The intracellular distribution of glutathine (GSH) in cultured hepatocytes hasbeen investigated by using the compound monochlorobimane (BmCI) Approximately 10-20% of total cellular GSH in rat liver is sequestered in the mitochondrial matrix (6, 7). The size ofthis pool depends on cytosolic GSH synthesis (8) and the active transport of GSH into mitochondria via a multicomponent system recently described (9).Conventional cell-fractionation studies have not provided evidence for the existence of functionally distinct pools of GSH in hepatocytes other than those in the cytosol and mitochondria. Despite the known functions of GSH in DNA synthesis (10) and protection from oxidative DNA damage (11), little is known about the nuclear localization ofGSH and the factors regulating the nuclear GSH level. Tirmenstein and Reed (12), using fractionation and centrifugation techniques in nonaqueous medium, measured the nuclear GSH content in rat kidney and found values similar to those in the cytosol. Other fractionation techniques (such as selective permeabilization of cell constituents with various detergents) provided equivocal results (13,14). However, the latter investigations have suggested that a nuclear pool of GSH may exist in intact cells.Recent advances in image-analysis technology, together with the development of additional, nontoxic fluorescent indicators that can be used in intact cells, have facilitated an enormous input into the study of various aspects of cell physiology (15,16 ITo whom reprint requests should be addressed. 4412The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Glutathione (GSH), a tripeptide particularly concentrated in the liver, is the most important thiol reducing agent involved in the modulation of redox processes. It has also been demonstrated that GSH cannot be considered only as a mere free radical scavenger but that it takes part in the network governing the choice between survival, necrosis and apoptosis as well as in altering the function of signal transduction and transcription factor molecules. The purpose of the present review is to provide an overview on the molecular biology of the GSH system; therefore, GSH synthesis, metabolism and regulation will be reviewed. The multiple GSH functions will be described, as well as the importance of GSH compartmentalization into distinct subcellular pools and inter-organ transfer. Furthermore, we will highlight the close relationship existing between GSH content and the pathogenesis of liver disease, such as non-alcoholic fatty liver disease (NAFLD), alcoholic liver disease (ALD), chronic cholestatic injury, ischemia/reperfusion damage, hepatitis C virus (HCV), hepatitis B virus (HBV) and hepatocellular carcinoma. Finally, the potential therapeutic benefits of GSH and GSH-related medications, will be described for each liver disorder taken into account.

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Western blot analysis of protein extracts from rat liver revealed the presence of the mGlu5 receptor, one of the G-protein-coupled receptors activated by glutamate (named ''metabotropic glutamate receptors'' or mGlu receptors). mGlu5 expression was particularly high in extracts from isolated hepatocytes, where levels were comparable with those seen in the rat cerebral cortex. The presence of mGlu5 receptors in hepatocytes was confirmed by reversetranscription polymerase chain reaction (RT-PCR) analysis, immunohistochemistry in neonate or adult rat liver, as well as by immunocytochemical analysis in HepG2 hepatoma cells, where the receptor appeared to be preferentially distributed in cell membranes. Interestingly, mGlu1 receptors (which are structurally and functionally homologous to mGlu5 receptors) were never found in rat liver or hepatocytes. In hepatocytes exposed to anoxic conditions for 90 minutes, glutamate, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) and quisqualate, which all activate mGlu5 receptors, accelerated the onset and increased the extent of cell damage, while 4-carboxy-3-hydroxyphenylglycine (4C3HPG), an agonist of mGlu2/3 receptors, was inactive. 2-Methyl-6-(2-phenyl-1-ethynyl)-pyridine (MPEP), a novel, noncompetitive, highly selective mGlu5 receptor antagonist, not only abolished the toxic effect of 1S,3R-ACPD, but, unexpectedly, was protective by itself against anoxic damage. This suggests that hepatocytes express mGlu5 receptors and that activation of these receptors by endogenous glutamate facilitates the development of anoxic damage in hepatocytes. (HEPATOLOGY 2000; 31:649-655.)Glutamate is the major excitatory neurotransmitter in the central nervous system (CNS), and evidence for peripheral glutamatergic fibers in mammals is still lacking. However, glutamate receptors have been identified in peripheral organs, including taste buds, 1 myenteric plexus, 2 and pancreatic islet cells. 3 Glutamate receptors may either form membrane ion channels (ionotropic glutamate receptors) or couple to guanosine triphosphate-binding proteins (''metabotropic'' glutamate [mGlu] receptors). mGlu receptors form a family of 8 subtypes, which are subdivided into 3 groups on the basis of their structural homology, pharmacological profile, and transduction pathways. Group I includes mGlu1 and -5 receptors, which are coupled to polyphosphoinositide (PI) hydrolysis and activated by 3,5-dihydroxyphenylglycine (DHPG) and quisqualate; group II (mGlu2 and -3) and -III (mGlu4, -6, -7, and -8) mGlu receptors are instead negatively coupled to adenylyl cyclase activity. 4,5 1S,3R-ACPD behaves as a non-subtype-selective agonist, with a preferential activity on group I and -II mGlu receptors. 4 We were intrigued by the finding that 1S,3R-ACPD and quisqualate stimulate PI hydrolysis in cultured hepatocytes, and this effect is reduced by the mixed mGlu receptor antagonist, ␣-methyl-4-carboxyphenylglycine (MCPG). 6 This suggests that either mGlu1 or -5 (or a related unknown receptor subtype) are expressed and...

