Regulation of cytoplasmic pH in resident and activated peritoneal macrophages

Abstract

Cytoplasmic pH (pH i) has been shown to be an important determinant of the activity of the NADPH oxidase in phagocytic cells. We hypothesized that a difference in pH i and/or its regulation existed between activated and resident macrophages (RES MOs) which might explain the increased NADPH oxidase activity observed in the former. The pH i of RES and lipopolysaccharide (LPS)-elicited MOs was examined using the fluorescent dye BCECF. Resting pH i did not differ between resident (RES) and elicited (ELI) MOs (7.16 ± 0.05 and 7.20 ± 0.05, respectively). pH i recovery after intracellular acid loading was partially dependent on the presence of Na + in the extracellular medium, and was partially inhibited by the Na + H + antiport inhibitor, amiloride. At comparable pH i, the rate of acid extrusion during recovery was not different in RES and ELI MOs (1.48 ± 0.12 and 1.53 ± 0.06 mM/min, respectively). In both RES and ELI MOs, approx. 40% of total pH i recovery was insensitive to amiloride and independent of extracellular Na +. In both RES and ELI MOs, stimulation with TPA resulted in a biphasic pH i response: an initial acidification followed by a sustained alkalinization to a new steady-state pH i. This alkalinization was Na +-dependent and amiloride-sensitive, consistent with a TPA-induced increase in Na + H + antiport activity. The new steady-state pH i attained after TPA stimulation was equivalent in RES and ELI MOs (7.28 ± 0.04 and 7.31 ± 0.06, respectively), indicating comparable stimulated Na + H + antiport activity. However, the initial acidification induced by TPA was greater in ELI than in RES MOs (0.18 ± 0.02 vs. 0.06 ± 0.02 pH unit, respectively, P < 0.05). The specific NADPH oxidase inhibitor diphenylene iodonium (DPI) completely inhibited the respiratory burst but reduced the magnitude of this pH i reduction by only about 50%. This suggested that the TPA-induced pH i reduction was due in part to acid produced via the respiratory burst, and in part to other acid-generating pathways stimulated by TPA.