Burst In Rat Neutrophils Research Articles

Inhibitory effects of phenolic compounds from Artocarpus styracifolius on respiratory burst of rat neutrophils

Context: Searching for polymorphonuclear neutrophils (PMNs) respiratory burst inhibitors is an important topic in the treatment of human diseases associated with inflammation.Objective: To investigate the inhibitory effects of phenolics isolated from Artocarpus styracifolius Pierre (Moraceae) on respiratory burst induced by phorbol myristate acetate (PMA).Materials and methods: The anti-respiratory burst activities of eight phenolics (20 µM) were assessed by determining luminol-dependent chemiluminiscence in rat PMNs. Cytotoxicity of active compounds (1–1000 µM) was assayed by Trypan blue dye exclusion method. Cell-free models were employed to evaluate scavenging capacity of active compounds (20 µM) against reactive oxygen species.Results: The PMA-induced respiratory burst was significantly inhibited (p < 0.05) by six isoprenylated phenolics (AS1–6) at the concentration of 20 µM (below the toxic concentration) with the inhibition rate ranging from 25.0 to 99.6%. The inhibitory potency estimated by IC50 was in the order of AS1 (3.1 µM) >AS6 (5.9 µM) >AS2 (9.1 µM) >AS3 (10.0 µM) >AS5 (29.7 µM) >AS4 (57.7 µM). AS1–4, four isoprenylated flavones, potently quenched superoxide anion, hydroxyl radical, and hydrogen peroxide at the concentration of 20 µM with their scavenging rates in the range of 30.1–78.1%, 35.4–69.7%, and 65.5–86.3%, respectively. In contrast, AS5–6, two isoprenylated 2-arylbenzofurans, showed less effect than that exhibited by AS1–4.Conclusion and discussion: The isoprenylated phenolics from A. styracifolius can potently inhibit PMA-induced respiratory burst in rat neutrophils without showing cytotoxicity. The inhibitory effects of these isoprenylated phenolics on the respiratory burst might depend on their different types of structure.

Inhibition by magnolol of formylmethionyl-leucyl-phenyl alanine-induced respiratory burst in rat neutrophils.

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.

Activation of phosphoinositide 3-kinase and Src family kinase is required for respiratory burst in rat neutrophils stimulated with artocarpol A

Artocarpol A (ART), a natural product isolated from Artocarpus rigida, stimulated superoxide anion (O 2 −) generation, which was inhibited by 2-(4-morpholinyl)-8-phenyl-4 H-1-benzopyran-4-one (LY 294002), a phosphoinositide 3-kinase (PI3K) inhibitor, in rat neutrophils. ART stimulated phosphorylation of protein kinase B (PKB/Akt) on both T308 and S473 residues, and LY 294002 inhibited these effects. Rat neutrophils expressed both class IA PI3K subunits (p85, p110α, p110β, and p110δ) and a class IB PI3K subunit (p110γ) as assessed by a combination of Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) approaches. Stimulation of neutrophils with ART evoked phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5) P 3) formation, which reached a maximal level at 2 min and was attenuated by LY 294002, as evidenced by immunofluorescence microscopy and by flow cytometry. Detectable membrane-association of class IA PI3Ks, class IB PI3K and Ras was seen as early as 1.5, 0.5 and 1.5 min, respectively, after stimulation with ART. The kinetics of ART-induced Ras activation paralleled the kinetics of class IA PI3Ks recruitment to membrane caused by ART, and the p85 and p110γ immunoprecipitates contain Ras. ART stimulated Src family kinase activation, which was detectable within 1.5 min of incubation with ART. Both Src kinase activity and PtdIns(3,4,5) P 3 formation in ART-stimulated neutrophils were inhibited by 4-amino-1- tert-butyl-3-(1′-naphthyl)pyrazolo[3,4-d]pyrimidine (PP1 analog). PP1 analog also attenuated the ART-stimulated O 2 − generation in rat neutrophils. These results indicate that the stimulation of respiratory burst by ART in neutrophils implicates PI3K signaling.

