Priming effect of calcium ionophores on phorbol ester-induced respiratory burst in mouse peritoneal neutrophils

The abilities of three calcium ionophores (A23187; 4-bromo-A23187, and iono mycin)to modulate the respiratory burst of neutrophils induced by phorbol e ster and to increase the concentration of free intracellular Ca2+ ([Ca2+])( i) were compared. The production; of; reactive oxygen species (ROS) was det ermined by luminol-dependent chemiluminescence and [Ca2+](i) was determined with the Fura-2 fluorescent probe. A23187 (0.05-2 mu M) and ionomycin (0.0 01-0.5 mu M) but not 4-bromo-A23187 amplified 3-4-fold the respiratory burs t induced by phorbol ester. The integral response (total production of ROS over 6 min) had a hell-shaped dependence on the concentration of ionomycin and A23187 with increase and decrease at low and high concentrations of the ionophores, respectively. The maximal effect was found at 0.5 mu M ionomyc in and 2 mu M A23187, these concentrations resulting in transient increases in [Ca2+](i) to 1776 +/- 197 and 955 +/- 27 nM, respectively. The ionophor es had no effect in calcium-free media, though they increased [Ca2+](i) to similar to 400 nM through the mobilization of intracellular Ca2+. In cells; with exhausted stores of Ca2+, the addition of 1.5 mM Ca2+ combined with p horbol ester amplified twofold the production of ROS. The inhibition of pho spholipase A(2) with 4-bromophenacyl bromide significantly decreased the pr oduction of ROS. Thus, the entrance of Ca2+ and generation of arachidonic a cid under the influence of phospholipase A(2) are necessary for the ionopho re-induced priming of production of ROS during cell activation. with phorbo l esters.