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.     

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.     

Endotoxin inhibition of distension-stimulated gastric acid secretion in rat: mediation by NO in the central nervous system.

1. The involvement of nitric oxide in the acute inhibitory effects of low doses of endotoxin, following intracerebroventricular (i.c.v.) or intravenous (i.v.) administration, on gastric acid secretion stimulated by distension or i.v. infusion of pentagastrin has been investigated in the continuously perfused stomach of the anaesthetized rat. 2. The i.c.v. administration of E. coli endotoxin (800 ng kg-1) abolished the acid secretory response induced by gastric distension (20 cm water intragastric pressure) within 30 min of administration. 3. By contrast, submaximal rates of acid secretion induced by i.v. infusion of pentagastrin (8 micrograms kg-1 h-1) were not inhibited by i.c.v. administration of endotoxin (800 ng kg-1). 4. Prior i.c.v. administration of the NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 800 micrograms kg-1) restored the acid secretory responses to distension in rats treated with endotoxin (i.c.v.). 5. Likewise, i.v. administration of endotoxin (5 micrograms kg-1) abolished the acid secretory response induced by gastric distension within 30 min of administration. Prior i.c.v. injection of L-NAME (800 micrograms kg-1) or its i.v. administration (10 mg kg-1) restored acid secretory responses in rats receiving i.v. endotoxin. 6. The reversal by L-NAME (i.v.) of the acid inhibitory effects of endotoxin (i.v.) was prevented by L-arginine (12 mg kg-1, i.c.v. or 100 mg kg-1, i.v.), but not by its enantiomer D-arginine. 7. The present results imply the existence of an acute response to endotoxin involving NO synthesis in the brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitric oxide mediates the inhibition by interleukin-1 beta of pentagastrin-stimulated rat gastric acid secretion.

Bolus injection of interleukin-1 beta (2 micrograms kg-1, i.v.) inhibited acid secretion induced by intravenous infusion of pentagastrin (8 micrograms kg-1 h-1) in the continuously perfused stomach of the anaesthetized rat. Administration of interleukin-1 beta did not modify mean systemic arterial blood pressure. Pretreatment with NG-nitro-L-arginine methyl ester (L-NAME, 2-10 mg kg-1, i.v.), but not dexamethasone (5 mg kg-1, s.c. twice over 16 h), restored the acid secretory responses to pentagastrin. The actions of L-NAME were reversed by the prior administration of L-arginine (100 mg kg-1, i.v.), but not by its enantiomer D-arginine (100 mg kg-1, i.v.). L-NAME (5 mg kg-1, i.v.) increased blood pressure but this was not the mechanism by which interleukin-induced acid inhibition was prevented, since similar systemic pressor responses induced by phenylephrine (10 micrograms kg-1 min-1, i.v.), had no such effect. These findings suggest that interleukin-induced inhibition of acid responses to pentagastrin involves synthesis of NO from L-arginine.     

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.     

Effects of Endotoxin on Neurally-mediated Gastric Acid Secretion in the Rat

The effects of a peripheral administration of E. coli endotoxin on neurally-mediated gastric acid secretion and the role of endogenous opioids or PAF receptors in endotoxin effects have been evaluated in the continuously perfused stomach of the anaesthetized rat. Gastric acid secretion stimulated by distension (20 cm H₂O) was reduced dose-dependently by single intravenous bolus injection of endotoxin (0.1–10 μg kg^?1). Doses of 5 μg kg^?1 induced a peak reduction of distension-stimulated acid output and significantly reduced the secretory response induced by an intravenous bolus of 2-deoxy-d -glucose (150 mg kg^?1). This dose of endotoxin did not significantly modify mean systemic arterial blood pressure throughout the experimental period. Pretreatment with the opioid receptor antagonist naloxone (1 mg kg^?1, i.v.) or the platelet-activating factor (PAF) receptor antagonist WEB 2086 (2 mg kg^?1, i.v.) did not reverse the inhibitory effects of endotoxin (5 μg kg^?1, i.v.) on acid secretion stimulated by both distension and 2-deoxy-d -glucose. These findings suggest that endotoxin-induced acute inhibition of neurally-mediated acid responses, stimulated by gastric distension or administration of 2-deoxy-d -glucose, do not involve the activation of endogenous opioids or PAF receptors.     

