KINETICS OF THE SARCOLEMMAL LACTATE CARRIER IN SINGLE HEART-CELLS USING BCECF TO MEASURE PH(I)

The pH-sensitive fluorescent indicator 2',7'-bis(carboxyethyl)-5(6)car boxyfluorescein (BCECF) was used to measure lactate transport in singl e cardiac myocytes. Addition of lactate externally caused a rapid fall of intracellular pH (pH(i)), which was largely inhibited by 5 mM alph a-cyano-4-hydroxycinnamate (CHC), a specific inhibitor of the lactate carrier. Stilbene disulfonates such as 4,4'-dibenzamidostilbene-2,2'-d isulfonate (DBDS) only partially inhibited the response, with inhibiti on being greater in guinea pig than rat myocytes. The data are consist ent with two isoforms of the lactate carrier, one sensitive and one in sensitive to DBDS, coexisting within a single myocyte and both having a stoichiometry of 1 lactate:1 proton. The initial rate of pH(i) fall was used to determine carrier kinetics. Rat myocytes had a Michaelis c onstant (K-m) for external L-lactate of 2.74 mM and a K-m for external pyruvate of 0.2 mM. Guinea pig cells had a K-m for external. L-lactat e of 2.2 mM. Kinetics of lactate efflux were also evaluated using the rate of pH(i) recovery on removing external lactate. The K-m and maxim al rate values for efflux were both threefold higher than for influx a nd were related to each other and the transmembrane pH gradient as pre dicted by the Haldane relationship. It is suggested that under hypoxic conditions, the carrier may be the rate-limiting factor for lactate e xtrusion.