2-benzyloxybenzaldehyde inhibits formyl-methionyl-leucyl-phenylalanine stimulation of phospholipase D activation in rat neutrophils

2-Benzyloxybenzaldehyde (CCYla) inhibited the formyl-methionyl-leucyl-phenylalanine (RALP)-stimulated phospholipase D (PLD)mediated products, phosphatidic acid (PA) and phosphatidylethanol (PEt) formation in rat neutrophils in a concentration-dependent manner with IC50 values of 15.8 +/- 2.5 and 13.9 +/- 2.0 muM, respectively. The underlying cellular signaling mechanism of CCYla inhibition was investigated. CCYla inhibited the plateau phase but not the initial Ca2+ spike of fMLP-stimulated Ca2+ signal. CCYla did not inhibit the [Ca2+](i) change in Ca2+-free medium in response to fMLP, but inhibited the [Ca2+](i) change by the subsequent addition of Ca2+. In addition, CCYla treatment attenuated the fMLP-induced protein tyrosine phosphorylation. The membrane translocation of ADP-ribosylation factor (ARF) and Rho A proteins in neutrophils stimulated with fMLP was attenuated by CCYla in a concentration-dependent manner. In a cell-free system, neither the membrane association of ARF and Rho A caused by GTPgammaS nor the phorbol myristate acetate-stimulated membrane translocation of Rho A was suppressed significantly by CCYla. These results indicate that the attenuation of protein tyrosine phosphorylation, blockade of Ca2+ entry, and the suppression of ARF and Rho A membrane translocation are probably obligatory for the CCYla inhibition of PLD activity in rat neutrophils in response to fMLP. (C) 2002 Elsevier Science B.V. All rights reserved.