Rebamipide suppresses formyl-methionyl-leucyl-phenylalanine (fMLP)-inducedsuperoxide production by inhibiting fMLP-receptor binding in human neutrophils

The purpose of the present work was to investigate the mechanism underlying the inhibitory action of rebamipide on superoxide anion (O-2(radical anion )) production induced by the chemotactic peptide formyl-methionyl-leucyl-ph enylalanine (fMLP) in human neutrophils. Phosphatidylinositol 3,4,5- trisph osphate (PIP3), a product of phosphoinositide 3-OH-kinase (PI 3-kinase) acc umulated in response to fMLP and this accumulation was well correlated with O-2(radical anion). production in human neutrophils. Rebamipide inhibited PIP3 production in parallel with the inhibition of fMLP-induced O-2(radical anion) production. PI 3-kinase activity in anti-PI 3-kinase p85 immunoprec ipitates was not affected by the presence of rebamipide, therefore rebamipi de did not have a direct inhibitory action on PI 3-kinase activity. Since r ebamipide had no inhibitory effect on O-2(radical anion) production induced by NaF, a direct activator of G protein, the target of the inhibitory acti on of rebamipide appears to be a component of the signal transduction pathw ay upstream of G protein. Scatchard analysis of [H-3] fMLP binding to human neutrophil membrane revealed that rebamipide increased the KD value of [H- 3] fMLP without altering the number of [H-3] fMLP binding sites, suggesting that rebamipide has a competitive antagonistic action against the fMLP-rec eptor. The competitive antagonistic action was further confirmed by the fin ding that rebamipide caused a parallel shift to the right in the dose-respo nse curve of O-2(radical anion) production induced by fMLP. These results p rovide evidence that the competitive inhibitory action of rebamipide on the fMLP-receptor plays a main role in its inhibitory action on fMLP-induced O -2(radical anion) production.