PAF-acether Antagonists Research Articles

ChemInform Abstract: 4-Alkyl-1,4-dihydropyridines Derivatives as Specific PAF-Acether Antagonists.

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Bacillus pasteurii urease shares with plant ureases the ability to induce aggregation of blood platelets

Ureases (EC 3.5.1.5) are highly homologous enzymes found in plants, bacteria and fungi. Canatoxin, an isoform Canavalia ensiformis urease, has several biological properties unrelated to its ureolytic activity, like platelet-aggregating and pro-inflammatory effects. Here, we describe that Bacillus pasteurii urease (BPU) also induces aggregation of rabbit platelets, similar to the canatoxin-induced effect (ED 50 0.4 and 0.015 mg/mL, respectively). BPU induced-aggregation was blocked in platelets pretreated with dexamethasone and esculetin, a phospholipase A 2 and a lipoxygenase inhibitor, respectively, while platelets treated with indomethacin, a cyclooxygenase inhibitor, showed increased response to BPU. Methoxyverapamil (Ca 2+ channel blocker) and AMP (ADP antagonist) abrogated urease-induced aggregation, whereas the PAF-acether antagonist Web2170 had no effect. We concluded that platelet aggregation induced by BPU is mediated by lipoxygenase-derived eicosanoids and secretion of ADP from the platelets through a calcium-dependent mechanism. Potential relevance of these findings for bacterium–plant interactions and pathogenesis of bacterial infections are discussed.

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.

Autocrine amplification of PAF-acether formation in immunologically activated murine macrophages

When murine macrophages activated in vivo with bacille Calmette-Guérin were triggered with either acetyl-CoA or propionyl-CoA to form PAF-acether (PAF), similar amounts of platelet-aggregating product were recovered. Liquid chromatographic purification and reversed-phase analysis showed that the composition of PAF molecular species formed in the presence of acetyl-CoA was an equimolar mixture of PAF bearing C16:0 alkyl chain (57% +/- 7, mean +/- SD, n = 3) and PAF C18:1. The PAF-like material obtained from the propionyl-CoA-supplemented macrophages was a mixture of the propionyl analogue of PAF (66% +/- 11, n = 3) and native PAF. The rate of lyso-PAF:acetyl-CoA acetyltransferase (EC 2.3.1.67) reaction in a macrophage lysate was similar for either substrate in the presence of an equimolar mixture of propionyl-CoA and acetyl-CoA. We conclude that the exogenously added propionyl-CoA is transferred to lyso-PAF acceptor to form propionyl-PAF by the PAF-forming acetyltransferase. Propionyl-PAF triggers the formation of native PAF probably from the endogenous acetyl-CoA pool. Two specific PAF antagonists, BN 52021 (60 microM) and WEB 2086 (3 microM), did not influence the rate of PAF synthesis in the presence of either acetyl-CoA or propionyl-CoA and did not prevent native PAF formation when propionyl-CoA was added alone, suggesting that the classical PAF receptors are not involved. This is the first description of a possible mechanism of autocrine amplification of PAF biosynthesis in macrophages.

Variations of blood PAF-acether levels during coronary artery surgery

Extracorporeal circulation (ECC) is associated with thrombocytopenia and transient leukopenia. After ECC and coronary artery bypass graft (CABG) surgery, some patients can develop pulmonary and cardiac dysfunction, which might be related to the release of various mediators such as thromboxane A 2, C 5a, and C 3a anaphylatoxins. The involvement of PAF-acether (PAF), a potent vasoactive thrombocytopenic and leukoneutropenic agent, has not been determined. Therefore, 10 patients were studied during and after CABG. The release of PAF, lipo PAF (PAF bound to blood lipoproteins), and lypo PAF (PAF precursor and metabolite) was measured in blood from the left atrium, radial artery, and pulmonary artery before and after CABG. PAF, lipo PAF, and lyso PAF were also determined during ECC at the entry and exit points of the oxygenator. Hemodynamic parameters, platelet, and leukocyte counts in the pulmonary artery were measured simultaneously. PAF did not increase significantly during ECC; it showed a transitory six-fold increase immediately after CABG in the radial artery (0.18 ± 0.13 v 1.09 ±0.36 ng/ mL, P < 0.05), but not in the pulmonary artery (0.10 ± 0.03 v 0.56 ± 0.21 ng/mL, P > 0.05). Blood PAF amounts in the radial artery were significantly higher than in the left atrium following ECC (1.09 ± 0.36 v 0.06 ± 0.04, P < 0.05), probably indicating PAF production in the heart. No variation of blood lipo PAF and lyso PAF was observed. No correlation was seen between PAF amounts and blood cell count. The positive correlation observed between PAF levels in the left atrium and the pulmonary arterial pressure (r = 0.80, P < 0.001) strengthens the putative physiologic role of this agent in pulmonary artery hypertension. The clinical use of PAF antagonists may help to determine PAF involvement in human physiology and pathology.

