Partial agonist effect of the platelet-activating factor receptor antagonists, WEB 2086 and WEB 2170, in the rat perfused heart.

1. WEB 2086 and WEB 2170 are potent platelet-activating factor (PAF) receptor antagonists and have been used widely as pharmacological tools to investigate the actions of PAF in a variety of biological systems. 2. Low concentrations of WEB 2086 and WEB 2170 blocked the vasoconstrictor action of PAF in the rat perfused heart. In this study, we observed that moderate concentrations of WEB 2086 and WEB 2170 increased the perfusion pressure in rat isolated hearts under constant flow perfusion. The vasoconstrictor actions of WEB 2086 and WEB 2170 were not observed with a structurally different PAF receptor antagonist, FR-900452. 3. To determine whether this vasoconstrictor action of WEB 2086 involved non-specific effects or was via the activation of PAF receptors, hearts were pretreated with 1000 pmol PAF or 50 microM FR-900452. These pretreatments attenuated the vasoconstrictor action of 1 microM WEB 2086, suggesting that the action of WEB 2086 may be mediated via PAF receptors. Pretreatment with the leukotriene receptor antagonist (L-649,923, 5 microM) and the leukotriene synthesis inhibitor (MK-886, 10 microM) that are known to block the vasoconstrictor action of PAF receptor activation also attenuated the vasoconstrictor action of WEB 2086. Pretreatment with PAF or MK-886 attenuated the vasoconstrictor action of 0.5 microM WEB 2170. 4. When PAF receptors were activated by PAF in the perfused heart, significant amounts of leukotriene C4 and leukotriene C4/D4/E4 were detected in the coronary effluent. However, no significant amount of these leukotrienes was detected in the coronary effluent when hearts were perfused with 1 microM WEB 2086 or 0.5 microM WEB 2170.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the actions of some platelet-activating factor antagonists on platelets and aortic smooth muscles.

The pharmacological actions of five platelet-activating factor (PAF) antagonists were compared in rabbit platelets and rat thoracic aorta. In PAF (2 ng/ml)-induced aggregation of washed rabbit platelets, WEB 2086 and WEB 2170 much were more potent inhibitors than BN 52021, kadsurenone and denudatin B, and the IC50 values were calculated to be 0.1, 0.3, 5, 8 and 10 micrograms/ml, respectively. WEB 2086, WEB 2170 and BN 52021 did not affect the platelet aggregation caused by collagen (10 micrograms/ml), ADP (20 microM), arachidonic acid (100 microM) or thrombin (0.1 U/ml). Kadsurenone and denudatin B suppressed ATP release, thromboxane B2 formation and the rise in intracellular calcium of washed rabbit platelets caused by collagen and thrombin, while WEB 2086, WEB 2170 and BN 52021 did not have an effect. Norepinephrine (3 microM) induced a sustained contraction in rat thoracic aorta. Pretreatment with these PAF antagonists (20-100 micrograms/ml) caused inhibition of the aortic contraction in the following order: kadsurenone greater than denudatin B greater than WEB 2086 greater than BN 52021 greater than WEB 2170. In high potassium (60 mM)-induced contraction of rat aorta, kadsurenone and denudatin B caused marked relaxation, while WEB 2086, WEB 2170 and BN 52021 had only a slight effect. It is concluded that WEB 2086, WEB 2170 and BN 52021 are specific PAF antagonists in rabbit platelets, and weak relaxants in rat aorta. Two other PAF antagonists, kadsurenone and denudatin B, may inhibit some aspects of signal transduction, e.g., thromboxane formation or intracellular Ca2+ mobilization in rabbit platelets, and cause vasorelaxation in rat aorta by inhibiting calcium influx.     

