RESUMO
The effect of hypoxia on subsequent susceptibility of porcine pulmonary artery endothelial cells (PAEC) to hydrogen peroxide (H2O2) injury was studied. Preexposure of PAEC to hypoxia for 3 or more h significantly increased susceptibility to subsequent H2O2 challenge. Analysis of the activities of antioxidant enzymes and xanthine oxidase/dehydrogenase suggested that changes in these enzymes in hypoxic PAEC were not responsible for the increased susceptibility. However, hypoxia resulted in significant time-dependent decreases in total glutathione at 12 h or more. The rate of glutathione regeneration in diethylmaleate-treated PAEC and the rate of uptake of cystine and glycine were significantly lower during hypoxia. Hypoxia also caused depletion of ATP and NADPH levels in PAEC, but these did not occur until well after hypoxia-enhanced susceptibility to H2O2 injury was demonstrable. Alterations in glutathione levels and enhanced susceptibility were reversible when hypoxic PAEC were returned to normoxia. These results indicate that hypoxia increased the susceptibility to H2O2 injury by decreasing the ability of PAEC to maintain and regenerate cellular glutathione content in response to H2O2 challenge.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Hipóxia/fisiopatologia , Artéria Pulmonar/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Aminoácidos/metabolismo , Animais , Células Cultivadas , Meios de Cultura , Suscetibilidade a Doenças , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Glutationa/análogos & derivados , Glutationa/metabolismo , Dissulfeto de Glutationa , Peróxido de Hidrogênio/metabolismo , NAD/metabolismo , NADP/metabolismo , Oxidantes/farmacologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologiaRESUMO
Relatively small but persistent amounts of L-lactate dehydrogenase (LDH) activity were found in mitochondrial preparations isolated from liver of the rat. Using a variety of cytosolic markers, it was found that essentially no cytosolic contamination was present. Respiratory velocities and respiratory control with L-lactate were somewhat lower than with glutamate, but equal or superior to those with pyruvate. Agarose gel electrophoresis showed LDH isoenzymes in mitochondria similar to that in corresponding cytosol. Subtilisin BPN', a bacterial protease, was incubated with intact mitochondria and enzyme activities were measured. Following mitochondrial disruption, the proteolytic treatment was repeated. Digitonin was also used in the fractionation of mitochondria. These techniques helped to determine the location of the LDH in the mitochondria as being mainly in the outer membrane and periplasmic space.
