The superoxide release and the change in the intracellular free calcium ions on stimulation with N-formyl-methionyl-leucyl-phenylalanine were studied in human neutrophils deprived of divalent cations by treatment of the cells with ionophore A23187 in the presence of EGTA. The depleted cells showed no release of superoxide on stimulation with the chemotactic peptide when calcium ions were absent in the medium, but the activity was completely recovered when the cells were preincubated with calcium for at least 3 min before the stimulation. The cells pretreated with Cd2+ showed slight activity of the release, but no recovery was observed with other divalent cations such as Mg2+, Sr2+, Co2+, Ba2+ and Zn2+. The recovery with calcium ions was dependent on the time of the addition relative to the time of the stimulation with the chemotactic peptide: a simultaneous addition of both calcium and the peptide elicited about half of the full activity, while no release was observed when calcium was added later than 2 min after the stimulation with the peptide, though a marked elevation of the intracellular free calcium monitored by quin-2 fluorescence was found. Comparison of the time-courses of the superoxide release and the change in the fluorescence suggest that, besides the elevation of intracellular free calcium, a transient reaction which is also dependent on calcium is required for the full induction of the superoxide-producing activity.
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