Antiinflammatory and antiallodynic actions of the lignan niranthin isolated from Phyllanthus amarus. Evidence for interaction with platelet activating factor receptor

Previous studies have shown that the extracts obtained from Phyllanthus amarus, and some of the lignans isolated from it, exhibit pronounced antiinflammatory properties. In the present study, we have assessed whether the antiinflammatory actions of these lignans can be mediated by interaction with platelet activating factor (PAF) receptor or interference with the action of this lipid. The local administration of nirtetralin, phyltetralin or niranthin (30 nmol/paw), similar to WEB2170 (a PAF receptor antagonist, 30 nmol/paw), significantly inhibited PAF-induced paw oedema formation in mice. The extracts of P. amarus (100 microg/ml) and niranthin (30 microM), but not nirtetralin or phyltetralin (30 microM), decreased the specific binding of [(3)H]-PAF in mouse cerebral cortex membranes. Furthermore, both niranthin and WEB2170 displaced, in a concentration-dependent manner, the [(3)H]-PAF binding sites. The mean IC(50) values from these effects were 6.5 microM and 0.3 microM, respectively. Additionally, both niranthin and WEB2170 (30 nmol/paw) inhibited the increase of myeloperoxidase activity induced by PAF injection in the mouse paw. When assessed the mouse model of pleurisy induced by PAF, pretreatment with niranthin (100 micromol/kg, p.o.) or WEB2170 (1.7 micromol/kg, i.p.) significantly inhibited PAF-induced protein extravasations. Moreover, in the rat model of PAF-induced allodynia, both niranthin (30 nmol/paw) and WEB2170 (30 nmol/paw) treatment significantly inhibited PAF-induced allodynia. In addition, niranthin had a rapid onset and long-lasting antiallodynic action when compared with WEB2170. Collectively, the present findings suggest that niranthin exhibits antiinflammatory and antiallodynic actions which are probably mediated through its direct antagonistic action on the PAF receptor binding sites.     

Mechanisms underlying the modulatory action of platelet activating factor (PAF) on the upregulation of kinin B1 receptors in the rat paw

1. The present study evaluated the ability of the administration of platelet activating factor (PAF) to induce the upregulation of B(1) receptors in the rat paw. 2. Local treatment with PAF resulted in a time-dependent increase of oedema formation induced by the B(1) receptor agonist des-Arg(9)-BK (des-Arg(9)-bradykinin), but not by the B(2) receptor agonist tyrosine(8)-bradykinin. Functional upregulation of B(1) receptors was accompanied by a prominent increase of B(1) receptor mRNA expression in the rat paw. 3. In PAF-treated paws, des-Arg(9)-BK-induced oedema formation was significantly inhibited by the B(1) receptor antagonists des-Arg(9)-[Leu(8)]-BK and R-715. The effects of PAF pretreatment were receptor operated, as assessed by the effects of the PAF receptor antagonist WEB2086 or by desensitisation of PAF receptors. 4. The protein synthesis inhibitor cycloheximide, the anti-inflammatory steroid dexamethasone or the nuclear factor (NF-kappaB) blockers pyrrolidine-dithiocarbamate (PDTC) and Nalpha-tosyl-L-chloromethylketone significantly blocked the functional upregulation of B(1) receptors. 5. The selectin inhibitor fucoidin, an anti-CD18 antibody or an anti-rat neutrophil antiserum, also significantly prevented des-Arg(9)-BK-induced paw oedema in rats pretreated with PAF. 6. Intradermal injection of PAF induced a 25-fold increase of myeloperoxidase activity in the rat paw, a response that was significantly inhibited by fucoidin, anti-CD-18, anti-rat neutrophil antiserum or PDTC. 7. Local treatment with PAF also resulted in a marked increase of NF-kappaB activation, an effect largely prevented by PDTC or by the anti-rat neutrophil antiserum. 8. Collectively, the present results indicate that the induction of B(1) receptors following treatment with the chemotatic mediator PAF is dependent on the recruitment of neutrophils, an event that is under the control of adhesion molecules, protein synthesis and NF-kappaB activation. These findings provide new insights into the role played by cell migration and chemotatic factors on B(1) receptor upregulation in vivo.     