Protective effects of yangambin on cardiovascular hyporeactivity to catecholamines in rats with endotoxin-induced shock

The protective effects of a new, selective, plant-derived platelet-activating factor (PAF) antagonist, yangambin, on the cardiovascular alterations and mortality due to endotoxic shock were investigated in anaesthetized rats. We also studied the involvement of PAF in the induction of the vascular and cardiac hyporesponsiveness to adrenergic stimulation observed during endotoxaemia. The animals were sensitized to the lethal effects of Escherichia coli lipopolysaccharide (LPS) with D(+)-galactosamine (50 mg/kg, i.v.) 15 min before LPS injection. LPS (3 mg/kg, i.v.) induced a progressive and marked decrease in mean arterial blood pressure from 85+/-4 to 30+/-3 mmHg and a reduction of cardiac output (CO) from 180+/-7 to 37+/-3 ml/min (120 min) accompanied by a maintenance of systemic vascular resistance, suggesting that cardiovascular collapse resulted mainly from myocardial depression. The maximum pressor responses to noradrenaline (0.3-3.0 microg/kg, i.v.) fell from 72+/-9 (control) to 5+/-1 mmHg (LPS) while the CO responses decreased from 81+/-5 to 8+/-3 ml/min. Pre-treatment with yangambin (30 mg/kg, i.v.) or with WEB 2086 (5 mg/kg, i.v.), a reference PAF receptor antagonist, completely prevented the LPS-induced cardiovascular collapse and abolished the sharp reductions of the arterial blood pressure and CO responses to noradrenaline observed during endotoxaemia. Post-treatment with yangambin 90 min after LPS administration did not reverse the arterial hypotension, cardiac failure or cardiovascular hyporesponsiveness to catecholamines. Finally, the acute (150 min) survival rates of endotoxic shock increased from 0% (LPS group) to 100% in the groups pretreated with either yangambin or WEB 2086. The long-term (7-day) survival also increased from 0% (LPS group) to 85% (yangambin pre-treatment group). In conclusion, these data suggest a role for PAF in the pathogenesis of endotoxin-induced vascular and cardiac hyporesponsiveness to catecholamines and confirm its involvement in the complex cascade of multiple mediators released during endotoxic/septic shock. Yangambin proved to be an effective pharmacological agent against cardiovascular collapse and mortality in endotoxin shock.     

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.     

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.     

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.     

Involvement of neuronal processes and nitric oxide in the inhibition by endotoxin of pentagastrin-stimulated sastric acid secretion

Administration of E. coli endotoxin (1 mg kg^–1, i.v.) abolished the acid response induced by the i.v. infusion of pentagastrin (8 g kg^–1, h^-1) in the continuously perfused stomach of the anaesthetized rat. Local serosal application of tetrodotoxin (36 ng per rat) completely restored acid responses to pentagastrin in endotoxin-treated rats. However, pretreatment with atropine (0.5 mg kg^–1, s.c.), capsaicin (20, 30, and 50 mg kg^–1, s.c. 2 weeks before the study) or guanethidine (16 mg kg^–1, s.c. 3 and 16h before) did not influence the inhibitory effects of endotoxin. Continuous i.v. infusion with N^G-nitro arginine methyl ester (L-NAME, 10 mg kg^–1, h^–1) restored the secretory responses to pentagastrin in endotoxin treated rats. The effects of L-NAME were reversed by L-arginine (100 mg kg^–1, h^–1, i.v.), but not by its enantiomer D-arginine (100 mg kg^–1, h^–1, i.v.). The secretory responses elicited by pentagastrin (10^–10–10^–6 M) in the isolated lumen perfused stomach of the rat were not influenced by incubation (100 min) with endotoxin (10 g ml ^–1). These observations with tetrodotoxin indicate that inhibition of acid secretion by endotoxin in vivo involves neuronal activity, while inhibition of NO synthesis had a comparable inhibitory action. Activation of a systemic non-adrenergic non-cholinergic neuronal pathway involving NO could thus mediate the acute acid inhibitory effects of endotoxin. Correspondence to: J. V. Esplugues at the above address     

Biological properties of the antagonist SRI 63-441 in the PAF and endotoxin models of hypotension in the rat and dog

Intravenous administration of platelet-activating factor (PAF) produces dose-dependent hypotension in several species. We have evaluated a recently developed PAF antagonist, SRI 63-441, for its ability to inhibit the hypotensive effect of PAF in the rat and dog. In the rat, 100 ng/kg PAF produced a 38.6 +/- 5.1% decrease in carotid mean arterial pressure (MAP), followed by a 3.2 +/- 0.7 min recovery period for MAP to return to baseline values. SRI 63-441 reduced the hypotension response in the rat, where the ED50 values for inhibition of MAP were 0.16 mg/kg i.v. and 0.19 mg/kg i.v. for the recovery period. Dogs challenged with 1.5 micrograms/kg PAF i.v. demonstrated a 52 +/- 8% decrease in MAP that persisted for at least 15 min. The ED50 for inhibition of MAP by SRI 63-441 was 0.20 mg/kg i.v. Following injection of tritium-labeled SRI 63-441, 56.8 +/- 2.4% of the dose was recovered in the urine and 43.2 +/- 8.9% in the feces in the rats while in dogs 38.7 +/- 5.6% and 60.9 +/- 23.5% of the dose was excreted in the urine and feces, respectively. In the rat model of endotoxin-induced hypotension, SRI 63-441 given 1 min after a 5 mg/kg endotoxin challenge (which produced a 52 +/- 7% decrease in MAP), reversed the systemic effects, with an ED50 of 0.18 mg/kg i.v. The ED50 for reversal 6 min after endotoxin injection was 0.01 mg/kg. These results of inhibition and reversal by SRI 63-441 strongly implicate PAF as a pivotal mediator of hypotension and shock.     

Interactions of constitutive nitric oxide with PAF and thromboxane on rat intestinal vascular integrity in acute endotoxaemia.

1. The involvement of endogenous platelet activating factor (PAF) and thromboxane A2 in the acute microvascular damage in the ileum and colon induced by the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) following endotoxin administration was investigated in the rat over a 1 h period. 2. Administration of L-NAME (1-10 mg kg-1, s.c.) concurrently with E. coli lipopolysaccharide (LPS; 3 mg kg-1, i.v.) dose-dependently increased vascular permeability in the ileum and colon, as determined by the leakage of radiolabelled albumin, and caused macroscopic mucosal damage in the ileum determined 1 h later. Neither LPS administration nor L-NAME (5 mg kg-1) alone affected resting vascular permeability. 3. Infusion of phenylephrine (10 micrograms kg-1 min-1, i.v. for 1 h) caused an elevation in blood pressure similar to that found following L-NAME administration (5 mg kg-1, i.v. or s.c.), but did not increase intestinal vascular permeability, when administered with LPS (3 mg kg-1, i.v.). 4. The increased vascular permeability in the ileum and colon and macroscopic damage in the ileum, induced by L-NAME (5 mg kg-1, s.c.) and LPS (3 mg kg-1, i.v.) was dose-dependently inhibited following s.c. pretreatment (15 min before challenge) with the thromboxane synthase inhibitors, OKY 1581 (5-25 mg kg-1) or 1-benzyl-imidazole (1-50 mg kg-1), or with the thromboxane receptor antagonist, BM 13177 (0.2-2 mg kg-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Diadenosine tetraphosphate-gating of cardiac K(ATP) channels requires intact actin cytoskeleton

The effects of endotoxin on gastric emptying of a solid nutrient meal and the neural mechanisms involved in such a response were investigated in conscious rats. The intraperitoneal (i.p.) administration of E. coli endotoxin (40 microg/kg) significantly reduced the 4-h rate of gastric emptying of a standard solid nutrient meal. Ablation of primary afferent neurons by systemic administration of high doses of capsaicin (20+30+50 mg/kg s.c.) to adult rats did not modify the rate of gastric emptying in control animals but prevented the delay in gastric transit induced by endotoxin. Local application of capsaicin to the vagus nerve rather than application of capsaicin to the celiac ganglion significantly repressed endotoxin-induced delay in gastric emptying. Neither treatment modified the rate of gastric emptying in vehicle-treated animals. Blockade of CGRP receptors (CGRP 8-37, 100 microg/kg i.v.) did not alter gastric emptying in control animals but significantly prevented endotoxin-induced inhibition of gastric emptying. In contrast, a tachykinin receptor antagonist ([D-Pro2, D-Trp7.9]-substance P, 2 mg/kg i.p.) significantly reduced the rate of gastric emptying in control animals and did not modify the inhibitory effects of endotoxin. Adrenergic blockade with phentolamine (3 mg/kg i.p.) +/- propranolol (5 mg/kg i.p.) or muscarinic antagonism with atropine (0.1 mg/kg i.p.) failed to reverse the delay in gastric emptying induced by endotoxin. These observations indicate that endotoxin-induced delay in gastric emptying of a solid nutrient meal is mediated by capsaicin-sensitive afferent neurons.     

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.     

The booster injection of antigen during active sensitization of guinea-pig modifies the anti-anaphylactic activity of the PAF antagonist WEB 2086.

1. Serum IgG levels of sensitized guinea-pigs bled at various times after the booster injection were evaluated and its capacity to sensitize passively lung strips from normal guinea-pigs assessed. Following the booster injection, both serum IgG and the ability to sensitize passively lung strips increased during the first week and decreased slowly thereafter. 2. The PAF antagonist WEB 2086 (3 mg kg-1, i.v.) blocked the anaphylactic bronchoconstriction induced by intravenous administration of ovalbumin (1 mg kg-1) when guinea-pigs were challenged 2 and 4 days after the booster injection, but became ineffective when tested in guinea-pigs challenged 7, 28 and 56 days after the booster injection. 3. The ability of WEB 2086 to reduce anaphylactic bronchoconstriction of guinea-pigs challenged 2 and 4 days after the booster injection was unrelated to either the selective involvement of one type of immunoglobulin, low IgG titres in sera or a reduced sensitizing capacity. 4. The booster injection, which accounts for the loss of efficacy of WEB 2086 from the fourth day thereafter, probably operates as a PAF-independent inflammatory challenge. 5. The protocol for immunisation and the day of experiment after the booster injection determines the sensitivity of the anaphylactic bronchoconstriction to inhibition of PAF antagonists.     

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.     

Mechanisms of L-NG-nitro arginine methyl ester-induced antinociception in mice: a role for serotonergic and adrenergic neurons.

1. The mechanisms involved in the antinociceptive action of L-NG-nitro arginine methyl ester (L-NAME) were investigated in mice. 2. Intraperitoneal administration of L-NAME produced a dose-dependent antinociception in the tail-flick, hot-plate and phenyl-p-quinone-induced writhing tests. 3. Pretreatment with the catecholamine depletors 6-hydroxydopamine (5 micrograms i.c.v.) or reserpine (5 mg/kg i.p.) or the serotonin synthesis inhibitor, p-chlorophenylalanine methyl ester (200 mg/kg i.p. on 2 consecutive days) resulted in a significant decrease in the antinociceptive effect of L-NAME. 4. Similarly, pretreatment with the selective alpha 1-adrenoceptor antagonist prazonin (2.5 mg/kg, i.p.), or the non-selective alpha-adrenoceptor blocker, phentolamine (5 mg/kg, i.p.) antagonized the antinociceptive effect of L-NAME. 5. However, the administration of the selective alpha 2-adrenoceptor antagonist, idazoxan (2.5 mg/kg i.p.) was without effect. 6. Likewise, pretreatment with the serotonin 5-HT2 receptor blocker, ketanserin (1 mg/kg, i.p.), the D2 dopamine receptor antagonist (+/-) sulpiride (30 mg/kg, i.p.) or the opioid antagonist naloxone (5 mg/kg, i.p.) did not inhibit the antinociceptive effect of L-NAME. 7. These results suggest that L-NAME produces antinociception in the mouse probably by an action on adrenergic and serotonergic synapses.     

