BW A4C Research Articles

Deneysel Astım Modeli Oluşturulmuş Kobay Trakeası Üzerinde 5-Lipoksijenaz İnhibitörlerinin Etkileri

Objective: The 5-lipoxygenase products, especially sulfidopeptide leukotrienes take an important role in the pathogenesis of asthma. The effects of two selective 5-lipoxygenase inhibitors, acetohydroxamic acid derivative BWA 4C and a naphtokinon derivative CGS 8515, were investigated for their effect on antigen-induced contraction in ovalbumin sensitized guinea-pig isolated tracheal tissues. Material and Methods: The study included 15 male guinea pigs. They were actively sensitized by the Modified Andersson Method, including two standard 0.5 mL injections of a solution containing 20 µg ovalbumin and 50 mg aluminium hydroxide in saline on day 0 and day 14. Twenty-one to 27 days after the injection of ovalbumin, the tracheal rings from sensitized guinea pigs were exposed to antigen, and the possible inhibitor effects of 5-lipoxygenase inhibitors on the antigen-induced contraction were examined in water-jacketed tissue baths containing Krebs buffer. Results: Both BWA 4C and CGS 8515 significantly reduced antigen-induced tracheal contraction compared to control groups. Conclusion: As both CGS 8515 and BWA 4C inhibit antigen induced tracheal smooth muscle contractions, they may take an important role in the treatment of asthma and related inflammatory events.

Reactive oxygen species and lipoxygenases regulate the oncogenicity of NPM-ALK-positive anaplastic large cell lymphomas

The chimera nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), the tyrosine kinase activity of which is constitutively upregulated, is the causative agent of 75% of the anaplastic large-cell lymphomas (ALCLs). We have demonstrated that NPM-ALK induces the production of reactive oxygen species (ROS) by a pathway involving the arachidonic acid-metabolizing enzymes of the lipoxygenase (LOX) family. The use of the LOX inhibitor nordihydroguaiaretic acid (NDGA) and of the anti-oxidant N-acetylcysteine (NAC) demonstrated that ROS are important in maintaining the ALK kinase active. Consistent with this, NDGA treatment resulted in the inhibition of key pathways, such as Akt, signal transducer and activator of transcription factor 3 (STAT3) and extracellular signal-regulated kinase (ERK), which are involved in NPM-ALK antiapoptotic and pro-mitogenic functions. Conversely, the stress-activated kinase p38, described in some instances as a mediator of apoptosis, was activated. Interestingly, 5-LOX, an isoform involved in many cancers, was found to be activated in NPM-ALK(+) cells. Functional studies have shown that transforming properties, namely proliferation and resistance to apoptosis, were abrogated by treatment with either NDGA or the 5-LOX inhibitor (N-(3-phenoxycinnamyl)-acetohydroxamic acid) (BW A4C). Together, these data point to the ROS/LOX pathway as a potential new target for therapy in NPM-ALK-positive tumors.

Blockade of leukotriene B 4 prevents articular incapacitation in rat zymosan-induced arthritis

We investigated whether leukotrienes mediate cell influx and articular incapacitation in zymosan-induced arthritis. Rats received 1 mg zymosan intra-articularly (i.a.). The hyperalgesia was measured using the rat articular incapacitation test. Cell influx, leukotriene B 4 and prostaglandin E 2 levels were assessed in the joint exudate, at 6 h. Groups received either the leukotriene B 4 synthesis inhibitor MK 886 (3-[1-(4-chlorobenzyl)-3- t-butyl-thio-5-isopropylindol-2-yl)]-2,2-dimethylpropanoic acid 30 min before or 2 h after the zymosan; 0.3–3 mg kg −1 i.p.), the leukotrienes synthesis inhibitor BWA 4C ( N-(3-phenoxycinnamyl)-acetohydroxamic acid—2 h after the zymosan; 10 μg i.a.) or the peptido-leukotrienes antagonist sodium montelukast (30 min before and 2 h after the zymosan; 10 mg kg −1 per os). MK 886 inhibited the articular incapacitation and cell influx, while reducing leukotriene B 4, but not prostaglandin E 2 levels. BWA 4C inhibited the articular incapacitation. Sodium montelukast did not affect either of the parameters. The data suggest that leukotriene B 4 is involved in cell influx and articular incapacitation in zymosan arthritis.