血小板激活因子拮抗剂WEB2086对小鼠腹腔巨噬细胞释放肿瘤坏死因子的影响(英文)

小鼠ip3%TG 4天后取其腹腔巨噬细胞,血小板激活因子拮抗剂WEB 2086对LPS诱导的巨噬细胞释放肿瘤坏死因子(TNF)有显著的抑制作用。时效研究表明,TNF的产生和WEB 2086的抑制作用从LPS刺激后4 h开始,持续到22 h,在16 h时达到高峰。本文还首次采用一种用放线菌素D和NaF处理的L-929细胞测定TNF的新方法,此法与另外3种生物测定方法进行比较的结果显示此法具有更敏感、方便的特点。     

血小板激活因子拮抗剂WEB2086对小鼠腹腔巨噬细胞释放肿瘤坏死因子的影响(英文)

小鼠ip3%TG 4天后取其腹腔巨噬细胞,血小板激活因子拮抗剂WEB 2086对LPS诱导的巨噬细胞释放肿瘤坏死因子(TNF)有显著的抑制作用。时效研究表明,TNF的产生和WEB 2086的抑制作用从LPS刺激后4 h开始,持续到22 h,在16 h时达到高峰。本文还首次采用一种用放线菌素D和NaF处理的L-929细胞测定TNF的新方法,此法与另外3种生物测定方法进行比较的结果显示此法具有更敏感、方便的特点。     

Hypoxic pulmonary vasoconstriction in isolated blood-perfused rat lung; modulation by thromboxane A2, platelet-activating factor,cysteinyl leukotrienes and endothelin-1

Recent evidence suggests that hypoxic pulmonary vasoconstriction (HPV) is mediated by hypoxia-induced closure of voltage-gated potassium channels in pulmonary vascular smooth muscle cells. It is also claimed that various vasoconstrictor mediators such as thromboxane A2 (TXA2), platelet activating factor (PAF), cysteinyl leukotrienes (cys-LTs) or endothelin-1 (ET-1) contribute to HPV. Their role, however, has not been unequivocally accepted. On the contrary, it is well known that endothelium-derived nitric oxide negatively modulates HPV. Since NO counteracts action of vasoconstrictor mediators, we tested the hypothesis that modulatory role of TXA2 PAF, cys-LTs and ET-1 in HPV would become apparent in absence of endogenous NO. For that purpose we assessed contribution of these mediators to HPV in the isolated blood-perfused rat lung pretreated with a non-selective NOS inhibitor, L-NAME. HPV, which was greatly augmented by L-NAME (300 microM) alone, was inhibited neither by a TXA2 synthase inhibitor (Camonagrel, 300 pM), nor by a PAF receptor antagonist (WEB 2170, 100 microM), nor by an inhibitor of five-lipooxygenase-activating protein (MK 886, 10 microM), nor by a non-selective ET-1 receptor antagonist (LU 302872, 30 pM). In summary, in isolated blood-perfused rat lung, TXA2, PAF, cys-LTs and ET-1 seem not to be involved in HPV, whereas we confirm the dominant role of endogenous NO in blunting HPV.     

Effect of BN 52256 and other mediator antagonists on ouabain-induced cardiac arrhythmia in a model of anaphylaxis in guinea-pigs.

