A glutamine synthetase inhibitor increases survival and decreases cytokine response in a mouse model of acute liver failure.

BACKGROUND: Acute liver failure (ALF) can be induced in mice by administering Escherichia coli lipopolysaccharide (LPS) and D-galactosamine (D-GalN), which induce an inflammatory response involving tumour necrosis factor (TNF)-α production and a hepatocyte-specific transcriptional block. Under these conditions, binding of TNF-α to its cognate receptor on hepatocytes eventually leads to their apoptosis. AIMS: As part of an effort to identify drugs to treat this disease model, we have investigated whether the glutamine synthetase inhibitor methionine sulfoximine (MSO) could play a protective role, given its effectiveness in the inhibition of brain swelling associated with hyperammonaemia. METHODS: Mouse survival, glutamine synthetase activity, hepatocyte apoptosis and induction of inflammatory cytokines were measured in mice treated with MSO before an intraperitoneal injection of LPS/D-GalN. The effect of MSO on viability and on TNF-α release was also assessed on inflammatory and liver cells. RESULTS: We have found that, in mice treated with LPS/D-GalN, MSO (i) drastically increases animal survival; (ii) sharply reduces glutamine synthetase activity, without inhibiting its other target, γ-glutamyl cysteine synthetase; (iii) inhibits death receptor-mediated apoptosis in hepatocytes upstream to cytokine binding; (iv) strongly reduces the overall inflammatory cytokine response, including a significant decrease in TNF-α induction in vivo and ex vivo, and in the interferon-γ level and signalling. CONCLUSIONS: These results demonstrate that the MSO target glutamine synthetase is required for the early steps of the cytokine response to endotoxins, and that its pharmacological inhibition may be exploited to treat inflammation.

Mitochondrial deoxynucleotide pool sizes in mouse liver and evidence for a transport mechanism for thymidine monophosphate.

Dividing cultured cells contain much larger pools of the four dNTPs than resting cells. In both cases the sizes of the individual pools are only moderately different. The same applies to mitochondrial (mt) pools of cultured cells. Song et al. [Song S, Pursell ZF, Copeland WC, Longley MJ, Kunkel TA, Mathews CK (2005) Proc Natl Acad Sci USA 102:4990-4995] reported that mt pools of rat tissues instead are highly asymmetric, with the dGTP pool in some cases being several-hundred-fold larger than the dTTP pool, and suggested that the asymmetry contributes to increased mutagenesis during mt DNA replication. We have now investigated this discrepancy and determined the size of each dNTP pool in mouse liver mitochondria. We found large variations in pool sizes that closely followed variations in the ATP pool and depended on the length of time spent in the preparation of mitochondria. The proportion between dNTPs was in all cases without major asymmetries and similar to those found earlier in cultured resting cells. We also investigated the import and export of thymidine phosphates in mouse liver mitochondria and provide evidence for a rapid, highly selective, and saturable import of dTMP, not depending on a functional respiratory chain. At nM external dTMP the nucleotide is concentrated 100-fold inside the mt matrix. Export of thymidine phosphates was much slower and possibly occurred at the level of dTDP.

Desensitization of the permeability transition pore by cyclosporin a prevents activation of the mitochondrial apoptotic pathway and liver damage by tumor necrosis factor-alpha.

We studied the effects of cyclosporin A (CsA) administration 1) on the properties of the permeability transition pore (PTP) in mitochondria isolated from the liver and 2) on the susceptibility to hepatotoxicity induced by lipopolysaccharide of Escherichia coli (LPS) plus D-galactosamine (D-GalN) in rats. CsA exerted a marked PTP inhibition ex vivo, with an effect that peaked between 2 and 9 h of drug treatment and decayed with an apparent half-time of about 13 h. Administration of LPS plus D-GalN to naive rats caused the expected increased serum levels of tumor necrosis factor (TNF)-alpha, liver inflammation with BID cleavage, activation of caspase 3, appearance of terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling-positive nuclei, and release of alanine aminotransferase and aspartate aminotransferase into the bloodstream. Treatment with CsA before or within 5 h of the administration of LPS plus D-GalN protected rats from hepatotoxicity despite the normal increase of serum TNF-alpha and BID cleavage. These results indicate that CsA prevents the hepatotoxic effects of TNF-alpha by blocking the mitochondrial proapoptotic pathway through inhibition of the PTP and provides a viable strategy for the treatment of liver diseases that depend on increased production and/or liver sensitization to TNF-alpha.