Activation of the neutrophil respiratory burst requires both intracellular and extracellular calcium.
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.
Kim-Park
W K
WK
Indiana University, Department of Oral Biology, Indianapolis 46202-5186, USA.
Moore
M A
MA
Hakki
Z W
ZW
Kowolik
M J
MJ
D002118:Calcium / Q000097:blood* / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008163:Luminescent Measurements; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D019255:NADPH Oxidases / Q000097:blood; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*; D016897:Respiratory Burst / Q000187:drug effects / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology
D014638:Vanadates; D004533:Egtazic Acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D019833:Genistein; D019255:NADPH Oxidases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium