title: Differential role of extra- and intracellular calcium in bradykinin and interleukin 1 alpha stimulation of arachidonic acid release from A549 cells.
abstract: The release of arachidonic acid in A549 cells was stimulated in a time- and dose-dependent manner by the Ca2+ ionophore ionomycin (t1/2 = 4 min), thapsigargin (t1/2 = 8 min), bradykinin (t1/2 = 12 min, EC50 = 3 nM), and interleukin 1 alpha (t1/2 = 28 min, EC50 = 0.3 ng/ml). Bradykinin (10 nM) and interleukin 1 alpha (1 ng/ml) stimulation was blocked by the bradykinin B2 receptor antagonist, D-Arg,[Hyp3,Thi5,8, D-Phe7]bradykinin and interleukin 1 receptor antagonist (IC50 = 30 mM and 20 ng/ml, respectively), suggesting receptor mediation. Diacylglycerol release was < 10% of total arachidonic acid release in all cases, suggesting activation of phospholipase A2 activity was greater than phospholipase C activation by these agents. The effects of ionomycin (3 microM) and thapsigargin (0.3 microM) were abolished in Ca(2+)-free buffer with and without 0.5 mM EGTA. Bradykinin (10 nM) stimulation was reduced by 50% in Ca(2+)-free buffer whereas interleukin 1 alpha (1 ng/ml) stimulation remained unaffected. However, the presence of EGTA completely abolished bradykinin stimulation and partially blocked the effect of interleukin 1 alpha (43% inhibition). In the presence of extracellular Ca2+, ionomycin (3 mM), thapsigargin (0.3 mM), bradykinin (10 nM), and interleukin 1 alpha (1 ng/ml) stimulation of arachidonic acid release was blocked by the Ca2+ influx blocker LaCl3 (29, 44, 35, and 41% inhibition, respectively). Nifedipine also blocked ionomycin and thapsigargin stimulation but only partially blocked bradykinin and interleukin 1 alpha stimulation. These results suggest that following B2 receptor activation, cytosolic phospholipase A2 is stimulated by a rise in intracellular Ca2+ levels which are sensitive to the action of EGTA, whereas interleukin 1 alpha stimulation of cytosolic phospholipase A2 is mediated by a rise in intracellular Ca2+ from both EGTA-sensitive and resistant pools. Furthermore the results of ionomycin and thapsigargin indicate that extracellular Ca2+ is important for activation of cytosolic phospholipase A2 in A549 cells.
authors.affiliation: Ono Pharmaceutical Co., Minase Research Institute, Osaka, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Tokumoto
authors.forename: J D
authors.initials: JD
authors.lastname: Croxtall
authors.forename: R J
authors.initials: RJ
authors.lastname: Flower
mesh_terms: D016718:Arachidonic Acid / Q000378:metabolism*; D001920:Bradykinin / Q000378:metabolism*; D002118:Calcium / Q000502:physiology*; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D007375:Interleukin-1 / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D007811:Lanthanum / Q000494:pharmacology; D009543:Nifedipine / Q000494:pharmacology; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
chemical_list: D007375:Interleukin-1; D013729:Terpenes; D016718:Arachidonic Acid; D015759:Ionomycin; D019284:Thapsigargin; D007811:Lanthanum; D010741:Phospholipases A; D054467:Phospholipases A2; D009543:Nifedipine; D001920:Bradykinin; D002118:Calcium