Cyclosporin A protects hepatocytes subjected to high Ca2+ and oxidative stress.

Hepatocytes incubated with 0.8 mM t-butylhydroperoxide are protected by cyclosporin A when the medium Ca2+ concentration is 10 mM, but not when it is 2.5 mM. The highest Ca2+ level is associated with an inhibition of t-butylhydroperoxide-dependent malondialdehyde accumulation and with mitochondrial Ca2+ loading within the cells. These findings are new evidence that t-butylhydroperoxide can kill cells by peroxidation-dependent and -independent mechanisms, and suggest that the mitochondrial permeability transition and the resultant de-energization are components of the peroxidation-independent mechanism. Cyclosporin A may have considerable utility for the protection of cells subjected to oxidative stress.

Involvement of glutathione in 1-naphthylisothiocyanate (ANIT) metabolism and toxicity to isolated hepatocytes.

1-Naphthylisothiocyanate (ANIT) is a model compound which causes cholestasis in laboratory animals. Various biochemical and morphological changes including biliary epithelial and parenchymal cell necrosis occur in the liver of animals treated with ANIT. Although the mechanism(s) for these effects is not understood, a role for glutathione (GSH) in toxicity has been implicated. The possible role of GSH in hepatocellular toxicity caused by ANIT was investigated in this study. Treatment of freshly isolated rat hepatocytes with ANIT caused a concentration- and time-dependent depletion of cellular GSH that preceded lactate dehydrogenase (LDH) leakage. Analysis of the incubation medium indicated that the majority of the cellular GSH which was lost was present extracellularly as GSH or as a GSH-releasing compound. Mixing ANIT with GSH at pH 7.5 yielded a compound that was characterized by HPLC and fast atom bombardment-mass spectrometry (FAB-MS) S-(N-naphthyl-thiocarbamoyl)-L-glutathione (GS-ANIT). When dissolved in aqueous solutions at neutral pH, 95% of GS-ANIT dissociated to yield free ANIT and GSH. Under conditions designed to maximize formation and stability of GS-ANIT, GS-ANIT was found in the extracellular medium of hepatocytes treated with ANIT. Treatment of hepatocytes with the GS-ANIT caused GSH depletion and LDH leakage similar to that observed with equimolar amounts of ANIT. These data suggest that ANIT depletes hepatocytes of GSH through a reversible conjugation process. Such a process may play a role in the toxicity of ANIT.

Toxicity to isolated hepatocytes caused by the intracellular calcium indicator, Quin 2.

To determine whether incubation for several hours with intracellular Ca++ indicators caused toxicity to freshly isolated hepatocytes from rats, cells were incubated under 95% O2-5% CO2 in medium containing 2 mM Ca++ and the acetoxymethyl (AM) esters of Quin 2, Indo 1, Fluo 3, 5,5'-Dimethyl BAPTA, 4,4'-Difluoro BAPTA or Fura 2 for up to 5 hr. Quin 2-AM and Indo 1-AM (2.5 microM) induced lipid peroxidation in the cells after 1 or 3 hr of treatment, respectively. Additional experiments with Quin 2-AM (25 microM) revealed that it also caused lactate dehydrogenase leakage, cell blebbing and vitamin E loss in cells, but did not affect reduced glutathione or intracellular Ca++ content. The ability of Quin 2-AM to cause toxicity was dependent on the amount of Quin 2 which was present in the cell. Ca++ appeared to be involved in the mechanism of Quin 2-AM toxicity, for modulation of the extracellular Ca++ concentration partially inhibited lipid peroxidation, vitamin E loss, cell blebbing and lactate dehydrogenase leakage.

Involvement of calcium and iron in Quin 2 toxicity to isolated hepatocytes.

