Occult intracellular calcium pools: Relevance to neutrophil oxidant production

Human neutrophils use divalent cations for the stimulus-response coupling leading to the respiratory burst. We have studied the relevance of the extracellular versus intracellular calcium stores necessary for superoxide generation in neutrophils and neutrophil cytoplasts stimulated by N-formyl-methionyl-leucylphenylalanine (FMLP) and phorbol ester and our findings are as follows. (1) FMLP triggered superoxide anion (O₂^-) generation only in the presence of extracellular calcium, but phorbol myristate acetate (PMA) activation was independent of extracellular calcium. (2) Inhibition of calcium transport channels with verapamil produced a dose-related inhibition of FMLP- and PMA-stimulated O₂^- production. Verapamil inhibition of FMLP-stimulated but not PMA-stimulated O₂^- was reversed simply by increasing extracellular calcium concentration. Cytosolic calcium loading with substimulatory concentrations of A23187 (0.3 to 0.6 μmol/L) (only in calcium-replete medium) could reverse the verapamil inhibition of PMA-triggered O₂^- generation. An inhibitor of intracellular calcium availability, 3,4,5-trimethoxybenzoic acid (TMB-8), similarly inhibited FMLP-stimulated and PMA-stimulated O₂^- production; this inhibition was reversible with A23187 calcium loading. (3) Verapamil, although it inhibited the functional oxidant response, had no inhibitory effect on fura 2-monitored cytosolic calcium rise induced by FMLP stimulation. (4) No cytosolic calcium increment was observed with PMA. O₂^- generation from enucleated polymorphonuclear leukocyte (PMN) cytoplasts was sensitive to inhibition by verapamil but was less so than O₂^- generation from intact PMNs. Cytoplasts, nearly depleted of cytoplasmic organelles and cytoskeleton, the potential sites of intracellular calcium stores, were markedly insensitive to the inhibitory effects of TMB-8. These data demonstrate that FMLP-stimulated O₂^- production and PMA-stimulated O₂^- production are sensitive to calcium channel blockade with verapamil and are sensitive to intracellular calcium flux inhibition with TMB-8. Transmembrane flux of extracellular calcium is necessary for FMLP-stimulated generation of O₂^-, but PMA triggers calcium mobilization from essential, occult, and A23187-accessible calcium pools during the oxidant response. These occult calcium pools are probably intracellular and may be associated with cytoskeletal or cytoplasmic organelles that are depleted from PMN cytoplasts.