Evaluation of Antioxidant, Anti-inflammatory, and Antinociceptive Properties of Aerial Parts of Vicia sativa and its Flavonoids

Objective: In this study, we aimed to identify the minor flavonoids of the areal parts of Vicia sativa , a biologically active plant growing in El-Hassana, North Sinai, Egypt, and to monitor the antioxidant, anti-inflammatory, and antinociceptive activities of the plant extract and its purified flavonoids. Methods: antioxiant activity of V. sativa was assessed by four different assays including DPPH assay, quenching of ROO radicals, respiratory burst in rat neutrophils, and lipid peroxidation. On the other hand, antiinflammatory and antinociceptive effects of V. sativa and its flavonoids were assessed by carrageenaninduced inflammation in mice, acetic acid-induced writhing and formalin-licking test. Results: Nine flavonoids were isolated from the ethanol extract of the aerial parts of V. sativa and identified as active components: apigenin 4'-O-β-D-glucopyranoside [1], apigenin 6,8-di-C-β-D-glucopyranoside [2], apigenin 6-C-α-L-arabinopyranoside-8-C-β-D-glucopyranoside [3], kaempferol-3-O-β-Ldirhamnopyranosyl (1→2, 1→6)- β-D-glucopyranoside [4], kaempferol 3-O-(4-β-D-xylopyranosyl)-α-L-rhamnopyranoside-7-O-L-rhamnopyranoside [5], kaempferol-3-O- α-L-rhamnopyranosyl (1→6)- β-D-glucopyranoside [6], Luteolin-7-O- β-D-glucopyranoside [7] quercetin 3-O-β-glucopyranoside [8], and quercetin 3,7 di-O- glucopyranoside [9]. Our findings indicated a potent antioxidant activity of V. sativa extract against different radicals (DPPH, superoxide anion, and peroxyl) and an inhibitory activity against carbon tetrachloride-induced lipid peroxidation. compound 6 showed effective inhibitory activity of both peroxyl radicals; compounds 1, 2, 3, 6, 8 and 9 decreased both superoxide anion radical production in PMA-stimulated neutrophil, and compounds 2, 3, 5, and 7 were dramatically effective in inhibition of CCl 4 -induced lipid peroxidation. Additionally, our results revealed that V. sativa extract exhibited inhibitory activity against different inflammatory and nociceptive chemical agents. The purified flavonoids generally showed low anti-inflammatory and antinociceptive activities in a range of 0.9-18.3 % of inhibition, whereas compounds 1, 3, 4, and 8 were the most effective flavonoids. Structure-activity correlation was also discussed. Conclusion: V. sativa extract possessed antioxidant, anti-inflammatory, and antinociceptive activities to variable extents, which may be attributed to the radical scavenging and anti-inflammatory activity of some of its flavonoid constituents. These activities may help in prevention and/or treatment of the pathological disorders in which free radical oxidation, inflammation, and cellular activation are suggested to play a fundamental pathogenic role.

Inhibition of formyl-methionyl-leucyl-phenylalanine-stimulated respiratory burst by cirsimaritin involves inhibition of phospholipase D signaling in rat neutrophils.

In this study, the cellular localization of the inhibitory effect of a natural flavonoid cirsimaritin against formyl-methionyl-leucyl-phenylalanine (fMLP)-induced respiratory burst in rat neutrophils was investigated. Cirsimaritin concentration-dependently inhibited the superoxide anion (O(*-)(2))generation and O(2) consumption (IC(50) 11.5+/-2.2 micro M and 17.0+/-3.9 micro M, respectively) of neutrophils. Cirsimaritin did not reduce, but slightly enhanced the O(*-)(2) generation in phorbol 12-myristate 13-acetate (PMA)-activated or arachidonic acid-stimulated NADPH oxidase preparation as well as during the autoxidation of dihydroxyfumaric acid. Cirsimaritin did not elevate cellular cAMP levels, and only partially inhibited the fMLP-induced [Ca(2+)](i) changes in the presence or absence of extracellular Ca(2+). The phosphorylation of protein tyrosine, extracellular signal-regulated protein kinase and Akt caused by fMLP were attenuated by cirsimaritin in a concentration-dependent manner. In contrast, cirsimaritin had no effect on the phosphorylation of p38 mitogen-activated protein kinase. Cirsimaritin produced a concentration-dependent reduction in the formation of phosphatidic acid and phosphatidylethanol, in the presence of ethanol, from fMLP-stimulated neutrophils (IC(50) 15.1+/-6.5 micro M and 15.6+/-3.4 micro M, respectively), but did not affect the phosphatidylethanol formation in response to PMA. Under the similar concentration range, cirsimaritin attenuated the membrane translocation of ARF and Rho A. However, the GTPgammaS-stimulated membrane-associated ARF and Rho in cell lysate were unaffected by cirsimaritin. Collectively, these results indicate that the inhibition of fMLP-induced respiratory burst by cirsimaritin in rat neutrophils is likely mainly through the blockade of phospholipase D signaling pathway.

Effect of isorhapontigenin on respiratory burst of rat neutrophils

The effect of Isorhapontigenin (Iso) isolated from Belamcanda chinensis on respiratory burst of rat neutrophils was investigated. Iso (1, 10, 100 mmol/l) showed an inhibitory effect on superoxide anion and hydrogen peroxide production in phorbol myristate acetate (PMA) activated rat neutrophils in a concentration-dependent manner. Scanning electron microscopy detected that Iso (100 mmol/l) protected against surface changes in rat neutrophils stimulated with PMA. Also, 100 mmol/l Iso inhibited the release of beta-glucuronidase from the activated neutrophils. Electron-spin resonance (ESR) detected that Iso scavenged oxygen free radicals generated in the PMA activated Neutrophils. These results suggest that Iso inhibits respiratory burst of PMA-activated rat neutrophils by scavenging oxygen free radicals.

Cellular mechanisms of inhibition of superoxide anion generation in rat neutrophils by the synthetic isoquinoline DMDI

This study was undertaken to assess the cellular localization of the inhibitory effect of a chemically synthetic isoquinoline compound 1-(3′,4′-dimethoxybenzyl)-6,7-dichloroisoquinoline (DMDI) on the formyl-methionyl-leucyl-phenylalanine (fMLP)-induced respiratory burst in rat neutrophils. The DMDI concentration dependently inhibited the superoxide anion (O 2 ⋅−) generation and O 2 consumption (IC 50 12.2±4.9 and 15.2±8.4 μM, respectively) of neutrophils. DMDI did not scavenge the O 2 ⋅− generated during the autoxidation of dihydroxyfumaric acid in a cell-free system. DMDI did not elevate cellular cyclic AMP levels. Inhibition of O 2 ⋅− generation by DMDI in neutrophils was not reversed by a cyclic AMP-dependent protein kinase inhibitor, (8 R,9 S,11 S)-(−)-9-hydroxy-9-hexoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1 H,8 H,11 H-2,7b,11a-triazadibenzo[ a,g]cycloocta[ cde]trinden-1-one (KT5720). The DMDI concentration dependently inhibited the late plateau phase but not the initial spike of fMLP-induced [Ca 2+] i changes in the presence of extracellular Ca 2+. However, DMDI had no effect on the fMLP-induced [Ca 2+] i changes in the absence of extracellular Ca 2+. In addition, DMDI did not affect the fMLP-stimulated phosphatidylinositol 3-kinase (PI3-kinase) activation. DMDI produced a concentration-dependent reduction in the formation of phosphatidic acid and phosphatidylethanol in the presence of ethanol from fMLP-stimulated neutrophils (IC 50 13.3±4.0 and 9.4±4.3 μM, respectively). On the basis of the immunoblot analysis of the phosphorylation of the mitogen-activated protein (MAP) kinase, DMDI attenuated the fMLP-stimulated MAP kinase phosphorylation in a similar concentration range. Collectively, these results indicate that the inhibition of the respiratory burst by DMDI in rat neutrophils is mediated through the blockade of phospholipase D and MAP kinase signaling pathways.

Effect of low-dose prednisone in vivo on the ability of complement receptor to mediate an oxidative burst in rat neutrophils

Glucocorticoids have been used in the treatment of a variety of inflammatory processes including autoimmune diseases. However, the influence of low-dose glucocorticoids on the respiratory burst activity of neutrophils has not been studied. The aim of this work was to study the effect of treatment with low-dose prednisone on the oxidative burst of rat peripheral blood neutrophils. Wistar male rats were treated with prednisone by gavage (28, 87 or 257 μg/animal/day) for 7 or 15 days. These doses are equivalent to 10, 30 or 90 mg/adult human (∼70 kg)/day, respectively. Sera from normal rats were used to opsonize zymosan (opZy). Neutrophils (1×105) were stimulated by opZy and the oxidative burst of control or treated rat cells was measured by luminol-dependent chemiluminescence (CL). Prednisone did not affect the CL of rat neutrophils for either period of treatment, or any studied doses, when compared with controls. These results suggest that the low-dose prednisone has no effect on the oxidative burst mediated by complement receptors during the rat neutrophil phagocytosis of complement-opZy.

Effects of Schisanhenol on respiratory burst in rat neutrophils

Objective: To explore the effects of Schisanhenol (Sal) on generation of reactive oxygen species (ROS) including superoxide anion, hydrogen peroxide and hydroxyl radical as well as on the activity of superoxide dismutase (SOD) and catalase in respiratory burst of rat neutrophils.Methods: Rat neutrophils were prepared from male Wistar rats. Superoxide anion, hydrogen peroxide, hydroxyl radical, SOD and catalase activity were measured.Results: Sal (1, 10, 100 μmol/L) inhibited generation of ROS of rat neutrophils during respiratory burst induced by formyl-methionyleucylphenylalanine (FMLP) and phorbol myristate acetate (PMA) dosedependently, and it enhanced SOD and catalase activities.Conclusion: Sal may inhibit generation of ROS and enhance activity of anti-oxidant enzymes of neutrophils to reduce oxidative injury to tissues.

Stimulation of respiratory burst by cyclocommunin in rat neutrophils is associated with the increase in cellular Ca 2+ and protein kinase C activity

In this study, the underlying mechanisms of stimulation by cyclocommunin, a natural pyranoflavonoid, of respiratory burst in rat neutrophils was investigated. Cyclocommunin evoked a concentration-dependent stimulation of superoxide anion (O 2 − ) generation with a slow onset and long lasting profile. The maximum response (16.4 ± 2.3 nmol O 2 − /10 min per 10 6 cells) was observed at 3–10 μM cyclocommunin. Cyclocommunin did not activate NADPH oxidase in a cell-free system. Cells pretreated with pertussis toxin or n-butanol did not affect the cyclocommunin-induced O 2 − generation. However, a protein kinase inhibitor staurosporine and EGTA greatly reduced the O 2 − generation caused by cyclocommunin. Treatment of neutrophils with phorbol 12-myristate 13-acetate (PMA), but not with formylmethionyl-leucyl-phenylalanine (fMLP), for 20 min significantly reduced the O 2 − generation following the subsequent stimulation of cells with cyclocommunin. Cyclocommunin did not affect the cellular mass of phosphatidic acid (PA). Neither the tyrosine kinase inhibitor, genistein, nor the p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, affected cyclocommunin-induced O 2 − generation. The enzyme activities of neutrophil cytosolic and membrane-associated protein kinase C (PKC) were both increased significantly with 100 μM cyclocommunin. The membrane-associated PKC-θ and PKC-β were increased following the stimulation of neutrophils with 30 and 100 μM cyclocommunin, respectively. Cyclocommunin reduced the [ 3H]phorbol 12,13-dibutyrate ([ 3H]PDB) binding to cytosolic PKC in a concentration-dependent manner. Cyclocommunin (≥3 μM) significantly evoked a slow and long lasting [Ca 2+] i elevation in neutrophils, and a phospholipase C (PLC) inhibitor U73122 greatly inhibited these Ca 2+ responses. Moreover, the increase in cellular inositol bis- and trisphosphate (IP 2 and IP 3) levels were observed in neutrophils stimulated with 30 μM cyclocommunin for 3 min. Collectively, these results indicate that the stimulation of respiratory burst by cyclocommunin is probably mediated by the synergism of PKC activation and [Ca 2+] i elevation in rat neutrophils.

Involvement of cyclic AMP generation in the inhibition of respiratory burst by 2-phenyl-4-quinolone (YT-1) in rat neutrophils

The inhibitory effect of 2-phenyl-4-quinolone (YT-1) on respiratory burst in rat neutrophils was investigated, and the underlying mechanism of action was assessed. YT-1 caused a concentration-dependent inhibition of the rate of O 2 ·− release from rat neutrophils in response to formylmethionyl-leucyl-phenylalanine (fMLP), but not to phorbol 12-myristate 13-acetate (PMA), with an ic 50 value of 60.7 ± 8.2 μM. A comparable effect was also demonstrated in the inhibition of O 2 consumption. Unlike superoxide dismutase, YT-1 had no effect on O 2 ·− generation in the xanthine-xanthine oxidase system and during dihydroxyfumaric acid autoxidation. The fMLP-induced inositol trisphosphate (IP 3) formation was unaffected by YT-1. In addition, YT-1 did not affect the initial spike of [Ca 2+] i, but it accelerated the rate of [Ca 2+] i decline in cells in response to fMLP. YT-1 was found to have little effect on the activity of neutrophil cytosolic protein kinase C (PKC). YT-1 increased the cellular cyclic AMP level, while having no effect on the cyclic GMP level. In addition, YT-1 increased neutrophil cytosolic protein kinase A (PKA) activity, but had no direct effect on the enzyme activity of pure porcine heart PKA. When neutrophils were treated with (8 R,9 S,11 S)-(−)-9-hydroxy-9-hexoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1 H,8 H,11 H-2,7b,11a-triazadibenzo[ a, g]cycloocta[ cde]trinden-1-one, (KT 5720), a PKA inhibitor, the inhibition of O 2 ·− generation by YT-1, as well as by prostaglandin E 1 (PGE 1) and dibutyryl cyclic AMP, was attenuated effectively. YT-1 did not activate the adenylate cyclase associated with neutrophil particulate fraction but inhibited the cytosolic phosphodiesterase (PDE) activity in a concentration-dependent manner. Neutrophils treated with YT-1 had a more pronounced increase in cellular cyclic AMP level by PGE 1. Moreover, the ability of PGE 1 to inhibit the respiratory burst in neutrophils was greatly enhanced by YT-1. These results suggest that the increase in cellular cyclic AMP levels by YT-1 through the inhibition of PDE (probably PDE4 isoenzyme) activity is involved in its inhibition of fMLP-induced respiratory burst in rat neutrophils.

Investigation of the inhibition by acetylshikonin of the respiratory burst in rat neutrophils

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 (O2.-) generation with IC50 values of 0.48 +/- 0.03 and 0.39 +/- 0.03 microM, respectively. Acetylshikonin also inhibited the O2 consumption in neutrophils in response to fMLP/CB as well as to PMA. 3. Acetylshikonin did not scavenge the generated O2.- in the xanthine-xanthine oxidase system or during dihydroxyfumaric acid (DHF) autoxidation but, on the contrary, acetylshikonin enhanced the O2.- generation in these cell-free oxygen radical generating systems. 4. Acetylshikonin inhibited the formation of inositol trisphosphate (IP3) (39.0 +/- 7.8% inhibition at 10 microM, P < 0.05) 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 unaffected by acetylshikonin. 6. Acetylshikonin did not affect 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 microM, both P < 0.05). Moreover, acetylshikonin attenuated the fMLP/CB-induced protein tyrosine phosphorylation (about 90% inhibition at 1 microM). 8. In PMA-activated neutrophil particulate NADPH oxidase preparations, acetylshikonin did not inhibit, but enhanced, the O2.- generation in the presence of NADPH. However, acetylshikonin decreased the membrane associated p47phox in PMA-activated neutrophils (about 60% inhibition at 1 microM). 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.