Neutralization of the oedematogenic activity of Bothrops jararaca venom on the mouse paw by an antibothropic fraction isolated from opossum (Didelphis marsupialis) serum.

The pharmacological modulation of mice paw oedema produced by Bothrops jararaca venom (BJV) has been studied. Intraplantar injection of BJV (1-30 micrograms/paw) produced a dose- and time-related oedema, which was maximal 30 min after injection, reduced gradually thereafter and disappeared over 48 h. BJV heated at 100 degrees C for 5 or 15 min blocked local hemorrhage and caused partial inhibition of its oedematogenic activity. The BJV oedema was not inhibited by the anti-histamine meclizine, the inhibitor of histamine and serotonin, cyproheptadine, PAF-acether antagonist WEB 2170 or by the anti-leukotrienes C4/D4, LY 171883. Dexamethasone, aspirin, indomethacin, and the dual cyclooxygenase and lipoxygenase inhibitor BW 755C inhibited BJV-induced oedema indicating that arachidonic acid metabolism products via the cyclooxygenase pathway participate in its genesis and/or maintenance. The antibothropic fraction (ABF) (25-200 micrograms/paw) isolated from Didelphis marsupialis serum neutralized the oedema induced by the venom with and without heating, the hemorrhage induced by BJV and partially blocked the oedema induced by bradykinin and by cellulose sulphate. The oedema produced by histamine, serotonin, PAF-acether or leukotriene C4 was not inhibited.

PAF-acether and acetylhydrolase in stool of patients with Crohn's disease

PAF-acether (PAF) is a phospholipid mediator with potent biological effects on the digestive tract. We report the presence of PAF in stool of patients with active Crohn's disease (39.1 +/- 13.5 ng/g of stool, mean +/- SEM, N = 19) and its absence in patients with irritable bowel syndrome with diarrhea and diarrhea with malabsorption. Fecal PAF acetylhydrolase activity was higher (P less than 0.04) in patients with Crohn's disease as compared to patients with irritable bowel syndrome with diarrhea and diarrhea with malabsorption. We also report a solid-phase extraction of fecal PAF using silica minicolumns, which yielded results highly correlated with those obtained with a high-performance liquid chromatography method (r = 0.86, P less than 0.001, N = 16). These findings may allow us to implicate PAF in the onset and perpetuation of digestive tract inflammatory symptoms observed during Crohn's disease. They would warrant to investigate the influence of various therapeutic agents, including PAF antagonists, on fecal PAF levels during inflammatory digestive ailments.

Increase in the rat blood leukocyte counts induced by PAF-acether is suppressed by general anesthesia

Blood leukocyte count alterations induced by PAF-acether in anesthetized and nonanesthetized rats were investigated. Intravenous injection of increasing amounts of PAF-acether (1.5-8 micrograms/kg) in nonanesthetized animals induced dose-dependent hemoconcentration and leukocytosis. The former was apparent within 10 min, peaked from 30 min to 1 h, and diminished thereafter. The leukocytosis was noted within 30 min, was maximal at 1 h, and was over 4 h after injection of PAF-acether (4 micrograms/kg). It was characterized by a marked increase in the blood neutrophil counts under conditions in which the number of lymphocytes, monocytes, and eosinophils remained unchanged. PAF-acether-induced leukocytosis occurred in parallel with a marked decrease in the number of bone marrow nucleated cells, suggesting that the latter phenomenon may determine the former one. Leukocytosis by PAF-acether was inhibited dose-dependently by specific PAF-acether antagonists including BN 52021 (median effective dose ED50 = 4.99 mg/kg), WEB 2086 (ED50 = 4.59 mg/kg), and 48740 RP (ED50 = 9.02 mg/kg). General anesthesia by either pentobarbital, urethane, or ether inhalation, but not by ketamine, also impaired the PAF-acether-induced blood leukocytosis under conditions in which the hemoconcentration was not modified. In addition, pentobarbital-anesthetized rats did not have reduced bone marrow nucleated cell counts after PAF-acether stimulation. These findings are consistent with the assessment that PAF-acether-induced rat leukocytosis is accounted for by a bone marrow neutrophil mobilization process that is clearly suppressed in animals anesthetized by pentobarbital.

