The effect of partial and complete dissipation of the membrane potential and partial depletion of cellular ATP content on the efflux of dinitrophenyl-glutathione and oxidized glutathione was examined in hepatocytes isolated from normal and mutant (TR−) rats exhibiting defective organic anion transport. Whereas alterations in the membrane potential difference had no effect on the initial efflux rate of dinitrophenyl-glutathione and oxidized glutathione, depletion of cellular ATP inhibited dinitrophenyl-glutathione and oxidized glutathione efflux and a linear relationship between the cellular ATP content and the initial efflux rate of dinitrophenyl-glutathione was observed in normal isolated rat hepatocytes. In contrast, depletion of cellular ATP content had no significant effect on the slower rate of dinitrophenyl-glutathione efflux from TR− rat hepatocytes. These findings implicate an ATP-dependent hepatic transport system for oxidized glutathione and glutathione conjugates that is absent in TR− mutants.
Fluorescence image analysis reveals normal secretion of a fluorescent bile acid fluorescein isothiocyanate glycocholate into the canalicular lumen of isolated normal and TR− mutant rat hepatocyte couplets, but negligible canalicular accumulation of a non-bileacid organic anion (carboxydichlorofluorescein diacetate) in TR− hepatocyte couplets. Canalicular membrane vesicles derived from normal rats exhibited saturable temperature- and ATP-dependent transport of sulfobromophthalein and sulfobromophthalein-glutathione that was absent in canalicular membrane vesicles from TR− rats. However, ATP-dependent daunomycin transport, reflecting transport mediated by the multidrug resistance gene product, p-glycoprotein, was present in canalicular membrane vesicles from both normal and TR− rats. Canalicular membrane vesicles from normal and TR− rats contained equal amounts of p-glycoprotein on immunoblots. These studies demonstrate that the conjugated hyperbilirubinemia in TR− mutant rats is the result of a functional absence of an ATP-dependent organic anion transport system on the canalicular membrane.