Protective effect of WEB 2086, a novel antagonist of platelet activating factor, in endotoxin shock

WEB 2086, a novel specific platelet activating factor (PAF) antagonist derived from triazolodiazepines, inhibited in a dose-related manner the hypotensive and lethal effect of PAF as well as of E. coli endotoxin in the rat. The hypotension induced by endotoxin (15 mg/kg i.v.) or PAF (30 ng/(kg X min) i.v.) in anaesthetized rats was prevented by oral (1-10 mg/kg) and inhibited or reversed by i.v. (0.1-5.0 mg/kg or 0.1-1.0 mg/kg) doses of WEB 2086. Similar oral and i.v. doses of WEB 2086 protected conscious rats from PAF (15 micrograms/kg i.v.)- and endotoxin (7.5 mg/kg i.v.)-induced death. The results obtained with WEB 2086 confirm that PAF has an important role in the pathophysiology of endotoxin shock. This compound may have a therapeutic effect in human septic shock.     

Effects of WEB 2086, a novel antagonist of platelet activating factor, in active and passive anaphylaxis

WEB 2086, a novel specific platelet activating factor (PAF) antagonist derived from triazolodiazepines, inhibited in a dose-related manner the PAF-dependent component of anaphylaxis as well as PAF-induced effects in mice and guinea pigs. In mice a lethal anaphylactic shock and a PAF-induced (100 micrograms/kg i.v.) death was inhibited by i.v. WEB 2086. The ED50 values were 13.6 and 0.37 mg/kg i.v., respectively. In actively sensitized guinea pigs, the anaphylactic lung reaction (bronchoconstriction), but not the corresponding hypotension, was prevented by oral (0.05-0.5 mg/kg) doses of WEB 2086. In contrast, in passively sensitized animals a dose-dependent inhibition of the pulmonary (bronchoconstriction) and blood pressure (hypotension) reaction due to anaphylaxis was achieved by i.v. WEB 2086. Similarly, oral (0.05-0.5 mg/kg) and i.v. (0.005-0.05 mg/kg) WEB 2086 inhibited PAF-induced reduction in respiratory flow (bronchoconstriction) and hypotension in guinea pigs. The ED50 values were 0.070 and 0.066 mg/kg p.o., and 0.017 and 0.015 mg/kg i.v., respectively. In conclusion, PAF seems to play a more major role in passive than in active anaphylaxis in guinea pigs. These results provide further evidence for an important role of PAF in anaphylaxis and support the hypothesis that PAF is involved in asthma and other allergic diseases.     

[3H]Ketanserin labels 5-HT2 receptors and α1-adrenoceptors in human and pig brain membranes

The binding characteristics of [3H]ketanserin (a reported selective radioligand for serotonin 5-HT2 receptors) and [125I]BE 2254 (which labels selectively alpha 1-adrenoceptors) were characterized in brain frontal cortex membranes of pig and man. Saturation experiments indicated that both radioligands label apparently a homogeneous class of binding sites in human and pig fontal cortex membranes. Competition experiments with [125I]BE 2254 using 17 agonists and antagonists showed monophasic and steep curves in human and pig frontal cortex membranes. The pharmacological profile of these sites is typical of alpha 1-adrenoceptors. In competition experiments with [3H]ketanserin, most of the tested compounds displayed shallow or biphasic curves. In particular, alpha 1-adrenoceptor-selective antagonists (prazosin, WB 4101, BE 2254...) displaced with nanomolar affinity about 15 and 40% of the specific [3H]ketanserin binding in human and pig frontal cortex membranes, respectively. The minor component of [3H]ketanserin binding correlated highly significantly with [125I]BE 2254 binding in both membrane preparations. The major component of [3H]ketanserin binding to pig and human frontal cortex membranes correlated significantly with [3H]ketanserin binding in rat brain cortex membranes (which is essentially to 5-HT2 receptors). The present data demonstrate that [3H]ketanserin in nanomolar concentrations binds significantly to alpha 1-adrenoceptors in human and pig frontal cortex membranes; this suggests a rather limited degree of selectivity of ketanserin for 5-HT2 receptors in pig and human tissues.     