Pharmacological modulation of the late eosinophilia induced by antigen in actively sensitized rats.

The intrathoracic injection of ovalbumin (12 micrograms/cavity) into actively sensitized rats led to a long-lasting eosinophil recruitment, which appeared 24 h after stimulation. In this study, pharmacological antagonists were used in order to evaluate the potential involvement of arachidonic acid metabolites and PAF-acether in the pleural eosinophil accumulation by antigen. Administration of the cyclooxygenase inhibitor indomethacin (2 mg/kg, i.p.), 1 h before the antigen challenge, failed to modify the 24-hour eosinophilia. In contrast, the dual cyclooxygenase and lipoxygenase inhibitor BW 755C and the more selective inhibitor BW A4C (5 and 10 micrograms/cavity, i.t.), injected 1 h before the antigen, were effective. Similarly, the PAF-acether antagonists BN 52021 and WEB 2086 (20 mg/kg, i.p.) abrogated the eosinophil accumulation, which was also sensitive to the topical treatment with the glucocorticoid dexamethasone (5 and 10 micrograms/cavity). Our findings suggest that the antigen-induced eosinophil mobilization is dependent on lipoxygenase derivatives and PAF-acether, but not on prostaglandins.

Production of cytokines by monocytes stimulated with gram positive bacteria: [formula omitted] University of Messina, I-98100 Messina, Italy

The intraplantar injection of PAF-acether (PAF), induced acute oedema in the rat paw, and desensitized it to subsequent challenges with the same agonist, but not to serotonin. The desensitization was maximal (up to 80% of initial response) after seven consecutive daily injections. In this condition, PAF-induced oedema of the contralateral paw was maintained. The analogue 2-methyl carbamate-PAF (2MC-PAF) was more effective than PAF as a desensitizing agent. Furthermore, the PAF-desensitized paw was refractory to challenges with 2-MC and vice-versa. PAF-acether, but not serotonin-induced rat paw oedema was inhibited by previous intravenous injection of PAF. Intravenous injections of serotonin were also effective in inhibiting selectively serotonin-induced paw oedema, but it was not possible to induce desensitization by repeated intraplantar injections of serotonin. Desensitization to PAF or the pre-treatment with the PAF antagonist BN 52021 did not block the edematogenic response induced by carrageenan.

Enterolobin induces rat paw oedema independently of PAF-acether

The potential participation of PAF-acether (PAF) on the paw oedema triggered by enterolobin was investigated. Intraplantar injections of enterolobin (5-20 micrograms/paw) yielded a dose response curve for oedema which appeared after 30 min, peaked in the interval between 2-4 h and faded after 24 h. The pre-treatment with BN 52021, but not with other PAF antagonists such as PCA 4248 or WEB 2086, significantly blocked enterolobin-induced oedema. To clarify better the discrepant results obtained with the PAF antagonists, desensitization to PAF was performed. The oedema triggered by enterolobin was not modified in PAF desensitized animals. It was concluded that the paw inflammation induced by enterolobin does not require PAF mechanism.

LTB4, a potent chemotactic factor for purified guinea-pig eosinophils: Interference of PAF-acether antagonists

Two populations of eosinophils were separated upon a discontinuous metrizamide gradient from peritoneal lavages of polymyxin B-treated guinea-pigs. One population was of low density (between 20 and 22% of metrizamide, purity: 63 ± 3%, n = 27) and another of normal density (between 22 and 24% of metrizamide, purity: 87 ± 2%, n = 26). Responses to chemotactic stimuli were studied using a micro-Boyden chamber, results being expressed as the chemotactic index (CI, mean ± S.E.M.), i.e. the ratio between the number of eosinophils migrating at 40 μm through a cellulose nitrate filter in the presence of the agonist and the number of cells migrating in the presence of the solvent alone. The normal density eosinophils responded more to LTB4 (CI = 19.4 ± 4.6 with LTB4 10 −8M; P<0.05; n = 9) than to PAF-acether (CI = 6.2 ± 1.4 with PAF-acether 10 −8M; P<0.05; n = 20). By contrast, low density eosinophils responded less intensely to LTB4 (CI = 7.6 ± 1.8 with LTB4 10 −8M; P<0.01; n = 6) and to PAF-acether (CI = 2.4 ± 1.4 with PAF-acether 10 −8M; P<0.05; n = 14). Guinea-pig eosinophils failed to migrate in response to FMLP and lyso PAF-acether. Migration induced by PAF-acether was inhibited by the PAF-acether antagonists BN 52021 (9 ± 4 vs 45 ± 7 with 100 μM BN 52021 against 10 nM PAF-acether; P<0.05, n = 4, 20 min exposure, number of migrating eosinophils in presence of inhibitor vs number of migrating eosinophils in presence of solvent alone) or WEB 2086 (24 ± 7 vs 53 ± 12 with 0.1 μM WEB 2086 against 100 nM PAF-acether, P<0.05, n = 4, 20 min exposure). WEB 2086 also reduced the migration induced by LTB4 (52 ± 14 vs 80 ± 18 with 0.1 μM WEB 2086 against 10 nM LTB4; P<0.05, n = 4, 20 min exposure). Our study demonstrated that LTB4 and PAF-acether, to a lesser degree, are potent chemoattractant factors on a pure guinea-pig eosinophil preparation. In contrast to their efficacy on normal density eosinophils, LTB4 and PAF-acether were very poorly effective on low density eosinophils. Cross-desensitization studies showed that PAF-acether does not mediate the migration of eosinophils induced by LTB4.

Bullous Pemphigoid Antigen Expression in Pam 212 Cells Induced by the Addition of Platelet Activating Factor

Platelet activating factor (Paf-acether) is a phospholipid which has various activities including platelet and neutrophil aggregation and eosinophil chemotaxis. We have previously reported that the blister fluids of bullous pemphigoid possess platelet aggregation activity and suggested that Paf-acether might be concerned with the accumulation and activation of neutrophils and eosinophils. In this study, we examined the influence of Paf-acether on BP antigen expression on Pam 212 cells. Paf-acether enhanced the expression of BP antigen on Pam 212 cells and this expression was blocked by Paf-acether antagonist. Our observations might suggest that Paf-acether contributes to the blister formation not only by the activation of inflammatory cells but the enhancement of BP antigen.

4-Alkyl-1,4-dihydropyridine derivatives as specific PAF-acether antagonists

A new series of 4-alkyl-1,4-dihydropyridines (1,4-DHP) were synthesized and evaluated for their ability to inhibit washed rabbit platelet aggregation induced by PAF-acether (1-O-hexadecyl/octadecyl-2-O-acetyl-sn-glycero-3-phosphorylcholine) and to reverse PAF-induced hypotension in anesthetized rats. Additionally, compounds were evaluated for their ability to inhibit the binding of radiolabeled PAF to its receptor on rabbit platelets. Among these compounds, 6I and 6L were the most potent and specific antagonists. At concentrations up to 100 microM, neither compound 6I nor compound 6L caused platelet aggregation nor did they inhibit platelet aggregation induced by collagen or adenosine diphosphate. Compound 6L did not show in vitro calcium channel blocker activity measured on vascular smooth muscle preparations of rabbit aorta and on [3H]nitrendipine binding assays. The compound did not show any cardiovascular effects in anesthetized rat at iv doses up to 1000 micrograms/kg, and the Ki value was 568.62 nmol. These results indicate that compound 6L is a potent and specific PAF antagonist with 1,4-dihydropyridine structure but devoid of a significant cardiovascular activity related to calcium-antagonist properties.

Distinct stimulatory effect of platelet-activating factor on prostaglandin I 2 and thromboxane A 2 biosynthesis by rat dental pulp

Platelet-activating factor (PAF-acether), but not lyso PAF, stimulated the production of both PGI 2 and TXA 2 by rat dental pulp tissue in vitro. However, there were differences in the dose- and time-dependence of the stimulatory effects. PAF-acether antagonists, BN 52021, CV 3988 and kadsurenone, dose dependently inhibited PAF-acether-induced PG production. BN 52021 and CV 3988 also dose dependently inhibited TX production, but kadsurenone was almost without effect on TX production. Pretreatment of the tissues with PAF-acether or phorbol 12-myristate 13-acetate completely abolished the effect of the second challenge with PAF-acether. The stimulatory effects of PAF-acether and the calcium ionophore A23187 on PGI 2 production were completely blocked by removal of extracellular calcium, whereas the effects on TXA 2 production were not. TMB-8, an intracellular calcium antagonist, completely inhibited PAF-acether-induced PG production, whereas it slightly inhibited TX production. H-7, a protein kinase C inhibitor, and neomycin, a phospholipase C inhibitor, completely inhibited PAF-acether-induced PG and TX production, whereas W-7, a calmodulin inhibitor, did not. These results suggest that PAF-acether stimulates PGI 2 and TXA 2 production in rat dental pulp by interacting with distinct PAF-acether receptors, and that these receptors are coupled to independent signal transduction pathways which have a different dependence on extra- and intracellular calcium.

