DIFFERENTIAL ROLE OF EXTRACELLULAR AND INTRACELLULAR CALCIUM IN BRADYKININ AND INTERLEUKIN-1-ALPHA STIMULATION OF ARACHIDONIC-ACID RELEASE FROM A549 CELLS

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 1alpha (t1/2 = 28 min, EC50 = 0.3 ng/ml). Bradyk inin (10 nM) and interleukin 1alpha (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 re lease was < 10% of total arachidonic acid release in all cases, sugges ting activation of phospholipase A, activity was greater than phosopho lipase C activation by these agents. The effects of ionomycin (3 muM) and thapsigargin (0.3 muM) were abolished in Ca2+-free buffer with and without 0.5 mM EGTA. Bradykinin (10 nM) stimulation was reduced by 50 % in Ca2+-free buffer whereas interleukin 1alpha (1 ng/ml) stimulation remained unaffected. However, the presence of EGTA completely abolish ed bradykinin stimulation and partially blocked the effect of interleu kin 1alpha (43% inhibition). In the presence of extracellular Ca2+, io nomycin (3 mM), thapsigargin (0.3 mM), bradykinin (10 nM), and interle ukin 1alpha (1 ng/ml) stimulation of arachidonic acid release was bloc ked by the Ca2+ influx blocker LaCl3 (29, 44, 35, and 41% inhibition, respectively). Nifedipine also blocked ionomycin and thapsigargin stim ulation but only partially blocked bradykinin and interleukin 1alpha s timulation. These results suggest that following B2 receptor activatio n, cytosolic phospholipase A2 is stimulated by a rise in intracellular Ca2+ levels which are sensitive to the action of EGTA, whereas interl eukin 1alpha stimulation of cytosolic phospholipase A2 is mediated by a rise in intracellular Ca2+ from both EGTA-sensitive and resistant po ols. Furthermore the results of ionomycin and thapsigargin indicate th at extracellular Ca2+ is important for activation of cytosolic phospho lipase A2 in A549 cells.