Inhibition of phospholipase D activation by CYL-26z in formyl peptide-stimulated neutrophils involves the blockade of RhoA activation

5-[4-Acridin-9-ylamino]phenyl]-5-methyl-3-methylenedihydrofuran-2-one (CYL-26z) inhibited the formyl-Met-Leu-Phe (fMLP)-stimulated phospholipase D (PLD) activity, which was assessed by the production of phosphatidylethanol (PEt) in the presence of ethanol, in rat neutrophils (IC50 1.2 ± 0.2 μM). CYL-26z caused a slight but significant attenuation of the global protein tyrosine phosphorylation stimulated by fMLP only at concentrations of CYL-26z up to 30 μM. CYL-26z blocked the membrane recruitment of protein kinase C-α (PKC-α) at concentrations of CYL-26z ≥3 μM, but failed to affect the membrane association of PKC-βI and -βII. The translocation of RhoA to the membrane was attenuated by CYL-26z (IC50 3.8 ± 0.8 μM) in fMLP-stimulated neutrophils, whereas CYL-26z caused no significant inhibition of the membrane recruitment of ADP-ribosylation factor (Arf). CYL-26z inhibited the activation of RhoA and dissociation of the RhoA-Rho guanine nucleotide dissociation inhibitor (GDI) complex in fMLP-stimulated neutrophils (IC50 1.8 ± 1.0 μM and 1.8 ± 0.9 μM, respectively). In a cell-free system, CYL-26z effectively attenuated the membrane association of RhoA in response to GTPγS (IC50 1.3 ± 0.5 μM). In contrast, the GTPγS-stimulated translocation of Arf to membrane was suppressed only at concentrations of CYL-26z up to 30 μM. CYL-26z inhibited the fMLP-stimulated membrane expression of CD11b, CD45 and CD63, and the release of lysozyme and β-glucuronidase. These results indicate that CYL-26z inhibited the fMLP-stimulated PLD activity, mainly through the blockade of RhoA activation, and degranulation in rat neutrophils.