Several laboratories have investigated monocarboxylate transport in a variety of cell types. The characterization of the cloned transporter isoforms in a suitable expression system is nevertheless still lacking. H+/monocarboxylate co-transport was therefore investigated in monocarboxylate transporter 1 (MCT1)-expressing Xenopus laevis oocytes by using pH-sensitive microelectrodes and [14C]lactate. Superfusion with lactate resulted in intracellular acidification of MCT1-expressing oocytes, but not in non-injected control oocytes. The basic kinetic properties of lactate transport in MCT1-expressing oocytes were determined by analysing the rates of intracellular pH changes under different conditions. The results were in agreement with the known properties of the transporter, with respect to both the dependence on the lactate concentration and the external pH value. Besides lactate, MCT1 mediated the reversible transport of a wide variety of monocarboxylic acids including pyruvate, D,L-3-hydroxybutyrate, acetoacetate, alpha-oxoisohexanoate and alpha-oxoisovalerate, but not of dicarboxylic and tricarboxylic acids. The inhibitor alpha-cyano-4-hydroxycinnamate bound strongly to the transporter without being translocated, but could be displaced by the addition of lactate. In addition to changes in the intracellular pH, lactate transport also induced deviations from the resting membrane potential.

The transport of lactate is an essential part of the concept of metabolic coupling between neurons and glia. Lactate transport in primary cultures of astroglial cells was shown to be mediated by a single saturable transport system with a K m value for lactate of 7.7 mM and a V max value of 250 nmol/(min ؋ mg of protein). Transport was inhibited by a variety of monocarboxylates and by compounds known to inhibit monocarboxylate transport in other cell types, such as ␣-cyano-4-hydroxycinnamate and p-chloromercurbenzenesulfonate. Using reverse transcriptase-polymerase chain reaction and Northern blotting, the presence of mRNA coding for the monocarboxylate transporter 1 (MCT1) was demonstrated in primary cultures of astroglial cells. In contrast, neuron-rich primary cultures were found to contain the mRNA coding for the monocarboxylate transporter 2 (MCT2). MCT1 was cloned and expressed in Xenopus laevis oocytes. Comparison of lactate transport in MCT1 expressing oocytes with lactate transport in glial cells revealed that MCT1 can account for all characteristics of lactate transport in glial cells. These data provide further molecular support for the existence of a lactate shuttle between astrocytes and neurons.The transport of lactate is an essential part of the concept of metabolic coupling between neurons and glia (1, 2). It has been demonstrated that glutamate at concentrations around 200 M strongly increases the rates of glycolysis and lactate release in cultured astroglial cells (3). It has further been shown that neurons are able to take up lactate and to use this compound as an energy substrate (1, 4, 5). In the mammalian retina, direct evidence has been provided for a transfer of lactate between Mü ller glial cells and photoreceptors (6). Besides its role as an exchangeable metabolic fuel, lactate also interferes with pH and volume regulation in neural cells (7).There is a considerable debate over the types of transporters involved in the uptake and release of lactate by astroglial cells.Nedergaard and Goldman (8) characterized lactate transport in cultured astrocytes and determined a low K m value of 0.4 mM. The carrier-mediated transport could not be inhibited by ␣-cyano-3-hydroxycinnamate or pCMBS, 1 both being typical inhibitors of monocarboxylate transport in other cell types. The transport process was reversible and accompanied by a cotransport of protons. Diffusion of protonated lactate could not be detected. In contrast to these results, Tildon et al. (9) identified two carrier-mediated processes for lactate uptake, characterized by K m values of 0.5 mM and 11 mM, respectively. The maximum velocity of the low-affinity transporter was 170 nmol/(min ϫ mg of protein), whereas only 10% of this value was found for the high affinity component. Transport was only partially inhibited by ␣-cyano-4-hydroxycinnamate and mersalyl. Acidic pH strongly increased transport activity, a finding consistent with a lactate/proton cotransport mechanism. Dringen et al. (10) detected solely non-saturable lactate transport ...

Transport of lactate, pyruvate, and the ketone bodies, acetoacetate and beta-hydroxybutyrate, is mediated in many mammalian cells by the monocarboxylate transporter MCT1. To be accepted as a substrate, a carboxyl group and an unpolar side chain are necessary. Site-directed mutagenesis of the rat MCT1 was used to identify residues which are involved in substrate recognition. Helices 8 and 10 but not helix 9 were found to contain critical residues for substrate recognition. Mutation of arginine 306 to threonine in helix 8 resulted in strongly reduced transport activity. Concomitantly, saturable transport was transformed into a nonsaturable dependence of transport activity on lactate concentration, suggesting that binding of the substrate was strongly impaired. Furthermore, proton translocation in the mutant was partially uncoupled from monocarboxylate transport. Mutation of phenylalanine 360 to cysteine in helix 10 resulted in an altered substrate side chain recognition. In contrast to the wild-type transporter, monocarboxylates with more bulky and polar side chains were recognized by the mutated MCT1. Mutation of selected residues in helix 9 and helix 11 (C336A, H337Q, and E391Q) did not cause alterations of the transport properties of MCT1. It is suggested that substrate binding occurs in the carboxy-terminal half of MCT1 and that helices 8 and 10 are involved in the recognition of different parts of the substrate.

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