Increasing Intracellular calcium of guinea pig ventricular myocytes induced by platelet activating factor through IP3 pathway

We used Fluo-3/AM to examine the effect of platelet-activating factor on the intracellular Ca2+([Ca2+]i) levels in isolated myocytes of guinea pig ventricle. Myocytes were isolated with Langendorff perfusion technique and were challenged with platelet-activating factor. Addition of platelet-activating factor (1 pM to 10 nM) significantly increased the [Ca2+]i in the presence and absence of extracellular Ca2+. The notion that increases in intracellular Ca2+ induced by platelet-activating factor is the result of stimulation of intracellular Ca2+ pool rather than increasing Ca2+ influx was further supported by the whole cell patch-clamp experiments in which the platelet-activating factor did not alter the activity of L-type of Ca2+ channels (I(Ca-L)). Treatment of myocytes with ryanodine failed to abolish the stimulatory effect of platelet-activating factor on [Ca2+]i. In contrast, inhibition of IP3-sensitive Ca2+ release pool with 2-aminoethoxydiphenyl borate (2-APB) blocked the effect of platelet-activating factor. We conclude that the platelet-activating factor-induced increase in intracellular Ca2+ is mediated by stimulation of IP3 receptor but not by stimulation of I(Ca-L) and ryanodine-sensitive receptor.     

Pharmacological characterization of histamine H3 receptors in human saphenous vein and guinea pig ileum

Studies were performed to assess the functional activity of histamine H3 receptors on neurogenic sympathetic end organ responses in cryopreserved human saphenous vein. (R)-alpha-methylhistamine inhibited electrical field stimulation-evoked contractile responses in a dose dependent manner (pD2 = 8.20). Prazosin (1 microM) and tetrodotoxin (1 microM) blocked the electrical field stimulation-evoked contractile responses in human saphenous vein indicating a sympathetic neural origin of these contractions. The histamine H3 antagonists thioperamide (pA2 = 8.41) and clobenpropit (pA2 = 10.10) produced parallel rightward shifts in the concentration response curve to (R)-alpha-methylhistamine in human saphenous vein and guinea pig ileum (pA2 = 8.59 and 9.83, respectively). Pretreatment with (R)-alpha-methylhistamine (1 microM) did not alter contractions to exogenous norepinephrine in human saphenous vein. In addition, clonidine (pD2 = 10.28) inhibited electrical field stimulation-evoked contractile responses in human saphenous vein which were blocked by yohimbine (30 nM, pA2 = 9.92) but did not alter the (R)-alpha-methylhistamine dose response curve. These results demonstrate the presence of functional presynaptic histamine H3 heteroreceptors on cryopreserved human saphenous vein sympathetic nerves that, upon activation, attenuate electrical field stimulation-evoked contractile responses in this vessel.     

3H]rauwolscine labels alpha 2-adrenoceptors and 5-HT1A receptors in human cerebral cortex

[3H]Rauwolscine binds with high affinity to alpha 2-adrenoceptors (Kd = 4.8 +/- 1.3 nM, Bmax = 79 +/- 26 fmol/mg protein, micromolar affinity for 5-HT) as well as to 5-HT1-like receptors (Kd = 13 +/- 2.7 nM, Bmax = 147 +/- 11.4 fmol/mg protein, nanomolar affinity for 5-HT) in human brain cortex membranes. The Ki values of 11 serotonergic compounds for the latter receptors agreed closely with those previously reported for 5-HT1A sites but not with those for 5-HT1B, 5-HT1C and 5-HT1D sites.     

Solubilization of [3H]leukotriene D4 receptor complex from guinea pig lung membranes

Guinea pig lung membrane leukotriene D4 (LTD4) receptors were prelabeled with [3H]LTD4 and solubilized using digitonin, 3-[(3-cholamidopropyl)- dimethylammonio]-1-propane sulfonate, and other non-ionic, zwitterionic, and ionic detergents. [3H]LTD4 remains tightly associated with the receptor complex in the digitonin solubilized state. The dissociation rate of [3]LTD4 from the soluble receptor complex was increased in the presence of guanine nucleotides and sodium ions in a manner similar to that observed for the receptors in the membrane-bound state. The soluble [3H]LTD4 receptor complex was retained on wheat germ lectin affinity columns and destabilized by heat (40 +/- 4 degrees), trypsin, and chymotrypsin treatment, suggesting that the receptor is a glycoprotein. Size exclusion high pressure liquid chromatography of the soluble receptor complex showed that an apparent molecular weight of the soluble receptor complex, in the presence of digitonin, is in the range of 240,000-500,000. An approximately 20-fold enrichment of receptor-radioligand complex was achieved by passing the solubilized LTD4 receptor preparation successively through size exclusion and wheat germ lectin chromatography columns. These data provide the first step toward the purification and chemical characterization of LTD4 receptors.     