Different mediators released by anaphylaxis seem to be involved in different pathophysiological conditions, including cardiac arrhythmia. Histamine, 5-HT and platelet-activating factor (PAF) could participate in the enhanced arrhythmogenicity during anaphylaxis in guinea-pigs. The threshold dose of ouabain-induced arrhythmia is decreased in actively sensitized guinea-pigs by i.p. administration of ovalbumin. The purpose of the present paper was to investigate the effect of different mediator antagonists. Antagonists of PAF (WEB 2170), histamine (clemastine) and 5-HT (cyproheptadine) in doses of 5.0 mg/kg, 5.0 mg/kg and 0.5 mg/kg, respectively, can increase the threshold dose of ouabain-induced arrhythmias signalling an antiarrhythmic effect. A combination of WEB 2170 and clemastine, each of them in inactive doses (2.0 mg/kg and 1.0 mg/kg, respectively) showed a statistically significant antiarrhythmic effect. A combination of the same dose of WEB 2170 and cyproheptadine (0.1 mg/kg) under the same conditions induced an antiarrhythmic effect, too. BN 52256 is a new antiallergic drug synthesized on the basis of a novel concept of combining inhibitory activity against various inflammatory mediators in one molecule. BN 52256 in doses of 20-80 micrograms/kg exhibited a statistically significant antiarrhythmic effect. BN 52256 needed a 12.5-125 fold lower dose to induce the same antiarrhythmic effect compared to the antagonists of PAF, histamine or 5-HT investigated in this study. Depending on the pathophysiological conditions, different mediators seem to be involved in the occurrence of cardiac arrhythmia. A complex inhibition of these mediators could induce a more specific influence on such kinds of cardiac arrhythmias.     

Investigation of the effects of platelet-activating factor (PAF) on ion transport and prostaglandin synthesis in human colonic mucosa in vitro

1. We have investigated the effects of platelet-activating factor (PAF), an endogenous mediator of inflammation, on ion transport and prostaglandin synthesis in the human isolated colon.
2. Application of PAF to the serosal surface of human colonic mucosa induced a marked, concentration-dependent increase in ion transport. Mucosal application was without effect.
3. The secretory response to PAF was significantly inhibited by prior application of a specific PAF receptor antagonist WEB 2170, indicating that the response is dependent on PAF receptor activation.
4. The response to PAF was attenuated by prior application of indomethacin or piroxicam, implicating products of the cyclo-oxygenase pathway in the response.
5. The response to PAF was attenuated by the loop diuretic bumetanide, indicating an involvement of chloride ion secretion in the response.
6. Addition of PAF to the serosal surface induced a significant increase in serosal prostaglandin E₂ (PGE₂), but not 6-oxo-PGF_1α release. There was no effect on mucosal application of PAF.
7. In summary, we have shown that PAF is a potent secretagogue in isolated preparations of human colon and that the response is dependent on a specific PAF receptor, cyclo-oxygenase products and bumetanide-sensitive chloride ion transport.

     

Action of the racemate and the isomers of the platelet-activating factor antagonist bepafant (WEB 2170) after oral administration to guinea-pigs and rats

Summary The aim of the present study was to clarify whether there is a difference in terms of potency and pharmacodynamic half time between the isomers and the racemate of the platelet-activating factor antagonist WEB 2170 (bepafant) after oral administration to guineapigs or rats.The following experiments were performed in the guinea-pig. Infusion of platelet-activating factor at 30 ng/ (kg × min) for 30 min to anaesthetized guinea-pigs induced a decrease of respiratory flow and mean arterial blood pressure. Oral pretreatment with WEB 2170 or isomers, respectively, 60 min before infusion of plateletactivating factor inhibited these changes in a dose-dependent manner. The ED₅₀S for inhibition of respiratory flow were: (–) WEB 2170 = 0.018 (0.009–0.036) mg/kg p.o.; (±) WEB 2170 = 0.021 (0.015–0.03) mg/kg p. o.; (+) WEB 2170 = 1.55 (1.01–3.05) mg/kg p. o. Similar ED₅₀ values were obtained for inhibition of decrease of MAP. Doses of isomers and racemate of WEB 2170 that provided almost complete protection against platelet-activating factor at 1 h after administration were chosen for determination of the duration of the protective effect after oral administration. Oral (–) WEB 2170 or (±) WEB 2170 showed almost identical time-response curves for inhibition (t_1/ = 14–17 h; 0.1 mg/kg p. o.) in the guinea-pig, whereas the duration of action of (+) WEB 2170 (3–6 h; 8 mg/kg) was significantly shorter. The following experiments were conducted in the rat. Oral pretreatment with WEB 2170 racemate and isomers resulted in a dose-dependent reversal of plateletactivating factor-induced hypotension. The (–) isomer was more potent than the racemate, whereas the (+) isomer had significantly less platelet-activating factorantagonistic activity. The duration of action of the isomers did not significantly differ from that of the racemate under the experimental conditions used.In conclusion, after oral administration to guinea-pigs there is no relevant difference between the (–) isomer and the racemate of WEB 2170 with respect to their potency to inhibit platelet-activating factor or to their duration of action. In contrast the (+) isomer of WEB 2170 is significantly less potent as a platelet-activating factor-antagonist, with a shorter duration of action. The same applies in principle to the rat, except that (–) WEB 2170 was significantly more potent and there was no difference between isomers and racemate in the duration of action. Send offprint requests to H. O. Heuer at the above address     

