ROLE OF ALTERED MEMBRANE FUNCTION IN XENOBIOTIC TOXICITY

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We have examined the mechanism, energetics, and xenobiotic sensitivity of renal organic anion (OA) transport, the major system which governs the elimination of many toxic xenobiotics. Isolated basolateral membrane (BLM) vesicles studies demonstrated that BLM OA transport is a complex, tertiary active process, indirectly coupled to metabolic energy through 1) the Na pump, 2) Na/alpha-ketoglutarate (alpha-KG) cotransport and 3) OA/alpha-KG exchange. BLM vesicles also provided a model for mechanistic assessment of the membrane effects of xenobiotics. For example, dehydroabietic acid (DHAA), a major component of pulp mill effluent, was shown to be a potent competitive inhibitor of OA transport. However, it also markedly reduced passive membrane permeability. thus, it slowed decay of imposed ion gradients, and, secondarily, stimulated gradient driven Na/alpha-KG cotransport, effects which increased the driving force for Na/alpha-KG coupled OA transport and partially compensated for its direct inhibitory effects. Luminal exit of OA was shown to occur predominantly via a carrier mediated, potential driven pathway. Luminal anion exchange, e.g., OH/OA, was shown to play only a limited role in OA secretion, playing instead an important role in urate reabsorption. Extending the isolated membrane studies, indirect Na-coupling of OA transport was demonstrated in intact epithelia from several species. Furthermore, the OK cell, a cultured cell line displaying many proximal tubular functions, was also shown to display Na/alpha-KG/OA transport, the first such demonstration in a cell line. Thus, OK cells provide a means to study metabolic control of alpha-KG levels and OA transport. Intact renal epithelia also showed discrete vesicular accumulation of OA within the cells during transport, raising the possibility that intracellular compartmentalization may play a role in transepithelial OA transport and/or in protection of intracellular integrity during transport. Finally, renal mRNA coding for the OA transport system was isolated, size fractionated, and expressed in Xenopus oocytes, setting the stage for preparation of the nucleotide probes needed for biochemical characterization of this system and assessment of its development and regulation.