Vol. 288, Issue 3, 1101-1106, March 1999

Mechanisms of Prostaglandin E₂ Release by Intact Cells Expressing Cyclooxygenase-2: Evidence for a `Two-Component' Model¹

Michael A. Saunders², Maria G. Belvisi^{2 3}, Guiseppe Cirino⁴, P. J. Barnes², Timothy D. Warner⁵ and Jane A. Mitchell⁶

Unit of Critical Care Medicine, Royal Brompton Hospital, Imperial College School of Medicine, National Heart and Lung Institute, London, England

Prostaglandin (PG) release in cells expressing constitutive cyclooxygenase-1 is known to be regulated by liberation of arachidonic acid by phospholipase A₂ followed by metabolism by cyclooxygenase. However, the relative contribution of phospholipase A₂ to the release of PGs in cells expressing cyclooxygenase-2 is not clear. We addressed this question by using radioimmunoassay to measure PGE₂ release by human cells (A549) induced to express cyclooxygenase-2 (measured by Western blot analysis) by interleukin-1. Cells were either unstimulated or stimulated with agents known to activate phospholipase A₂ (bradykinin, Des-Arg¹⁰-kallidin, or the calcium ionophore A23187) or treated with exogenous arachidonic acid. When cells were treated to express cyclooxygenase-2, the levels of PGE₂ released over 15 min were undetectable; however, in the same cells stimulated with bradykinin, A23187, or arachidonic acid, large amounts of prostanoid were produced. Using selective inhibitors/antagonists, we found that the effects of bradykinin were mediated by B₂ receptor activation and that prostanoid release was due to cyclooxygenase-2, and not cyclooxygenase-1, activity. In addition, we show that the release of PGE₂ stimulated by either bradykinin, A23187, or arachidonic acid was inhibited by the phospholipase A₂ inhibitor arachidonate trifluoromethyl ketone. Hence, we have demonstrated that PGE₂ is released by two components: induction of cyclooxygenase-2 and supply of substrate, probably via activation of phospholipase A₂. This is illustrated in A549 cells by a clear synergy between the cytokine interleukin-1 and the kinin bradykinin.