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

Abstract

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 (O2.-) generation (IC50 4.9 ± 0.7 μM) and O2 consumption (IC50 4.9 ± 1.5 μM). Concanavalin A (Con A)- and NaF-induced O2.- 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 O2(.-) 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 μM) and phosphatidylethanol (PEt) (IC50 22.6 ± 1.9 μM) 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 O2.- 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.