Prostaglandin D2 modulates human neutrophil intracellular calcium flux and inhibits superoxide release via its ring carbonyl.

We compared the effects of prostaglandin D2 (PGD2), prostaglandin F2 alpha (PGF2) and various ketones on superoxide (OX) release by human neutrophils, which had been stimulated by N-formyl methionyl leucyl phenylalanine (FMLP). Our data suggested that the ring carbonyl of PGD2 is essential to its inhibitory effect on OX release, but the carbonyl group as a ketone, alone is not sufficient. Using the fluorescent Ca2+ probe, Fura-2AM, we found that PGD2 increased the rate of decline of FMLP stimulated intracellular free Ca2+ (Ca)i, but that PGF2 had no effect. cAMP altered FMLP stimulated (Ca)i, in a pattern similar to PGD2. Furthermore, the ring carbonyl of PGD2 is crucial to its effect on OX as well as on (Ca)i.

The effect of enkephalins and prostaglandins on O-2 release by neutrophils.

Derivatives of superoxide (O-2), produced by phagocytic cells, are thought to play a role in the adult respiratory distress syndrome (ARDS) and other disease states. Control of the release of O-2 may prove beneficial. Using human neutrophils as a source of O-2, and an assay for O-2 based upon the reduction of cytochrome C, we found that prostaglandin D2 (PGD2), leucine enkephalin (LE), and methionine enkephalin (ME) inhibited O-2 release. The Escherichia coli product, N-formyl methionyl leucyl phenylalanine (FMLP), was employed to stimulate O-2 release. PGD2 was most potent while there was no significant difference between LE and ME. Another peptide, thyrotropin releasing hormone (TRH), had no effect on O-2 release. There was no correlation between the potency of the inhibitory effect on O-2 release and the effect of these agents on the binding of [3H] FMLP to human neutrophils. Comparison of different but structurally related prostaglandins (PGD2, PGE2, and PGF2 alpha) revealed that PGD2 was more potent than PGE2 in inhibiting O-2 and that PGF2 alpha had no effect. This result suggested that the presence and position of the carbonyl group was an important determinant of the magnitude of inhibition.

Inhibition by naloxone of neutrophil superoxide release: a potentially useful antiinflammatory effect.

The working hypothesis of many studies of shock has been that naloxone acts by blocking centrally and/or peripherally located opioid receptors. At plasma concentrations used to treat experimental shock (10(-6) M and above), naloxone inhibited the in vitro release of superoxide (O2-) by human neutrophils that were stimulated by the E. coli peptide N-formyl methionyl leucyl phenylalanine (FMLP). Superoxide release stimulated by phorbol 12,13-dibutyrate (PDB) was also inhibited by naloxone. Naloxone had no effect on the FMLP-stimulated release of beta-glucuronidase or lysozyme. Naloxone had no effect on 3H FMLP receptor binding. Studies utilizing 3H naloxone revealed the presence of a ligand-specific naloxone binding site on human neutrophils with a Kd of 1.2 X 10(-5) M, which is close to the ID50 of the inhibitory effect upon O2- release (1.8 X 10(-5). Thyrotropin releasing factor (TRF) had no effect upon 3H naloxone binding or on O2- release. Verapamil, a calcium channel blocker, inhibited 3H naloxone binding, and O2- release while nifedipine, another calcium channel blocker had no effect on either assay except at 10(-4) M, at which concentration 3H naloxone binding as well as the release of O2- were increased. These experiments suggest that the inhibitory effect of naloxone upon O2- release is mediated via a specific binding site.