The metabolic, digestive, and excretory functions of the liver are dependent on the uptake from blood and/or secretion into bile of circulating amphipathic organic solutes, including bile acids, nonbile acid organic anions such as bilirubin, and fatty acids. Accumulation of solutes such as bilirubin or bile acids in the bloodstream largely account for the biochemical or clinical manifestations of certain hereditary disorders of hepatic organic anion transport, as well as of cholestasis, a common manifestation of acquired liver disease. We and others have characterized the hepatocyte transport mechanisms for bile acids, nonbile acid organic anions such as bilirubin, and long-chain fatty acids. Progress toward the isolation of certain transport proteins has been reported; however, attempts at purification and antibody production have been frustrated by the low abundance of such proteins and/or the presence of multiple membrane proteins of similar molecular weight. Moreover, important aspects of the function of these transport proteins remain undefined. For example, our preliminary observations suggest that the uptake of taurocholate and of the long-chain fatty acid oleate is electrogenic, yet little is known regarding the relationship between transport of bile acids or fatty acids and transmembrane electrical potential difference, which we have shown to vary in response to physiologic stimuli and intracellular pH. Certain hormones alter transport of bile acids or fatty acids, but it is not known whether these changes reflect a direct effect on transport, mediated possibly by Ca2+ or cAMP, or hormonal effects on transmembrane electrical potential difference. The role of transmembrane ion gradients and ATP in the transport of certain organic anions is also unclear. The proposed studies will define the function and regulation of several liver-specific transport proteins which mediate the basolateral uptake or canalicular secretion of certain bile acids and nonbile acid organic anions. We will utilize a variety of approaches including electrophysiological and fluorescence techniques which have been developed over the previous grant period, are ideally suited to address the questions posed, and have had limited application to liver. These techniques will be applied to hepatocytes, as well as to expression systems including Xenopus laevis oocytes and transfected mammalian cells, in which the function of a single transport protein can be studied in isolation. To accomplish this objective, we and our collaborators will utilize expression cloning in Xenopus laevis oocytes, a strategy which does not depend on antibodies or synthetic nucleotides, has been successfully used to clone ion channels, receptors, and transporters, and, as demonstrated by our collaborators and our own preliminary studies, can be applied to liver specific membrane transport proteins. The proposed studies will provide new insights into mechanisms of canalicular bile formation and will advance our understanding of the pathophysiology of certain hereditary hepatic disorders and of cholestatic liver disease.
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