Regulation of N-.formyl-methionyl-leucyl-phenylalanine

Neutrophil responses to a-N-formyl-LMetL-Leu-L-Phe (IMLF) are modulated by inhibitors of surface membrane neutral endopeptidase (NEP), such as phosphoramidon (PPAD). Because receptor recycling is presumably required for a sustained cellular response, the effect of PPAD on receptor reexpression was examined. After down-regulation of surface fMLF receptors by IMLF, PPAD blocked the normal reexpression ofsurface receptors in a manner that was related to the time ofprior exposure to fMLF. Internalized fML[3H]F was hydrolyzed by NEP at a rate comparable to the rate of receptor reexpression at the cell surface, suggesting that ligand hydrolysis is rate limiting. To test this hypothesis, cells were incubated with fluorescein-labeled formyl-Met-Leu-PheNie-Tyr-Lys at 15#{176}C. After binding was complete, but before internalization of receptor-ligand complexes, high-affinity antifluorescein antibody F(ab’)2 fragments were added and the cells incubated at 37#{176}Cfor 60 mm in the presence of PPAD. Under these conditions, the inhibitory effects of PPAD were largely reversed and nommmune F(ab’)2 fragments were without effect. J. Leukoc. Biol. 58: 468-476; 1995.