Beta-naphthylamine induces anion superoxide production in rat peritoneal macrophages.

Rat peritoneal macrophages were incubated in the presence of beta-naphthylamine (beta-NA), a well known carcinogenic agent, and some parameters of respiratory burst were studied. beta-NA induced a time- and dose-dependent stimulation of superoxide anion (O-2) production, and this enhancement was suppressed by the addition of superoxide dismutase enzyme. Also, no cooperative effect between beta-NA and phorbol 12-myristate 13-acetate was observed. Other observations were as follows: (i) the simultaneous presence of polymyxin B, and staurosporine inhibitors of protein kinase C, inhibited beta-NA-dependent O-2 production; (ii) NADPH-oxidase contained in postnuclear fraction from beta-NA-incubated macrophages showed a greater activity than control fractions; (iii) the stimulation of O-2 production elicited by beta-NA was several-fold enhanced in activated macrophages compared to resident cells. These data suggest that beta-NA produces the activation of NADPH-oxidase through protein kinase C.     

Rhein: an anthraquinone that modulates superoxide anion production from human neutrophils

Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid), the active metabolite of diacetylrhein, which has been reported as an effective antirheumatic drug in man, inhibited superoxide anion production from human neutrophils challenged with N-formylmethionyl-leucyl-phenylalanine (FMLP: IC50, 2 x 10(-5) M) and A23186 (IC50, 10(-5) M), but not with phorbol myristate acetate. In the same concentration range (10(-6)-10(-3) M), the drug did not affect oxy-radical production by a cell-free hypoxanthine-xanthine oxidase system and exerted weak inhibitory effects on FMLP-evoked lysosomal enzyme release. Rhein inhibitory effects on neutrophil functioning may contribute to the overall therapeutic activity of the parent drug, diacetylrhein.     

The signal transduction mechanism involved in kazinol B-stimulated superoxide anion generation in rat neutrophils

1. In this study, the underlying mechanism of stimulation of respiratory burst by kazinol B, a natural isoprenylated flavan, in rat neutrophils in vitro was investigated.
2. Kazinol B concentration-dependently stimulated the superoxide anion (O₂[dot over 2]⁻) generation, with a lag but transient activation profile, in neutrophils but not in a cell-free system. The maximum response (13.2±1.4 nmol O₂[dot over 2]⁻ 10 min⁻¹ per 10⁶ cells) was observed at 10 μM kazinol B.
3. Pretreatment of neutrophils with phorbol 12-myristate 13-acetate (PMA) or formylmethionyl-leucyl-phenylalanine (fMLP) significantly enhanced the O₂[dot over 2]⁻ generation following the subsequent stimulation of cells with kazinol B.
4. Cells pretreated with EGTA or a protein kinase inhibitor staurosporine effectively attenuated the kazinol B-induced O₂[dot over 2]⁻ generation. However, a p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and a phosphoinositide 3-kinase (PI3K) inhibitor wortmannin had no effect on the kazinol B-induced response.
5. Kazinol B significantly stimulated [Ca²⁺]_i elevation in neutrophils, with a lag and slow rate of rise activation profile, and this response was attenuated by a phospholipase C (PLC) inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122. Kazinol B also stimulated the inositol bis- and trisphosphate (IP₂ and IP₃) formation with a 1 min lag time.
6. The membrane-associated PKC-α and PKC-θ but not PKC-ι were increased following the stimulation of neutrophils with kazinol B. It was more rapid and sensitive in the activation of PKC-θ than PKC-α by kazinol B. Kazinol B partially inhibited the [³H]phorbol 12,13-dibutyrate ([³H]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 O₂[dot over 2]⁻ 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 [Ca²⁺]_i elevation in rat neutrophils.

     

Inhibition of superoxide anion and elastase release in human neutrophils by 3'-isopropoxychalcone via a cAMP-dependent pathway

1 Chalcone is abundantly present in the plant kingdom and has various biological activities such as anti-inflammatory and antioxidant. In this study, the semisynthetic chalcone derivative, 3'-isopropoxychalcone (H2O7D), was demonstrated to inhibit the generation of superoxide and the release of elastase, as well as to accelerate resequestration of cytosolic calcium in formyl-L-methionyl-L-leucyl-L-phenylalanine-activated human neutrophils. 2 H2O7D displayed no antioxidant or superoxide-scavenging ability, and it failed to alter the subcellular NADPH oxidase activity. 3 H2O7D induced a substantial increase in cAMP but not cGMP levels. The elevation of cAMP formation by H2O7D was inhibited by adenosine deaminase (ADA). Furthermore, The inhibitory effects of H2O7D were reversed by protein kinase (PK)A inhibitors, as well as ADA and a selective A2a-receptor antagonist. 4 H2O7D inhibited phosphodiesterase (PDE) activities, but it did not alter adenylyl cyclase and soluble guanylyl cyclase activities. These results show that the cAMP-elevating effect of H2O7D results from the inhibition of PDE activity and not from the stimulation of cyclase function. Consistent with this, H2O7D potentiated the PGE(1)-caused inhibitory effects and cAMP formation. 5 In summary, these results indicate that the inhibitory effect of H2O7D is cAMP/PKA dependent, and that it occurs through inhibition of cAMP PDE, which potentiates the autocrine functions of endogenous adenosine. Inhibition of respiratory burst and degranulation in human neutrophils may give this drug the potential to protect against the progression of inflammation.     

Effect of okadaic acid on histamine release from rat peritoneal mast cells activated by anti-IgE

Abstract— The effect of okadaic acid, a potent inhibitor of protein phosphatase 1 and 2A, on histamine release from mast cells has been investigated. Okadaic acid strongly and dose-dependently inhibited histamine release from mast cells induced by anti-IgE. The IC50 value of okadaic acid on histamine release induced by anti-IgE was 3·2 Nm. However, okadaic acid failed to inhibit histamine release induced by A23187 and compound 48/80. Moreover, okadaic acid showed no effect on the initial rise in intracellular Ca²⁺, Ca²⁺-mobilization from intracellular Ca²⁺-stores and the generation of inositol trisphosphate. These results suggest a possible involvement of protein phosphatase 2A in the histamine release from mast cells induced by anti-IgE.     

Inhibition of superoxide anion generation by YC-1 in rat neutrophils through cyclic GMP-dependent and -independent mechanisms

3-(5'-Hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), a soluble guanylyl cyclase (sGC) activator, inhibited formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide anion (O(2)*(-)) generation and O(2) consumption in rat neutrophils (IC(50) values of 12.7+/-3.1 and 17.7+/-6.9 microM, respectively). Inhibition of O(2)*(-) generation by YC-1 was partially reversed by the cyclic GMP-lowering agent 6-anilinoquinoline-5,8-quinone (LY83583) and by the Rp isomer of 8-(4-chlorophenylthio)guanosine-3',5'-monophosphorothioate (Rp-8-pCPT-cGMPS), a cyclic GMP-dependent protein kinase inhibitor. In cell-free systems, YC-1 failed to alter O(2)*(-) generation during dihydroxyfumaric acid autoxidation, phorbol 12-myristate 13-acetate (PMA)-activated neutrophil particulate NADPH oxidase preparation, and arachidonic acid-induced NADPH oxidase activation. YC-1 increased cellular cyclic GMP levels through the activation of sGC and the inhibition of cyclic GMP-hydrolyzing phosphodiesterase activity. The plateau phase, but not the initial spike, of fMLP-induced [Ca(2+)](i) changes was inhibited by YC-1 (IC(50) about 15 microM). fMLP- but not PMA-induced phospholipase D activation was inhibited by YC-1 (IC(50) about 28 microM). Membrane-associated ADP-ribosylation factor and Rho A in cell activation was also reduced by YC-1 at a similar concentration range. Neither cytosolic protein kinase C (PKC) activity nor PKC membrane translocation was altered by YC-1. YC-1 did not affect either fMLP-induced phosphatidylinositol 3-kinase activation or p38 mitogen-activated protein kinase phosphorylation, but slightly attenuated the phosphorylation of extracellular signal-regulated kinase. Collectively, these results indicate that the inhibition of the fMLP-induced respiratory burst by YC-1 is mediated by cyclic GMP-dependent and -independent signaling mechanisms.     