When treated with the cytosolic Ca++ indicator Quin 2-acetoxymethyl ester (Quin 2-AM), isolated hepatocytes exhibited signs of toxicity, such as extensive lipid peroxidation and vitamin E loss and release of lactate dehydrogenase. Lipid peroxidation induced by this agent was blocked completely by cotreatment of the cells with ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid, EDTA, ruthenium red, carbonyl cyanide m-chlorophenylhydrazone, desferal and trifluoperazine, and was partially inhibited by quinacrine and indomethacin. With the exception of carbonyl cyanide m-chlorophenylhydrazone and quinacrine, these agents also inhibited lactate dehydrogenase leakage. Although the results with ruthenium red suggested that Quin 2-AM may cause toxicity by altering handling of Ca++ by mitochondria, mitochondrial membrane potential was not altered in cells treated with Quin 2-AM until after toxicity occurred. Evidence of a direct, potentiative effect of Quin 2 on iron-induced lipid peroxidation was gained from experiments with liposomes. Treatment of cells with Quin 2-AM did not enhance nitro blue tetrazolium reduction, suggesting that Quin 2 did not stimulate O2- production by the cells. Direct chelation of Ca++ did not appear to be involved in the mechanism of Quin 2 toxicity, for an analog of Quin 2 that is virtually nonhydrolyzable, which greatly limits the binding of Ca++, also caused lipid peroxidation and cell death. These results suggest that Quin 2 causes toxicity by chelating iron or by activating some cellular process(es) that is dependent on the presence of iron or Ca++.

Cyclooxygenase inhibition in lungs or in neutrophils attenuates neutrophil-dependent edema in rat lungs perfused with phorbol myristate acetate.

Results from previous studies indicate that injury in isolated rat lungs perfused with buffer containing phorbol myristate acetate (PMA) and rat neutrophils (PMNs) is dependent on the production of reactive oxygen species and thromboxane (Tx) A2. The purpose of this study was to determine whether the lung or the PMN was the source of TxA2 required to produce lung injury in this model. Prostanoid synthesis by rat lungs or PMNs was inhibited selectively by pretreatment of either rats or isolated PMNs with aspirin (100 mg/kg p.o. or 100 microM, respectively). Unbound aspirin was removed from the lungs and PMNs before use in experiments. Lungs from vehicle-pretreated rats that were perfused with PMA and untreated PMNs exhibited increases in weight, lavage fluid albumin content and TxB2 production with respect to lungs perfused with PMA but no PMNs. Increases in these markers were prevented when cyclooxygenase from either the lungs or the PMNs was inhibited. These results indicate that TxA2 is produced by both PMNs and by lung cells in this preparation, and that TxA2 production by both of these sources is required for the manifestation of edema.

The role of cyclooxygenase products in the acute airway obstruction and airway hyperreactivity of ponies with heaves.

Airway obstruction and hyperreactivity are characteristics of human asthma and of "heaves," a naturally occurring respiratory disorder of horses and ponies. To document the role of cyclooxygenase products of arachidonic acid metabolism in the pathogenesis of heaves, we measured plasma and bronchoalveolar lavage (BAL) fluid concentrations of metabolites of thromboxane (TX)A2 and prostaglandins (PG) I2 and D2 in five affected ponies and their age- and gender-matched controls prior to and during acute airway obstruction precipitated by housing the ponies in a barn and exposing them to hay dust. Pulmonary resistance increased significantly and dynamic compliance and arterial oxygen tension decreased significantly in affected ponies that were placed in the barn. At this time, histamine aerosol challenge demonstrated the presence of airway hyperresponsiveness in the affected ponies. Plasma TXB2 was the only metabolite that increased significantly during the acute disease state. In a subsequent experiment, the ponies were treated with flunixin meglumine, a cyclooxygenase inhibitor, to determine if this would alter the onset or development of clinical disease. At a dose of 1.1 mg/kg intramuscularly, 3 times daily, flunixin meglumine inhibited TXB2 production but did not alter the degree of airway obstruction or airway hyperreactivity measured at pasture and in the barn. We conclude that cyclooxygenase products of arachidonic acid metabolism are altered but do not play a role in the airway obstruction and hyperreactivity observed in ponies with heaves.