The influence of the plant product magnolol on neutrophil superoxide anion (O2-*) generation has been investigated in the rat. Intraperitoneal injection of magnolol (30mg kg(-1)) significantly inhibited the formylmethionyl-leucyl-phenylalanine (fMLP)-induced respiratory burst in rat whole blood ex-vivo. Magnolol also inhibited the 02-* generation with an IC50 (concentration resulting in 50% inhibition) of 15.4+/-1.6 microM and O2 consumption in rat neutrophils in-vitro. Magnolol weakly inhibited the O2-* generation in the xanthine-xanthine oxidase system, decreased cellular cyclic AMP level and had no effect on cyclic GMP levels. It weakly inhibited neutrophil cytosolic protein kinase C activity but did not alter porcine heart protein kinase A activity. Magnolol attenuated fMLP-induced protein tyrosine phosphorylation with an IC50 of 24.0+/-1.9 microM and the phosphorylation of mitogen-activated protein kinase p42/44 with an IC50 of 28.5+/-4.5 microM. However, magnolol alone activated neutrophil phospholipase D activity as determined by the formation of phosphatidic acid and phosphatidyl-ethanol in the presence of ethanol. In the presence of NADPH, the arachidonate-activated NADPH oxidase activity in a cell-free system was weakly suppressed by magnolol. These results suggest that the inhibition of respiratory burst in fMLP-activated neutrophils by magnolol is probably attributable mainly to the attenuation of protein tyrosine phosphorylation and p42/44 mitogen-activated protein kinase activation, and partly to the suppression of protein kinase C and NADPH oxidase activities.

1 The possible mechanisms of the inhibitory e ect 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 ⋅7 ) generation (IC 50 4.9+0.7 mM) and O 2 consumption (IC 50 4.9+1.5 mM). Concanavalin A (Con A)-and NaF-induced O 2 ⋅7 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 ⋅7 generation in the xanthine-xanthine oxidase system and dihydroxyfumaric acid (DHF) autoxidation. 4 HAJ11 showed no activity on fMLP-induced inositol phosphates formation and [Ca 2+ ] i elevation in intact neutrophils. In addition, HAJ11 had no e ect on neutrophil cytosolic phospholipase C (PLC) activity. 5 HAJ11 reduced fMLP-induced phosphatidic acid (PA) (IC 50 29.1+6.5 mM) and phosphatidylethanol (PEt) (IC 50 22.6+1.9 mM) 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 e ect on brain PKC. Cellular cyclic nucleotides levels were not altered by HAJ11. In addition, HAJ11 did not a ect protein kinase A (PKA) activity. 7 HAJ11 had no e ect on the O 2 ⋅7 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.

1 The ability of acetylshikonin to inhibit the respiratory burst in rat neutrophils was characterized and the underlying mechanism of action was also assessed in the present study. 2 Acetylshikonin caused an irreversible and a concentration-dependent inhibition of formylmethionylleucyl-phenylalanine (fMLP) plus dihydrocytochalasin B (CB)-and phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O 2 .7) generation with IC 50 values of 0.48+0.03 and 0.39+0.03 mM, respectively. Acetylshikonin also inhibited the O 2 consumption in neutrophils in response to fMLP/CB as well as to PMA. 3 Acetylshikonin did not scavenge the generated O 2.7 in the xanthine-xanthine oxidase system or during dihydroxyfumaric acid (DHF) autoxidation but, on the contrary, acetylshikonin enhanced the O 2 .7 generation in these cell-free oxygen radical generating systems. 4 Acetylshikonin inhibited the formation of inositol trisphosphate (IP 3 ) (39.0+7.8% inhibition at 10 mM, P50.05) in neutrophils in response to fMLP. 5 Both the neutrophil cytosolic protein kinase C (PKC) activity and the PMA-induced PKC associated with the membrane were una ected by acetylshikonin. 6 Acetylshikonin did not a ect the porcine heart protein kinase A (PKA) activity. Upon exposure to acetylshikonin, the cellular cyclic AMP level was decreased in neutrophils in response to fMLP. 7 The cellular formation of phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt) induced by fMLP/CB were inhibited by acetylshikonin (60.1+7.3 and 63.2+10.5% inhibition, respectively, at 10 mM, both P50.05). Moreover, acetylshikonin attenuated the fMLP/CB-induced protein tyrosine phosphorylation (about 90% inhibition at 1 mM). 8 In PMA-activated neutrophil particulate NADPH oxidase preparations, acetylshikonin did not inhibit, but enhanced, the O 2 .7 generation in the presence of NADPH. However, acetylshikonin decreased the membrane associated p47 phox in PMA-activated neutrophils (about 60% inhibition at 1 mM). 9 Collectively, these results suggest that the attenuation of protein tyrosine phosphorylation and a failure in the assembly of a functional NADPH oxidase complex probably contribute predominantly to the inhibition of respiratory burst in neutrophils by acetylshikonin. In contrast, the blockade of phospholipase C (PLC) and phospholipase D (PLD) pathways play only a minor role in this respect.

1 In this study, the underlying mechanism of stimulation of respiratory burst by kazinol B, a natural isoprenylated¯avan, in rat neutrophils in vitro was investigated. 2 Kazinol B concentration-dependently stimulated the superoxide anion (O 2 ⋅7 ) generation, with a lag but transient activation pro®le, in neutrophils but not in a cell-free system. The maximum response (13.2+1.4 nmol O 2 ⋅7 10 min 71 per 10 6 cells) was observed at 10 mM kazinol B.3 Pretreatment of neutrophils with phorbol 12-myristate 13-acetate (PMA) or formylmethionyl-leucylphenylalanine (fMLP) signi®cantly enhanced the O 2 ⋅7 generation following the subsequent stimulation of cells with kazinol B. 4 Cells pretreated with EGTA or a protein kinase inhibitor staurosporine e ectively attenuated the kazinol B-induced O 2 ⋅7 generation. However, a p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and a phosphoinositide 3-kinase (PI3K) inhibitor wortmannin had no e ect on the kazinol Binduced response. 5 Kazinol B signi®cantly stimulated [Ca 2+ ] i elevation in neutrophils, with a lag and slow rate of rise activation pro®le, and this response was attenuated by a phospholipase C (PLC) inhibitor U73122. Kazinol B also stimulated the inositol bis-and trisphosphate (IP 2 and IP 3 ) formation with a 1 min lag time. 6 The membrane-associated PKC-a and PKC-y but not PKC-i were increased following the stimulation of neutrophils with kazinol B. It was more rapid and sensitive in the activation of PKC-y than PKC-a by kazinol B. Kazinol B partially inhibited the [ 3 H]phorbol 12,13-dibutyrate ([ 3 H]PDB) binding to the neutrophil cytosolic PKC. 7 Neither the cellular mass of phosphatidic acid (PA) and phosphatidylethanol (PEt), in the presence of ethanol, nor the protein tyrosine phosphorylation were stimulated by kazinol B. In addition, the kazinol B-induced O 2 ⋅7 generation remained relatively unchanged in cells pretreated with ethanol or a tyrosine kinase inhibitor genistein. 8 Collectively, these results indicate that the stimulation of the respiratory burst by kazinol B is probably mediated by the synergism of PKC activation and [Ca 2+ ] i elevation in rat neutrophils.

1 The possible mechanisms of action of the inhibitory eect of abruquinone A on the respiratory burst in rat neutrophils in vitro was investigated. 2 Abruquinone A caused an irreversible and a concentration-dependent inhibition of formylmethionylleucyl-phenylalanine (fMLP) plus dihydrocytochalasin B (CB)-and phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O 2 . 5 Abruquinone A did not aect the enzyme activities of neutrophil cytosolic protein kinase C (PKC) and porcine heart protein kinase A (PKA). 6 Abruquinone A had no eect on intracellular guanosine 3' : 5'-cyclic monophosphate (cyclic GMP) levels but decreased the adenosine 3' : 5'-cyclic monophosphate (cyclic AMP) levels. 7 The cellular formation of phosphatidic acid (PA) and phosphatidylethanol (PEt) induced by fMLP/ CB was inhibited by abruquinone A with IC 50 values of 2.2+0.6 mg ml 71 and 2.5+0.3 mg ml 71 , respectively. Abruquinone A did not inhibit the fMLP/CB-induced protein tyrosine phosphorylation but induced additional phosphotyrosine accumulation on proteins of 73 ± 78 kDa in activated neutrophils. 8 Abruquinone A inhibited both the O 2. 7 generation in PMA-activated neutrophil particulate NADPH oxidase (IC 50 0.6+0.1 mg ml 71) and the iodonitrotetrazolium violet (INT) reduction in arachidonic acid (AA)-activated cell-free system (IC 50 1.5+0.2 mg ml 71 ). 9 Collectively, these results indicate that the inhibition of respiratory burst in rat neutrophils by abruquinone A is mediated partly by the blockade of phospholipase C (PLC) and phospholipase D (PLD) pathways, and by suppressing the function of NADPH oxidase through the interruption of electron transport.