DSM-RX78, a new phosphodiesterase inhibitor, suppresses superoxide anion production in activated human neutrophils and attenuates hemorrhagic shock-induced lung injury in rats

Neutrophils are activated following hemorrhagic shock and the accumulation of neutrophils in the lung is associated with lung injury. This research investigated the effects of a semisynthetic 2-benzoylaminobenzoic acid derivative, methyl 2-(2-fluorobenzamido)benzoate (DSM-RX78), on superoxide anion (O₂⁻) production in formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP)-activated human neutrophils, and on lung injury in Sprague-Dawley rats subjected to trauma-hemorrhage. DSM-RX78 concentration-dependently inhibited O₂⁻ production, but not elastase release, in FMLP-activated human neutrophils. DSM-RX78 displayed no superoxide-scavenging ability, and it failed to alter the subcellular NADPH oxidase activity. Significantly, DSM-RX78 increased cAMP formation and protein kinase (PK)A activity in FMLP-activated neutrophils, which occurred through the selective inhibition of cAMP-specific phosphodiesterase (PDE) activity but not an increase in adenylate cyclase function or cGMP-specific PDE activity. These results show that DSM-RX78 is a new inhibitor of cAMP-specific PDE. Moreover, DSM-RX78 reduced FMLP-induced phosphorylation of protein kinase B (Akt), but not calcium mobilization. The inhibitory effects of DSM-RX78 on O₂⁻ production and Akt phosphorylation were reversed by PKA inhibitors, suggesting that DSM-RX78 regulates O₂⁻ production of human neutrophils by promoting cAMP/PKA-dependent inhibition of Akt activation. On the other hand, administration of DSM-RX78 significantly attenuated the increase in myeloperoxidase activity and edema in the lung, as well as protein concentrations in bronchoalveolar lavage fluid in rats after trauma-hemorrhagic shock. In summary, these results strongly suggest that DSM-RX78 exerts anti-inflammatory effects, which result from the elevation of cAMP levels and PKA activity through its inhibition of cAMP-specific PDE. Also, our findings show that DSM-RX78 attenuates hemorrhagic shock-induced lung injury in rats.     

(-)-Xanthienopyran, a new inhibitor of superoxide anion generation by activated neutrophils, and further constituents of the seeds of Xanthium strumarium

The dried seeds of XANTHIUM STRUMARIUM (Asteraceae) are used after thorough stir-frying as an ingredient in traditional Chinese medicines for relieving allergy. Two new compounds, xanthialdehyde ( 2) and (-)-xanthienopyran ( 7), as well as 26 known compounds were isolated in the present study. The structures of the isolates were elucidated by spectroscopic methods. Among them, compound 7 exhibited significant selective inhibition of superoxide anion generation by human neutrophils induced by formyl- L-methionyl- L-leucyl- L-phenylalanine, with an IC50 value of 1.72 microg/mL.     

Peptides and histamine release from rat peritoneal mast cells

Various vasoactive peptides were compared for their histamine releasing effects on rat mast cells. Neurotensin, substance P (SP), and kallidin were the most active natural peptides, followed by bradykinin; neurokinin A and B, bombesin, angiotensin and tuftsin were practically inactive. Several kinins and tachykinin-related peptides were tested in an attempt to characterize the receptors mediating histamine liberation. The order of potency of the kinins was the following: kallidin greater than [Tyr(Me)8]bradykinin = bradykinin greater than [desArg10]kallidin greater than desArg9-bradykinin, the same as that found in smooth muscle possessing receptors of the B2 type. Tachykinin-related peptides were potent stimulants and followed the order: [D-Tryp7,9,10]SP-(1-11) greater than [D-Pro2,D-Tryp7,9,10]SP-(1-11) greater than SP-(1-11) greater than SP-(1-9) greater than [D-Pro4,D-Tryp7,9,Leu11]SP-(4-11) greater than SP-(1-7) greater than SP-(4-11) greater than neurokinin A = neurokinin B, indicating that: (a) undecapeptide antagonists of SP behave as superagonists; (b) both N- and C-terminal portions of SP-(1-11) are essential for activity; and (c) receptors for the tachykinins mediating histamine release appear to be of the SP-P type.     

