The roles of extracellular and intracellular calcium in lysosomal enzyme release and superoxide anion generation by human neutrophils

Abstract

The relative importance of intracellular and extracellular calcium in the stimulation of human neutrophils was studied by use of chelators and calcium antagonists. In the first series of experiments, EGTA was used to see if extracellular calcium was an absolute requirement for neutrophil activation. Stimulated neutrophils secreted lysosomal enzymes and generated superoxide anion radicals in the presence or absence of 5 mM EGTA or Mg-EGTA. Seven different stimuli were employed: the chemotactic peptide N-formylmethionylleucylphenylalanine (10 −7 M), calcium ionophore A23187 (10 −5 M), concanavalin A (30 μg/ml), serum-treated zymosan (2 mg/ml), zymosan-treated serum serum (10%), an immune complex (150 μg/ml) and phorbol myristate acetate (1 μg/ml). Release of the lysosomal enzymes β-glucuronidase and lysozyme and generation of superoxide anions in response to these stimuli were readily demonstrable in the presence of EGTA. Each response of neutrophils was variably, but not completely, reduced in the presence of the chelator; EGTA did not completely block the responses elicited by any of the stimuli. Consequently, it is unlikely that an influx of divalent cations from the extracellular medium is obligatory for stimulus-response coupling in human neutrophils. The role of intracellular calcium was then studied using calcium antagonists. Inhibition of neutrophil responses was readily obtained using TMB-8, an antagonist of the mobilization of intracellular calcium, and trifluoperazine and W-7, inhibitors of calmodulin. These results suggest that intracellular, but not extracellular, calcium plays an obligatory role in the responses of neutrophils to a variety of soluble and insoluble stimuli.