Lactate transport in L6 skeletal muscle cells and vesicles: allosteric or multisite mechanism and functional membrane marker of differentiation.
Membrane lactate transport was studied in skeletal muscle cells and membrane vesicles from the L6 line in relation to in vitro myogenesis. In myoblasts, lactate was transported by simple diffusion and insensitive to classical inhibitors: a positive correlation between onset of creatine kinase activity and lactate transport in differentiated myotubes was observed and could be considered to be a functional marker of cell differentiation. In myotubes, complete analysis of the velocity curves (direct coordinates, Eadie-Scatchard plots, Hill plots) gave parameters showing that lactate was carried by an allosteric or multisite system. This was confirmed by using sarcolemmal vesicles and specific inhibitors. In whole cells, alpha-cyano-4-hydroxycinnamic acid (CIN) and parachloromercuribenzylsulphonic acid (pCMBS) inhibited the maximal velocity without modifying the global cooperativity of the system. The weak effect of 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), which has a low affinity constant (Ki = 22.5 microM), implicated the monocarboxylate system rather than the anionic exchanger as a carrier system in muscle cells. CIN and DIDS exhibited one type of interaction with lactate carriers, and the curvilinear shape of the lactate Hill plot with or without inhibitors suggested that inhibitors were active at the same family of interaction sites and had a common range of affinities. The apparent competitive inhibition of pyruvate (Ki = 3.2 mM) did not modify the transport pathway of lactate in L6 myotubes. In conclusion, kinetic analysis of lactate transport in the presence or absence of inhibitors gave evidence for a multisite lactate carrier activity in myotubes composed of two systems at least, related to two or three isoforms of lactate carriers.
Beaudry
M
M
Laboratoire de Physiologie des Adaptations UFR Medicine Cochin, Université René Descartes, Paris, France.
Mouaffak
N
N
el Abida
K
K
Rieu
M
M
Mengual
R
R
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000494:Allosteric Regulation; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D019343:Citric Acid / Q000494:pharmacology; D003373:Coumaric Acids / Q000494:pharmacology; D003402:Creatine Kinase / Q000378:metabolism; D007700:Kinetics; D019344:Lactic Acid / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D010663:Phenylmercury Compounds / Q000494:pharmacology; D019289:Pyruvic Acid / Q000494:pharmacology; D051381:Rats; D012508:Sarcolemma / Q000378:metabolism; D019802:Succinic Acid / Q000494:pharmacology
D003373:Coumaric Acids; D010663:Phenylmercury Compounds; C002910:4-hydroxymercuribenzenesulfonate; C007175:alpha-cyano-4-hydroxycinnamate; D019343:Citric Acid; D019344:Lactic Acid; D019289:Pyruvic Acid; D019802:Succinic Acid; D003402:Creatine Kinase; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid