Assessment of Na+-H+ exchange activity in phagosomal membranes of human neutrophils

To assess the presence of Na+-H+ exchange in internalized membranes, the phagosomal pH was monitored in suspensions of intact human neutrophils by measuring the fluorescence of ingested bacteria (Micrococcus lysodeikticus) prelabeled with a pH-sensitive dye. Uptake of fluoresceinated bacteria was confirmed by flow cytometry and by phase-contrast and electron microscopy. Manipulation of the cytoplasmic ion content was accomplished by permeabilization of the plasma membrane with nystatin, which did not alter phagosomal permeability. At 37 degrees C, the phagosomal interior acidified at a maximal rate of 0.135 +/- 0.003 pH units/min (n = 10). The endogenous Na+-H+ exchanger does not affect phagosomal acidification, since the rate and extent of the pH change were not altered by 1) omission of intraphagosomal Na+ and 2) addition of the permeant inhibitor methylisobutylamiloride or by trapping amiloride in the phagosome during bacterial ingestion. Moreover, amiloride-sensitive Na+-H+ exchange was not detectable when Na+ or H+ gradients were imposed across the phagosomal membrane. Under comparable conditions, Na+-H+ exchange could be readily detected across the surface membrane. These data imply that the Na+-H+ antiporters are either inactivated in the phagosome or are segregated and not internalized into the phagosomal membrane.