title: Oxidant stress inhibits pH regulatory mechanisms in murine peritoneal macrophages.
abstract: BACKGROUND
  Maintenance of cytoplasmic pH (pHi) close to the physiologic range is vital to normal cellular homeostasis. We have 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 pHi regulatory mechanism. Since the inflammatory microenvironment is frequently characterized by the influx of cells known to release reactive oxygen metabolites, we performed studies to examine the effect of oxidant stress on pHi regulation in peritoneal macrophages. Specifically, the effect of hydrogen peroxide on V-ATPase-mediated proton extrusion from acid-loaded macrophages was investigated.
  
  
  METHODS
  Thioglycollate-elicited murine peritoneal macrophages were exposed to varying concentrations of hydrogen peroxide and examined for their ability to recover from an acid-load. pHi was studied by preloading cells with the pH-sensitive fluorescent dye, bis-carboxyethyl-carboxyfluorescein, and monitoring changes in fluorescence under various conditions using a fluorescence spectrometer.
  
  
  RESULTS
  Hydrogen peroxide caused a time- and dose-dependent decrease in proton pump-mediated pHi 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 hypothesized that ATP depletion may underlie the effect. These studies showed that hydrogen peroxide-mediated ATP depletion was both necessary and sufficient for the effect. Finally, depletion of intracellular glutathione in vivo by using diethyl maleate increased the sensitivity of V-ATPase activity to oxidant stress.
  
  
  CONCLUSIONS
  Oxidant stress within the inflammatory milieu impairs macrophage pHi regulation. This effect is magnified by depletion of intracellular antioxidants, as occurs during sepsis. This represents another mechanism whereby oxidants may contribute to cellular dysfunction associated with inflammatory states.
authors.affiliation: Department of Surgery, University of Toronto, Ontario, Canada.
authors.forename: G F
authors.initials: GF
authors.lastname: Brisseau
authors.forename: O
authors.initials: O
authors.lastname: Tsai
authors.forename: T
authors.initials: T
authors.lastname: Nordström
authors.forename: J C
authors.initials: JC
authors.lastname: Marshall
authors.forename: S
authors.initials: S
authors.lastname: Grinstein
authors.forename: O D
authors.initials: OD
authors.lastname: Rotstein
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D005260:Female; D006861:Hydrogen Peroxide / Q000633:toxicity*; D006863:Hydrogen-Ion Concentration; D017737:Macrophages, Peritoneal / Q000378:metabolism*; D051379:Mice
chemical_list: D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide