SC-41930, a leukotriene B4 receptor antagonist, inhibits 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) binding to epidermal cells.

SC-41930, 7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxyl]-3, 4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid, a potent leukotriene-B4 (LTB4) receptor antagonist, inhibits in vivo 12-hydroxyeicosatetraenoic acid (12-HETE)-induced neutrophil infiltration, suggesting a potential 12-HETE receptor antagonist effect, as well. Since 12-HETE is assumed to have a pathophysiological role in inflammatory skin diseases, and epidermal cells possess high affinity binding sites for 12(S)-HETE, we studied the effect of SC-41930 on 12(S)-HETE binding to the human epidermal cell line, SCL-II. SC-41930 antagonized the 12(S)-HETE binding to SCL-II cells with a Ki of 480 nM. This Ki value is similar to that obtained for the inhibition of LTB4 binding to human neutrophils. Our results show that SC-41930, in addition to its LTB4 receptor antagonist effect, exhibits 12-HETE receptor antagonist effect as well, and therefore may be of benefit in skin diseases with elevated 12-HETE levels.     

Benzophenone dicarboxylic acid antagonists of leukotriene B4. 2. Structure-activity relationships of the lipophilic side chain

A series of lipophilic benzophenone dicarboxylic acid derivatives were found to inhibit the binding of the potent chemotaxin leukotriene B4 (LTB4) to its receptor on intact human neutrophils. Activity at the LTB4 receptor was determined by using a [3H]LTB4-binding assay. The structure-activity relationship for the lipophilic side chain was systematically investigated. Compounds with n-alkyl side chains of varying lengths were prepared and tested. Best inhibition of [3H]LTB4 binding was observed with the n-decyl derivative. Analogues with alkyl chains terminated with an aromatic ring showed improved activity. The 6-phenylhexyl side chain was optimal. Substitution on the terminal aromatic ring was also evaluated. Methoxyl, methylsulfinyl, and methyl substituents greatly enhanced the activity of the compound. For a given substituent, the para isomer had the best activity. Thus the nature of the lipophilic side chain can greatly influence the ability of the compounds to inhibit the binding of LTB4 to its receptor on intact human neutrophils. The most active compound from this series, 84 (LY223982), bound to the LTB4 receptor with an affinity approaching that of the agonist.     

omega-[(4,6-Diphenyl-2-pyridyl)oxy]alkanoic acid derivatives: a new family of potent and orally active LTB4 antagonists.

A series of omega-[(4,6-diphenyl-2-pyridyl)oxy]alkanoic acid derivatives was prepared which inhibited the binding of leukotriene B4 to its receptors on guinea pig spleen membranes and on human polymorphonuclear leukocytes (PMNs) and selectively antagonized the LTB4-induced elastase release in human PMNs. On the basis of these three screens, a structure-activity relationship was investigated. alpha-Substitution on the carboxylic acid side chain led to only small changes in the binding affinities but greatly enhanced the LTB4 antagonist activity. Substitution on the phenyl rings was also evaluated. The terminal carboxylic acid function can be replaced by a tetrazole ring without loss in activity. The best in vitro LTB4 antagonists of this series were investigated in vivo in the inhibition of LTB4-induced leukopenia in rabbits. Compound 9b (RP69698) displayed potent LTB4 antagonist activity, after oral administration, with an ED50 value of 6.7 mg/kg.     

Adenosine A2 receptor-induced inhibition of leukotriene B4 synthesis in whole blood ex vivo.

1. Engagement of adenosine A2 receptors suppresses several leukocyte functions. In the present study, we examined the effect of adenosine on the inhibition of leukotriene B4 (LTB4) synthesis in heparinized human whole blood, pretreated with lipopolysaccharide (LPS) and tumour necrosis factor alpha (TNF-alpha) and stimulated with the chemotactic peptide, N-formyl-Met-Leu-Phe (FMLP). 2. The FMLP-induced synthesis of LTB4 in whole blood pretreated with LPS and TNF-alpha was dose-dependently inhibited by adenosine analogues in the following order of potency; 5'(N-ethyl)carboxamidoadenosine (NECA) approximately equal to CGS 21680 > 2-Cl-adenosine > N6-cyclopentyladenosine (CPA), indicating the involvement of the adenosine A2 receptor subtype. The IC50 values for NECA, CGS 21680, 2-Cl-adenosine, and CPA were 6 nM, 9 nM, 180 nM, and 990 nM, respectively. 3. Dipyridamole, an agent that blocks the cellular uptake of adenosine by red cells and causes its accumulation in plasma, also inhibited the synthesis of LTB4 in LPS and TNF-alpha-treated whole blood stimulated by FMLP; moreover, this inhibition was reversed upon addition of adenosine deaminase. 4. A highly selective antagonist of the adenosine A2 receptor, 8-(3-chlorostyryl)caffeine (CSC), reversed the inhibition of LTB4 synthesis by 2-Cl-adenosine and dipyridamole in LPS and TNF-alpha-treated whole blood, stimulated by FMLP. 5. LTB4 synthesis in whole blood originates predominantly from neutrophils and to a lesser extent from monocytes. 2-Cl-adenosine also inhibited the synthesis of LTB4 induced by FMLP in these isolated LPS and TNF-alpha-treated cells; however, 2-Cl-adenosine was a more potent inhibitor of LTB4 synthesis in neutrophils than monocytes. 6. The present data demonstrate that adenosine, acting through A2 receptors, exerts a potent inhibitory effect on the synthesis of LTB4 and thus contribute to the understanding of its anti-inflammatory properties.     

Effect of auranofin on eicosanoids and protein kinase C in human neutrophils

Auranofin (AF), a lipophilic chrysotherapeutic agent, was investigated for its effect on the formation of lipoxygenase products and the activity of protein kinase C in human neutrophils. We have previously shown that inhibition of LTB4 formation by 5-lipoxygenase (5-LO) inhibitors is intimately associated with a marked increased in 15-HETE in excess of arachidonic acid. The calcium- and phospholipid-dependent protein kinase, protein kinase C, is activated in FMLP- and A23187-stimulated neutrophils, is hypothesized to stimulate superoxide generation, and plays an essential role in eicosanoid production. AF dose-dependently inhibited the generation of leukotriene B4(LTB4) in FMLP-stimulated neutrophils, the ID50 was approximately 4.5 micrograms/ml. Unlike known 5-LO inhibitors, AF did not enhance the production of 15-HETE. In neutrophils stimulated with the calcium ionophore, A23187, AF did not inhibit the generation of LTB4 nor did AF change the 15-HETE levels. AF inhibited superoxide generation in FMLP-stimulated neutrophils dose-dependently, but did not change the activation of protein kinase C in the cells. We therefore conclude, that AF inhibition of LTB4 production in neutrophils is different from 5-lipoxygenase inhibitors and is elicited at a step distal to protein kinase C activation.     

Inhibition of human neutrophil leukotriene B4 synthesis by combination auranofin and eicosapentaenoic acid.

It has been demonstrated that both auranofin and eicosapentaenoic acid (EPA) have anti-inflammatory properties and both inhibit neutrophil leukotriene B4 (LTB4) synthesis. In the present study, we examined interactions between auranofin and EPA with regard to inhibition of human neutrophil LTB4 synthesis. Auranofin inhibited A23187-stimulated LTB4 synthesis, but the dose required for inhibition of LTB4 was greater than that required for inhibition of other 5-lipoxygenase metabolites; namely, the all-trans isomers of LTB4 and 5-hydroxyeicosatetraenoic acid. These results were explained after a comparison of the rates of synthesis of these 5-lipoxygenase metabolites in the presence and absence of added arachidonic acid which led to the conclusion that leukotriene A hydrolase, the enzyme catalysing the formation of LTB4, was saturated with substrate and rate-limiting for LTB4 synthesis during A23187 stimulation. In combination, auranofin and EPA had a simple additive effect on inhibition of the 5-lipoxygenase pathway. Favorable drug/EPA combinations have the potential to provide a beneficial anti-inflammatory effect with lower levels of each component than are required when used individually.     

Stimulation of protein kinase C redistribution and inhibition of leukotriene B4-induced inositol 1,4,5-trisphosphate generation in human neutrophils by lipoxin A4.

1. To test the hypothesis that protein kinase C (PKC) is involved in the inhibitory actions of lipoxin A4 (LXA4) on second messenger generation, we studied the effects of LXA4 on PKC in human neutrophils and on leukotriene B4 (LTB4)-stimulated inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) generation. 2. LXA4, 1 microM, caused a fall in cytosolic PKC-dependent histone phosphorylating activity to 23.5% of basal levels. 3. LXA4, caused an increase in particulate PKC-dependent histone phosphorylating activity with a bell-shaped dose-response fashion; maximal stimulation was observed at 10 nM LXA4. 4. Western blot analysis with affinity-purified antibodies to alpha- and beta-PKC showed that only the beta-PKC isotype was translocated by LXA4. 5. LXA4 inhibited LTB4-stimulated Ins(1,4,5)P3 generation in a bell-shaped fashion with maximal inhibition at 1 nM LXA4. The observed inhibition was dose-dependently removed by pre-incubation with a PKC inhibitor (Ro-31-8220). 6. These results show that LXA4 activates PKC in whole cells and supports a role for PKC activation in the inhibitory action of LXA4 on LTB4-induced Ins(1,4,5)P3 generation. 7. LXA4 (1-1000 nM) pre-incubation did not affect specific binding of [3H]-LTB4 to neutrophils. Thus, the inhibitory effect of LXA4 on LTB4-stimulated Ins(1,4,5)P3 generation could not be attributed to an effect on LTB4 receptors.     

Auranofin stimulates LTA hydrolase and inhibits 5-lipoxygenase/LTA synthase activity of isolated human neutrophils

The effect of auranofin on the 5-lipoxygenase pathway was studied in human neutrophils stimulated with either fMLP or A23187 (with or without arachidonic acid). The synthesis of leukotriene B4 (LTB4), 5-HETE and the all-trans isomers of LTB4 was measured by HPLC. At low concentrations (0.5-2.0 microM), auranofin stimulated LTB4 synthesis, but inhibited it at higher concentrations (100% inhibition at less than 10 microM). In contrast auranofin caused dose-dependent inhibition of the synthesis of 5-HETE and the all-trans isomers of LTB4. Similar observations were made with each agonist. The stimulation of LTB4 synthesis and inhibition of the trans isomer production suggests that auranofin at low concentrations stimulates LTA hydrolase--the enzyme that converts LTA4 to LTB4, whereas the inhibition of synthesis of all lipoxygenase products at higher auranofin concentrations, suggests inhibition of 5-lipoxygenase/LTA synthase.     

Inhibition of leukotriene B4 in neutrophils by lipoxins A4 and B4.

Lipoxins are derived from the oxygenation products of arachidonic acid in human leukocytes. They have exhibited selective biological effects different from those of other eicosanoids. We have examined the effect of lipoxin A4 and B4 (LXA4, LXB4) on the production of leukotriene B4 (LTB4) in human neutrophils. Cultured human polymorphonuclear leukocytes were preincubated with LXA and B and their ability to inhibit LTB4 generation was assessed after incubation with calcium ionophore A23187. We found that the pretreatment of neutrophils with lipoxins inhibit the release of LTB4 by A23187 stimulated PMNs. Our data suggests that LXA4 and B4 can contribute to immunosuppression in an inflammatory state via the inhibition of LTB4 synthesis.     

Autocrine enhancement of leukotriene synthesis by endogenous leukotriene B4 and platelet-activating factor in human neutrophils.

1. Platelet-activating factor (PAF) and leukotriene B4 (LTB4), two potent lipid mediators synthesized by activated neutrophils, are known to stimulate several neutrophil functional responses. In this study, we have determined that endogenous LTB4 and PAF exert autocrine effects on LT synthesis, as well as the underlying mechanism involved. 2. Pretreatment of neutrophils with either pertussis toxin (PT), or with receptor antagonists for LTB4 and PAF, resulted in an inhibition of LT synthesis induced by calcium ionophore, A23187. This inhibition was most marked at submaximal (100-300 nM) A23187 concentrations, whilst it was least at ionophore concentrations which induce maximal LT synthesis (1-3 microM). Thus newly-synthesized PAF and LTB4 can enhance LT synthesis induced by A23187 under conditions where the LT-generating system is not fully activated. 3. In recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF)-primed neutrophils, LT synthesis in response to chemoattractants (fMet-Leu-Phe or rhC5a) was also significantly inhibited by the LTB4 receptor antagonist, and to a lesser extent by PAF receptor antagonists. 4. Further investigation revealed that LTB4 and/or PAF exert their effects on LT synthesis via an effect on arachidonic acid (AA) availability, as opposed to 5-lipoxygenase (5-LO) activation. Indeed, the receptor antagonists, as well as PT, inhibited LT synthesis and AA release to a similar extent, whereas 5-LO activation (assessed with an exogenous 5-LO substrate) was virtually unaffected under the same conditions. Accordingly, we showed that addition of exogenous LTB4 could enhance AA availability in response to chemoattractant challenge in rhGM-CSF-primed cells, without significantly affecting the 5-LO activation status.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pulmonary actions of LY255283, a leukotriene B4 receptor antagonist.

The actions of LY255283, a leukotriene (LT) B4 receptor antagonist, were examined on guinea pig lung. LTB4 and LY255283 displaced [3H]LTB4 from its binding site on lung membranes with pKi values of 9.9 and 7.0, respectively. In the functional correlate of the binding studies, LY255283 competitively reduced contractile responses of lung parenchyma to LTB4 (pA2 = 7.2). LTB4 produced airway obstruction which was reduced by LY255283 administered i.v. (ED50 = 2.8 mg/kg) or orally (ED50 = 11.0 mg/kg). Contractile responses to histamine, LTD4 and the thromboxane mimetic, U46619, were not reduced by LY255283. The compound also did not inhibit cyclooxygenase or 5-lipoxygenase enzymes. We conclude that LY255283 selectively antagonized pharmacologic responses to LTB4 on lung tissue and appears to be a useful tool to investigate the role of LTB4 in pulmonary disease.     

Inhibition of neutrophil and eosinophil induced chemotaxis by nedocromil sodium and sodium cromoglycate.

1. Neutrophils and eosinophils infiltrate the airways in association with the allergen-induced late phase asthmatic reaction. Mobilization of these cells takes place via lipid-like and protein-like chemotactic factors. In this study platelet-activating factor (PAF), leukotriene B4 (LTB4), zymosan-activated serum (ZAS) and N-formyl-methionyl-leucyl-phenylalanine (FMLP) were used as illustrative examples of both groups. Chemotaxis was studied in human neutrophils and eosinophils. The inhibitory effects of nedocromil sodium and sodium cromoglycate were evaluated. 2. All chemotactic factors tested attracted neutrophils with the following rank order of activity: ZAS greater than PAF identical to FMLP identical to LTB4. Eosinophils were only mobilized by PAF, LTB4 and ZAS with the following rank order of activity: ZAS greater than PAF greater than LTB4. 3. Nedocromil sodium and sodium cromoglycate were equally active as the PAF antagonist BN 52021 in inhibiting the PAF-induced chemotaxis of neutrophils (IC50 approximately 10(-8) M). Both drugs were also equally active in inhibiting the chemotaxis of neutrophils induced by ZAS (IC50 approximately 10(-7)-10(-6) M), FMLP (IC50 approximately 10(-7) M) and LTB4 (IC50 approximately 10(-6) M). 4. Nedocromil sodium significantly inhibited the chemotaxis of eosinophils induced by PAF (IC50 approximately 10(-6) M) and LTB4 (IC50 approximately 10(-7) M). The inhibitory potency of BN 52021 was similar to that of nedocromil sodium on the PAF-induced chemotaxis of eosinophils. Sodium cromoglycate was incapable of eliciting significant inhibition of these chemotactic responses. However, sodium cromoglycate significantly inhibited the chemotaxis of eosinophils induced by ZAS (IC50 approximately 10(-7) M), whereas nedocromil sodium was ineffective.     

Synthesis and structure-activity relationships of new 1, 3-disubstituted cyclohexanes as structurally rigid leukotriene B(4) receptor antagonists

A series of 1-hydroxy-3-?3-hydroxy-7-phenyl-1-hepten-1-yl cyclohexane acetic acid derivatives was designed based on postulated active conformation of leukotriene B(4) (LTB(4)) and evaluated as human cell surface LTB(4) receptor (BLTR) antagonists. Binding was determined through ?(3)HLTB(4) displacement from human neutrophils and receptor antagonistic assays by in vitro measurements of inhibition of leukocyte chemotaxis induced by LTB(4). On the basis of these assays, a structure-affinity relationship was investigated. Optimization of the acid chain length and omega-substitution of a phenyl group on the lipophilic tail were shown to be critical for binding activity. These modifications led to the discovery of compounds with submicromolar potency and selective BLTR antagonism. The most potent compound 3balpha (IC(50) = 250 nM) was found to significantly inhibit oedema formation in a topical model of phorbolester-induced inflammation. Substantial improvement of in vitro potency was achieved by modification of the carboxylic acid function leading to the identification of the N,N-dimethylamide series. Compound 5balpha, free of agonist activity, displayed higher potency in receptor binding with an IC(50) of 40 nM. These results support the hypothesis that the spatial relationship between the carboxylic acid and allylic hydroxyl functions is crucial for high binding affinity with BLTR.     

The effects of BW755C and other anti-inflammatory drugs on eicosanoid concentrations and leukocyte accumulation in experimentally-induced acute inflammation

BW755C (3-amino-1-[m-trifluoromethyl)phenyl]-2-pyrazoline HCl) reduced the concentration of leukotriene B4 (LTB4), thromboxane B2 (TXB2) and prostaglandin E2 (PGE2) in exudate derived from the subcutaneous implantation in rats of 0.5% carrageenan-impregnated polyester sponges. Polymorphonuclear leukocyte (PMN) migration into the inflammatory exudate was also decreased. The inhibition of LTB4 may, in part, account for the lower number of cells in the exudate since LTB4 is a potent leukotactic agent. Inhibition of LTB4-formation and cell migration by BW755C was dose-related, but the two dose-response curves were not parallel. Cell influx still occurred at doses of BW755C that completely inhibited formation of LTB4: this indicates that, although LTB4 may have a chemotactic role in-vivo, other factors must also contribute to cell migration into the inflammatory exudate. Treatment of rats with dexamethasone also caused a reduction in leukocytes and eicosanoids in the exudate. As with BW755C, there was a differential effect on PMN and LTB4: dexamethasone (1 mg kg-1) reduced PMN accumulation by 40% but LTB4 formation was inhibited by 70%. Leukocyte accumulation was also inhibited by the non-steroidal anti-inflammatory drugs (NSAID's), indomethacin and flurbiprofen. These drugs reduced the concentration of both PGE2 and TXB2 in exudate but that of LTB4 was unchanged. This suggests that reduction of PMN accumulation by indomethacin and flurbiprofen is mediated by a mechanism other than inhibition of LTB4-synthesis. Aspirin also reduced the levels of PGE2 and TXB2 in the exudate but did not consistently affect PMN influx, thereby confirming that inhibition of cyclo-oxygenase does not reduce cell migration in inflammation.     

Inhibition by troglitazone of the antigen-induced production of leukotrienes in immunoglobulin E-sensitized RBL-2H3 cells

1. The effect of troglitazone, an anti-diabetic drug with insulin-sensitizing action, on antigen-induced production of leukotriene (LT) B(4), C(4) and E(4) and prostaglandin D(2) (PGD(2)) was examined in dinitrophenol (DNP)-specific immunoglobulin E (IgE)-sensitized RBL-2H3 mast cells following stimulation by the antigen, DNP-conjugated human serum albumin. Levels of LTB(4), C(4) and E(4) and PGD(2) in the conditioned medium were enzyme-immunoassayed. 2.Troglitazone inhibited the antigen-induced production of LTB(4), C(4) and E(4) and the potency of the inhibition was comparable to that of zileuton, a specific inhibitor of 5-lipoxygenase (5-LOX) and a clinically used anti-asthmatic drug. Neither troglitazone nor zileuton affected antigen-induced production of PGD(2), arachidonic acid release from membrane phospholipids and degranulation. 3.Troglitazone inhibited LTB(4) production by the supernatant fraction of RBL-2H3 cell lysate with similar potency to zileuton, suggesting that troglitazone inhibits LT production by direct inhibition of 5-LOX activity. 4. Furthermore, it was shown that troglitazone as well as zileuton inhibited LTB(4) production in A23187-stimulated rat peritoneal neutrophils. 5. These findings suggest that troglitazone inhibits antigen-induced LT production in the IgE-sensitized RBL-2H3 cells and A23187-stimulated rat peritoneal neutrophils by direct inhibition of 5-LOX activity.     

Specific inhibition of leukotriene B4 (LTB4)-induced neutrophil emigration by 20-hydroxy LTB4: implications for the regulation of inflammatory responses.

1. The interaction between leukotriene B4 (LTB4) and its metabolite, 20-hydroxy LTB4 in the control of neutrophil emigration was examined in guinea-pig skin. 2. Leukotriene B4 (10-300 ng) elicited a dose-dependent increase in neutrophil infiltration (as measured by myeloperoxidase activity) 4 h after injection into guinea-pig skin. In contrast, 20-hydroxy LTB4 (30-1000 ng) displayed only weak inflammatory activity in this assay. 3. Although 20-hydroxy LTB4 had low agonist activity, this metabolite caused a potent dose-dependent inhibition of responses to LTB4 (100 ng), when administered systemically (ED50 = 1.3 micrograms kg-1, s.c.) without significantly affecting neutrophil infiltration in response to C5a (2 micrograms). Systemic administration of 20-carboxy LTB4 (10 micrograms) did not affect neutrophil accumulation in response to LTB4 or C5a. In addition, neither 15(S)-hydroxy 5(S)-HPETE(10 micrograms) nor lipoxin A4 (10 micrograms) inhibited responses to LTB4. 4. Addition of 20-hydroxy LTB4 (10(-11)-10(-8) M) to human blood prior to isolation of the neutrophils led to concentration-dependent decrease in the number of LTB4 receptors and decreased chemotactic responsiveness to LTB4 without affecting responses to C5a. Incubation of blood with 20-carboxy LTB4 (10(-8) M) did not reduce LTB4 receptor number of chemotactic responsiveness to LTB4. 5. These data indicate that although 20-hydroxy LTB4 is a weak agonist at LTB4 receptors, it can desensitize neutrophils to the effects of LTB4 via down-regulation of the high affinity receptor and thus provides evidence for a mechanism whereby inflammatory responses may be regulated.     

Linoleic acid and dihomogammalinolenic acid inhibit leukotriene B4 formation and stimulate the formation of their 15-lipoxygenase products by human neutrophils in vitro. Evidence of formation of antiinflammatory compounds.

Enzymatic transformation of the n-6 polyunsaturated fatty acid (PUFA) arachidonic acid (AA) by the 5-lipoxygenase (LO) enzyme results in the formation of leukotrienes (LTs) including leukotriene B4 (LTB4), which is a potent mediator of inflammation. The purpose of the present study was to determine the effect of other n-6 fatty acids on the formation of LTB4 by human neutrophils and to determine if these n-6 fatty acids themselves may be transformed into products with antiinflammatory capacity. Purified neutrophils isolated from heparinized human venous blood were incubated with A23187 (5 microM) and different concentrations (0-100 microM) of the n-6 fatty acids linoleic acid (LA) and dihomo-gamma-linolenic acid (DGLA). LO products were determined by use of quantitative reversed-phase high performance liquid chromatography (RP-HPLC) and mass spectrometry. The formation of LTB4 was dose dependently inhibited by both LA (IC50 = 45 microM) and DGLA (IC50 = 40 microM). This inhibition of LTB4 formation was associated with a dose dependent increase in the formation of the respective 15-LO products of LA (13-hydroxy-octadecadienoic acid; 13-HODE) and DGLA (15-hydroxy-eicosatrienoic acid; 15-HETrE). To determine whether these 15-LO products themselves might inhibit LTB4 formation, neutrophils were incubated with 13-HODE and 15-HETrE. Both 15-LO products lead to a dose-dependent inhibition of LTB4 formation (IC50 = 7.5 microM and IC50 = 0.2 microM). For comparison the 15-LO product of AA, 15-hydroxy-eicosatetraenoic acid (15-HETE), also inhibited LTB4 formation (IC50 = 0.75 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the inhibitory prostanoid receptors on human neutrophils.