Carvedilol, a new beta-adrenoceptor antagonist and vasodilator antihypertensive drug, inhibits superoxide release from human neutrophils.

Carvedilol produced a dose-dependent inhibition of superoxide (O2-) release from human neutrophils (PMNs) (IC50 = 28 microM) and scavenged O2- generated during dihydroxyfumaric acid (DHF) autooxidation (IC50 = 41 microM). Other beta-blockers, such as celiprolol, labetalol and atenolol, or the antioxidant, 'lazaroid', U74500A had no effect on O2- either released from PMNs or generated during DHF autooxidation. Propranolol, at 0.3 mM, inhibited O2- release from PMNs (73%) but failed to scavenge O2- generated from DHF. The novel free radical-scavenging effect of carvedilol may contribute to the cardioprotective activity of the compound.     

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 microM, 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, (8R,9S,11S)-(-)-9-hydroxy-9-hexoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-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 microM, 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.     

Investigation of the cellular mechanism of inhibition of formyl-methionyl-leucyl-phenylalanine-induced superoxide anion generation in rat neutrophils by 2-benzyloxybenzaldehyde

The inhibition of formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide anion (O2(.-)) generation by 2-benzyloxybenzaldehyde (CCY1a) was investigated in rat neutrophils, and the underlying mechanism of this inhibition was assessed. CCY1a concentration-dependently inhibited O2(.-) generation (IC(50)=18.5+/-4.3 microM). In cell-free systems, CCY1a failed to alter O2(.-) generation during dihydroxyfumaric acid autoxidation, in phorbol 12-myristate 13-acetate (PMA)-activated neutrophil particulate NADPH oxidase preparations, or during arachidonic acid-induced NADPH oxidase activation. CCY1a increased cellular cyclic AMP (cAMP) levels in a time- and concentration-dependent manner, and this cAMP-elevating effect was inhibited by the adenylyl cyclase inhibitor 9-(tetrahydro-2'-furyl)adenine (SQ22536), adenosine deaminase (ADA), and the adenosine receptor antagonist 8-(p-sulfophenyl)theophylline. In neutrophils, inhibition of O2(.-) generation by CCY1a was partially reversed by the protein kinase A inhibitor (9R,10S,12S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-l][1,6]benzodiazocine-10-carboxylic acid, hexyl ester (KT5720). CCY1a did not affect fMLP-induced p38 mitogen-activated protein kinase phosphorylation, but concentration-dependently attenuated the phosphorylation of extracellular signal-regulated kinase (ERK) and Akt (IC(50) about 31.3 and 19.4 microM, respectively). The plateau phase, but not the initial spike, of fMLP-induced [Ca2+](i) changes was inhibited by CCY1a in a concentration-dependent manner. CCY1a inhibition of Ca2+ entry, ERK, and Akt phosphorylation was not prevented by SQ22536 or ADA. fMLP-induced phospholipase D (PLD) activation was inhibited by CCY1a (IC(50)=13.9+/-2.0 microM). ADA and KT5720 did not prevent the inhibition of PLD activation by CCY1a. Collectively, these results indicate that the inhibition by CCY1a of fMLP-induced O2(.-) generation in rat neutrophils can probably be attributed to the increase in cAMP levels, and to the blockade of Ca2+ entry, suppression of Akt, and PLD activation via cAMP-independent mechanisms.     

Prostaglandin and histamine release from stimulated rat peritoneal mast cells

The production of five prostanoids (PGD2, PGE2, PGF2 alpha, 6-oxo-PGF1 alpha, and TxB2) was examined after mast cell activation. Prostaglandin D2 (PGD2) was the major prostanoid produced after stimulation of rat peritoneal mast cells with the calcium ionophore A 23187, compound 48/80 or anti-rat IgE. The amount of PGD2 generated was not dependent on the quantity of histamine released. The time course of PGD2 production paralleled the release of histamine following activation with A 23187 or anti-rat IgE but with compound 48/80 release of histamine reached a maximum within 15 sec, whereas production of PGD2 was apparent only after 5 min and was still increasing at 30 min.     

