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Mechanisms of cytoplasmic pH recovery in acid-loaded macrophages
C J Swallow1, O D Rotstein, S Grinstein
1Department of Surgery, Toronto General Hospital, Canada.
Abstract:
In the acidic microenvironment of an abscess, efficient antimicrobial function is dependent upon the phagocyte's maintenance of its physiological intracellular pH. To determine the mechanisms by which macrophages recover from an intracellular acid load, the cytoplasmic pH of murine peritoneal macrophages was measured using the pH-sensitive cytoplasmic fluorescent dye bis(carboxyethyl)-5(6)-carboxyfluorescein. These studies showed that pH recovery was primarily mediated by a Na+/H+ antiport in the plasma membrane which exchanged intracellular H+ for extracellular Na+. The proportion of pH recovery mediated by this exchanger was determined by measuring the rate of acid extrusion in the presence of the Na+/H+ antiport inhibitor, amiloride. Mean rate of acid extrusion (in mM/min) was reduced from 4.4 +/- 0.2 in control cells to 1.6 +/- 0.2 in the presence of amiloride (mean +/- SEM, n = 8, P less than 0.01), demonstrating the presence of a second mechanism for pHi recovery. Inhibition of this residual recovery both by the sulhydryl reagent N-ethylmaleimide and by ATP depletion suggested that the additional mechanism was an ATP-dependent proton extrusion pump. Thus, macrophages have at least two efficient mechanisms for maintaining physiological pH when exposed to an intracellular acid load. Since the Na+/H+ antiport is inhibited at low extracellular pH, the maintenance of physiological cytoplasmic pH, and of normal cell function, within the acidic milieu of a tumor or abscess may depend on the alternate mechanism of pH recovery demonstrated here.
Insights
Macrophages use two main mechanisms to maintain their internal pH balance, crucial for fighting infections in acidic environments like abscesses. One is a sodium-hydrogen exchanger, and the other is an ATP-dependent proton pump.
Area of Science:
- Cellular Biology
- Immunology
- Physiology
Background:
- Phagocyte antimicrobial function depends on maintaining intracellular pH, especially in acidic abscess environments.
- Macrophages need efficient mechanisms to recover from intracellular acid loads to function properly.
Purpose of the Study:
- To identify and characterize the mechanisms by which macrophages recover their cytoplasmic pH after an acid load.
- To investigate the roles of different transporters and energy-dependent processes in macrophage pH regulation.
Main Methods:
- Cytoplasmic pH measurement in murine peritoneal macrophages using the fluorescent dye bis(carboxyethyl)-5(6)-carboxyfluorescein.
- Assessment of acid extrusion rates with and without the Na+/H+ antiport inhibitor amiloride.
- Evaluation of residual pH recovery mechanisms using the sulhydryl reagent N-ethylmaleimide and ATP depletion.
Main Results:
- pH recovery was primarily mediated by a plasma membrane Na+/H+ antiport.
- Inhibition of the Na+/H+ antiport with amiloride significantly reduced acid extrusion rates.
- A second, ATP-dependent proton extrusion pump was identified as responsible for residual pH recovery.
Conclusions:
- Macrophages possess at least two efficient mechanisms for maintaining physiological intracellular pH under acidic conditions.
- The Na+/H+ antiport is a primary pH recovery mechanism, but an ATP-dependent proton pump provides an alternative.
- This alternative mechanism may be critical for macrophage function in acidic microenvironments like tumors and abscesses where the Na+/H+ antiport may be inhibited.