Kinetics of the sarcolemmal lactate carrier in single heart cells using BCECF to measure pHi.
The pH-sensitive fluorescent indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was used to measure lactate transport in single cardiac myocytes. Addition of lactate externally caused a rapid fall of intracellular pH (pHi), which was largely inhibited by 5 mM alpha-cyano-4-hydroxycinnamate (CHC), a specific inhibitor of the lactate carrier. Stilbene disulfonates such as 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) only partially inhibited the response, with inhibition being greater in guinea pig than rat myocytes. The data are consistent with two isoforms of the lactate carrier, one sensitive and one insensitive to DBDS, coexisting within a single myocyte and both having a stoichiometry of 1 lactate:1 proton. The initial rate of pHi fall was used to determine carrier kinetics. Rat myocytes had a Michaelis constant (Km) for external L-lactate of 2.74 mM and a Km for external pyruvate of 0.2 mM. Guinea pig cells had a Km for external L-lactate of 2.2 mM. Kinetics of lactate efflux were also evaluated using the rate of pHi recovery on removing external lactate. The Km and maximal rate values for efflux were both threefold higher than for influx and were related to each other and the transmembrane pH gradient as predicted by the Haldane relationship. It is suggested that under hypoxic conditions, the carrier may be the rate-limiting factor for lactate extrusion.
Wang
X
X
Department of Physiology, School of Medical Sciences, University of Bristol, United Kingdom.
Levi
A J
AJ
Halestrap
A P
AP
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D005452:Fluoresceins; D006168:Guinea Pigs; D006352:Heart Ventricles; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007773:Lactates / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D027501:Monocarboxylic Acid Transporters; D009206:Myocardium / Q000378:metabolism*; D015388:Organelles / Q000378:metabolism*; D051381:Rats; D012508:Sarcolemma / Q000378:metabolism*; D013045:Species Specificity
D002352:Carrier Proteins; D005452:Fluoresceins; D007773:Lactates; D008565:Membrane Proteins; D027501:Monocarboxylic Acid Transporters; C057433:2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid