Inhibition by HAJ11 of respiratory burst in neutrophils and the involvement of protein tyrosine phosphorylation and phospholipase D activation

1 The possible mechanisms of the inhibitory effect of ethyl 2-(3-hydroxyanilino)-4-oxo-4,5-dihydrofuran-3-carboxylate (HAJ11) on the respiratory burst of rat neutrophils in vitro was investigated. 2 HAJ11 caused a reversible and a concentration-dependent inhibition of formyl-Met-Leu-Phe (fMLP)-induced superoxide anion (O-2(.-)) generation (IC50 4.9+/-0.7 mu M) and O-2 consumption (IC50 4.9+/-1.5 mu M). Concanavalin A (Con A)- and NaF-induced O-2(.-) generation were also suppressed by HAJ11. However, HAJ11 was a weak inhibitor of the phorbol 12-myristate 13-acetate (PMA)-induced responses. 3 HAJ11 did not scavenge the O-2(.-) generation in the xanthine-xanthine oxidase system and dihydroxyfumaric acid (DHF) autoxidation. 4 HAJ11 showed no activity on fMLP-induced inositol phosphates formation and [Ca2+](i) elevation in intact neutrophils. In addition, HAJ11 had no effect on neutrophil cytosolic phospholipase C (PLC) activity. 5 HAJ11 reduced fMLP-induced phosphatidic acid (PA) (IC50 29.1+/-6.5 mu M) and phosphatidylethanol (PEt) (IC50 22.6+/-1.9 mu M) formation in a concentration-dependent manner. HAJ11 also reduced protein tyrosine phosphorylation in neutrophils stimulated by fMLP. 6 HAJ11 was a weak inhibitor of neutrophil cytosolic protein kinase C (PKC) activity, and had a negligible effect on brain PKC. Cellular cyclic nucleotides levels were not altered by HAJ11. In addition, HAJ11 did not affect protein kinase A (PKA) activity. 7 HAJ11 had no effect on the O-2(.-) generation of PMA-activated and arachidonic acid (AA)-activated NADPH oxidase preparations. 8 Taken together these results indicate that the inhibition of respiratory burst by HAJ11 probably mainly occurs through inhibition of protein tyrosine phosphorylation and phospholipase D (PLD) activity.