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Chemotactic peptide‐induced cytoplasmic pH changes in incubated human monocytes
Author(s) -
Bernardo John,
Brennan Lisa,
Brink Holly F.,
Ortiz Maria F.,
Newburger Peter E.,
Simons Elizabeth R.
Publication year - 1993
Publication title -
journal of leukocyte biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.819
H-Index - 191
eISSN - 1938-3673
pISSN - 0741-5400
DOI - 10.1002/jlb.53.6.673
Subject(s) - chemotaxis , incubation , biology , n formylmethionine leucyl phenylalanine , microbiology and biotechnology , in vitro , superoxide , stimulation , cytoplasm , peptide , biochemistry , cytosol , neutrophile , endocrinology , receptor , enzyme
Stimulation of phagocytic leukocytes with chemotactic factors results in transient acidification, followed by alkalinization of the cytosol. Human monocytes are known to alter their functional responses to the chemotactic peptide N ‐formylmethionyl‐leucyl‐phenylalanine (fMLP) in a complex fashion as they mature in vitro to macrophages. To examine the evolution of the cytoplasmic pH (pH ; ) response of monocytes to fMLP as they mature into macrophages, we incubated cells for 0, 24, 48, and 96 h (Medium‐199 + 10% fetal bovine serum; 37°C) and examined pH i using the fluorescent probe 2′, 7′‐bis(carboxyethyl)‐5(6)‐carboxyfluorescein (BCECF; 1 μΜ) and a Perkin‐Elmer 650/10 spectrofluorimeter (λ em = 530 nm, λ_ex = 500, 450 nm) as previously described. The resting pH; of fresh (0 h) monocytes was 7.07 ± 0.16 (SD) and was unchanged after incubation for 24, 48, or 96 h (7.09, 7.11, 7.05, respectively). Cells exhibited an fMLP dose‐dependent cytoplasmic acidification, with maximal δpHi occurring 30‐60 s after exposure to 10 −7 M fMLP. The response to fMLP did not change with the duration of incubation and, as with neutrophils, cytoplasmic realkalinization was blocked by dimethyl‐ amiloride (20 μΜ). Incubation with 2‐deoxyglucose (10 min, 5 mM), sufficient to inhibit by more than 90% the formyl peptide‐stimulated superoxide generation by monocytes, slowed fMLP‐induced acidification and abrogated the alkalinization. In addition, monocytes isolated from the blood of a patient with X‐linked chronic granulomatous disease (CGD) underwent fMLP‐induced acidification that was unmasked further by coincubation with di‐ methylamiloride, in a manner quantitatively similar to that of normal monocytes, despite the inability of the CGD cells to produce superoxide. The chemotactic factor‐induced cytoplasmic pH responses of mono‐ cytes/macrophages remained constant as the cells matured in vitro and exhibited a dimethylamiloride‐independent acidification and dependent alkalinization, as did the response in neutrophils. The cytoplasmic acidification of these cells thus did not correlate with the cells' production of superoxide and with the concomitant hexose monophosphate shunt activation, as has been suggested for other leukocyte types.