Evidence for the extracellular reduction of ferricyanide by rat liver. A trans-plasma membrane redox system

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Abstract
Reduction of ferricyanide by the isolated perfused rat liver and by isolated rat hepatocytes was studied. Ferricyanide was reduced to ferrocyanide by the perfused liver at a linear rate of 0.22 .mu.mol/min per g of liver. Ferricyanide was not taken up by the liver and the perfusate concentration of ferricyanide + ferrocyanide remained constant throughout the perfusion. Perfusate samples from livers perfused without ferricyanide did not reduce ferricyanide. Isolated hepatocytes reduced ferricyanide in a biphasic manner. The initial rate of 2.3 .mu.mol/min per g of cells proceeded for .apprx. 3 min and derived from low-affinity sites (apparent Km > 1.3 mM). The secondary rate of 0.29 .mu.mol/min per g of cells was maintained for the remainder of the incubation and derived from higher affinity sites (apparent Km = 0.13 mM). Disruption of the cells resulted in an increase in the low-affinity rate and a decrease in the high-affinity rate. Ferrocyanide was oxidized by isolated hepatocytes but not by perfused liver. The apparent Km for ferrocyanide oxidation by hepatocytes was 1.3 mM. Oxidized cytochrome c was reduced by isolated hepatocytes in the presence of 1 mM-KCN but at a rate less than that of the reduction of ferricyanide. Properties of the ferricyanide-reducing activities of intact hepatocytes and the perfused liver were examined. The low-affinity rate, present only in cell and broken cell preparations, was inhibited by 1 .mu.M-rotenone and 0.5 mM-ferrocyanide and stimulated by 0.1 mM-KCN. The mitochondrial substrate, succinate, also stimulated this rate. The perfused liver showed only a high-affinity activity for ferricyanide reduction. This activity was also present in liver cells and was unaffected by rotenone, antimycin A, KCN, NaN3 or p-hydroxymercuribenzoate but was inhibited by 2.6 mM-CaCl2, 2-heptyl-4-hydroxyquinoline-N-oxide and ferrocyanide. These results are consistent with the occurrence of a transplasma membrane redox system of liver that reduces extracellular ferricyanide to ferrocyanide. The reduction process shows properties which are similar to that of the NADH:ferricyanide oxidoreductase found in isolated liver plasma membranes but different from that of mitochondria.