Inhibitory effects of ent-kauranes from the stems of Annona squamosa on superoxide anion generation by human neutrophils

Eleven ent-kauranes, isolated from the fresh stems of Annona squamosa L. (Annonaceae), were subjected to assays on the generation of superoxide anion (O2.--) by human neutrophils. Except for ent-kaur-16-en-19-oic acid, 16beta,17-dihydroxy- ent-kauran-19-al, and 16alpha,17-dihydroxy -ent-kauran-19-al, all ent-kauranes showed significant inhibitory effect on O2.-- generation in response to formyl- L-methionyl- L-leucyl- L-phenylalanine (fMLP/CB). In contrast, phorbol myristate acetate (PMA)-induced O2.-- generation was not suppressed by any ent-kauranes. Especially, ent-kaur-16-en-19-oic acid could significantly increase O2.-- production. The structure-activity relationship of these compounds is also discussed herein. Furthermore, the effect of ent-kauranes on nitric oxide generation by NR8383 macrophages in response to lipopolysaccharide (LPS) was examined. None of the compounds showed an inhibitory effect on nitric oxide generation.     

超氧阴离子对脑片谷氨酸释放的增强及依布硒的抑制

超氧阴离子对脑片谷氨酸释放的增强及依布硒的抑制易永杨祥良徐辉碧赵西龙１张亨山１秦钰慧１（华中理工大学化学系，武汉４３００７４；１中国预防医学科学院环境卫生监测研究所毒理室，北京１０００２１）兴奋性毒性是神经系统疾病中神经元损伤的一种重要病理机理，其特...     

Carbon tetrachloride-induced eicosanoid synthesis and enzyme release from rat peritoneal leucocytes

When rat peritoneal leucocytes were incubated with carbon tetrachloride, a PLA2 was activated, eicosanoids were generated and lysosomal and cytoplasmic enzymes were released. The predominant eicosanoid generated was TXB2 with lesser amounts of PGE2, 6-keto PGF1 alpha and LTB4. Preincubation of the cells with two structurally unrelated thromboxane synthetase inhibitors reduced PLA2 activity and enzyme release and also reduced the total amounts of eicosanoids liberated. An anti-PGI2 antibody partially reversed the effects of thromboxane synthetase inhibitors indicating a role for endogenous PGI2 generation in the cytoprotective effects of these agents in this system. Exogenous PGI2 was also cytoprotective but the timing of its administration was critical. The cytoprotective effect of PGI2 was potentiated by a phosphodiesterase inhibitor, indicating a possible pivotal role of cAMP in cell protection.     

GABA and baclofen potentiate the K+-evoked release of methionine-enkephalin from rat striatal slices

GABA potentiates the potassium-evoked release of methionine-enkephalin (ME) from slices of rat corpus striatum. This potentiation is observed only when a submaximal concentration (30 mM) of K+ is used to evoke release. The effect of GABA is dose-dependent between 100 and 100 micrometers. The basal release of ME is not altered by these concentrations of GABA. Before, but not muscimol, mimics the effect of GABA on the evoked release of ME. This effect is not stereoselective as both the (+)- and (-)-isomers of baclofen enhance ME release. Picrotoxin (100 micrometers) blocks the enhancement of ME release produced by both GABA and baclofen. Bicuculline methiodide (100 micrometers) does not block the effect of GABA. The effect of GABA on ME release may be mediated by an atypical GABA receptor which is activated by baclofen.     

Analysis of agonist-evoked nitric oxide release from human endothelial cells: role of superoxide anion

1. Dichlorofluorescein oxidation and electrochemical monitoring of in situ nitric oxide (NO) release from cultured human endothelial cells reveals that agonists such as thrombin and histamine simultaneously stimulate transient superoxide production. 2. The duration of *NO release was increased only in the simultaneous presence of extracellular L-arginine and exogenous superoxide dismutase. In contrast, the inhibition of membrane reduced nicotinamide adenine dinucleotide (phosphate) oxidases, the major source of *O2- in endothelial cells, did not prolong *NO release, although extracellular L-arginine was also present. Comparison of these two experimental conditions suggested that H2O2 was involved in the extension of the *NO signal. 3. The present study demonstrates that, in the absence of external L-arginine, *O2- production does not constitute the major pathway controlling the duration of agonist-induced *NO signal. These results suggest that L-arginine and H2O2 act jointly to maintain nitric oxide synthase in an activated form.     

