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Activation of the neutrophil respiratory burst requires both intracellular and extracellular calcium.

Authors
Type
Published Article
Journal
Annals of the New York Academy of Sciences
Publication Date
Volume
832
Pages
394–404
Identifiers
PMID: 9704067
Source
Medline
License
Unknown

Abstract

Activation of neutrophil oxidases, including NADPH oxidase, is Ca2+ dependent. The aim of this study was to determine the roles of intra- and extracellular Ca2+, leading to generation of the respiratory burst, as monitored by luminol-dependent chemiluminescence (CL). All results were recorded as integrals (millivolt.min) and compared by a two-tail Student's t test. Preincubation of cells with chelators of intra- or extracellular Ca2+ inhibited N-Formyl-Met-Leu-Phe (FMLP)-stimulated burst activity (p < 0.01). In contrast, stimulation by phorbol myristate acetate (PMA), while inhibited by extracellular Ca2+ chelation with EGTA (p < 0.001), was potentiated by intracellular Ca2+ chelation with BAPTA (p < 0.01). This suggests that the protein kinase C (PKC)-mediated burst may be diminished by intracellular Ca(2+)-dependent phosphatase. A selective inhibitor of tyrosine phosphatase, sodium vanadate, potentiated CL generation by both FMLP and PMA, indicating a dominant phosphatase activation with transiently increased Ca2+, masking the kinase-mediated respiratory burst. The selective inhibitors of PKC or tyrosine kinase prevented PMA and vanadate/PMA stimulation (p < 0.005). Furthermore, the putative Ca2+ channel agonists glutamate (10(-5)M) and N-methyl-D-aspartate (NMDA) (10(-5)M) alone failed to influence CL output, but produced marked potentiation following pre-treatment with vanadate. Again this indicates a dominant activation of phosphatase triggered by the glutamate-mediated Ca2+ influx, so masking the kinase-dependent NADPH oxidase activity. A competitive antagonist of NMDA, AP7, significantly decreased vanadate-mediated CL in an EGTA-sensitive manner (p < 0.001). The data confirm a requirement for intra- and extracellular Ca2+ in neutrophil respiratory burst activation via the kinase/phosphatase cycle, and an agonist effect by NMDA within the Ca2+ cascade mechanism.

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