A microelectrode study of the mechanisms of L‐lactate entry into and release from frog sartorius muscle.

Abstract

Changes in intracellular pH and intracellular anion levels were monitored in frog sartorius muscle fibres during exposure to extracellular L-lactate, using ion-sensitive microelectrodes. Resting intracellular pH (pHi) in 20 mmol l-1 HEPES buffer was 7.18 .+-. 0.015 (S.E. of mean, n =62). Exposure to an extracellular solution at pH 6.5 buffered with 20 mmol l-1 3-(N-morpholino)propanesulphonic acid (MOPS) resulted in a slow intracellular acidification. A reversible decrease in pHi and an increase in intracellular anion levels was observed when L-lactate replaced chloride in equimolar amounts. The increase in intracellular anion level is consistent with intracellular accumulation of L-lactate ion. The rate and steady-state change in pHi and anion level was a function of both extracellular pH and L-lactate concentration, providing evidence for the coupled movement of lactate and proton equivalents. The initial rate of uptake of L-lactate, as measured by the change of pHi, was a non-linear function of the extracellular L-lactate concentration at extracellular pH 6.8 and 7.35. No saturation was observed iwth concentrations of L-lactate between 5 and 60 mmol l-1 at pH 7.35 and 2.5 and 40 mmol l-1 at pH 6.8. The non-linear relationship between the initial rate of change in pHi and extracellular L-lactate was well fitted by a curve defining uptake as the sum of a carrier process displaying Michaelis-Menten kinetics and a passive diffusion component. The apparent Km of the carrier was 10 mmol l-1 at pH0 7.35 and 4 mmol l-1 at pH0 6.8. The initial rate of change of pHi in the presence of L-lactate was significantly inhibited 39.1.+-.6.2% by 2-5 mmol l-1 .alpha.-cyano-4-hydroxycinnamate (n = 9; P < 0.05, paired t test). .alpha.-Cyano-4-hydroxycinnamate had no detectable effect on the initial rate of change of pHi induced by propionate exposure. The initial rate of change of phi induced by L-lactate was not affected by 20-100 .mu.mol l-1 4-acetamido-4-isothiocyanostilbene-2,2''-disulphonic acid (SITS). We conclude that L-lactate crosses the membrane of the frog sartorius muscle with proton equivalents via (1) a carrier-mediated process, and (2) passive diffusion of lactic acid. In the physiological range of L-lactate concentrations and pH the transport process dominates.