Our overall objective is to understand the mechanisms of hepatic GSH transport at the sinusoidal, canalicular and inner mitochondria membrane, and the zonation of GSH and precursor sulfur amino acid transport.
The specific aims of: (1.) Determine the hepatic acinar heterogeneity of both the availability of sulfur amino acids and the efflux of GHS. We will use quantitative densitometry of autoradiographs and HPLC of biopsies of the perfused rat liver to study the zonation of sulfur amino acid availability, to localize radiolabeled GSH in the acinus, and to determine zonal heterogeneity of GSH efflux into perfusate and bile. To explain the lower maximal GSH levels in perivenular cells, we will compare the kinetics of GSH efflux in isolated periportal and perivenular - enriched rat hepatocytes. In addition, to determine the role of sinusoidal membrane- glutamyl-transpeptidase in certain species other than rat, we will assess the formation and fate of products of transpeptidation, such as glutamylcystine and cysteinyl-glycine, and the zonation of their availability in perfused liver and isolated hepatocytes of the guinea pig: (2.) Define the kinetics, symmetry and specificity of GSH transport and its inhibition in isolated hepatocytes and bLPM and cLPM vesicles. We will examine trans-stimulation of GSH efflux and GSH uptake in isolated rat hepatocytes. In addition, we will examine the influence of inhibition of GSH efflux by methionine and its analogues on the rate of GSH repletion from cysteine in isolated hepatocytes. Finally, we will use inside-out bLPM (basolateral) and cLPM (canalicular membrane vesicles) to precisely define kinetic parameters of GSH transport, sidedness, induction, and kinetics of inhibition and trans-stimulation by organic anions, methionine and various analogues, including GSH monethyl ester: (3.) Reconstitute, identify and purify the bLPM GSH transporter. We will reconstitute GSH transport from bLPM in proteoliposomes and determine its kinetics and specificity. We will use GSH affinity chromatography, chromatofocusing, and other techniques to purify the transporter while reconstituting at each stage to determine function with the use of BSP-GSH as a specific inhibitor of bLPM transport. As an alternative strategy to characterize the bLPM GSH transporter, we will inject rat liver mRNA in Xenopus laevis oocytes and examine the expression of GSH transport: (4.) Characterize the kinetics and specificity of GSH transport in mitochondria. Using permeabilized rat hepatocytes, we will determine the characteristics of mitochondrial GSH transport and effect of inhibitors of transport on net mitochondrial GSH repletion in intact rat hepatocytes. Finally, we will attempt to reconstitute the mitochondrial GSH transporter and initiate work on its purification. The ultimate goal of this work is the prevention and treatment of liver disease and its extrahepatic complications by exploiting and manipulating hepatic GSH.
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