Platelet activating factor-induced clinical and histopathologic responses in atopic skin and their modification by the platelet activating factor antagonist BN52063

The clinical and histopathologic responses to intradermal platelet-activating factor (PAF-acether) in atopic subjects, without evidence of atopic dermatitis are documented. An immediate acute wheal and flare reaction was observed in all volunteers. Histopathologically, the reaction was characterized by a predominantly neutrophilic response, which was seen at 30 minutes and was maximal at 4 hours. Eosinophils were observed in the infiltrate as early as 30 minutes after injection, and were maximal by 12 hours. The specific PAF-acether antagonist BN52063 antagonized the acute flare response to intradermal PAF-acether but had little effect on cellular recruitment at the site of injection.

The problem of the recognition and coding of xenobiotics images by organism: pharmacological aspects

Two populations of eosinophils were separated upon a discontinuous metrizamide gradient from peritoneal lavages of polymyxin B-treated guinea-pigs. One population was of low density (between 20 and 22% of metrizamide, purity: 63 ± 3%, n = 27) and another of normal density (between 22 and 24% of metrizamide, purity: 87 ± 2%, n = 26). Responses to chemotactic stimuli were studied using a micro-Boyden chamber, results being expressed as the chemotactic index (CI, mean ± S.E.M.), i.e. the ratio between the number of eosinophils migrating at 40 μm through a cellulose nitrate filter in the presence of the agonist and the number of cells migrating in the presence of the solvent alone. The normal density eosinophils responded more to LTB4 (CI = 19.4 ± 4.6 with LTB4 10−8M; P<0.05; n = 9) than to PAF-acether (CI = 6.2 ± 1.4 with PAF-acether 10−8M; P<0.05; n = 20). By contrast, low density eosinophils responded less intensely to LTB4 (CI = 7.6 ± 1.8 with LTB4 10−8M; P<0.01; n = 6) and to PAF-acether (CI = 2.4 ± 1.4 with PAF-acether 10−8M; P<0.05; n = 14). Guinea-pig eosinophils failed to migrate in response to FMLP and lyso PAF-acether. Migration induced by PAF-acether was inhibited by the PAF-acether antagonists BN 52021 (9 ± 4 vs 45 ± 7 with 100 μM BN 52021 against 10 nM PAF-acether; P<0.05, n = 4, 20 min exposure, number of migrating eosinophils in presence of inhibitor vs number of migrating eosinophils in presence of solvent alone) or WEB 2086 (24 ± 7 vs 53 ± 12 with 0.1 μM WEB 2086 against 100 nM PAF-acether, P<0.05, n = 4, 20 min exposure). WEB 2086 also reduced the migration induced by LTB4 (52 ± 14 vs 80 ± 18 with 0.1 μM WEB 2086 against 10 nM LTB4; P<0.05, n = 4, 20 min exposure).Our study demonstrated that LTB4 and PAF-acether, to a lesser degree, are potent chemoattractant factors on a pure guinea-pig eosinophil preparation. In contrast to their efficacy on normal density eosinophils, LTB4 and PAF-acether were very poorly effective on low density eosinophils. Cross-desensitization studies showed that PAF-acether does not mediate the migration of eosinophils induced by LTB4.

Effect of platelet-activating factor on hypothalamic and hypophyseal pro-opiomelanocortin-related peptides and hypothalamo-pituitary-adrenal axis in the rat

To examine the effect of platelet-activating factor (PAF-acether) on pro-opiomelanocortin (POMC)-related peptides and on the hypothalamo-pituitary-adrenal axis, we administered PAF-acether and BN 52021, a selective PAF-acether antagonist, to freely moving rats. Minipumps loaded with either PAF-acether (30 μg/kg) or the vehicle alone were connected to the jugular vein for 7 days and positioned under the back skin of rats. A group of animals treated with PAF-acether also received 15 mg/kg of BN 52021 orally twice a day. In vivo treatment with PAF-acether alone or in association with BN 52021 did not affect the hypothalamic concentrations of corticotropin-releasing factor (CRF), α-melanocyte-stimulating hormone (α-MSH) and β-endorphin. Using a perifusion system for rat hypothalamic slices, we did not observe any effect of PAF-acether on spontaneous or potassium-induced release of α-MSH in vitro. In addition, treatment of rats with PAF-acether alone or in association with BN 52021 did not modify the α-MSH or β-endorphin concentration in the neurointermediate lobe of the pituitary. In contrast, in vivo administration of PAF-acether caused a significant reduction of ACTH concentration in the anterior lobe of the pituitary and a marked decrease in the corticosterone level in plasma and adrenal glands. The inhibitory effect of PAF-acether was reversed by concomitant administration of BN 52021. The ineffectiveness of PAF-acether to modulate in vitro ACTH release from perifused anterior pituitary fragments ruled out a direct effect of PAF-acether on corticotrophs. These findings support the view that PAF-acether exerts a specific inhibitory effect on the hypothalamo-pituitary-adrenal axis.