Species difference in the specific receptors of platelet activating factor

Relative potencies of platelet activating factor (PAF) and PAF analogs and several PAF receptor antagonists when inhibiting the [3H]PAF specific binding to human and rabbit platelet membranes and membrane fragments of human lung tissues were compared. In rabbit platelets, L-652,731 was found to be most potent in the list of PAF receptor antagonists with an equilibrium inhibition constant (Ki) of 9.83 (+/- 2.92) X 10(-9) M followed by L-653,150 greater than kadsurenone congruent to Ono-6240 greater than ginkgolide B greater than CV-3988 greater than L-651,142, whereas in human platelets the relative potencies of these PAF receptor antagonists were as follows: Ono-6240 greater than L-653,150 congruent to L-652,731 congruent to kadsurenone greater than ginkgolide B greater than CV-3988 greater than L-651,142. Ono-6240 was the most potent one with a Ki of 4.86 (+/- 1.44) X 10(-8) M which was roughly two times more potent than that in rabbit platelets, whereas the affinity of L-652,731 was about ten times less in human platelets (Ki = 1.03 (+/- 0.15) X 10(-7) M) compared to that in rabbit platelets (Ki = 9.83 (+/- 2.92) X 10(-9) M). These variations between species among PAF antagonists strongly suggest that there exists a species difference at or near the binding site of the receptor of platelet activating factor. The relative potency of these PAF receptor antagonists in human lung membranes differed very little from that in human platelets and was found to be Ono-6240 greater than L-653,150 congruent to kadsurenone congruent to L-652,731 greater than ginkgolide B greater than CV-3988 greater than L-651,142. Even though C16-PAF showed slightly higher potency in human lung, and CV-3988 and Ono-6240 showed slightly lower, the difference was too small to suggest that there is a difference in the PAF receptors between human platelets and human lung tissues.     

己酮可可碱减轻血小板激活因子致离体豚鼠肺通透性水肿(英文)

血小板激活因子(PAF,1 nmol·L~(-1))可使离体灌注豚鼠肺重量和微血管液体滤过系数明显增加,已酮可可碱(Pen,0.5和1.0mmol·L~(-1))对此有明显抑制作用,但对PAF引起的肺毛细血管压和静脉阻力的增高无明显影响。各组动物肺水肿的程度与灌注液中白细胞数无相关关系,提示Pen有直接抗肺血管通透性的作用。     

三磷酸肌醇信号途径参与血小板激活因子诱导豚鼠心室肌细胞钙浓度增高

目的观察血小板激活因子(PAF)是否影响心室肌细胞钙离子浓度([Ca~(2+)]i)以及通过哪条信号转导通路起作用。方法采用酶法分离豚鼠心室肌细胞,用PAF刺激细胞;用Fluo-3/AM和共聚焦显微镜观察细胞[Ca~(2+)]i;用2-氨乙基硼酸二苯酯(2-APB)和雷诺定(ryanodine)分别阻断IP3和雷诺定信号通路。全细胞膜片钳技术观察心室肌细胞L型钙电流(ICa-L)。结果无论在有钙还是无钙台氏液,PAF(1pmol·L-1~10nmol·L-1)都能增加心肌细胞[Ca~(2+)]i,其作用能够被2-APB2μmo·lL-1阻断,而不能被雷诺定200μmol·L-1阻断。PAF1nmol·L-1对ICa-L没有明显影响,对照组和实验组电流密度分别为-(11.0±1.9)和-(10.5±1.3)pA·pF-1。结论PAF能增加豚鼠心室肌细胞[Ca~(2+)]i,IP3信号通路可能参与了这一过程。     

[3H]-Spiroperidol labels dopamine receptors in membranes from rabbit mesenteric artery

