Cytoplasmic pH in pulmonary macrophages: recovery from acid load is Na+ independent and NEM sensitive

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Abstract
The pulmonary macrophage plays a primary role in the immunological defense of the lung. Although many studies have been devoted to elucidation of its phagocytic and secretory functions, little is known of its membrane transport properties or of how it regulates intracellular pH (pHi). The purpose of this study, therefore, was to determine base-line pHi and the mechanism(s) by which the cell recovers pHi when challenged with an intracellular acid load. Through the use of the pH-sensitive fluorescent dye, 2,7-biscarboxyethyl-5(6)-carboxy-fluorescein (BCECF), base-line pHi was estimated to be 7.24 +/- 0.03. Cells were acidified by two methods, nigericin and weak acids, while recovery (dpHi/dt) was monitored. The rate of recovery was found to be independent of external Na+ and K+ and was insensitive to amiloride. Pretreatment with 4,4'-diiso-thiocyanatostilbene-2,2'-disulfonic acid, an inhibitor of Cl- -HCO3- exchange, was also without effect on recovery from an intracellular acid load in these cells, under nominally HCO3- -free conditions. In contrast, N-ethylmaleimide (NEM) and N,N'-dicyclohexylcarbodiimide, nonspecific inhibitors of proton adenosinetriphosphatases (ATPases), virtually abolished pHi recovery. Efflux of H+ equivalents by pulmonary macrophages was measured by techniques involving both pH stat titration and the effect on fluorescence of extracellular BCECF. Basal H+ extrusion was approximately 2.75 +/- 0.64 nmol H+.min-1.10(6) cells-1 and was enhanced to approximately 26.0 +/- 6.95 nmol H+.min-1.10(6) cells-1 in acid-loaded cell suspensions. The basal rate of H+ extrusion was reduced to approximately 0.84 +/- 0.31 nmol H+.min-1.10(6) cells-1 in the presence of 1 mM NEM. These results suggest that recovery of cytoplasmic pH from an intracellular acid load, as well as regulation of pHi, under the conditions examined, is not mediated by a Na+-H+ exchanger in these cells. Rather, the data are consistent with the presence of an H+-ATPase in the plasma membrane of pulmonary macrophages.