Platelet-activating factor increases mucosal permeability in rat intestine via tyrosine phosphorylation of E-cadherin

1. Platelet-activating factor (PAF), an inflammatory mediator, plays an important role in mediating intestinal injury. However, it remains unclear whether PAF has a function in the intestine. The production of PAF by normal intestine and by unstimulated intestinal epithelial cell lines suggests that PAF may have a regulatory function in the normal bowel. 2. In this study we investigated the role of PAF in modulating intestinal mucosal permeability in rats. Lumen-to-blood transit of FD-4 (dextran 4400), (an index of intestinal permeability), was assessed in sham-operated rats and rats injected with PAF (1.25 microg kg(-1), i.v., a dose insufficient to induce intestinal injury). 3. PAF-induced villus cytoskeletal changes were examined by staining the intestine for F-actin. The effect of PAF on tyrosine phosphorylation of the junctional protein E-cadherin was examined by immunoprecipitation. Some rats were pretreated with AG1288 (a tyrosine kinase inhibitor) before PAF injection, and mucosal permeability change was assessed. 4. To investigate the role of endogenous PAF upon mucosal permeability, we studied the effect of PAF antagonists on (intraluminal) glucose-induced increase in mucosal permeability. 5. We found that low dose PAF: (a) alters the cytoskeletal structure of intestinal epithelium, (b) causes the influx of FD4 from intestinal lumen to systemic circulation, (c) induces tyrosine phosphorylation of E-cadherin and cadherin-associated proteins. Glucose-induced mucosal permeability increase is abolished by using two structurally different PAF antagonists. 6. These results suggest that endogenous PAF modulates macromolecular movement across the intestinal mucosal barrier, probably via tyrosine phosphorylation of E-cadherin and cytoskeletal alteration of enterocytes.     

Platelet-activating factor antagonists in experimental shock.

Platelet-activating factor (PAF) at 10 micrograms/kg i.v. induced profound hyperkalemia, changes of hematological parameters, patterns of ECG, and acid-base balance in rats. In separate experiments infusions of PAF at 30 ng/kg/min or injection of endotoxin from E. coli (15 mg/kg i.v.) induced a marked drop in blood pressure. All these changes were antagonized by pretreatment or post-treatment (in case of hypotension) by the selective hetrazepinoic PAF antagonists apafant (WEB 2086, CAS 105219-56-5; 0.1-5 mg/kg i.v.), bepafant (WEB 2170, CAS 114776-28-2; 0.05-1.0 mg/kg i.v.), or STY 2108 (0.01-0.1 mg/kg), respectively. The results support the view that PAF can mimic features of endotoxin shock and that the hetrazepines (like apafant, bepafant) are useful tools to clarify the role of PAF in such conditions.     

Antagonism of platelet-activating factor-induced increase in cytosolic free calcium concentration in human endothelial cells.

