1 The possible mechanisms of action of the inhibitory effect of gomisin C on the respiratory burst of rat neutrophils in vitro was investigated. 2 The peptide formyl-Met-Leu-Phe (FMLP) induced superoxide anion (02 -) formation and 02 consumption, which was inhibited by gomisin C in a concentration-dependent manner (IC50 21.5 ± 4.2 fg ml-' for 02-formation). Gomisin C also suppressed 02-formation and 02 consumption at low concentrations of phorbol myristate acetate (PMA) with an IC50 value of 26.9 ± 2.1 Pg ml-' for°2-formation. However, gomisin C did not affect the responses induced by a high concentration of PMA. 3 Gomisin C had no effect on°2-generation and uric acid formation in the xanthine-xanthine oxidase system, and failed to alter 02-generation during dihydroxyfumaric acid (DHF) autoxidation, indicating that it does not scavenge superoxide. 4 Like trifluoperazine (TFP), gomisin C attenuated the activity of PMA-activated neutrophil particulate NADPH oxidase in a concentration-dependent manner.5 Gomisin C reduced the elevations of cytosolic free Ca2+ in neutrophils stimulated by FMLP in the presence or absence of EDTA. Cyclopiazonic acid (CPA) induced the release of Ca2+ from intracellular stores and this was also reduced by gomisin C. However, the Ca2+ influx pathway activated by CPA was not affected by gomisin C. 6 The cellular cyclic AMP level was markedly increased by forskolin, but not by gomisin C. Moreover, the inositol phosphate levels in FMLP-activated neutrophils were not affected by gomisin C. 7 These results show that the inhibitory action of gomisin C on the respiratory burst is not mediated by changes in cellular cyclic AMP or in inositol phosphates, or by scavenging°2 released from neutrophils, but may be mediated partly by the suppression of NADPH oxidase and partly by the decrease of cytosolic Ca2+ released from an agonist-sensitive intracellular store.

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.

Cycloheterophyllin, a prenylflavone, inhibited the superoxide anion (O2-) generation from formylmethionyl-leucyl-phenylalanine (fMLP)- and phorbol 12-myristate 13-acetate (PMA)-stimulated rat neutrophils in a concentration-dependent manner with IC50 values of 47.0 +/- 5.0 and 1.7 +/- 0.4 microM, respectively. Cycloheterophyllin had no effect on O2- generation in xanthine-xanthine oxidase system and during dihydroxyfumaric acid (DHF) autoxidation. Cycloheterophyllin exerted a concentration-dependent inhibition of neutrophil cytosolic protein kinase C (PKC) and rat brain PKC, but had no effect on porcine heart protein kinase A (PKA). Unlike staurosporine, cycloheterophyllin did not affect the trypsin-treated rat brain PKC. [3H]Phorbol 12,13-dibutyrate ([3H]PDB) binding to neutrophil cytosolic PKC was significantly suppressed by cycloheterophyllin. However, cycloheterophyllin had negligible effect on the PMA-induced membrane translocation of PKC-beta and PKC-delta in neutrophils. Moreover, the fMLP-induced [Ca2+]i elevation and inositol trisphosphate (IP3) formation of neutrophils were not affected by cycloheterophyllin at concentrations which significantly suppressed the O2- generation. In cell-free system, addition of arachidonate (AA) into the mixture of cytosol and membrane fractions of the resting neutrophils to make NADPH oxidase assembly and activation. Cycloheterophyllin had no effect on O2- generation in AA-activated cell-free system. These results suggest that the suppression of PKC activity through the interaction with the regulatory region of PKC is involved in the inhibition by cycloheterophyllin of the O2- generation in rat neutrophils.

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