Simultaneous regulation of amino acid influx and efflux by system A in the hepatoma cell HTC. Ouabain simulates the starvation-induced derepression of system A amino acid transport.

In the cultured hepatoma cell HTC, amino acid starvation stimulated both influx and efflux of 2-(methylamino)isobutyric acid (MeAIB) across the plasma membrane with little effect on the ultimate cellular accumulation of this amino acid. In agreement, prior amino acid starvation had little effect on the cellular steady state levels reached for various natural amino acids during subsequent incubation in an amino acid-rich medium containing cycloheximide. Furthermore, efflux of [14C]MeAIB was markedly increased by amino acid starvation. These findings do not mean that adaptive regulation of neutral amino acid transport is pointless. If membrane transport rather than metabolism is the rate-limiting step for net amino acid production or consumption, or becomes so during times of elevated formation or catabolism of an amino acid, then proportionate changes of both the opposed fluxes should enhance its net generation or consumption. Amino acid starvation enhances MeAIB-dependent Na+ influx. Alteration of the external [Na+] changes the Km, not the Vmax, for MeAIB influx when the degree of System A derepression is stabilized with cycloheximide. In both starved and unstarved cells, Km/Vmax for MeAIB entry yields a linear function with the reciprocal of the external [Na+], supporting at least for influx a rapid equilibrium-ordered kinetic model in which Na+ binds to the carrier site before the amino acid. Elevated cellular [Na+] obtained by ouabain treatment increased MeAIB efflux in parallel. Trans-inhibition of MeAIB influx by accumulated MeAIB, and as a related phenomenon by cellular Na+, was as effective in unstarved as in starved cells, showing independence of this kinetic phenomenon from adaptive regulation. The decreased MeAIB accumulation resulting from decreased influx and increased efflux occurring at high internal [Na+] applies both to unstarved and starved cells. We conclude that cellular Na+ accumulations, produced by increasing levels of ouabain, reversibly reduce the ability of MeAIB to repress System A because its interior concentration is prevented from rising, although transport in both directions continues; accordingly, the repressive signal appears to come from the internal amino acid levels rather than from occupation of the carrier site for System A flux.