Summary The potent dopamine receptor antagonist [³H]-spiroperidol was used to label binding sites in a membrane fraction derived from rabbit mesenteric artery which had characteristics expected for dopamine receptors. The binding was of high affinity with an equilibrium dissociation constant (K_D) of 13.1 nM; it was saturable with 110 fmol of [³H]-spiroperidol bound/mg protein at maximal occupancy of the sites. Binding at 37° C was rapid and readily reversible with rate constants of 0.0154 nM^–1 min^–1 and 0.114 min^–1 for forward and reverse reaction, respectively. Dopamine receptor antagonists were about 100–200 times more potent than -adrenolytic drugs in competing for the [³H]-spiroperidol binding sites and dopamine was much more potent than (–)-noradrenaline, adrenaline, (–)-isoprenaline, clonidine or serotonin. It is concluded that in a membrane fraction of the rabbit mesenteric artery there exist binding sites for [³H]-spiroperidol indistinguishable from dopamine receptors. Thus the present results support the view that in vascular smooth muscle there exist specific dopamine receptors.This work was supported by the Deutsche Forschungsgemeinschaft     

Metitepine distinguishes two receptors mediating inhibition of [3H]-5-hydroxytryptamine release in guinea pig hippocampus

Summary Inhibition of [³H]-5-hydroxytryptamine ([³H]-5-HT) release from guinea pig brain slices via activation of the terminal 5-HT autoreceptor has previously been characterised as a model of 5-HT_1D receptor activation, based on the rank potencies of a range of agonists, and the potent antagonism of the inhibitory effects of 5-HT by metitepine. The present study uses this model, in slices of the guinea pig hippocampus, to examine the antagonist potency of metitepine against the 5-HT receptor agonists sumatriptan, 5-carboxamidotryptamine (5-CT) and 5-HT Addition of metitepine to the perfusion buffer (30, 300 and 1000 nmol/l) significantly shifted the concentration-response curve to 5-HT, producing a Schild slope of 1.1, and a pA₂ value of 7.6. However, the ability of metitepine to antagonise the effects of sumatriptan or 5-CT in this model was less marked. A clear-cut shift in the concentration-response curve to sumatriptan was only achieved at1000 nmol/l metitepine (apparent pA₂ = 6.7),and this was similar to the ability of metitepine to attenuate the effects of 5-CT (apparent pA₂ 7.0 at 300 nmol/l and 6.7 at 1000 nmol/l). These findings suggest heterogeneity in the receptor mediating inhibition of [³H]-5-HT release in guinea pig hippocampus.Send offprint requests to L. O. Wilkinson at the above address     

血小板活化因子和肾上腺素对人血小板凝集的钙依赖性协同作用(英文)

AIM: To investigate the mechanism (s) involved in the synergistic interaction of platelet activating factor (PAF) and epinephrine. METHODS: Blood was obtained from healthy human subjects reported to be free of medications for at least two weeks before sampling. Aggregation was monitored at 37℃ using Dual-channel Lumi-aggregometer.The resulting aggregation was recorded for 5 min by the measurement of light transmission as a function of time.RESULTS: Platelet aggregation mediated by subthreshold concentrations of PAF (5-8 nmol/L) plus epinephrine(0.5-2 μmol/L) was inhibited by α_2-receptor blocker, yohimbine, and PAF receptor antagonist WEB 2086. This synergism was inhibited by calcium channel blockers, verapamil and diltiazem. In addition, platelet aggregation by co-addition of PAF and epinephrine was also inhibited by very low concentrations of phospholipase C (PLC) inhibitor (U73122; IC_(50)=0.2 μmol/L), the MAP kinase inhibitor, PD 98059 (IC_(50)=3 μmol/L), and cyclooxygenase(COX-1) inhibitors in     

Unusual binding of [3H] MDL 72222 in guinea pig hippocampus

[³H] MDL 72222 labeled a non-homogeneous population of sites in guinea pig hippocampal membranes (K_d1 = 1 nM; K_d2 = 60 nM). The binding was not sodium dependent. Competition studies with a variety of characterizing agents showed displacement of [³H] MDL 72222 binding by 5-HT uptake inhibitors. [³H] MDL 72222 binding was not effectively displaced by established 5-HT₃ antagonists. MDL 72222, fluoxetine, fluvoxamine and citalopram competitively inhibited the uptake of [³H] 5-HT into guinea pig hippocampal synaptosomes with K_i values of 1.97, 0.02, 0.023, 0.049 μM, respectively. The results demonstrate that [³H] MDL 72222 labels a non-homogeneous population of sites in guinea pig brain, as well as inhibiting 5-HT uptake into synaptosomal preparations.