Eosinophil Accumulation in the Rat Pleural Cavity after Mast Cell Stimulation with Compound 48/80 Involves Protein Synthesis and Is Selectively Suppressed by Dexamethasone

Alterations in the local mast cell population and the eosinophil accumulation in the rat pleural cavity were studied using pleurisy induced by compound 48/80, a standard mast-cell-degranulating agent. Twenty-four hours after the intrathoracic injection of compound 48/80 (1-50 micrograms/cavity), a dose-dependent eosinophil enrichment of the exudate was noted, concomitantly with a drastic reduction in the total number of undamaged mast cells recovered from the pleural washing. At 24 h, neutrophil counts were not modified, and the number of mononuclear cells was increased, but only at the highest dose of compound 48/80. The temporal analysis showed that mast cell degranulation, exudation and neutrophil infiltration were maximal at the interval of 1-6 h after compound 48/80 (25 micrograms/cavity), whereas eosinophil accumulation peaked within 24 h, persisting elevated at least until 96 h. Since compound 48/80 was itself unable to induce eosinophil migration in vitro, attempts were made to investigate the potential involvement of recognized eosinophil chemo-attractants, such as histamine, leukotriene B4 (LTB4) and platelet-activating factor (PAF-acether). The intraperitoneal pretreatment with either cyproheptadine (2 mg/kg), meclizine (40 mg/kg), BW755C (25 mg/kg) or with the PAF-acether receptor antagonist WEB 2086 (20 mg/kg) had no effect on the eosinophil recruitment induced by compound 48/80 (25 micrograms/cavity). However, the treatment with the corticosteroid dexamethasone or the local inhibition of protein biosynthesis with cycloheximide (0.04-200 nmol/cavity) blocked the eosinophil pleural accumulation, but not the mast cell degranulation induced by compound 48/80. Our findings indicate that the pleural eosinophil accumulation induced by compound 48/80 is sensitive to dexamethasone, requires local protein biosynthesis and is independent of histamine, LTB4 and PAF-acether.

Effects of a PAF‐antagonist (BN 52063) on bronchoconstriction and platelet activation during exercise induced asthma.

1. The effects of a specific PAF acether antagonist (BN 52063) on the response to isocapnic hyperventilation with dry cold air (ISH study) and exercise (EIA study) were assessed in a single dose and short term treatment study in 10 patients with exercise induced asthma. 2. ISH challenge was performed twice within 1 h after administration of either placebo, 240 mg BN 52063 p.o. or inhalation of 2.4 mg BN 52063. Hyperventilation increased Raw from 0.30 +/- 0.02 to 0.89 kPa s l-1 (P less than 0.001) after the first challenge and from 0.28 +/- 0.04 to 0.84 +/- 0.06 kPa s l-1 (P less than 0.001) after the second challenge. Oral pretreatment with BN 52063 did not result in a reduction of bronchoconstriction during both challenges. A significant increase of Raw was noted immediately after inhalation of BN 52063. An inhibition of PAF induced platelet aggregation (by a factor of 2) occurred after oral administration of BN 52063 after both ISH challenges (P less than 0.05). No significant inhibition of PAF induced platelet aggregation was seen after inhalation of BN 52063. At concentrations up to 30 microM in vitro, BN 52063 inhibited PAF induced platelet aggregation in a dose dependent manner. The IC50 of BN 52063 against the aggregating effect of 1 microM PAF was 7.0 +/- 2.1 microM. 3. In the EIA study the patients were challenged on the third day of treatment with either placebo or 240 mg BN 52063 p.o. or 5 mg BN 52063 by inhalation. Peak expiratory flow rates (PEFR) fell by 155 +/- 37 1 min-1 after exercise.(ABSTRACT TRUNCATED AT 250 WORDS)