Effects of the inhibition of cyclo-oxygenase 1 or 2 or 5-lipoxygenase on the activation of the hypothalamic-pituitary-adrenal axis induced by interleukin-1beta in the male Rat.

The limited entry of interleukin-1beta (IL-1beta) into the central nervous system has led to the hypothesis that IL-1beta acts, through IL-1beta receptors located notably on endothelial cells, on the release of prostaglandins which in turn stimulate the hypothalamic-pituitary-adrenal (HPA) axis. We used cyclo-oxygenase-1 (COX-1) and cyclo-oxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) inhibitors, before the injection of IL-1beta, to explore the role of arachidonic acid metabolic pathways on HPA axis activation. Adult male rats were i.m injected 20 min before i.p injection of IL-1beta, with (i): a COX-1/COX-2 inhibitor (ketoprofen); (ii) a COX-2 selective inhibitor (NS 398); or (iii) a 5-LOX inhibitor (BW A4C). Following this, rats were killed 90 min after i.p. IL-1beta injection and analysis for plasma adrenocorticotropic hormone (ACTH) and corticosterone concentrations and determination of anterior pituitary pro-opio melanocortin (POMC) gene transcription was conducted. Administration of the COX-1/COX-2 inhibitor led to a complete blockage of ACTH and corticosterone secretion and POMC gene transcription. The COX-2 inhibitor led to a complete blockade of ACTH secretion and POMC gene transcription but had no effect on corticosterone secretion. The 5-LOX inhibitor had no significant effect on any parameter. These results demonstrate the crucial role of eicosanoid pathways in mediating the stimulation of the HPA axis induced by IL-1beta. Moreover, we found a clear dissociation of the effect of the blockage of COXs upon ACTH and corticosterone secretion, suggesting that IL-1beta may act at the brain as well as at the adrenal cortex to stimulate the secretion of corticosterone.

Leukotriene receptor antagonists and synthesis inhibitors reverse survival in eosinophils of asthmatic individuals.

Eosinophilia is a feature of airway inflammation associated with asthma. Leukotriene antagonists provide therapeutic benefit in asthma, but their potential antiinflammatory actions have not been fully explored. We have examined the role of eosinophil-derived cysteinyl leukotrienes in the maintenance of eosinophil survival, and the involvement of leukotrienes in the paracrine stimulation of eosinophil survival by mast cells and lymphocytes. We obtained eosinophils and autologous lymphocytes from peripheral blood of asthmatic subjects. Leukotriene (LT)-B(4), LTC(4) and LTD(4), granulocyte-macrophage colony-stimulating factor (GM-CSF), and fibronectin promoted eosinophil survival. LTD(4) (10(-)(6) M) was as effective as GM-CSF (5 ng/ml) and fibronectin (400 ng/ml) in promoting survival. Lymphocytes and conditioned medium from a human mast cell line (HMC-1) induced eosinophil survival. Blockade of cysteinyl leukotriene receptors with SKF 104353 (pobilukast, 3 nM), and inhibition of 5-lipoxygenase (5-LO) with BW A4C (1 microM) and of 5-LO activating protein with MK 886 (1 microM), all increased basal rates of eosinophil apoptosis and reversed GM-CSF-induced eosinophil survival. Fifty percent reversal of GM-CSF- induced survival was achieved with SKF 104353 at 0.3 nM. The potency of SKF 104353 was two orders of magnitude greater than that of the LTB(4) receptor antagonist SB 201146. Mast cell- and lymphocyte-induced eosinophil survival were completely reversed by SB 201146, SKF 104353, BW A4C, and MK 886. These findings provide evidence for the involvement of an autocrine cysteinyl leukotriene pathway that supports eosinophil survival in response to a range of survival stimuli. They also suggest that LTB(4) could act as a paracrine stimulus of eosinophil survival.

