title: Dissociation of the PAF-receptor from NADPH oxidase and adenylate cyclase in human neutrophils results in accelerated influx and delayed clearance of cytosolic calcium.
abstract: The magnitude and duration of the abruptly occurring increases in cytosolic Ca2+ in human neutrophils following activation with PAF (20 and 200 nM) and FMLP (1 microM), have been compared and related to alterations in NADPH oxidase activity, membrane potential and intracellular cyclic AMP. Cytosolic Ca2+ and membrane potential were measured by spectrofluorimetry, transmembrane fluxes of Ca2+ by radiometric procedures, and NADPH oxidase activity and cyclic AMP by chemiluminescence and radioimmunoassay respectively. Activation of neutrophils with both PAF (200 nM) and FMLP (1 microM) was accompanied by an abrupt increase in cytosolic Ca2+, which was of similar magnitude for each activator (393+/-9 and 378+/-17 nM respectively). Unlike FMLP-activated cells in which Ca2+ was rapidly removed from the cytosol, peak levels of cytosolic Ca2+ were sustained for longer (0.14+/-0.02 vs 1.16+/-0.04 min, P<or=0.0001) and declined at a slower rate in PAF-treated neutrophils. The prolonged elevation of cytosolic Ca2+ in PAF-treated cells was due to accelerated store-operated influx of extracellular cation and was attenuated by dibutyryl cyclic AMP (4 mM), the Ca2+-chelator, EGTA (5 mM), and SKF 96365 (10 microM). In contrast to FMLP, basal levels of superoxide production and cyclic AMP were unaltered in PAF-activated neutrophils, while only moderate membrane depolarization was detected. These observations demonstrate that mechanisms which restore Ca2+ homeostasis to FMLP-activated neutrophils, viz. activation of NADPH oxidase and adenylate cyclase, are not operative in PAF-treated cells, presenting the potential hazard of Ca2+ overload and hyperactivity.
authors.affiliation: Medical Research Council Unit for Inflammation and Immunity, Department of Immunology, Institute for Pathology, University of Pretoria, Republic of South Africa. hsteel@postillion.up.ac.za
authors.forename: H C
authors.initials: HC
authors.lastname: Steel
authors.forename: R
authors.initials: R
authors.lastname: Anderson
mesh_terms: D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism; D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism*; D003994:Bucladesine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D006706:Homeostasis; D006801:Humans; D066298:In Vitro Techniques; D019272:Leukocyte Elastase / Q000378:metabolism; D008163:Luminescent Measurements; D008564:Membrane Potentials; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D019255:NADPH Oxidases / Q000378:metabolism*; D018375:Neutrophil Activation; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D010627:Phenethylamines / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010972:Platelet Activating Factor / Q000378:metabolism* / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D058906:Purinergic P1 Receptor Agonists; D011863:Radioimmunoassay; D043705:Receptor, Adenosine A2A; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D020889:Rolipram / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013481:Superoxides / Q000378:metabolism
chemical_list: D010627:Phenethylamines; D010726:Phosphodiesterase Inhibitors; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D058906:Purinergic P1 Receptor Agonists; D043705:Receptor, Adenosine A2A; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C039351:platelet activating factor receptor; D013481:Superoxides; C061282:2-(4-(2-carboxyethyl)phenethylamino)-5'-N-ethylcarboxamidoadenosine; D009240:N-Formylmethionine Leucyl-Phenylalanine; D003994:Bucladesine; D000242:Cyclic AMP; D019255:NADPH Oxidases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D019272:Leukocyte Elastase; D000262:Adenylyl Cyclases; D020889:Rolipram; D000241:Adenosine; D002118:Calcium