Lactate and pyruvate transport is dominated by a pH gradient-sensitive carrier in rat skeletal muscle sarcolemmal vesicles

Abstract

The mechanisms of lactate and pyruvate transport across the plasma membrane of rat skeletal muscle under various pH and ionic conditions were studied in skeletal muscle sarcolemmal (SL) membrane vesicles purified from 22 female Sprague-Dawley rats. Transport by SL vesicles was measured as uptake of l(+)-[U-¹⁴C] lactate and [U-¹⁴C] pyruvate. Lactate (La⁻) transport is pH-sensitive; stimulations to fivefold overshoot above equilibrium values were observed both directly by a proton gradient directed inward, and indirectly by a monensin- or nigericin-stimulated exchange of Na⁺ or K⁺ for H⁺ across the SL. Isotopic pyruvate could utilize the transporter, and demonstrated pH gradient-stimulated overshoot and cis-inhibition characteristics similar to those of lactate. Overshoot kinetics were also demonstrated by pH gradient formed by manipulation of external media at pH 5.9, 6.6, and 7.4 and intravesicular media at 6.6, 7.4, and 8.0, respectively. Carbonyl cyanide m-chlorophenylhydrazone, an H⁺ ionophore, was used as a “pH clamp” to return all stimulated uptake courses back to equilibrium values. Lactate uptake was depressed when internal pH was lower than external pH. These data strongly suggest that La⁻ and H⁺ are either cotransported by the carrier, or transported as the undissociated HLa, and can account for the majority of the lactate uptake at pH 7.4. The mechanism does not require cotransport of either K⁺ or Na⁺. However, an inwardly directed Na⁺ gradient without ionophore in the absence of a pH gradient doubled La⁻ transport; treatment with amiloride, an inhibitor of the $\text{Na}{}^{\mathrm{+}}\text{H}{}^{\mathrm{+}}$ exchanger, abolished this stimulation, suggesting that this transporter may be an important coregulator of intracellular pH, and could disrupt 1:1 H⁺ and La⁻ efflux stoichiometry in vivo. We conclude that the majority of La⁻ crosses the skeletal muscle SL by a specific carriermediated process that is saturable at high La⁻ concentrations, but flux is passively augmented at low intracellular pH by undissociated lactic acid. In addition, a $\text{Na}{}^{\mathrm{+}}\text{H}{}^{\mathrm{+}}$ exchange mechanism was confirmed in skeletal muscle SL, does affect both lactate and proton flux, and is potentially an important coregulator of intracellular pH and thus, cellular metabolism.