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

W. K. Kim-Park, M. A. Moore, Z. W. Hakki, M. J. Kowolik

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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 Ca2+-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 (10-5M) and N-methyl-D-aspartate (NMDA) (10-5M) 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.

Original languageEnglish (US)
Pages (from-to)394-404
Number of pages11
JournalAnnals of the New York Academy of Sciences
Volume832
DOIs
StatePublished - Dec 15 1997

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ASJC Scopus subject areas

  • Neuroscience(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • History and Philosophy of Science

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