting a special role in lactic acid oxidation. By contrast, MCT4 is most evident in white muscle and other cells with a high glycolytic rate, such as tumour cells and white blood cells, suggesting it is expressed where lactic acid efflux predominates. MCT2 has a ten-fold higher affinity for substrates than MCT1 and MCT4 and is found in cells where rapid uptake at low substrate concentrations may be required, including the proximal kidney tubules, neurons and sperm tails. MCT3 is uniquely expressed in the retinal pigment epithelium. The mechanisms involved in regulating the expression of different MCT isoforms remain to be established. However, there is evidence for alternative splicing of the 5'- and 3'-untranslated regions and the use of alternative promoters for some isoforms. In addition, MCT1 and MCT4 have been shown to interact specifically with OX-47 (CD147), a member of the immunoglobulin superfamily with a single transmembrane helix. This interaction appears to assist MCT expression at the cell surface. There is still much work to be done to characterize the properties of the different isoforms and their regulation, which may have wide-ranging implications for health and disease. In the future it will be interesting to explore the linkage of genetic diseases to particular MCTs through their chromosomal location. (+info)