The objectives of this proposal are to elucidate and characterize the processes involved in (i) delivery from plasma to the sinusoidal membrane (bLPM), (ii) intracellular transport, and (iii) selective access to enzymes in the endoplasmic reticulum (E.R.) of an array of small hydrophobic and amphipathic molecules (e.g., organic anions, bile salts, endogenous and xenobiotic substrates). Studies will focus specifically on dynamic functions of selected membrane structures, soluble proteins and membrane-carrier proteins involved in these processes. The proposed series of interrelated projects will employ high-resolution biochemical/biophysical and molecular biological techniques to extend and re-define existing concepts of hepatic uptake, hepatocellular transport and transmembrane E.R. transfer of small molecules. Stopped-flow fluorimetry and ultra-rapid filtration will be applied to study rapid ligand transfer processes between model and native membranes, lipid particles and soluble binding proteins. The concept that ligands are transported by spontaneous intermembrane diffusion (vs. protein-mediated transfer) will be examined. To characterize the intravascular (extracellular) binding and delivery of small molecules to the bLPM, the contribution of circulating lipid assemblies (HDL, LDL, erythrocyte membranes) and serum albumin will be investigated. The kinetics of intermembrane (bLPM to E.R.) and cytosolic protein-mediated transfer will be defined, and the hypothesis that an inherent membrane lipid (e.g., cholesterol) gradient directs substrates from the bLPM to the E.R. will be examined. Utilizing these data an integrated model of intracellular transport will then be derived. The crucial mechanisms by which a variety of hydrophobic substrates and the nucleotide-sugar, UDP-glucuronic acid (the essential cosubstrate for all glucuronidation reactions), traverse the E.R. membrane to the lumenal binding sites on the UDP- glucuronosyltransferases (UGTs), and whether an E.R. carrier (e.g., antiport) system exports nascent glucuronides to the cytosol, also will be investigated. We propose to clone a cDNA encoding for the UDP-glucuronic acid transport protein, using a combined approach of IAM-HPLC, photoaffinity labeling protein isolation, and several alternative cloning strategies (e.g., functional expression in oocytes). Transfection into a COS cell (microsomal) expression system and development of polyclonal antibodies will further validate the function and regulation of the cloned transporter. These findings will facilitate studies on protein expression and gene regulation, and will enhance our understanding of the integrated microsomal UGT enzyme system. Collectively, these data should provide new insights into the mechanisms of extracellular transport and delivery to the hepatocyte, intracellular trafficking, and detoxification of a wide variety of small hydrophobic molecules. In addition, these findings should contribute to our understanding of membrane pathobiology, and the regulation and function of complex membrane-bound (microsomal) enzymes, and will likely have important implications for drug metabolism and pharmacokinetics, and the pathophysiology of hepatotoxicity and cholestasis.
Showing the most recent 10 out of 27 publications
