title: Mechanism of arachidonic acid liberation in platelet-activating factor-stimulated human polymorphonuclear neutrophils.
abstract: Upon stimulation of human polymorphonuclear neutrophils with platelet-activating factor (PAF), arachidonic acid (AA) is released from membrane phospholipids. The mechanism for AA liberation, a key step in the synthesis of biologically active eicosanoids, was investigated. PAF was found to elicit an increase in the cytoplasmic level of free Ca2+ as monitored by fluorescent indicator fura 2. When [3H] AA-labeled neutrophils were exposed to PAF, the enhanced release of AA was observed with a concomitant decrease of radioactivity in phosphatidylinositol and phosphatidylcholine fractions. The inhibitors of phospholipase A2, mepacrine and 2-(p-amylcinnamoyl)-amino-4-chlorobenzoic acid, effectively suppressed the liberation of [3H]AA from phospholipids, indicating that liberation of AA is mainly catalyzed by the action of phospholipase A2. The extracellular Ca2+ is not required for AA release. However, intracellular Ca2+ antagonists, TMB-8 and high dose of quin 2/AM drastically reduced the liberation of AA induced by PAF, indicating that Ca2+ is an essential factor for phospholipase A2 activation. PAF raised the fluorescence of fura 2 at concentrations as low as 8 pM which reached a maximal level about 8 nM, whereas more than nM order concentrations of PAF was required for the detectable release of [3H]AA. Pretreatment of neutrophils with pertussis toxin resulted in complete abolition of AA liberation in response to PAF. However, the fura 2 response to PAF was not effectively inhibited by toxin treatment. In human neutrophil homogenate and membrane preparations, guanosine 5'-O-(thiotriphosphate) stimulated AA release and potentiated the action of PAF. Guanosine 5'-O-(thiodiphosphate) inhibited the effects of guanosine 5'-O-(thiotriphosphate). These results suggest several points: 1) PAF stimulates human polymorphonuclear neutrophils to liberate AA mainly by the action of phospholipase A2; 2) Ca2+ mobilization alone is not sufficient to stimulate AA release, although Ca2+ is the important factor for phospholipase A2 activation; and 3) a pertussis toxin-sensitive GTP-binding protein may be implicated in activation of phospholipase A2.
authors.affiliation: Department of Biochemistry, Gifu University School of Medicine, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Nakashima
authors.forename: A
authors.initials: A
authors.lastname: Suganuma
authors.forename: M
authors.initials: M
authors.lastname: Sato
authors.forename: T
authors.initials: T
authors.lastname: Tohmatsu
authors.forename: Y
authors.initials: Y
authors.lastname: Nozawa
mesh_terms: D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000097:blood*; D002118:Calcium / Q000378:metabolism / Q000502:physiology; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009504:Neutrophils / Q000378:metabolism* / Q000502:physiology; D037342:Pertussis Toxin; D010972:Platelet Activating Factor / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface / Q000032:analysis; D043562:Receptors, G-Protein-Coupled; D014316:Tritium; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D001095:Arachidonic Acids; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D010566:Virulence Factors, Bordetella; C039351:platelet activating factor receptor; D014316:Tritium; D016718:Arachidonic Acid; D037342:Pertussis Toxin; D002118:Calcium