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 ionomycin) to modulate the respiratory burst of neutrophils induced by phorbol ester and to increase the concentration of free intracellular Ca2+ ([Ca2+]i) were compared. The production of reactive oxygen species (ROS) was determined by luminol-dependent chemiluminescence and [Ca2+]i was determined with the Fura-2 fluorescent probe. A23187 (0.05-2 microM) and ionomycin (0.001-0.5 microM) but not 4-bromo-A23187 amplified 3-4-fold the respiratory burst induced by phorbol ester. The integral response (total production of ROS over 6 min) had a bell-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 microM ionomycin and 2 microM A23187, these concentrations resulting in transient increases in [Ca2+]i to 1776 +/- 197 and 955 +/- 27 nM, respectively. The ionophores had no effect in calcium-free media, though they increased [Ca2+]i to approximately 400 nM through the mobilization of intracellular Ca2+. In cells with exhausted stores of Ca2+, the addition of 1.5 mM Ca2+ combined with phorbol ester amplified twofold the production of ROS. The inhibition of phospholipase A2 with 4-bromophenacyl bromide significantly decreased the production of ROS. Thus, the entrance of Ca2+ and generation of arachidonic acid under the influence of phospholipase A2 are necessary for the ionophore-induced priming of production of ROS during cell activation with phorbol esters.