title: Calcium is required for PMA induced superoxide release from human neutrophils.
abstract: 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 (O2-) 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 O2-. In 1 mM Ca++, 2.0 million adherent neutrophils released 10.7 +/- 1.2 nmol O2- in 20 min (n = 4). O2- 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 O2- in 20 min. With cells stimulated at low density, PMA stimulated O2- release was significantly decreased (3.0 +/- 0.6 nmol O2- in 20 min) as was the initial rate of secretion of O2- 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 O2- release measured in the presence of calcium. Furthermore, PMA stimulated O2- 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 O2- 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 O2- and increasing superoxide release after a maximum stimulating dose of PMA.
authors.affiliation: Department of Physiology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107.
authors.forename: Y
authors.initials: Y
authors.lastname: Ishihara
authors.forename: D L
authors.initials: DL
authors.lastname: Rosolia
authors.forename: P J
authors.initials: PJ
authors.lastname: McKenna
authors.forename: S P
authors.initials: SP
authors.lastname: Peters
authors.forename: K H
authors.initials: KH
authors.lastname: Albertine
authors.forename: M H
authors.initials: MH
authors.lastname: Gee
mesh_terms: D002118:Calcium / Q000494:pharmacology* / Q000502:physiology; D002452:Cell Count; D006801:Humans; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
chemical_list: D013481:Superoxides; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium