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.     

Inhibition of Mac-1-dependent leukocyte adhesion by Y-24180, an anti-inflammatory agent, in mice

Y-24180 (4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]-diazepine), an antagonist of platelet-activating factor (PAF), has been already clarified to suppress the expression of an adhesion molecule, Mac-1, on human neutrophils in the previous in vitro study. In the present paper, we examined the effect of Y-24180 on in vivo Mac-1 expression on mouse neutrophils using a lipopolysaccharide-induced leukocyte reduction model in which Mac-1-dependent infiltration of neutrophils was involved. Prophylactic oral administration of Y-24180 inhibited the induction of Mac-1-strongly positive neutrophils by intraperitoneal injection of lipopolysaccharide and prevented the reduction of leukocyte number. In contrast, WEB 2086 (3-[4-(2-chlorophenyl)-9-methyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-2-yl]-1-(4-morpholinyl)-1-propanone), another PAF antagonist, showed little effect. PAF injection failed to induce the Mac-1-strongly positive neutrophils in peripheral blood and the reduction of circulating leukocytes, indicating that PAF was not concerned with the lipopolysaccharide-induced up-regulation of Mac-1 expression and leukocyte reduction. Y-24180 inhibited the leukocyte infiltration also in the thioglycollate medium-induced peritonitis, which was mediated by Mac-1-dependent leukocyte adhesion. These results indicate that Y-24180 inhibits the leukocyte infiltration into the inflamed sites by suppressing Mac-1 expression on leukocytes in vivo and can contribute to the improvement of inflammatory diseases in which the Mac-1-dependent leukocyte adhesion is involved.     

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.     

Interference of azelastine with anaphylaxis induced by ovalbumin challenge in actively sensitized rats.

The inhibition of the haematological alterations and prevention of death due to systemic anaphylaxis after antigen challenge were investigated in rats after various drug treatments. The i.v. injection of ovalbumin (250 micrograms/kg) into actively sensitized rats induced marked thrombocytopenia and haemoconcentration within 5 min and significant leukocytosis within 30 min, lasting for 2 h after the challenge. Pretreatment with meclizine or terfenadine (15-30 mg/kg i.p.) inhibited antigen-induced haemoconcentration, whereas WEB 2086 (2-10 mg/kg i.p.) and PCA 4248 (5-10 mg/kg p.o.), two platelet-activating factor (PAF) antagonists, interfered with thrombocytopenia only. Azelastine (1-20 mg/kg p.o.) dose dependently inhibited antigen-induced haemoconcentration and thrombocytopenia but failed to block leukocytosis. Azelastine also inhibited the thrombocytopenia observed after the i.v. administration of PAF (4 micrograms/kg). Administration of ovalbumin at a dose of 1.5 mg/kg resulted in a lethal anaphylactic reaction in about 85% of the rats. Pretreatment with WEB 2086 (10 mg/kg i.p.), meclizine (30 mg/kg i.p.) or both increased the survival rate from 15 to 57, 68 and 87%, respectively. Azelastine alone (20 mg/kg p.o.) completely blocked the lethal reaction. It was concluded that the ability of azelastine to antagonize histamine and PAF is important for its effectiveness against anaphylactic shock.     

Inhibitory effect of methyl 7-butyl-4,5,6,7-tetrahydro-3-methylamino-4,6-dioxo-5-propyl-2H-pyrazol o[3, 4-d]pyrimidine-2-carboxylate (AA-2379) on type III allergic (Arthus) reaction

Methyl 7-butyl-4,5,6,7-tetrahydro-3-methylamino-4,6-dioxo-5-propyl-2H- pyrazolo[3,4-d]pyrimidine-2-carboxylate (AA-2379), a non-steroidal, non-acidic agent, markedly inhibits type III allergic (Arthus) reaction; the ID50 values of AA-2379 in the rat reversed passive Arthus pleurisy, the rat active Arthus pleurisy, and the reversed passive Arthus reaction in rat skin were 5-10 mg/kg, p.o., and 30 mg/kg of AA-2379 inhibited the active Arthus reaction in rabbit skin by about 50%. Dexamethasone, but not acidic non-steroidal anti-inflammatory drugs and aminopyrine, inhibited the Arthus reaction. The vascular permeability in the reversed passive Arthus pleurisy is enhanced biphasically in the early response mediated by physiologically active amines, prostaglandins, and leukotrienes, and in the late response mediated by complements and polymorphonuclear leukocytes (PMNs). AA-2379 inhibited the late response more potently than the early one. Furthermore, when given after the early response was reduced, AA-2379 obviously inhibited the late response. Rat zymosan-induced paw edema and mouse zymosan-activated serum-induced peritonitis, mediated by complements, were dose-dependently inhibited by AA-2379; the ID50 values were 11.4 and 10.2 mg/kg, p.o., respectively. The results suggest that AA-2379 differs from non-steroidal anti-inflammatory agents in strongly inhibiting the late response of the Arthus reaction, which associated with PMNs.     

Antiinflammatory and antiallergic effects of a novel metabolite of Nocardiopsis sp. as a potent protein kinase C inhibitor from microbial origin

The antiallergic and antiinflammatory effects of the new protein kinase C and calmodulin inhibitor K-252a (8R, 9S, 11S)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11- epoxy-1H,8H,11H-2,7b,11a-triazadibenzo [a,g]cycl oocta [cde] trinden-1-one) were investigated in animal models, and the following results were obtained: 1. Oral administration of K-252a, ketotifen or oxatomide showed a dose-dependent inhibition on 48 h homologous passive cutaneous anaphylaxis in rats and anaphylactic bronchoconstriction in passively sensitized guinea pigs. 2. K-252a (1-100 mg/kg p.o.) exerted a dose-dependent protection against platelet-activating factor (PAF)-induced mortality in mice. This protective effect of K-252a was sustained for 7 h. 3. K-252a (1-100 mg/kg p.o.), as well as dexamethasone (1 mg/kg s.c.), showed remarkable inhibitory effects on rat paw edema induced by carrageenin, zymosan, PAF, and antigen-antibody reaction (passive Arthus reaction) and on the croton oil-induced rat ear edema. On the other hand, indomethacin (10 mg/kg p.o.) significantly inhibited carrageenin-induced edema, but did not inhibit edema induced by other phlogistic agents. Based on these results it is suggested that K-252a, by oral administration, has antiallergic and antiinflammatory effects.     

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.     

WEB 2086, a novel specific PAF antagonist, prevents PAF induced arrhythmogenicity

The hetrazepine derivative WEB 2086 (0.5-2 mg/kg, i.v.), a well known PAF receptor antagonist, was found to reduce the arrhythmogenic effect of PAF in a dose dependent manner on ouabain induced arrhythmias in guinea-pigs. There was no detectable substance-specific influence of WEB 2086 on the threshold doses of ouabain, since in a dose of 2 mg/kg given i.v. it was able to inhibit cardiac rhythm disturbances induced by PAF as well. Guinea-pigs actively sensitized with ovalbumin responded to antigenic challenge with an increased susceptibility to ouabain induced arrhythmias. This arrhythmogenic effect could also be inhibited by WEB 2086. In conclusion, WEB 2086 was found to exert marked protective effects against PAF related cardiac arrhythmias, indicating its potential usefulness for the treatment of cardiac anaphylaxis.     

Effect of KP-136 on allergic paw edema in the rat

The pharmacological properties of KP-136, an inhibitor of type I allergy, were studied in rat paw models. In four allergic responses, three of the immediate type and one delayed type, KP-136 (p.o.) produced potent inhibitions on the mast cell-mediated type I allergy (ID30: 1.0 mg/kg) and the neutrophil-infiltrated passive Arthus reaction (ID30: 1.6 mg/kg). In addition, KP-136 (10 mg/kg, 50 mg/kg, p.o.) inhibited the injury of bone tissues in rat adjuvant arthritis, confirming that it was effective on the allergic inflammation with tissue injury. Although KP-136 was a weak inhibitor of carrageenin-induced paw edema, prostaglandin synthesis and complement-mediated hemolysis, the compound inhibited the release of lysosomal enzymes such as lysozyme and beta-glucuronidase in the passive Arthus reaction, suggesting the blockage of inflammatory mediator release for its mode of action.     

Inhibition of platelet-activating factor fails to limit ischemia and reperfusion-induced myocardial damage.

To determine the role of platelet-activating factor (1-O-hexa-decyl-2-acetyl-sn-glyceryl-phosphoryl-choline, PAF) in myocardial ischemic and reperfusion-induced injury, the effects of a PAF receptor antagonist (WEB 2086) were studied in an anesthetized canine model of ischemia (90 min) and reperfusion (6 h). Thirty minutes after onset of ischemia, WEB 2086 was administered as a bolus (20 mg/kg intravenously, i.v.) followed by a continuous 6-h infusion (10 mg/kg/h i.v.). Controls received vehicle alone (0.9% saline). Platelet aggregation was studied at baseline and at 1, 2, 4, and 6 h of drug administration and at the end of the reperfusion period. WEB 2086 treatment did not significantly affect platelet aggregation stimulated by ADP or arachidonic acid (AA). After 1 h of drug infusion, the ex vivo aggregatory response to exogenous (200 nM) PAF was ablated in WEB 2086-treated animals. WEB 2086 administration did not affect heart rate (HR) or mean arterial blood pressure (MAP) during the occlusion or reperfusion phases. During reperfusion of the ischemic tissue, left circumflex coronary artery (LCX) blood flow of WEB 2086-treated animals increased (p < 0.05) above control value. The area of the left ventricle at risk of infarct was not different between control and WEB 2086-treated groups. Infarct size was not significantly reduced in WEB 2086-treated animals. The results of our investigation using a 90-min ischemic period followed by 6-h reperfusion show that pharmacologic antagonism of PAF by WEB 2086 does not protect the heart against ischemia and reperfusion-induced injury.     

Comparison of several new 5-lipoxygenase inhibitors in a rat Arthus pleurisy model.

The 5-lipoxygenase inhibitors WY-50,295 tromethamine, A-64,077, L-663,536 and ICI-207,968 were compared in a reverse passive Arthus reaction-induced pleurisy model of eicosanoid biosynthesis in the rat. When a 1 h pretreatment schedule was employed, all four inhibitors equivalently blocked leukotriene B4 (LTB4) production with ED50 values of 2.0-2.9 mg/kg p.o. Conversely, WY-50,295 tromethamine (225 mg/kg p.o.) and L-663,536 (100 mg/kg p.o.) did not significantly alter thromboxane B2 (TxB2) levels, whereas A-64,077 (50 mg/kg p.o.) and ICI-207,968 (100 mg/kg p.o.) significantly reduced TxB2 by 50 and 72%, respectively. When 3 and 18 h pretreatment schedules were employed, WY-50,295 tromethamine demonstrated a longer duration of action than the other 5-lipoxygenase inhibitors with ED50 values of 1.7 and 6.3 mg/kg p.o., respectively. At doses of 50 and 100 mg/kg p.o., all drugs tested significantly inhibited inflammatory cell influx by 15-27%, albeit in a non-dose-related manner. However, only A-64,077 significantly lowered fluid extravasation by 35%, presumably due to inhibition of cyclooxygenase product formation. These results demonstrate that in this rat reverse passive Arthus pleurisy model, WY-50,295 tromethamine potently and selectively inhibits 5-lipoxygenase in vivo, and possesses a longer duration of action than the other 5-lipoxygenase inhibitors employed.     

Antagonistic activity of etizolam on platelet-activating factor in vivo experiments

The ability of etizolam, 6-(o-chlorophenyl)-8-ethyl-1-methyl-4H-s-triazolo[3,4-c]thieno[2,3-e] [1,4]diazepine (Y-7131), an anti-anxiety drug, to inhibit platelet-activating factor (PAF)-induced reactions was investigated in experimental animals in vivo. Etizolam (0.01-0.3 mg/kg, i.v.) dose dependently inhibited PAF (0.3 microgram/kg, i.v.)-induced bronchoconstriction (Konzett and R?ssler's method) in guinea pigs, but even at doses as large as 3 mg/kg, i.v., it had no effect on bronchoconstriction induced by histamine, serotonin, acetylcholine, arachidonic acid, bradykinin, angiotensin l or leukotriene D4. Etizolam (0.1-1 mg/kg, i.v.) also dose-dependently reversed PAF (1 microgram/kg, i.v.)-induced hypotension in anesthetized rats. Injection of PAF into the tail veins of mice produced lethal shock within 10-30 min. Etizolam (0.1-3 mg/kg, i.v. and 1-10 mg/kg, p.o.) protected against the lethal effect of PAF (75 micrograms/kg, i.v.) in a dose-dependent manner. These results indicate that etizolam specifically inhibits the action of PAF in vivo.     

Effects of endothelin-1 on vascular permeability in the conscious rat: interactions with platelet-activating factor.

1. The objectives of the present experiments were to assess the effects of endothelin-1 on the macrovascular permeability in selected vascular beds, to study the involvement of platelet-activating factor (PAF) in vascular responses to endothelin-1 and to examine the vascular effects of combined administration of endothelin-1 and PAF in conscious rats. 2. Intravenous bolus injection of endothelin-1 (0.1-2 nmol kg-1) resulted in a dose-dependent biphasic change in mean arterial blood pressure (MABP) with initial transient hypotension followed by a prolonged pressor action. These changes were accompanied by a dose-dependent increase in haematocrit values. 3. Endothelin-1 (0.1 and 1 nmol kg-1) increased dose-dependently the vascular permeability of the trachea, upper and lower bronchi, stomach, duodenum, spleen and kidney (up to 240%) as measured by the extravasation of Evans blue dye. The permeability of pulmonary parenchyma, liver and pancreas was not affected significantly by endothelin-1 treatment. 4. Pretreatment of animals with the specific PAF receptor antagonist, WEB 2086 (1 mg kg-1, i.v.) or BN 52021 (10 mg kg-1, i.v.) reduced the endothelin-1 (1 nmol kg-1)-induced rise in haematocrit by about 50 and 30%, respectively. Both antagonists were highly effective at inhibiting protein extravasation in the stomach, duodenum and kidney. On the other hand, BN 52021, but not WEB 2086, significantly attenuated the effect of endothelin-1 on permeability in the lower bronchi and spleen. Neither WEB 2086 nor BN 52021 modified the changes in MABP evoked by endothelin-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role for intracellular platelet-activating factor in the circulatory failure in a model of gram-positive shock.

1. This study investigates the effects of two structurally different antagonists of platelet-activating factor (PAF), BN52021 and WEB2086, on the circulatory and renal failure elicited by lipoteichoic acid (LTA) from Staphylococcus aureus (an organism without endotoxin) in anaesthetized rats. 2. Administration of LTA (10 mg kg-1, i.v.) caused hypotension and vascular hyporeactivity to noradrenaline (1 microgram kg-1, i.v.) WEB2086 (5 mg kg-1, i.v., 20 min before and 150 min after LTA) inhibited the delayed fall in mean arterial blood pressure (at 300 min: 99 +/- 6 mmHg vs. 75 +/- 6 mmHg, P < 0.01) and prevented the decrease in pressor response to noradrenaline (at 300 min: 36 +/- 5 mmHg min vs. 17 +/- 5 mmHg min, P < 0.01). Surprisingly, BN52021 (20 mg kg-1, i.v., 20 min before and 150 min after LTA) neither prevented the hypotension (74 +/- 6 mmHg) nor the vascular hyporeactivity (21 +/- 5 mmHg min). However, BN52021 inhibited the hypotension to injections of PAF as well as the circulatory failure elicited by lipopolysaccharides (10 mg kg-1, i.v.). 3. LTA caused an increase in plasma concentration of creatinine from 39 +/- 5 microM (sham-operated) to 70 +/- 8 microM and urea from 4.7 +/- 0.1 to 13.1 +/- 1.6 mM. The renal failure elicited by LTA was significantly inhibited by WEB2086 (creatinine: 45 +/- 4 microM and urea: 5.7 +/- 0.7 mM), but not by BN52021. 4. The induction of nitric oxide synthase activity in lungs by LTA was attenuated by WEB2086 from 98 +/- 17 to 40 +/- 15 pmol L-citrulline 30 min-1 mg-1 protein (P < 0.01), but not by BN52021 (148 +/- 21 pmol L-citrulline 30 min-1 mg-1 protein). Similarly, WEB2086, but not BN52021, inhibited the increase in plasma nitrite concentration associated with the delayed circulatory failure caused by LTA. The release of tumour necrosis factor-alpha (TNF-alpha) after injection of LTA was not attenuated by WEB2086. 5. The induction of nitrite release by cultured macrophages activated with LTA (10 micrograms ml-1 for 24 h) was inhibited by 74 +/- 4% by WEB2086 (3 x 10(-4) M), but not by BN52021, indicating that only WEB2086 acts on intracellular PAF receptors. 6. Thus, the intracellular release of PAF contributes to the circulatory and renal failure and induction of nitric oxide synthase elicited by LTA in anaesthetized rats. The difference between the two structurally different PAF antagonists in our septic shock models using either LTA or lipopolysaccharide (LPS), shows the importance of models for Gram-positive sepsis in the elucidation of the pathophysiology of septic shock and for the evaluation of potential drugs.     

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.     

Interference by the novel PAF-acether antagonist WEB 2086 with the bronchopulmonary responses to PAF-acether and to active and passive anaphylactic shock in guinea-pigs

The interaction between the triazolothienodiazepine WEB 2086 and the in vitro and in vivo bronchopulmonary effects of PAF-acether and active/passive anaphylaxis in the guinea-pig was studied. WEB 2086 (1–100 nM) inhibited PAF-acether (10–100 ng)-induced bronchoconstriction and TXB₂ release from isolated and perfused guinea-pig lungs without affecting the response to 100 μg arachidonic acid. In addition, 1–10 μM WEB 2086 significantly reduced antigen-induced TXB₂ and histamine release from lungs from actively and passively sensitized guinea-pigs. In the presence of the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA), mepyramine, methysergide, indomethacin and atropine, WEB 2086 (20–50 μM) inhibited by 30–40% the residual contraction of lung parenchyma strips from guinea-pigs actively sensitized by 0.1–10 μg antigen. In vivo, WEB 2086 (0.1–1 mg/kg) reversed or abolished the bronchoconstriction, hypotension, thrombocytopenia and leukopenia evoked by perfusion of PAF-acether (3 or 44 ng/kg per min). At 3 mg/kg, WEB 2086 also markedly decreased the bronchoconstriction and leukopenia induced by 100 μg/kg antigen in mepyramine (5 μg/kg)-treated passively sensitized guinea-pigs. In contrast, WEB 2086 was ineffective against active anaphylaxis in vivo. These results demonstrate that WEB 2086 antagonizes the bronchopulmonary effects due to PAF-acether and to anaphylactic shock in the guinea-pig.     

The role of nitric oxide and platelet-activating factor in the inhibition by endotoxin of pentagastrin-stimulated gastric acid secretion.

Administration of E. coli endotoxin (1 mg/kg i.v.) abolished the acid secretory response induced by a bolus injection of pentagastrin (100 micrograms/kg i.v.) in the continuously perfused stomach of the anaesthetized rat. Endotoxin administration did not modify mean systemic arterial blood pressure. Pretreatment with NG-nitro-L-arginine methyl ester (L-NAME; 5-20 mg/kg i.v.), but not dexamethasone (5 mg/kg s.c. twice) or indomethacin (5 mg/kg i.m.), substantially restored the secretory responses to pentagastrin. The actions of L-NAME were reversed by the prior administration of L-arginine (100 mg/kg i.v.), but not by its enantiomer D-arginine (100 mg/kg i.v.). L-NAME (10 mg/kg i.v.) increased blood pressure but this does not seem to be the mechanism by which endotoxin-induced acid inhibition was prevented, since similar systemic pressor responses induced by noradrenaline (15 micrograms/kg per min i.v.) had no such effect. The platelet-activating factor (PAF) receptor antagonist, WEB 2086 (2 mg/kg), induced a partial reversal of the inhibition by endotoxin of acid responses to pentagastrin. In endotoxin-treated rats, the combined administration of L-NAME (10 mg/kg) and WEB 2086 (2 mg/kg) completely restored the degree of H+ output induced by pentagastrin to levels similar to those of control, vehicle-treated animals. These findings suggest that endotoxin-induced acute inhibition of acid responses to pentagastrin involves NO synthesis and the release of PAF.     

Antiallergic effects of astemizole on immediate type hypersensitivity reactions.

