Copper transport kinetics by isolated rat hepatocytes

Abstract

Uptake and efflux of 64Cu were examined to determine whether hepatic parenchymal cells exhibit the kinetic criteria of a specific transport system for copper and related trace metals. Saturation kinetics were clearly indicated by both v versus [Cu] and 1/v versus 1/[Cu] plots (Km = 11 +/- 0.6 microM and Vmax = 2.7 nmol Cu X min-1 X mg prot-1). Identical results were obtained by cold-copper analyses, and contributions from simple diffusion or nonspecific binding were not detected. Virtually all of the accumulated 64Cu was intracellular by 0.5 min (the initial velocity period), with approximately 40% in the cytosolic fraction. Several related trace metals inhibited 64Cu uptake, but Ni(II) at a 10:1 molar excess did not. Zn(II) acted as a simple competitive inhibitor of 64Cu uptake (Ki = 16 microM). Efflux from preloaded cells was biphasic, with an initial rapid phase of approximately 5 min. Approximately 35% of preloaded 64Cu was transported out of the cells by 40 min, and little efflux occurred thereafter. Thus, hepatocytes exhibit saturation kinetics, competition by related substrates, and countertransport criteria of specific facilitated transport. A wide variety of metabolic inhibitors have no effect on 64Cu uptake under the same conditions that inhibit the active transport of bile acids. Specific inhibitor tests for electrogenic coupling were also negative. Because the identical kinetic parameters were obtained for free 64Cu and the 1:1 64Cu-histidine complex, it is inferred that copper is probably transported as the free ion. Cells incubated with greater than or equal to 10 microM 64Cu showed a net loss of copper after 40- to 60-min incubation, which may involve specific hepatic mechanisms in copper homeostasis.

Uptake and efflux of copper-64 in Menkes'-disease and normal continuous lymphoid cell lines

“…5)]. However, the kinetic parameters of efflux have been derived for isolated rat hepatocytes, and they were found to correspond well with the parameters of uptake (Schmitt et al, 1983). This suggested that, in these cells, uptake and efflux are mediated by the same system and fit the model of symmetrical facilitated diffusion (Christensen, 1975).…”

“…The kinetic constants of copper uptake have been described for rat hepatocytes (Schmitt et al, 1983) and suggest a saturable process of facilitated diffusion with a similar Km but with a Vmax some 200-fold higher than for continuous lymphoid cells. These results are consistent with the conclusion that a component ofcopper transport is facilitated in continuous lymphoid cells.…”

“…Studies on copper uptake and accumulation in normal cells have been performed by using a variety of different extracellular media (Weiner & Cousins, 1980;Schmitt et al, 1983;Darwish et al, 1984b). The distribution between, and binding of copper to, the various components of the media are important in determining the chemical activity of free copper with respect to uptake.…”

“…Indeed, it is possible that the rate-determining step in uptake may be the dissociation of copper from copperbinding ligands in the medium. Schmitt et al (1983) used a balanced salt solution not containing any serum proteins for their measurements of copper uptake and accumulation in isolated rat hepatocytes. Uptake of copper was found to occur via a passive saturable process.…”

Characterization of Sandwich-Cultured Hepatocytes As an in Vitro Model to Assess the Hepatobiliary Disposition of Copper

“…Uptake studies in mouse and rat hepatocyte suspensions indicated a transporter-mediated process with saturable kinetics (Schmitt et al, 1983;Darwish et al, 1984;McArdle et al, 1988;Bingham and McArdle, 1994). Dijkstra et al (1995) measured Cu transport in plasma membrane vesicles enriched in canalicular or basolateral membranes and found that ATP stimulated uptake of Cu into canalicular plasma membrane vesicles and, to a lesser extent, into basolateral plasma membrane vesicles.…”