CHARACTERIZATION OF THE PGE RECEPTOR SUBTYPE MEDIATING INHIBITION OF SUPEROXIDE PRODUCTION IN HUMAN NEUTROPHILS

1 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)E(2). 2 PGE(2) (0.001-10 mu M) inhibited FMLP (100 nM)-induced O-2-generation from human peripheral blood neutrophils in a concentration-dependent manner, with an EC(50) of 0.15 +/- 0.03 mu M, and a maximum effect ranging from 36-84% (mean inhibition of 68.7 +/- 2.5%, n = 32). 3 The EP(2)-receptor agonists, misoprostol, 11-deoxy PGE(1), AH13205 and butaprost, all at 10 mu M, inhibited O-2- 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 PGE(2). Similarly, the EP(1)-receptor agonist, 17-phenyl PGE(2) (10 mu M), and the EP(3)/EP(1)-receptor agonist, sulprostone (10 mu M), also inhibited O-2- 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 PGE(2), misoprostol, 11-deoxy PGE(1), butaprost, and AH 13205 to the left, to give EC(50)s 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 mu M (n = 3) respectively, allowing equieffective concentration-ratios (EECs, PGE(2) = 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 EP(2) receptors. 5 By contrast, even in the presence of IBMX (0.25 mM), sulprostone and 17-phenyl PGE(2) were only effective at the highest concentration (10 mu M), and gave EECs of > 700 and 486 respectively, suggesting that EP(1) or EP(3) 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 PGE(2) to the left to give EC(50)s of 0.06 +/- 0.022, 0.015+/- 0.0, 0.012 +/- 0.006 mu M at 2, 10 and 50 nM respectively, compared to the control EC(50) of 0.27 +/- 0.09 mu M for PGE(2). 7 The EP(4)/TP receptor blocking drug, AH 23848B (10 mu M, 10 min) did not inhibit O-2- generation by PGE(2), but was found to potentiate significantly the effect of PGE(2) at the lower concentrations of PGE(2) tested (0.001-0.1 mu M). 8 The adenylate cyclase inhibitor, SQ 22,536 (0.1 mM, 2 min) reduced PGE(2)-induced inhibition of O-2- production, giving an EC(50) in the absence of SQ 22,536 of 0.24 +/- 0.1, and 1.9 +/- 1.1 mu M in its presence. 9 These results suggest that inhibition of superoxide generation by PGE(2) is mediated by stimulation of EP(2) receptors and activation of adenylate cyclase, leading to the elevation of intracellular levels of cyclic AMP.