Inhibition by unsaturated fatty acids of type II secretory phospholipase A2 synthesis in guinea-pig alveolar macrophages evidence for the eicosanoid-independent pathway.

The effect of arachidonic acid (C20:4) on the production of secretory type II phospholipase A2 (sPLA2-II) by guinea-pig alveolar macrophages was investigated. We show that incubation of these cells with 1-30 microM of arachidonic acid inhibits the synthesis of sPLA2-II in a concentration-dependent manner with an IC50 of approximately 7.5 microM. The inhibition by low concentrations (5 microM) of arachidonic acid was partially reduced by pretreatment of alveolar macrophages with cyclooxygenase or cytochrome P450 inhibitors (aspirin and 1-aminobenzotriazole, respectively), but not by lipoxygenase inhibitor, BW A4C. However, these inhibitors failed to interfere with the effect of high concentrations (30 microM) of arachidonic acid, suggesting that the latter may act on the expression of sPLA2-II, at least in part, independently of eicosanoid generation. Indeed, a similar inhibitory effect on sPLA2-II activity and mRNA expression was observed with other unsaturated fatty acids such as eicosapentaenoic (C20:5) and oleic (C18:1) acids, but not with the saturated fatty acid, palmitic acid (C16:0). In addition, arachidonic acid partially reduced the secretion of tumor necrosis factor alpha, an important intermediate in the induction of sPLA2-II synthesis by guinea-pig alveolar macrophages. However, addition of recombinant tumor necrosis factor alpha failed to reverse the inhibitory effect of arachidonic acid on sPLA2-II expression, suggesting that this process occurs downstream of tumor necrosis factor alpha secretion. We conclude that the expression of sPLA2-II in alveolar macrophages is down-regulated at the transcriptional level by arachidonic acid either directly or via its cyclooxygenase and cytochrome P450-derived metabolites.

Bothrops lanceolatus (Fer de lance) venom induces oedema formation and increases vascular permeability in the mouse hind paw

The ability of snake venoms to increase vascular permeability and to induce oedema through the release of pharmacologically active substances is well known. We have studied the oedema and vascular permeability induced by Bothrops lanceolatus venom in male Swiss white mice. Paw oedema was induced by the subplantar injection of B. lanceolatus venom (125–1000 ng/paw) and was quantified as the increase in paw weight. Changes in vascular permeability were assessed by measuring the amount of Evans blue dye extravasation. The oedema and the increase in vascular permeability were maximal within 2 h and had resolved after 24 h. The administration of the vasodilator iloprost (20 ng/paw) immediately after B. lanceolatus venom potentiated the oedema and the increase in vascular permeability by approximately four-fold. Pretreating the mice with indomethacin, dexamethasone, NDGA or BW A4C inhibited the venom-induced oedema and the increase in vascular permeability. In contrast, histamine, serotonin and PAF-acether antagonists (mepyramine, cyproheptadine and WEB 2086, respectively) were ineffective. Histological examination showed that B. lanceolatus venom (250 ng and 500 ng/paw) caused thickening of the inner dermal layers which was accompanied by extensive intercellular spaces indicative of oedema. In addition, there was a marked infiltration of inflammatory cells, particularly neutrophils, into the underlying muscle layer. The latter, however, remained morphologically unaffected during the 3 h of observation. Venom doses larger than 500 ng/paw produced intense haemorrhage. These results indicate that B. lanceolatus venom induces oedema and increases vascular permeability in the mouse hind paw. The principal mediators of this inflammatory response are cyclooxygenase and lipoxygenase products.

Effects of inhibitors of inflammatory mediators and cytokines on eosinophil and neutrophil accumulation induced by Mycobacterium bovis bacillus Calmette-Guérin in mouse pleurisy.

In this work we characterize the Mycobacterium bovis bacillus Calmette-Guerin (BCG) -induced pleurisy and investigate the role of chemical mediators and cytokines in BCG-induced granulocyte accumulation at 24 h. Intrathoracic injection of BCG in C57B1/6 mice induces a biphasic inflammatory reaction with intense leukocyte accumulation at 24 h and 15 days. Neutrophils were observed in the pleural cavity at 4-24 h, mononuclear cells and eosinophils after 24 h. A new wave of mononuclear cells and neutrophils were observed after 15 days. Pretreatments with dexamethasone, BW 755C, BW A4C, WEB 2170, L-NAME, and monoclonal antibody (mAb) anti-interleukin-5 (IL-5; TRFK-5) had inhibited the eosinophil accumulation. On the other hand, only the pretreatments with dexamethasone, L-NAME, or mAb anti-tumor necrosis factor alpha (TNF-alpha; MP6-XT3) had inhibited the neutrophil influx. These results suggest the involvement of leukotrienes, platelet-activating factor, nitric oxide, and IL-5 in the eosinophil accumulation, and a role for nitric oxide and TNF-alpha in the neutrophil influx induced by BCG.

Effects of a lipoxygenase inhibitor on digoxin-induced cardiac arrhythmias in the isolated perfused guinea-pig heart

1. 1. The effects of a lipoxygenase inhibitor, BW A4C, on digoxin-induced arrhythmias and cardiac dynamics (contractile force, perfussion pressure, heart rate) were investigated in Langen-dorff-perfused isolated guinea-pig hearts. In the control group, arrhythmias were induced by 25 μg/ml digoxin at a perfusion rate of 0.5 ml/min. In the treated groups, BW A4C (1 and 0.3 μM) perfused continuously from 15 min prior to digoxin until cardiac arrest occurred. Digoxin exposure (μg/g wet weight of heart) for the occurrence of arrhythmias and cardiac arrest were the parameters evaluated to assess cardiotoxicity. 2. 2. Digoxin caused a marked increase in leukotriene B 4 release in the coronary effluent, and was collected during tachyarrhythmias. BW A4C markedly inhibited the digoxin-induced elevation of LTB 4. 3. 3. BW A4C (1 and 0.3 μM) did not prevent the onset of ventricular fibrillation and ventricular tachycardia despite a slight delay in the occurrence of ventricular fibrillation and cardiac arrest at the 0.3 μM concentration. 4. 4. Contractile force increased significantly after digoxin infusion which was concomitant with the time of onset of arrhythmias. In the presence of BW A4C, the contractile force increased, but not significantly. Perfusion pressure increased initially after digoxin infusion in the absence and the presence of BW A4C, but not significantly. 5. 5. These findings show that the lipoxygenase inhibitor lacked any protective action on digoxin-induced arrhythmias despite its effective suppression of digoxin-induced elevation of LTB 4 in coronary effluent.

A test system for leukotriene synthesis inhibitors based on the in-vitro differentiation of the human leukemic cell lines HL-60 and Mono Mac 6.

Differentiation of HL-60 cells along the granulocytic lineage by DMSO in the presence of transforming growth factor-beta and low concentrations of 1,25-dihydroxyvitamin D3 leads to the upregulation of 5-lipoxygenase activity in 100,000 g supernatants and intact cells to levels which are comparable to normal granulocytes. Similarly, differentiation of the human monocytic cell line Mono Mac 6 by 1,25-dihydroxyvitamin D3 and transforming growth factor-beta strongly upregulates the 5-lipoxygenase pathway. Here, we describe an assay system for leukotriene biosynthesis inhibitors which is based on the in-vitro differentiation of HL-60 and Mono Mac 6 cells. Different leukotriene biosynthesis inhibitors like the nonredox type inhibitor ZM 230487, the redox type inhibitor BW A4C and the FLAP inhibitor MK886 were tested and the results were compared with an assay system based on normal human granulocytes. ZM 230487, BWA4C and MK886 showed similar potencies in these cell lines as compared to normal leukocytes. Thus, the in-vitro differentiation of HL-60 and Mono Mac 6 cells provides an excellent model for the screening of drugs affecting the 5-lipoxygenase pathway.