Astemizole (0.5-5 mg/kg, p.o.) dose-dependently inhibited heterologous and homologous PCA reactions in rats at ID50 values of 1.48 mg/kg and 2.37 mg/kg, respectively. The inhibitory effect of astemizole on heterologous PCA was most remarkable when this compound was given p.o. 2 h prior to antigen challenge. Astemizole (0.1-5 mg/kg, p.o.) dose-dependently inhibited experimentally-induced asthma in guinea pigs at an ID50 of 0.86 mg/kg. Ex vivo, astemizole (0.5-5 mg/kg, p.o.) inhibited antigen-induced histamine release from lung pieces of sensitized guinea pigs. In in vitro experiments, the drug dose-dependently inhibited antigen-induced histamine and SRS-A releases from guinea pig lung pieces at concentrations of 0.05-10 microM. Furthermore, astemizole (0.1-10 microM) inhibited the histamine release induced by compound 48/80 and antigen-antibody reaction from rat peritoneal mast cells, and at 0.1-500 nM inhibited both leukotriene C4- and platelet-activating factor (PAF)-induced contraction of isolated guinea pig trachea at submicromolar concentrations. Astemizole not only inhibited 45Ca uptake into rat mast cells but also prevented the Ca2+ release from the intracellular Ca store induced by compound 48/80, although this compound did not affect the histamine release from permeabilized mast cells induced by Ca2+. Our results suggest that one of the antiallergic mechanisms of astemizole may be an inhibition of signal transduction from the mast cell membrane to the intracellular systems.     

Novel effect of Y-24180, a presumed specific platelet-activating factor receptor antagonist, on Ca2+ levels and growth of human osteosarcoma cells.

In human osteosarcoma MG63 cells, the effect of Y-24180, a presumed specific platelet-activating factor (PAF) receptor antagonist, on intracellular Ca(2+) concentration ([Ca(2+)](i)) and proliferation was measured by using fura-2 and tetrazolium as fluorescent dyes, respectively. Y-24180 (1-5 microM) caused a rapid and sustained [Ca(2+)](i) rise in a concentration-dependent manner. The [Ca(2+)](i) rise was inhibited by 35% by dihydropyridines or removal of extracellular Ca(2+), but was not altered by verapamil and diltiazem. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca(2+)](i) rise, after which 5 microM Y-24180 failed to increase [Ca(2+)](i); conversely, depletion of Ca(2+) stores with 5 microM Y-24180 abolished thapsigargin-induced [Ca(2+)](i) rise. U73122, an inhibitor of phoispholipase C, inhibited histamine-induced, but not 5 microM Y-24180-induced [Ca(2+)](i) rise. Overnight treatment with 0.1-5 microM Y-24180 inhibited cell proliferation in a concentration-dependent manner. Together, these findings suggest that Y-24180 acts as a potent and cytotoxic Ca(2+) mobilizer in human osteosarcoma cells, by inducing both extracellular Ca(2+) influx and intracellular Ca(2+) release. Alterations in cytosolic Ca(2+) regulation may lead to interferences of various cellular functions; thus, attention should be exercised in using Y-24180 as a selective PAF receptor antagonist.     

The role of complement, platelet-activating factor and leukotriene B4 in a reversed passive Arthus reaction.

1. The mechanisms underlying oedema formation induced in a reversed passive Arthus (RPA) reaction and, for comparison, in response to zymosan in rabbit skin were investigated. 2. Oedema formation at skin sites was quantified by the accumulation of intravenously-injected 125I-labelled human serum albumin. 3. Recombinant soluble complement receptor type 1 (sCR1), administered locally in rabbit skin, suppressed oedema formation induced in the RPA reaction and by zymosan. 4. The platelet-activating factor (PAF) antagonists, WEB 2086 and PF10040 administered locally, inhibited oedema formation induced in the RPA reaction and by PAF but not by zymosan. 5. A locally administered leukotriene B4 (LTB4) antagonist, LY-255283, inhibited oedema formation induced by LTB4 but did not inhibit oedema responses to PAF, zymosan or the RPA reaction. 6. The results demonstrate a role for complement in oedema formation in both the RPA reaction and in response to zymosan. An important contribution by PAF is indicated in the RPA reaction but not in response to zymosan whereas no evidence was obtained to suggest a role for LTB4 in either inflammatory response.