The aims of this study were to characterize the EP receptor subtype mediating the inhibition of superoxide anion generation by formyl methionyl leucine phenylalanine (FMLP)-stimulated human neutrophils, and to test the hypothesis that adenosine 3':5'-cyclic monophosphate (cyclic AMP) is the second messenger mediating the inhibition of the neutrophil by prostaglandin (PG)E2. 2. PGE2 (0.001-10 microM) inhibited FMLP (100 nM)-induced O2-generation from human peripheral blood neutrophils in a concentration-dependent manner, with an EC50 of 0.15 +/- 0.03 microM, and a maximum effect ranging from 36-84% (mean inhibition of 68.7 +/- 2.5%, n = 32). 3. The EP2-receptor agonists, misoprostol, 11-deoxy PGE1, AH13205 and butaprost, all at 10 microM, inhibited O2- generation, causing 95.5 +/- 2.9%, 56.8 +/- 5.2%, 37.1 +/- 6.6% and 18.9 +/- 4.4% inhibition respectively, the latter two being much less effective than PGE2. Similarly, the EP1-receptor agonist, 17-phenyl PGE2 (10 microM), and the EP3/EP1-receptor agonist, sulprostone (10 microM), also inhibited O2- generation, causing 32.2 +/- 7.0% and 15.3 +/- 3.4% inhibition respectively. 4. The non-selective phosphodiesterase inhibitor, isobutyl methylxanthine (IBMX, 0.25 mM) inhibited the FMLP response by 54.5 +/- 5.0%. In addition, IBMX shifted concentration-effect curves for PGE2, misoprostol, 11-deoxy PGE1, butaprost, and AH 13205 to the left, to give EC50s of 0.04 +/- 0.03 (n = 13), 0.07 +/- 0.03 (n = 4), 0.08 +/- 0.03 (n = 4), 0.33 +/- 0.13 (n = 4) and 0.41 +/- 0.2 microM (n = 3) respectively, allowing equieffective concentration-ratios (EECs, PGE2 = 1) of 11.5, 5.3, 50.7 and 12.7 to be calculated. This agrees well with the relative potencies of these agonists at EP2 receptors.5. By contrast, even in the presence of IBMX (0.25 mM), sulprostone and 17-phenyl PGE2 were only effective at the highest concentration (10 microM), and gave EECs of > 700 and 486 respectively, suggesting that EP1 or EP3 receptors are not involved.6. The selective type IV phosphodiesterase inhibitor, rolipram at 2 and 10 nM did not inhibit the FMLP response, but at the higher concentration of 50 nM, it decreased the FMLP response by 46.6 +/-7.3%.However, rolipram shifted concentration-effect curves for PGE2 to the left to give EC50s of 0.06 +/-0.022,0.015 +/- 0.0, 0.012 +/- 0.006 microM at 2, 10 and 50 nM respectively, compared to the control EC50 of0.27+/- 0.09 microM for PGE2.7. The EP4/TP receptor blocking drug, AH 23848B (10 microM, 10 min) did not inhibit 02- generation by PGE2, but was found to potentiate significantly the effect of PGE2 at the lower concentrations of PGE2 tested (0.001-0.1 microM).8. The adenylate cyclase inhibitor, SQ 22,536 (0.1 mM, 2 min) reduced PGE2-induced inhibition of 02-production, giving an EC50 in the absence of SQ 22,536 of 0.24 +/- 0.1, and 1.9 +/- 1.1 AM in its presence.9. These results suggest that inhibition of superoxide generation by PGE2 is mediated by stimulation ofEP2 receptors and activation of adenylate cyclase, leading to the elevation of intracellular levels of cyclic AMP.