Role of resident mast cells and macrophages in the neutrophil migration induced by LTB4, fMLP and C5a des arg.

In the present study, we have investigated the participation of resident peritoneal cells (macrophages and mast cells) in the neutrophil migration induced in rats by the intraperitoneal administration of LTB4, fMLP or C5a des arg. The intraperitoneal injection of LTB4 (10 nmol), fMLP (10 nmol) and C5a des arg (zymosan-activated plasma, 1 ml) caused an intense neutrophil migration compared to the saline control (1,000, 1,500 and 2,000%, respectively). An 83% depletion in the number of resident cells following peritoneal lavage reduced the LTB4-stimulated neutrophil migration by 73.6% without affecting that caused by fMLP and C5a des arg. Increasing the peritoneal macrophage population (236%) by pretreating the cavities with thioglycollate enhanced the neutrophil migration induced by LTB4 (129%), but did not alter that induced by fMLP and C5a des arg. Similarly, reducing the population of peritoneal mast cells containing toluidine-blue-staining granules by subchronically pretreating the cavities with compound 48/80 diminished the LTB4-induced NM by 69% but had no effect on the responses to fMLP and C5a des arg. Pretreating the animals with dexamethasone strongly inhibited (70%) the neutrophil migration induced by the intraperitoneal injection of LTB4, fMLP and C5a des arg. Indomethacin, BW A4C and NDGA had no such effect. The incubating medium from peritoneal macrophages and mast cells stimulated with LTB4 induced neutrophil migration when injected into the peritoneal cavity of rats. This migration was strongly reduced (70%) by treating the cells with dexamethasone. In contrast, stimulating the cells with fMLP or C5a des arg did not result in the release of any promigratory activity into the incubating fluid. Our results suggest that LTB4 induces neutrophil migration via a mechanism dependent on resident mast cells and macrophages while that induced by C5a des arg and fMLP seems to be independent of such cellular involvement. The neutrophil migration induced by LTB4 is apparently mediated by factor(s) whose release is blocked by dexamethasone. fMLP and C5a appear to cause in vivo migration by the formation of a concentration gradient and by a glucocorticoid-sensitive mechanism different from that stimulated by LTB4.

Purification and Characterization of Linoleate 8-Dioxygenase from the Fungus Gaeumannomyces graminis as a Novel Hemoprotein

The fungus Gaeumannomyces graminis, which causes the major root disease of wheat known as "take-all," can metabolize linoleic acid to (8R)-hydroperoxylinoleic acid. The enzyme linoleate 8-dioxygenase abstracts hydrogen and introduces molecular oxygen in an antarafacial way at C-8. We have now purified the enzyme 1000-fold to a specific activity of 1.8 micronol/min/mg of protein. Acetone powder of mycelia of G. graminis was subjected to extraction and ammonium sulfate precipitation with solubilization. The 8-dioxygenase was purified by hydrophobic interaction chromatography, size-exclusion chromatography, anion-exchange chromatography, and immobilized metal ion affinity chromatography. The active enzyme appeared to consist of four subunits since the active enzyme had an apparent molecular mass of 520 kDa determined by gel filtration, while SDS-polyacrylamide gel electrophoresis showed a protein band of 130 kDa. Spectroscopy indicated the presence of heme. The characteristic pyridine ferrohemochrome alpha-band was found at 557 nm and the beta-band at 525 nm. The purified protein showed an absorption maximum at 408 nm (gamma, Soret). The absorption maximum shifted to 429 nm after reduction with dithionite and to 421 nm after treatment of the reduced enzyme with carbon monoxide. BW A4C, a hydroxamic acid derivative, inhibited the enzyme by >90% at 10 microM. The pH optimum was 7.2-7.4, the isoelectric point was 5.2 by chromatofocusing, and the Km values were 8 microM for linoleic acid and 30 microM for oxygen. We conclude that linoleate 8-dioxygenase appears to be a tetrameric hemoprotein distinct from other fatty-acid dioxygenases.