Nonalcoholic fatty liver disease (NAFLD) is a continuum of liver abnormalities often starting as simple steatosis and to potentially progress into nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis and hepatocellular carcinoma. Because of its increasing prevalence, NAFLD is becoming a major public health concern, in parallel with a worldwide increase in the recurrence rate of diabetes and metabolic syndrome. It has been estimated that NASH cirrhosis may surpass viral hepatitis C and become the leading indication for liver transplantation in the next decades. The broadening of the knowledge about NASH pathogenesis and progression is of pivotal importance for the discovery of new targeted and more effective therapies; aim of this review is to offer a comprehensive and updated overview on NAFLD and NASH pathogenesis, the most recommended treatments, drugs under development and new drug targets. The most relevant in vitro and in vivo models of NAFLD and NASH will be also reviewed, as well as the main molecular pathways involved in NAFLD and NASH development.

Although the use of melatonin in the transplantation field has been suggested, it has not been previously tested in a liver cold-storage model. We used a rat liver model to study (a) the dose-dependent effect of melatonin on bile production, and (b) the potential of melatonin to improve liver function after cold-storage. Male Wistar rats were perfused with Krebs-Henseleit bicarbonate buffer (KHB) at 37 degrees C without or with 25, 50, 100 and 200 microM melatonin. Each dose of melatonin stimulated bile production. For cold-storage studies, livers were flushed with either University of Wisconsin (UW) or Celsior solution and stored for 20 hr at 4 degrees C. Reperfusion (120 min) was performed with KHB at 37 degrees C. In subsequent studies, 100 microM melatonin were added to the perfusate during the reperfusion period. ATP and melatonin levels in the tissue were measured. Bile analysis was performed by measuring melatonin, bilirubin and gamma-glutamyl transpeptidase (gamma-GT) levels in the fluid. A dose-dependent increase in bile secretion, associated with an enhanced melatonin and bilirubin levels in the bile were observed. Also, tissue levels of melatonin increased in a dose-dependent manner. When melatonin was added during the reperfusion period, bile production and bile bilirubin levels increased both with UW and Celsior solutions. The analysis of gamma-GT in the bile showed an increase in the Celsior-preserved liver and the addition of melatonin to the perfusate reduced this effect. Tissue ATP levels were higher when melatonin was added to the perfusion medium. Higher levels of melatonin in bile than in tissue were found. In conclusion, we demonstrate that melatonin improves significantly the restoration of liver function after cold-storage and reperfusion.

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Studies assessing the effects of partial-hepatic ischemia/reperfusion (I/R) injury focused on the damage to the ischemic-lobe, whereas few data are available on non-ischemic lobe. This study investigated whether acute liver I/R does affect non-ischemic lobe function via modulation of extracellular matrix remodeling. Male Sprague-Dawley rats underwent left lateral-and median-lobe ischemia for 30 min and reperfusion for 60 min or sham operation. After reperfusion, blood samples and hepatic biopsies from both the ischemic (left-lobe, LL) and the non-ischemic lobe (right-lobe, RL) were collected. Serum hepatic enzymes and TNF-alpha, tissue matrix metalloproteinases (MMP-2, MMP-9), liver morphology, malondialdehyde (MDA), and myeloperoxidase (MPO) were evaluated. Liver I/R injury was confirmed by altered increased hepatic enzymes and TNF-alpha. I/R induced an altered morphology and an increase in MMP-2 and MMP-9 activity not only in left-ischemic lobe (LL) but also in the right-non-ischemic (RL) lobe. A lobar difference was detected for MDA formation and MPO activity in both sham and I/R submitted rats, with higher levels in the left lobe for both groups. This study indicates that an increase in MMPs, which may be TNF-alpha-mediated, occurs in both the ischemic-and the non-ischemic lobes; the heterogeneous lobe concentrations of MDA and MPO suggest that the random sampling of liver tissue should be avoided.