OXIDANT STRESS INHIBITS PH REGULATORY MECHANISMS IN MURINE PERITONEAL-MACROPHAGES

Background. Maintenance of cytoplasmic cytoplasmic pH (pH(i)) close to the physiologic range is vital to normal cellular homeostasis. We hav e previously reported that a vacuolar-type H+-adenosine triphosphatase (V-ATPase) situated in the plasma membrane of macrophages and poised to extrude protons from the cytoplasmic to the extracellular space is an important pH(i) regulatory mechanism. Since the inflammatory, micro environment is frequently characterized by the influx of cells known t o release reactive oxygen metabolites, we performed studies to examine the effect of oxidant stress on pH(i) regulation in peritoneal macrop hages. Specifically, the effect of hydrogen peroxide on V-ATPase-media ted proton extrusion from acid-loaded macrophages was investigated. Me thods. Thioglycollate-elicited murine peritoneal macrophages were expo sed to varying concentrations of hydrogen peroxide and examined for th eir ability to recover from an acid-load. pH(i) was studied by preload ing cells with the pH-sensitive fluorescent dye, bis-carboxyethyl-carb oxyfluorescein; and monitoring changes in fluorescence under various c onditions using a fluorescence spectrometer. Results. Hydrogen peroxid e caused a time- and dose-dependent decrease in proton pump-mediated p H(i) recovery in peritoneal macrophages. This effect occurred without cytotoxicity and was a specific effect as evidenced by the ability, of catalase to reverse the inhibition. Since hydrogen peroxide is known to deplete intracellular ATP, a substrate for V-ATPase activity, we hy pothesized that ATP depletion may underlie the effect. These studies s howed that hydrogen peroxide-mediated ATP depletion was both necessary and sufficient for the effect. Finally, depletion of intracellular gl utathione in vivo by using diethyl maleate increased the sensitivity o f V-ATPase activity to oxidant stress. Conclusions. Oxidant stress wit hin the inflammatory milieu impairs macrophage pH(i) regulation. This effect is magnified by depletion of intracellular antioxidants, as occ urs during sepsis. This represents another mechanism whereby oxidants may contribute to cellular dysfunction associated with inflammatory st ates.