Participation of lnterleukin-5 and lnterleukin-8 in the Eosinophil Migration Induced by a Large Volume of Saline

Recently we demonstrated that the eosinophil migration into rat peritoneal cavities induced by a large volume of saline is mediated by LTB4 released by the resident macrophages and mast cells. In the present study, we have investigated the involvement of IL-5 and IL-8 in this process. We observed that saline-stimulated mast cells released the eosinophil chemotactic cytokines IL-5 and IL-8, while macrophages released only IL-8. These observations were confirmed by the ability of antibodies against IL-5 and IL-8 block the eosinophil chemotactic activity of the mast cell supernatants while the chemotactic activity of the macrophage supernatants was inhibited only by the antibody to IL-8. Recombinant forms of IL-5 and IL-8, when injected intraperitoneally, induced a dose-dependent eosinophil accumulation in naïve rats. The mechanism by which these cytokines induce eosinophil migration seems to be dependent on the resident cell population since depleting the peritoneal cavities of the latter renders the animals unresponsive to the eosinophil recruitment when challenged with IL-5, IL-8 or the supernatants of saline-treated mast cells or macrophages. Dexamethasone and MK 886 blocked the eosinophil migration induced by both the supernatants of saline-stimulated mast cells or macrophages and by IL-5 or IL-8. The IL-5-induced eosinophil migration was also blocked by BW A4C, another lipoxygenase inhibitor. Together, these results suggest LTB4 to be the lipoxygenase metabolite involved in the eosinophil recruitment induced by IL-5 and IL-8. Our results indicate that the eosinophil migration induced by saline is a complex phenomenon which is dependent on the resident mast cells and macrophages and is mediated by LTB4, IL-5 and IL-8. Mast cells release LTB4, IL-5 and IL-8, whereas macrophages release mainly LTB4 and IL-8. The inhibition of one of these mediators (IL-5, IL-8 or LTB4) completely blocked the eosinophil migration induced by saline, suggesting that they act synergistically.

Colonic microvascular integrity in acute endotoxaemia: interactions between constitutive nitric oxide and 5-lipoxygenase products

Administration of the nitric oxide synthase inhibitor, N G-nitro- l-arginine methyl ester (5 mg/kg s.c.) provoked acute microvascular injury (assessed by the leakage of radiolabelled human serum albumin) in the rat colon within 1 h, when administered concurrently with endotoxin ( Escherichia coli lipopolysaccharide, 3 mg/kg i.v.). Pretreatment with the selective inhibitor of 5-lipoxygenase, BW A137C ( N-[4-benzyloxybenzyl] acetohydroxamic acid; 1–20 mg/kg s.c., 15 min before endotoxin) attenuated such damage in a dose-dependent manner. These findings suggest a balance between protective constitutive nitric oxide and the detrimental actions of 5-lipoxygenase products in the maintenance of vascular integrity in the early stage of sepsis.

Interleukin-1 receptor antagonist displays intrinsic agonist activity on rat gastric fundus motility in vitro

It has been shown previously that both forms of interleukin-1, 1α and 1β, produce dose-dependent relaxation of the rat gastric fundus in vitro, accompanied by an increased production and release of eicosanoids. This effect appears to be mediated, at least in part, by leukotrienes, since the inhibition of 5-lipoxygenase by specific drugs counteracts interleukin-1-induced gastric relaxation. In the present study, we attempted to antagonize interleukin-1-induced inhibition of gastric fundus motility with a interleukin-1 receptor antagonist. Surprisingly, the interleukin-1 receptor antagonist itself possessed interleukin-1-like agonist activity, since: (a) it produced rapid, dose-dependent relaxation of the rat gastric fundus, with an estimated EC 50 of 70 pg/ml and a maximal effect at 10 ng/ml; (b) interleukin-1 receptor antagonist-induced relaxation was dose dependently inhibited by N-(3-phenoxycinnamyl)acetohydroxamic acid (BW A4c), a specific inhibitor of 5-lipoxygenase; (c) in the first 5 min after its addition to the bath solution, interleukin-1 receptor antagonist produced a significant increase in prostaglandin E 2 release from the gastric strips. This evidence suggests that, shortly after receptor occupancy, in this experimental model interleukin-1 and interleukin-1 receptor antagonist share the same pattern of mechanical and biochemical activities.

