Investigation of the cellular mechanism of inhibition of formyl-methionyl-leucyl-phenylalanine-induced superoxide anion generation in rat neutrophils by 2-benzyloxybenzaldehyde

Abstract

The inhibition of formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide anion (O 2 −) generation by 2-benzyloxybenzaldehyde (CCY1a) was investigated in rat neutrophils, and the underlying mechanism of this inhibition was assessed. CCY1a concentration-dependently inhibited O 2 − generation ( ic 50=18.5±4.3 μM). In cell-free systems, CCY1a failed to alter O 2 − generation during dihydroxyfumaric acid autoxidation, in phorbol 12-myristate 13-acetate (PMA)-activated neutrophil particulate NADPH oxidase preparations, or during arachidonic acid-induced NADPH oxidase activation. CCY1a increased cellular cyclic AMP (cAMP) levels in a time- and concentration-dependent manner, and this cAMP-elevating effect was inhibited by the adenylyl cyclase inhibitor 9-(tetrahydro-2′-furyl)adenine (SQ22536), adenosine deaminase (ADA), and the adenosine receptor antagonist 8-( p-sulfophenyl)theophylline. In neutrophils, inhibition of O 2 − generation by CCY1a was partially reversed by the protein kinase A inhibitor (9 R,10 S,12 S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1 H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-l][1,6]benzodiazocine-10-carboxylic acid, hexyl ester (KT5720). CCY1a did not affect fMLP-induced p38 mitogen-activated protein kinase phosphorylation, but concentration-dependently attenuated the phosphorylation of extracellular signal-regulated kinase (ERK) and Akt ( ic 50 about 31.3 and 19.4 μM, respectively). The plateau phase, but not the initial spike, of fMLP-induced [Ca 2+] i changes was inhibited by CCY1a in a concentration-dependent manner. CCY1a inhibition of Ca 2+ entry, ERK, and Akt phosphorylation was not prevented by SQ22536 or ADA. fMLP-induced phospholipase D (PLD) activation was inhibited by CCY1a ( ic 50=13.9±2.0 μM). ADA and KT5720 did not prevent the inhibition of PLD activation by CCY1a. Collectively, these results indicate that the inhibition by CCY1a of fMLP-induced O 2 − generation in rat neutrophils can probably be attributed to the increase in cAMP levels, and to the blockade of Ca 2+ entry, suppression of Akt, and PLD activation via cAMP-independent mechanisms.