1. We have evaluated the effects of various prostanoid agonists on the release of leukotriene B4 (LTB4) and superoxide anions (O2-) from human neutrophils stimulated with opsonized zymosan (OZ) and formyl-methionyl-leucyl-phenylalanine (FMLP), respectively. 2. Prostaglandin E2 (PGE2) and PGD2 inhibited both OZ-induced LTB4 release (EC50 0.72 microM and 0.91 microM respectively), and FMLP-induced O2- release (EC50 0.42 microM and 0.50 microM respectively). PGF2 alpha, the TP-receptor agonist, U46619, and the IP-receptor agonist, iloprost, were also active, but were all at least an order of magnitude less potent than PGE2 and PGD2. 3. The EP2/EP3-receptor agonist, misoprostol, and the selective EP2-agonist, AH13205, were both effective inhibitors of LTB4 release, being approximately equipotent with and 16-times less potent than PGE2, respectively. In contrast, the EP1/EP3-receptor agonist, sulprostone, had no inhibitory activity at concentrations of up to 10 microM. 4. The selective DP-receptor agonist, BW245C, inhibited LTB4 release, (EC50 0.006 microM) being approximately 50 times more potent than PGD2. BW245C also inhibited O2- release, and this inhibition was antagonized competitively by the DP-receptor blocking drug, AH6809 (pA2 6.6). 5. These data indicate the presence of both inhibitory EP- and DP-receptors on the human neutrophil. The rank order of potency of EP-receptor agonists suggest that the EP-receptors are of the EP2-subtype.     

Effects of dauricine on the metabolism of arachidonic acid in rat pleural neutrophils

The effects of dauricine (Dau), an isoquinoline alkaloid and anti-arrhythmic agent used in China recently, on the biosynthesis of metabolites of arachidonic acid in rat pleural neutrophils, comparing with dazoxiben, indomethacin and BW-755 c, were studied. The major products of metabolism by 5-lipoxygenase (5-LPO), measured by HPLC, were LTB4 and 5-HETE, whereas the major cyclooxygenase products measured by HPLC and RIA, were HHT and TXB2. The formation of all products by neutrophils was significantly depressed by Dau in a dose-dependent manner. The concentration of Dau required to obtain 50% inhibition (IC50) of formation of HHT, TXB2, LTB4 and 5-HETE was 25.3, 59.3, 31.8 and 59.5 mumol/L, respectively. These results indicate that the two major metabolic pathways of arachidonic acid in rat pleural neutrophils are inhibited by Dau.     

Effect of dexamethasone on neutrophil accumulation and oedema formation in rabbit skin: an investigation of site of action.

1. The anti-inflammatory actions of dexamethasone on vascular and leukocyte responses in rabbit skin were investigated. 2. Neutrophil accumulation and oedema formation were simultaneously measured as the local accumulation of i.v. administered 111In-labelled neutrophils and 125I-labelled albumin. Systemically administered dexamethasone (3 mg kg-1) inhibited neutrophil accumulation induced by i.d. zymosan activated plasma (ZAP), N-formyl-methionyl-leucyl-phenylalanine (FMLP) and leukotriene B4 (LTB4) when co-injected with prostaglandin E2 (PGE2). Dexamethasone also inhibited oedema formation elicited by these stimuli and the responses induced by i.d. platelet activating factor (PAF)+PGE2 and bradykinin (BK)+PGE2. 3. Intradermal dexamethasone (2 x 10(-10) mol per site) but not indomethacin (10(-8) mol per site) inhibited oedema formation induced by i.d. ZAP+PGE2 and BK+PGE2. This inhibitory effect of dexamethasone was significant only with pretreatment periods of 4 h, shorter pretreatment periods resulting in greatly reduced effects. Intradermal dexamethasone had no effect on neutrophil accumulation induced by ZAP+PGE2. 4. Intradermal dexamethasone (2 x 10(-10) mol per site) had no effect on increase in blood flow induced by PGE2 as measured by 133Xenon clearance. 5. The accumulation of neutrophils isolated from donor rabbits pretreated with i.v. saline or dexamethasone (3 mg kg-1) was investigated in untreated recipient rabbits. The accumulation of neutrophils, induced by ZAP+PGE2, FMLP+PGE2 and LTB4+PGE2, from dexamethasone-pretreated donors was significantly smaller than the accumulation of neutrophils from saline-pretreated donors. 6. The results of this study suggest that dexamethasone can have a direct effect on vascular endothelial cells resulting in an inhibition of oedema formation.(ABSTRACT TRUNCATED AT 250 WORDS)