Interleukin-1β- and tumour-necrosis-factor-induced inhibition of rat gastric fundus motility in vitro

In this study, we compared the effects of interleukin-1β (IL-1β) and tumour necrosis factor (TNF) on in vitro rat gastric fundus motility. IL-1β and TNF produced rapid, concentration-dependent relaxation of the rat gastric fundus strips with maximal effect at 300 pg ml −1 and 10 ng ml −1 and estimated EC 50S at 10 and 450 pg ml −1, respectively. The relaxant effects of IL-1β and TNF were not influenced by the inhibition of cyclo-oxygenase or NO-synthase activities. IL-1β- and TNF-induced gastric relaxations were inhibited by BW 755c, which inhibits both cyclo-oxygenase and lipoxygenase (LO), BW A4c, which selectively inhibits the 5-LO pathway, and SC 41930, a selective leukotriene B4 (LTB4) receptor antagonist, providing pharmacological evidence that LTB4 is involved in the relaxant effects of both cytokines. The IL-1β- and TNF-induced activation of 5-LO pathway did not appear to be triggered by phospholipase A2. An alternative pathway could involve the activation of a phospholipase C, specific for phosphatidylcholine, from which, in sequence: the formation of diacylglycerol (DAG), DAG-induced activation of protein kinase C and the formation of free arachidonic acid from DAG would ensue. This mechanism is suggested by the finding that LTB4 is able to mimic cytokine-induced strip relaxation only in the presence of phorbol 12-myristate 13-acetate, which selectively activates protein kinase C.

Interleukin-1 beta and interleukin-6 stimulate neurohypophysial hormone release in vitro.

Interleukin-1 (IL-1) and interleukin-6 (IL-6) have been reported to stimulate the release of corticotrophin-releasing hormone (CRH) in vitro, the response being antagonized by the cyclo-oxygenase inhibitor, indomethacin. The effects of cytokines on the other major ACTH-releasing hormone, vasopressin (AVP), and the other neurohypophysial hormone, oxytocin, have been little studied, and the published data are conflicting. We have therefore used a previously validated rat hypothalamic explant model to evaluate whether IL-1 beta and IL-6 can directly activate the AVP and oxytocin neurosecretory system. In addition, we have also investigated the effects of inhibition of cyclo-oxygenase (CO) and lipoxygenase (LO) activities on the stimulated release of AVP and oxytocin by means of a series of antagonists, including a specific LO pathway inhibitor. The static rat hypothalamic incubation system used involves fresh hypothalamic explants with consecutive 20-min incubations, and estimation of AVP and oxytocin concentrations in the medium by specific and sensitive radioimmunoassays. It was found that IL-1 beta produced a dose-dependent increase in the release of AVP and oxytocin at doses of 10 and 100 U/ml (P < 0.005). Only at the higher dose of 100 U/ml was IL-6 able to increase significantly AVP and oxytocin release (P < 0.05). These stimulatory effects of IL-1 beta and IL-6 were blocked by cyclo-oxygenase inhibitors, indomethacin (28 microM) and ibuprofen (100 nM), but not by the lipoxygenase inhibitor, BW A4C (10 micrograms/ml), suggesting that prostaglandins are involved in this process.(ABSTRACT TRUNCATED AT 250 WORDS)

BW A4C and other hydroxamic acids are potent inhibitors of linoleic acid 8 R-dioxygenase of the fungus Gaeumannomyces graminis