The effects of platelet-activating factor (PAF) antagonists on the agonist-induced increase in cytosolic free calcium concentration, [Ca2+]i, in human vascular endothelial cells grown in monolayer were investigated by a continuous superfusion technique using a calcium fluorescent probe, fura-2. PAF caused a small but dose-dependent increase in [Ca2+]i. Seven structurally dissimilar PAF antagonists dose-dependently suppressed the peak response, among which WEB 2086 was the most potent, followed by WEB 2170 greater than FR 900452 not equal to ONO 6240 greater than BN 52021 not equal to kadsurenone not equal to CV 3988. These antagonists except for CV 3988 were specific for PAF, since they had no effects on calcium mobilization induced by thrombin or histamine, while CV 3988 had a non-specific effect. PAF in the same range of concentration increased prostacyclin release from human endothelial cells. WEB 2086 also inhibited the PAF-induced prostacyclin release, while it had not effect on the release induced by histamine and thrombin. These results demonstrate the specificity and dose-response characteristics of PAF antagonists in cultured human endothelial cells and suggest that a PAF antagonist could be a valuable therapeutic agent in certain human diseases where PAF activation of endothelial cells may have a critical role.     

银杏内酯B对脂多糖刺激的小鼠腹腔巨噬细胞TNFα生成及大鼠胸腔多形核白细胞NF-κB活化的影响

目的研究银杏内酯B对脂多糖刺激的小鼠腹腔巨噬细胞TNFα生成及大鼠胸腔多形核白细胞NF-κB活化的影响。方法用L929细胞结晶紫染色法检测TNFα的含量，用电泳迁移率改变检测法检测NF-κB的结合活性。结果1和10 μmol·L^-1银杏内酯B能够显著抑制LPS刺激的小鼠腹腔巨噬细胞TNFα的生成，其IC₅₀为0.26 μmol·L^-1；1 mg·L^-1 LPS和1 nmol·L^-1 PAF均可活化大鼠胸腔多形核白细胞NF-κB；银杏内酯B能够抑制LPS或 PAF刺激的NF-κB活化。结论银杏内酯B能够抑制LPS刺激的小鼠腹腔巨噬细胞TNFα生成及大鼠胸腔多形核白细胞NF-κB的活化。PAF参与LPS激活NF-κB的过程。     

Antagonists of PAF and histamine inhibit ouabain-induced cardiac arrhythmias in sensitized guinea-pigs

Platelet-activating factor (PAF) seems to be involved in different pathophysiological conditions, including cardiac arrhythmia. The arrhythmogenic potency of PAF has been shown experimentally by different methods. PAF antagonists inhibit the PAF-induced enhanced arrhythmogenicity. The present paper demonstrates that the threshold dose of ouabain-induced arrhythmia is decreased in sensitized guinea-pigs. Antagonists of PAF (BN 52021, WEB 2086, WEB 2170) and histamine antagonist clemastine can increase the threshold dose of ouabain-induced arrhythmia. A combination of WEB 2170 and clemastine, each of the drug is a low dose which is without effect when applying one of them only, shows a highly significant antiarrhythmic effect in this method. The threshold dose of ouabain necessary to induce ventricular flutter was increased from 89 micrograms/kg to 129 micrograms/kg and the threshold dose of ventricular fibrillation was enhanced from 101 micrograms/kg to 137 micrograms/kg. In dependence on the pathophysiological conditions, different mediators seem to be involved in the occurrence of cardiac arrhythmia. Therapeutically influencing these mediators could be a real chance to optimise the treatment of such kind of cardiac arrhythmias.     

PAF activation of a voltage-gated R-type Ca2+ channel in human and canine aortic endothelial cells.

By the use of fura-2 and digital imaging techniques, [K]o depolarization or PAF (10(-9) M) were shown to induce a sustained increase of [Ca]i in human or canine single aortic vascular endothelial cells (VEC) that was insensitive to nifedipine but sensitive to (-)-PN200-110 or to lowering of [Ca]o. The PAF-induced effect on [Ca]i was blocked by the PAF receptor antagonist, WEB2170. Our results suggest that [K]o depolarization and PAF increase [Ca]i via the activation of R-type Ca2+ channels.