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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DK030312-18
Application #
2872184
Study Section
Special Emphasis Panel (NSS)
Program Officer
Serrano, Jose
Project Start
1982-02-01
Project End
2000-05-31
Budget Start
1999-03-01
Budget End
2000-05-31
Support Year
18
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Southern California
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
041544081
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Han, Derick; Hanawa, Naoko; Saberi, Behnam et al. (2006) Hydrogen peroxide and redox modulation sensitize primary mouse hepatocytes to TNF-induced apoptosis. Free Radic Biol Med 41:627-39
Liu, Zhang-Xu; Han, Derick; Gunawan, Basuki et al. (2006) Neutrophil depletion protects against murine acetaminophen hepatotoxicity. Hepatology 43:1220-30
Gunawan, Basuki K; Liu, Zhang-Xu; Han, Derick et al. (2006) c-Jun N-terminal kinase plays a major role in murine acetaminophen hepatotoxicity. Gastroenterology 131:165-78
Liu, Zhang-Xu; Govindarajan, Sugantha; Kaplowitz, Neil (2004) Innate immune system plays a critical role in determining the progression and severity of acetaminophen hepatotoxicity. Gastroenterology 127:1760-74
Mittur, Aravind; Wolkoff, Allan W; Kaplowitz, Neil (2002) The thiol sensitivity of glutathione transport in sidedness-sorted basolateral liver plasma membrane and in Oatp1-expressing HeLa cell membrane. Mol Pharmacol 61:425-35
Mittur, A V; Kaplowitz, N; Kempner, E S et al. (2000) Radiation inactivation studies of hepatic sinusoidal reduced glutathione transport system. Biochim Biophys Acta 1464:207-18
Ookhtens, M; Kaplowitz, N (1998) Role of the liver in interorgan homeostasis of glutathione and cyst(e)ine. Semin Liver Dis 18:313-29
Mittur, A V; Kaplowitz, N; Kempner, E S et al. (1998) Novel properties of hepatic canalicular reduced glutathione transport revealed by radiation inactivation. Am J Physiol 274:G923-30
Lu, S C; Kuhlenkamp, J; Wu, H et al. (1997) Progressive defect in biliary GSH secretion in streptozotocin-induced diabetic rats. Am J Physiol 272:G374-82
Lu, S C; Cai, J; Kuhlenkamp, J et al. (1996) Alterations in glutathione homeostasis in mutant Eisai hyperbilirubinemic rats. Hepatology 24:253-8

Showing the most recent 10 out of 42 publications