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 neglibible 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 intra-cellular binding components. No transstimulation of uptake or efflux was detected. Copper efflux was strictly passive. Extra-cellular 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° 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 (K(m) = 5.5 ± 0.8 μM; V(max) = 1.1 ± 0.1 nmol.min^-1.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. Preloading with histidine had no specific effect on the rate or total efflux, and neither albumin nor histidine affected efflux when included in the extracellular media. Thus, the transport system is apparently specific for Cu(II) or the Cu(II) histidine complex rather than albumin or histidine.