Cellular localization of the inhibitory action of abruquinone A against respiratory burst in rat neutrophils.

1. The possible mechanisms of action of the inhibitory effect 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 (O2.-) generation with IC50 values of 0.33 +/- 0.05 microgram ml-1 and 0.49 +/- 0.04 microgram ml-1, respectively. 3. Abruquinone A also inhibited O2 consumption in neutrophils in response to fMLP/CB and PMA. However, abruquinone A did not scavenge the generated O2.- in xanthine-xanthine oxidase system and during dihydroxyfumaric acid (DHF) autoxidation. 4. Abruquinone A inhibited both the transient elevation of [Ca2+]i in the absence of [Ca2+]o (IC50 7.8 +/- 0.2 micrograms ml-1) and the generation of inositol trisphosphate (IP3) (IC50 10.6 +/- 2.0 micrograms ml-1) in response to fMLP. 5. Abruquinone A did not affect the enzyme activaties of neutrophil cytosolic protein kinase C (PKC) and porcine heart protein kinase A (PKA). 6. Abruquinone A had no effect 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 IC50 values of 2.2 +/- 0.6 micrograms ml-1 and 2.5 +/- 0.3 micrograms ml-1, 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 O2.- generation in PMA-activated neutrophil particulate NADPH oxidase (IC50 0.6 +/- 0.1 microgram ml-1) and the iodonitrotetrazolium violet (INT) reduction in arachidonic acid (AA)-activated cell-free system (IC50 1.5 +/- 0.2 micrograms ml-1) 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.

Examination of the Inhibitory Effect of Norathyriol in Formylmethionyl-Leucyl-Phenylalanine-Induced Respiratory Burst in Rat Neutrophils

Norathyriol, aglycone of a xanthone C-glycoside mangiferin isolated from Tripterospermum lanceolatum, concentration dependently inhibited the formylmethionyl-leucyl-phenylalanine (fMLP)-induced superoxide anion (O 2 ̇−) generation and O 2 consumption in rat neutrophils. In cell-free oxygen radical generating system, norathyriol inhibited the O 2 ̇− generation during dihydroxyfumaric acid (DHF) autoxidation and in hypoxanthine-xanthine oxidase system. fMLP-induced transient elevation of [Ca 2+] i and the formation of inositol trisphosphate (IP 3) were significantly inhibited by norathyriol (30 μM) (about 30 and 46% inhibition, respectively). Norathyriol concentration dependently suppressed the neutrophil cytosolic phospholipase C (PLC). In contrast with the marked attenuation of fMLP-induced protein tyrosine phosphorylation (about 70% inhibition at 10 μM norathyriol), norathyriol only slightly modulated the phospholipase D (PLD) activity as determined by the formation of phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt). Norathyriol did not modulate the intracellular cyclic AMP level. In the presence of NADPH, the phorbol 12-myristate 13-acetate (PMA)-activated particulate NADPH oxidase activity was suppressed by norathyriol in a concentration-dependent manner and the inhibition was noncompetitive with respect to NADPH. Norathyriol inhibited the iodonitrotetrazolium violet (INT) reduction in arachidonic acid (AA)-activated cell-free NADPH oxidase system at the same concentration range as those used in the suppression of PMA-activated particulate NADPH oxidase activity. Taken together, these results suggest that the scavenging ability of norathyriol contributes to the reduction of generated O 2 ̇−, however, the inhibition of O 2 ̇− generation from neutrophils by norathyriol is attributed to the blockade of PLC pathway, the attenuation of protein tyrosine phosphorylation, and to the suppression of NADPH oxidase through the interruption of electrons transport.

