(2R,3R)-2-(3′,4′-dihydroxybenzyl)-3-(3″,4″-dimethoxybenzyl)butyrolactone suppresses fMLP-induced superoxide production by inhibiting fMLP-receptor binding in human neutrophils

This study investigated the mechanism underlying the inhibiting effect of (2R,3R)-2-(3′,4′-dihydroxybenzyl)-3-(3″,4″-dimethoxybenzyl) butyrolactone (PP-6), a lignan from Piper philippinum, on superoxide anion production induced by the chemotactic peptide formly-methionyl-leucyl-phenylalanine (fMLP) in human neutrophils. Human neutrophils were stimulated with fMLP (1 μM), PMA (100 nM) or leukotriene B₄ (LTB₄; 1 μM) and induced superoxide anion release. PP-6 specifically inhibited fMLP-induced superoxide anion production in a concentration-dependent manner with an IC₅₀ value of 0.3 ± 0.1 μM. Intracellular signaling caused by fMLP, PMA or LTB₄ were evaluated. PP-6 specifically inhibited fMLP-induced intracellular calcium mobilization and ERK (p42/p44), Akt and p38 phosphorylation. Moreover, PP-6 specifically inhibited fMLP-induced Mac-1 expression without affecting this caused by LTB₄ or PMA. PP-6 did not increase cAMP level in human neutrophils. PP-6 did not inhibit superoxide anion production by NaF (20 mM), a direct activator of G-protein, the target of the inhibitory action of PP-6 appears to be a component of the signal transduction pathway upstream of G-protein. PP-6 inhibited FITC-fMLP binding to neutrophils in a concentration-dependent manner with an IC₅₀ of 1.5 ± 0.2 μM. PP-6 did not bring a parallel shift in the concentration response of fMLP-induced superoxide anion. Additionally, the inhibiting effect of PP-6 on fMLP-induced superoxide anion was reversed when PP-6 was washed out. These experimental results suggest that PP-6 exerts non-competitive and reversible antagonistic effect on fMLP receptor.