Calcium Is Required for PMA Induced Superoxide Release From Human Neutrophils

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.