ROLE OF ALTERED MEMBRANE FUNCTION IN XENOBIOTIC TOXICITY

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Summary of Work: Renal secretory transport of organic anions (OA) and organic cations (OC) controls the excretion of most foreign chemicals and/or their metabolites. We have previously characterized the mechanisms and energetics of both processes. Our current focus is the biochemistry of these transport proteins; their development, expression, and control; and their impact on the toxicity of xenobiotics. We have begun to identify the genes coding for these transport proteins using expression cloning in Xenopus oocytes. Because of their large size and ability to make efficient use of foreign mRNA, it is relatively easy to inject mRNA and assay for expressed transport activity in each egg. We have now cloned one of the OC transport proteins and one OA transporter as well. The cloned OC carrier (OCT2) has been fully sequenced and is distinct from the other recently cloned OC transporter (OCT1). OCT1 has been tentatively identified as the basolateral OC carrier. Functional data indicates that OCT2 shares many features with the luminal OC/proton exchanger, including proton-dependent transport as well as similar substrate specificity and kinetic constants. However, it can also mediate potential driven OC transport, a property of the basolateral carrier. Studies using monolayer cultures of transfected epithelial cells have been initiated to resolve this issue. The cloned OA transport protein has also been sequenced. Functional studies demonstrate that it is basolateral OA/ -ketoglutarate exchanger which mediates uphill entry of OA into the tubular cells. It is the first renal organic anion transporter to be cloned and has been designated as ROAT1. It's properties include a Km of about 100 muM, inhibition by a variety of OAs but not OCs, cis-inhibition and trans-stimulation by -ketoglutarate, and independence of membrane potential -- all properties of the basolateral OA carrier. A related study assessed the renal mechanisms which might contribute to the observation that phenolphthalein causes renal tumors. These studies, in both isolated membrane vesicles and intact tubules, demonstrated rapid and effective renal uptake of the parent molecule and its glucuronide conjugate via the OA system; thus, apparently rendering the kidney particularly at risk for the toxic effects of these compounds. In addition, both parent and metabolite were potent inhibitors of renal elimination of other anionic xenobiotics.