Mechanisms of cytoplasmic pH recovery in acid-loaded macrophages

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.