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Oxidant stress inhibits pH regulatory mechanisms in murine peritoneal macrophages
G F Brisseau1, O Tsai, T Nordström
1Department of Surgery, University of Toronto, Ontario, Canada.
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.
Insights
Oxidant stress impairs macrophage pH regulation by inhibiting proton pumps, a process worsened by depleted antioxidants. This cellular dysfunction is linked to inflammatory conditions like sepsis.
Area of Science:
- Cellular Physiology
- Immunology
- Biochemistry
Background:
- Cytoplasmic pH (pHi) maintenance is crucial for cellular homeostasis.
- Vacuolar-type H(+)-adenosine triphosphatase (V-ATPase) in macrophage plasma membranes regulates pHi by extruding protons.
- Inflammatory environments often involve reactive oxygen metabolites, necessitating study of oxidant stress effects on macrophages.
Purpose of the Study:
- To investigate the impact of oxidant stress, specifically hydrogen peroxide, on V-ATPase-mediated pHi regulation in peritoneal macrophages.
- To determine the mechanisms underlying oxidant-induced impairment of pHi recovery.
Main Methods:
- Murine peritoneal macrophages were acid-loaded and exposed to varying hydrogen peroxide concentrations.
- Intracellular pH was monitored using the fluorescent dye bis-carboxyethyl-carboxyfluorescein and a fluorescence spectrometer.
- Intracellular ATP levels and glutathione status were assessed.
Main Results:
- Hydrogen peroxide caused a time- and dose-dependent decrease in V-ATPase-mediated pHi recovery without cytotoxicity.
- Catalase reversed the inhibitory effect, indicating specificity.
- Hydrogen peroxide-induced ATP depletion was necessary and sufficient for impaired pHi recovery.
- Depletion of intracellular glutathione increased V-ATPase sensitivity to oxidant stress.
Conclusions:
- Oxidant stress in inflammatory conditions impairs macrophage pHi regulation.
- This impairment is exacerbated by depleted intracellular antioxidants, as seen in sepsis.
- Oxidants contribute to cellular dysfunction in inflammatory states via impaired pHi regulation.