Kinetics of lactate and pyruvate transport in cultured rat myotubes

Skeletal muscle transport of lactate and pyruvate was studied in primary cu ltures of rat myotubes, applying the pH-sensitive fluorescent indicator 2', 7'-bis(carboxyethyl)-5 (6) carboxyfluorescein. The initial rate of decrease in intracellular pH (pH(i)) upon lactate or pyruvate incubation was used t o determine total transport (carrier mediated and diffusion). Both lactate and pyruvate transport could be inhibited by a combination of 0.5 mM 4,4'-d iisothiocyanostilbene-2,2'-disulfonic acid, 5 mM mersalyl and 10 mM alpha-c yano-4-hydroxycinnamate. The kinetic parameters, K-m and V-max, 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, K-m and V-max were 4.4 +/- 1.3 mM a nd 0.30 +/- 0.5 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 t hat 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 ca talyzed by the monocarboxylate transporter isoform MCT3, J. Biol. Chem. 273 (1998) 15920-15926)), were also expressed in primary culture of myotubes. (C) 1999 Elsevier Science B.V. All rights reserved.