Glutathione and antioxidant enzymes serve complementary roles in protecting activated hepatic stellate cells against hydrogen peroxide-induced cell death.

BACKGROUND: In chronic liver disease, hepatic stellate cells (HSCs) are activated, highly proliferative and produce excessive amounts of extracellular matrix, leading to liver fibrosis. Elevated levels of toxic reactive oxygen species (ROS) produced during chronic liver injury have been implicated in this activation process. Therefore, activated hepatic stellate cells need to harbor highly effective anti-oxidants to protect against the toxic effects of ROS. AIM: To investigate the protective mechanisms of activated HSCs against ROS-induced toxicity. METHODS: Culture-activated rat HSCs were exposed to hydrogen peroxide. Necrosis and apoptosis were determined by Sytox Green or acridine orange staining, respectively. The hydrogen peroxide detoxifying enzymes catalase and glutathione-peroxidase (GPx) were inhibited using 3-amino-1,2,4-triazole and mercaptosuccinic acid, respectively. The anti-oxidant glutathione was depleted by L-buthionine-sulfoximine and repleted with the GSH-analogue GSH-monoethylester (GSH-MEE). RESULTS: Upon activation, HSCs increase their cellular glutathione content and GPx expression, while MnSOD (both at mRNA and protein level) and catalase (at the protein level, but not at the mRNA level) decreased. Hydrogen peroxide did not induce cell death in activated HSCs. Glutathione depletion increased the sensitivity of HSCs to hydrogen peroxide, resulting in 35% and 75% necrotic cells at 0.2 and 1mmol/L hydrogen peroxide, respectively. The sensitizing effect was abolished by GSH-MEE. Inhibition of catalase or GPx significantly increased hydrogen peroxide-induced apoptosis, which was not reversed by GSH-MEE. CONCLUSION: Activated HSCs have increased ROS-detoxifying capacity compared to quiescent HSCs. Glutathione levels increase during HSC activation and protect against ROS-induced necrosis, whereas hydrogen peroxide-detoxifying enzymes protect against apoptotic cell death.

Superoxide anions and hydrogen peroxide inhibit proliferation of activated rat stellate cells and induce different modes of cell death.

BACKGROUND: In chronic liver injury, hepatic stellate cells (HSCs) proliferate and produce excessive amounts of connective tissue causing liver fibrosis and cirrhosis. Oxidative stress has been implicated as a driving force of HSC activation and proliferation, although contradictory results have been described. AIM: To determine the effects of oxidative stress on activated HSC proliferation, survival and signalling pathways. METHODS: Serum-starved culture-activated rat HSCs were exposed to the superoxide anion donor menadione (5-25 micromol/L) or hydrogen peroxide (0.2-5 mmol/L). Haem oxygenase-1 mRNA expression, glutathione status, cell death, phosphorylation of mitogen-activated protein (MAP) kinases and proliferation were investigated. RESULTS: Menadione induced apoptosis in a dose- and time-dependent, but caspase-independent manner. Hydrogen peroxide induced necrosis only at extremely high concentrations. Both menadione and hydrogen peroxide activated Jun N-terminal kinase (JNK) and p38. Hydrogen peroxide also activated extracellular signal-regulated protein. Menadione, but not hydrogen peroxide, reduced cellular glutathione levels. Inhibition of JNK or supplementation of glutathione reduced menadione-induced apoptosis. Non-toxic concentrations of menadione or hydrogen peroxide inhibited platelet-derived growth factor- or/and serum-induced proliferation. CONCLUSION: Reactive oxygen species (ROS) inhibit HSC proliferation and promote HSC cell death in vitro. Different ROS induce different modes of cell death. Superoxide anion-induced HSC apoptosis is dependent on JNK activation and glutathione status.

Multidrug resistance-associated proteins are crucial for the viability of activated rat hepatic stellate cells.

UNLABELLED: Hepatic stellate cells (HSCs) survive and proliferate in the chronically injured liver. ATP-binding cassette (ABC) transporters play a crucial role in cell viability by transporting toxic metabolites or xenobiotics out of the cell. ABC transporter expression in HSCs and its relevance to cell viability and/or activation have not been reported so far. The aim of this study was to investigate the expression, regulation, and function of multidrug resistance-associated protein (Mrp)-type and multidrug resistance protein (Mdr)-type ABC transporters in activated rat HSCs. Rat HSCs were exposed to cytokines or oxidative stress. ABC transporter expression was determined by quantitative polymerase chain reaction and immunohistochemistry. HSCs were exposed to the Mdr inhibitors verapamil and PSC-833 and the Mrp inhibitor MK571. Mdr and Mrp transporter function was evaluated with flow cytometry. Apoptosis was determined by activated caspase-3 and acridine orange staining, and necrosis was determined by Sytox green nuclear staining. An in vivo model of carbon tetrachloride (CCl(4))-induced liver fibrosis was used. With respect to hepatocytes, activated HSCs expressed high levels of Mrp1 and comparable levels of Mrp3, Mrp4, Mdr1a, and Mdr1b but not the hepatocyte-specific transporters bile salt export pump, Mrp2, and Mrp6. Mrp1 protein staining correlated with desmin staining in livers from CCl(4)-treated rats. Mrp1 expression increased upon activation of HSCs. Cytokines induced Mdr1b expression only. Oxidative stress was not a major regulator of Mdr and Mrp transporter expression. Activated HSCs became necrotic when exposed to the Mrp inhibitors. CONCLUSION: Activated HSCs contain relatively high levels of Mrp1. Mrp-type transporters are required for the viability of activated HSCs. Mrp-dependent export of endogenous metabolites is important for the survival of activated HSCs in chronic liver diseases.

Mutation that dramatically alters rat titin isoform expression and cardiomyocyte passive tension.

Titin is a very large alternatively spliced protein that performs multiple functions in heart and skeletal muscles. A rat strain is described with an autosomal dominant mutation that alters the isoform expression of titin. While wild type animals go through a developmental program where the 3.0 MDa N2B becomes the major isoform expressed by two to three weeks after birth (approximately 85%), the appearance of the N2B is markedly delayed in heterozygotes and never reaches more than 50% of the titin in the adult. Homozygote mutants express a giant titin of the N2BA isoform type (3.9 MDa) that persists as the primary titin species through ages of more than one and a half years. The mutation does not affect the isoform switching of troponin T, a protein that is also alternatively spliced with developmental changes. The basis for the apparently greater size of the giant titin in homozygous mutants was not determined, but the additional length was not due to inclusion of sequence from larger numbers of PEVK exons or the Novex III exon. Passive tension measurements using isolated cardiomyocytes from homozygous mutants showed that cells could be stretched to sarcomere lengths greater than 4 mum without breakage. This novel rat model should be useful for exploring the potential role of titin in the Frank-Starling relationship and mechano-sensing/signaling mechanisms.