title: Regulation of cytoplasmic pH in resident and activated peritoneal macrophages.
abstract: Cytoplasmic pH (pHi) has been shown to be an important determinant of the activity of the NADPH oxidase in phagocytic cells. We hypothesized that a difference in pHi 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 pHi of RES and lipopolysaccharide (LPS)-elicited MOs was examined using the fluorescent dye BCECF. Resting pHi did not differ between resident (RES) and elicited (ELI) MOs (7.16 +/- 0.05 and 7.20 +/- 0.05, respectively). pHi 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 pHi, 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 pHi recovery was insensitive to amiloride and independent of extracellular Na+. In both RES and ELI MOs, stimulation with TPA resulted in a biphasic pHi response: an initial acidification followed by a sustained alkalinization to a new steady-state pHi. This alkalinization was Na(+)-dependent and amiloride-sensitive, consistent with a TPA-induced increase in Na+/H+ antiport activity. The new steady-state pHi 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 less than 0.05). The specific NADPH oxidase inhibitor diphenylene iodonium (DPI) completely inhibited the respiratory burst but reduced the magnitude of this pHi reduction by only about 50%. This suggested that the TPA-induced pHi reduction was due in part to acid produced via the respiratory burst, and in part to other acid-generating pathways stimulated by TPA.
authors.affiliation: Department of Surgery, Toronto General Hospital, Canada.
authors.forename: C J
authors.initials: CJ
authors.lastname: Swallow
authors.forename: S
authors.initials: S
authors.lastname: Grinstein
authors.forename: O D
authors.initials: OD
authors.lastname: Rotstein
mesh_terms: D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism*; D005260:Female; D005452:Fluoresceins; D005456:Fluorescent Dyes; D019298:Glucose-6-Phosphate; D005958:Glucosephosphates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases; D010529:Peritoneal Cavity / Q000166:cytology; D012964:Sodium / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D002352:Carrier Proteins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005958:Glucosephosphates; D008070:Lipopolysaccharides; D017923:Sodium-Hydrogen Exchangers; D013481:Superoxides; D019298:Glucose-6-Phosphate; D000584:Amiloride; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D012964:Sodium; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D013755:Tetradecanoylphorbol Acetate