The liver is the principal organ responsible for the metabolism and detoxification of a multitude of endogenous compounds and xenobiotics. However, relatively little is known about the mechanisms by which these substances are transported to their site(s) of biotransformation within the hepatocyte. It has generally been assumed that the intracellular transport of hydrophobic substrates is mediated largely via cytosolic binding proteins. However, there is evidence to suggest that membranes may facilitate the intracellular movement of hydrophobic molecular species. The objectives of this project are to define the mechanisms by which hydrophobic organic anions (e.g. bilirubin) and other small hydrophobic molecular species (e.g. bile salts) are transported within the hepatocyte and, specifically, to delineate the role of intracellular membranes and soluble binding proteins in this process. The initial studies, using. unconjugated bilirubin as a model substrate, will employ the technique of stopped-flow fluorescence quenching to determine the kinetics of transfer of this pigment between membrane vesicle populations. The rate of substrate transfer between membranes and soluble binding proteins (e.g. albumin, glutathione S-transferases) also will be measured. These data will permit the construction of a kinetic model of intracellular bilirubin transport. Similar techniques will then be utilized to study the membrane-to-membrane transfer of bilirubin conjugates and a series of fluorescent bile salt derivatives. The influence of ligand hydrophobicity, netcharge, surface activity, bilayer localization, and competition on these transfer processes will then be analyzed. The effect of alterations in membrane composition, charge, fluidity, source, and molecular packing on ligand transfer kinetics also will be assessed. Finally, the membrane-to- membrane transfer of a variety of relevant organic anions and xenobiotics will be measured in order to confirm the predictions of the previously developed kinetic model. Since many antibiotics, including some penicillins and cephalosporins, are metabolized by the liver via the same pathway as endogenous hydrophobic compounds such as bilirubin, these studies should help to clarify the processes involved in substrate access to membrane-bound enzymes and drug metabolism. Ultimately, an in vitro system will be developed in an attempt to precisely model the intracellular milieu of the hepatocyte. Additionally, information gained from this project may have direct implications for the pathophysiology of kernicterus (bilirubin-induced encephalopathy) and pigment gallstone formation. Collectively, the proposed studies should serve to enhance the understanding of the mechanisms which underlie the hepatocellular transport of hydrophobic molecular species, and may also have relevance to epithelial transport by other organ systems, such as the kidney and small intestine.
