Copper transport kinetics by isolated rat hepatocytes.
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.
D000818:Animals; D001692:Biological Transport; D003300:Copper / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008297:Male; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013347:Subcellular Fractions / Q000378:metabolism; D013696:Temperature; D014634:Valinomycin / Q000494:pharmacology
D014634:Valinomycin; D003300:Copper; D011188:Potassium