Effects of tenoxicam on superoxide anion formation, β-glucuronidase release and fMLP binding in human neutrophils: Comparison with other NSAIDs

Non-steroidal anti-inflammatory drugs (NSAIDs) are considered to exert their activity by interfering with the generation of arachidonate metabolites in various cells, mainly in neutrophils and monocytes. The inhibition of cellular cyclooxygenase enzyme, however, does not always correlate with the in vivo activity of these drugs. Recent evidence indicates that several NSAIDs may interfere with the stimulus-response coupling of inflammatory cells. In this study, the effects of tenoxicam, an oxicam derivative with a thienothiazine structure, on neutrophil activation were evaluated by the assessment of the following parameters: (1) superoxide anion generation by neutrophils and whole blood stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP), the calcium ionophore A23187 and serum treated zymosan (STZ); (2) beta-glucuronidase release from neutrophils stimulated with fMLP, A23187 and STZ; (3) binding of [3H]fMLP to intact neutrophils. The results were compared to those obtained using piroxicam and diclofenac. Tenoxicam, added in vitro to whole blood, at concentrations ranging between 10(-5) and 3 x 10(-4) M, significantly inhibited the generation of superoxide anion induced by fMLP, A23187 and STZ. The activity of tenoxicam on whole blood was similar to that of piroxicam, whereas diclofenac had only minimal effects on this experimental system. In isolated cells tenoxicam inhibited the generation of superoxide anion induced by A23187 and STZ. In addition, at the 3 x 10(-4) M concentration, tenoxicam and diclofenac similarly inhibited O2- generation by neutrophils stimulated with fMLP, whereas piroxicam only minimally affected this parameter. Tenoxicam also slightly, but not significantly, inhibited beta-glucuronidase release by isolated neutrophils induced by all the agonists used. Specific binding of [3H]fMLP to neutrophils was inhibited by the three NSAIDs tested in a dose-dependent fashion and tenoxicam was the most potent. The affinities (Kd) of tenoxicam, piroxicam and diclofenac were 1.11, 1.80 and 2.70 x 10(-5) M, respectively. The mechanism of inhibition of [3H]fMLP binding by tenoxicam was non-competitive. It is concluded that tenoxicam, at concentrations achievable in plasma at steady state, effectively inhibits some of the processes involved in neutrophil activation, which bear some relevance in the inflammatory disease.     

Inhibition of arachidonate release from rat peritoneal macrophage by biflavonoids

Biflavonoid is one of unique classes of naturally-occurring bioflavonoid. Previously, certain biflavonoids were found to possess the inhibitory effects on phospholipase A₂ activity and lymphocytes proliferation¹ suggesting their anti-inflammatory/immunoregulatory potential. In this study, effects of several biflavonoids on arachidonic acid release from rat peritoneal macrophages were investigated, because arachidonic acid released from the activated macrophages is one of the indices of inflammatory conditions. When resident peritoneal macrophages labeled with [³H]arachidonic acid were activated by phorbol 12-myristate 13-acetate (PMA) or calcium ionophore, A23187, radioactivity released in the medium was increased approximately 4.1∼7.3 fold after 120 min incubation compared to the spontaneous release in the control incubation. In this condition, biflavonoids (10 uM) such as ochnaflavone, ginkgetin and isoginkgetin, showed inhibition of arachidonate release from macrophages activated by PMA (32.5∼40.0% inhibition) or A23187 (21.7∼41.7% inhibition). Amentoflavone showed protection only against PMA-induced arachidonate release, while apigenin, a monomer of these biflavonoids, did not show the significant inhibition up to 10 uM. Staurosporin (1 uM), a protein kinase C inhibitor, showed an inhibitory effect only against PMA-induced arachidonate release (96.8% inhibition). Inhibition of arachidonate release from the activated macrophages may contribute to an anti-inflammatory potential of biflavonoidsin vivo.