Daphnoretin-induced respiratory burst in rat neutrophils is, probably, mainly through protein kinase C activation

Daphnoretin, a dicoumarin isolated from Wikstroemia indica C.A. Mey. ( Thymelaceae), induced superoxide anion (O 2 −) formation in rat neutrophils in a concentration-dependent manner. Addition of staurosporine reduced daphnoretin-induced respiratory burst. Removal of extracellular free Ca 2+ by EGTA did not affect the respiratory burst of neutrophils in response to daphnoretin. Prior exposure of neutrophils to phorbol 12-myristate 13-acetate (PMA) or daphnoretin reduced the O 2 − formation caused by a subsequent challenge with PMA and daphnoretin, but potentiated the response caused by a subsequent addition of formyl-Met-Leu-Phe (fMLP). Like PMA, daphnoretin did not increase the [Ca 2+] i during cell activation. In neutrophil suspension, daphnoretin increased the membrane associated protein kinase C activity. In the presence of Ca 2+ and phosphatidylserine, daphnoretin also activated protein kinase C isolated from cytosolic fraction of resting neutrophils. Staurosporine inhibited the direct activation of protein kinase C caused by daphnoretin as well as by PMA. Daphnoretin reduced the [ 3H]Phorbol-12,13-dibutyrate ([ 3H]PDB) binding to the neutrophil cytosolic protein kinase C in a concentration-dependent manner with an IC 50 value of 1.77±0.37 μM. These results indicate that daphnoretin, like PMA, may direct activation of protein kinase C which in turn activated NADPH oxidase and elicited respiratory burst.

Zinc hydroxide induced respiratory burst in rat neutrophils

The effects of zinc hydroxide on the respiratory burst and phagocytosis by rat neutrophils were examined. Zinc hydroxide induced an increase in oxygen consumption and O 2 − production. Electronmicroscopy showed that neutrophils engulfed zinc hydroxide particles by phagocytosis. Pertussis toxin (0.25, 0.5, 1.0 μ/ml) and EGTA (1, 2, 5 mM) inhibited zinc hydroxide-induced O 2 − production in a dose-dependent manner. The inhibitors of protein kinase C, 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide inhibited zinc hydroxide-induced O 2 − production with IC 50 values ranging between 10 μM and 25 μM. The inhibitory study using an inhibitor of myosin light chain kinase, 1-(5-iodonapthalene-1-sulfonyl)-1H- hexahydro-1,4-diazepine, showed IC 50 values ranging from 5 μM to 10 μM. These findings indicate that zinc hydroxide induces respiratory burst and phagocytosis by rat neutrophils.

Enhancement of zymosan-induced respiratory burst of rat neutrophils by lead in vitro.

The effect of Pb2+ on serum-treated zymosan (STZ)-induced O2 consumption of rat peritoneal neutrophils was studied. Pb2+ was found to mimic effectively the enhancing action of Ca2+ on the O2 consumption depending on the concentration up to about 80 microM. However, at concentrations over 80 microM, Pb2+ inhibited the O2 consumption. The enhancing effect of Pb2+ on the O2 consumption was further examined using the intracellularly Ca2(+)-depleted neutrophils. Pb2+ also enhanced the O2 consumption of the Ca2(+)-depleted cells as effectively as Ca2+. The Pb2(+)-enhanced O2 consumption of the Ca2(+)-depleted cells was inhibited by N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) based on its calmodulin antagonistic action. The effect of Pb2+ on the activity of activator-deficient 3',5'-cyclic nucleotide phosphodiesterase (PDE), a calmodulin-dependent enzyme, was examined. Pb2+ activated PDE as effectively as Ca2+ only in the presence of calmodulin. The Pb2(+)-activated PDE activity was also inhibited by W-7 only in the presence of calmodulin. These results indicated that Pb2+ could replace Ca2+ in the activation process(es) of the respiratory burst, suggesting a possible involvement of calmodulin in the enhancing mechanism of the O2 consumption by Pb2+.