New insights into canalicular organic anion secretion

Abstract

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.