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Calcium Is Required for PMA Induced Superoxide Release From Human Neutrophils
Author(s) -
Ishihara Y.,
Rosolia D.L.,
McKenna P.J.,
Peters S.P.,
Albertine K.H.,
Gee M.H.
Publication year - 1990
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.48.1.89
Subject(s) - superoxide , extracellular , calcium , incubation , biology , secretion , phorbol , stimulation , microbiology and biotechnology , n formylmethionine leucyl phenylalanine , biochemistry , biophysics , endocrinology , medicine , protein kinase c , enzyme
Experiments were designed to reexamine the relationship between extracellular calcium and superoxide generation in phorbol myristate acetate (PMA) stimulated neutrophils exploiting a newly adapted method to measure superoxide anion (O 2 ‐ ) generation from adherent cells stimulated at high and low cell density. Human neutrophils were plated in microtiter wells in cell densities of either 0.2 or 2.0 million cells/well. Superoxide release was measured sequentially over 60 min by reduction of ferricytochrome c. Cells were maintained in 1 mM Ca ++ or 0 mM Ca ++ Hanks' buffer for 60 min prior to activation as well as during measurement of O 2 ‐ . In 1 mM Ca ++ , 2.0 million adherent neutrophils released 10.7 ± 1.2 nmol O 2 ‐ in 20 min (n=4). O 2 ‐ release was not significantly different for high density cells incubated and stimulated in 0 mM Ca ++ . In the presence of 1 mM Ca ++ , 0.2 million adherent neutrophils released 6.3 ± 0.5 nmols O 2 ‐ in 20 min. With cells stimulated at low density, PMA stimulated O 2 ‐ release was significantly decreased (3.0 ± 0.6 nmol O 2 ‐ in 20 min) as was the initial rate of secretion of O 2 ‐ in the absence of extracellular calcium. Basal release of superoxide was also greater in the presence of 1 mM Ca ++ (0.96 nmol/20 min) compared to basal release in 0 mM Ca ++ (0.22 nmol/20 min). Additional experiments with 0.2 million cells/well showed that extracellular Ca ++ was required during stimulation with PMA and that prior incubation of cells for up to 60 min in 0 mM Ca ++ had no effect on O 2 ‐ release measured in the presence of calcium. Furthermore, PMA stimulated O 2 ‐ was independent of verapamil (10 ‐5 –10 ‐7 M), suggesting that voltage‐dependent calcium channels do not participate in this response. The planar areas for unstimulated neutrophils in 0 mM Ca ++ increased after addition of PMA. Unstimulated cells in 1 mM Ca ++ tended to be larger and planar areas did not increase after PMA. These studies demonstrate that PMA stimulated O 2 ‐ secretion is dependent on extracellular calcium particularly when adherent neutrophils are stimulated at low cell density. Furthermore, extracellular calcium at a concentration of 1 mM primes neutrophils by increasing basal secretion of O 2 ‐ and increasing superoxide release after a maximum stimulating dose of PMA.