Copper efflux kinetics from rat hepatocytes

The kinetics of copper efflux from rat hepatocytes were determined to further characterize the hepatic Cu(II) transport system. Efflux was biphasic. Net efflux was rapid for 1-5 min, and 35-45% of preloaded copper was lost by 40 min. Efflux was negligible after 40 min. The retained percentage was independent of the preloading concentration, but the total amount of intracellular copper that was available for efflux gradually decreased as the duration of the preloading period increased. Unlabeled extracellular Cu(II) displaced 64Cu from intracellular pools, but exchange with intracellular Cu was not required for uptake. Zinc also displaced copper but less effectively than copper. This implies some specificity in intracellular binding components. No transstimulation of uptake or efflux was detected. Copper efflux was strictly passive. Extracellular Cu(II) decreased the rate of efflux and preloading inhibited Cu(II) uptake. Metabolic inhibitors had no effect on the rate or total amount of 64Cu efflux, and significant efflux occurred at 4 degrees C. Moreover, when a steady state was attained at the end of a preloading period, the effective intracellular concentration of free copper approximated the Cu(II) concentration in the extracellular medium. By use of this estimate for the intracellular concentration of free copper, efflux kinetic parameters were obtained from initial (1-min) rate data (Km = 5.5 +/- 0.8 microM; Vmax = 1.1 +/- 0.1 nmol X min-1 X mg prot-1). These parameters are similar to those obtained for Cu(II) uptake, and the saturation kinetics observed are consistent with specific facilitated efflux by this copper transport system.(ABSTRACT TRUNCATED AT 250 WORDS)