Linoleic acid is converted to 8 R-hydroperoxylinoleic acid by the soluble 8 R-dioxygenase of the fungus Gaeumannomyces graminis. Effects of different lipoxygenase inhibitors on the 8 R-dioxygenase were evaluated. Three hydroxamic acid derivatives were investigated. BW A4C ( N-(3-phenoxycinnamyl)acetohydroxamic acid) was the most potent with an IC 50 of 0.2 μM, followed by zileuton (3–10 μM) and linoleate-hydroxamic acid (0.02 mM). Two other lipoxygenase inhibitors, nordihydroguaiaretic acid and eicosatetraynoic acid, were less potent (IC 50 0.09 and 0.15 mM, respectively). The 8 R-dioxygenase was also strongly inhibited by commonly used buffer additives, dithiothreitol, β-mercaptoethanol and phenylmethanesulfonyl fluoride. G. graminis also contains a hydroperoxide isomerase, which converts 8 R-hyroperoxylinoleic acid to 7S,8 S-dihydroxylinoleic acid. Ammonium sulphate precipitation and gel filtration indicated that the dioxygenase and the hydroperoxide isomerase activities could be seperated.

Steroidal and nonsteroidal drugs in endotoxin-induced uveitis.

Various classes of anti-inflammatory compounds like steroids (dexamethasone), cyclooxygenase inhibitors (indomethacin and flurbiprofen), 5-lipoxygenase inhibitors (BWA 218C and BWA 4C), immunosuppressive drugs (cyclosporin and rapamycin) and cod liver oil were tested for their antiinflammatory activities in endotoxin-induced uveitis model in rabbits. Intraocular inflammation was assessed in terms of two inflammatory responses i.e. breakdown of blood-aqueous barrier (BAB) and leukocyte infiltration into aqueous humor and iris ciliary body (ICB). Prostaglandin (PG) E2 and leukotriene (LT) B4 release into aqueous humor was also measured. Indomethacin significantly inhibited PGE2 release without affecting leukocyte or BAB response. Flurbiprofen prevented leukocyte, PGE2 and LTB4 release into aqueous humor but not ICB chemotaxis. BWA 218C and BWA 4C also significantly inhibited leukocyte and LTB4 release but not BAB responses. Dexamethasone (2mg/kg, i.m.) and cyclosporin A (25 mg/kg i.m.) significantly inhibited leukocyte infiltration into aqueous humor and ICB, and PGE2 release but they failed to inhibit breakdown of BAB and LTB4 release. On the other hand, rapamycin (10mg/kg i.m.) and cod liver oil (1 ml daily i.m. up to 15 days) significantly prevented leukocyte and BAB response. Cod liver oil also significantly inhibited PGE2 and LTB4 release but rapamycin affected only PGE2 release into aqueous humor. It is concluded that arachidonic acid metabolites may not play a vital role in this uveitis model and additional proinflammatory mediators like cytokines may be involved.

Role of resident peritoneal cells in eosinophil migration induced by saline.

In the present study, we investigated the role of resident peritoneal cells as well as the mediators involved in the eosinophil migration induced by large volumes of physiological saline. Two consecutive intraperitoneal injections of saline given 48 h apart, induced a selective recruitment of eosinophils into the cavity. This response was not observed with phosphate-buffered saline (PBS). Saline-induced eosinophil migration may be mediated at least in part by LTB4, since the lipoxygenase inhibitors BW A4C and MK 886 prevented the response. In the presence of saline, but not of PBS, mast cells and macrophages incubated in vitro released a factor which induced eosinophil migration when injected into the peritoneal cavity of rats. This release was inhibited by BW A4C and MK 886. These results indicate the importance of mast cells and macrophages in the eosinophil migration induced by saline and suggest the participation of LTB4 in this phenomenon. An abrupt reduction in the extracellular potassium concentration at the membrane of resident cells may be responsible for the saline effects since addition of potassium ions to saline abolished the eosinophil chemotactic activity of the same as well as its ability to stimulate the release of eosinophil chemotactic factor in vitro. Dexamethasone blocked both the saline-induced eosinophil migration and the release of eosinophil chemotactic factor by mast cells and macrophages. Pretreatment of the animals with dexamethasone inhibited the eosinophil migration induced either by the supernatants of saline-stimulated mast cells and macrophages or by LTB4. These results indicate that the release of additional mediators is necessary in order to account for the final eosinophil migration.(ABSTRACT TRUNCATED AT 250 WORDS)