Occult intracellular calcium pools: Relevance to neutrophil oxidant production

D. J. Weisdorf, M. S. Thayer

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10 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)260-265
Number of pages6
JournalJournal of Laboratory and Clinical Medicine
Issue number3
StatePublished - 1989


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