Characterization of cardiovascular events mediated by platelet activating factor during systemic anaphylaxis.

Previous studies have shown that an anaphylactic reaction in the isolated perfused heart is characterized by drastic coronary constriction, arrhythmias, and severe impairment of contractility. In vivo anaphylaxis is associated with myocardial ischemia and rapid cardiovascular failure. Recently, not only histamine but also platelet activating factor (PAF) has been implicated in cardiac manifestation of anaphylaxis. The present study was designed to separate the effects of PAF from those of histamine on cardiovascular function during systemic anaphylaxis. In guinea pigs, sensitization was produced by subcutaneous (s.c.) application of ovalbumin. Fourteen days after sensitization, the effects of an intravenous (i.v.) infusion of ovalbumin were tested in anesthetized artificially ventilated guinea pigs. The renewed application of the antigen induced severe cardiac dysfunction. Within 3 min, cardiac output (CO) had already decreased by 90% and left ventricular end-diastolic pressure (LVEDP) increased significantly, indicating left ventricular pump failure. Concurrently, ECG recordings uniformly showed signs of acute myocardial ischemia. In addition, arrhythmias occurred in terms of atrioventricular block. After 4 min, blood pressure (BP) rapidly decreased. All animals died within 10 min. Pretreatment with the H1-receptor antagonist mepyramine (1 mg/kg i.v.) in combination with the H2-receptor antagonist cimetidine (10 mg/kg i.v.) delayed onset of myocardial ischemia, arrhythmias and cardiac pump failure. After 10 min, however, LV contractility and BP steadily decreased, leading to severe hypotension within 30 min. If the selective PAF antagonist WEB 2086 (1 mg/kg i.v.) was administered in addition to cimetidine and mepyramine, myocardial ischemia and LV contractile failure were markedly inhibited further. In contrast, pretreatment with WEB 2086 alone had no beneficial effects on the anaphylactic cardiovascular changes. Therefore, we conclude that histamine is the predominant mediator during the early phase of systemic anaphylaxis whereas PAF-mediated effects are involved in cardiac dysfunction during the protracted late phase of anaphylaxis.     

Bradykinin causes inhibition of methacholine-induced bronchoconstriction in vivo in mice

In this study the influence of bradykinin on airway responses was investigated in anaesthetised and ventilated mice. Airway resistance in mice was monitored using whole body plethysmography. Intravenous (i.v.) administration of bradykinin (4-40 microg/kg) did not cause a direct effect on airway resistance. Also pretreatment with propranolol (1 mg/kg, i.v.), atropine (1 mg/kg, i.v.) or indomethacin (5 mg/kg, i.v.) did not result in any effect of intravenous bradykinin on baseline airway resistance. However, i.v. bradykinin (4-40 microg/kg) caused a dose-dependent inhibition of the (0.5 mg/kg, i.v.) methacholine-induced bronchoconstriction, with an ED50 value of 3.4 +/- 0.4 microg/kg. The maximal inhibition of the bronchoconstrictor response to methacholine was 65.5 +/- 2.0%. The inhibition of the methacholine-induced bronchoconstriction by bradykinin could be prevented by treatment with the B2 receptor antagonist icatibant (Hoe 140, 0.13 mg/kg, i.v.). Also pretreatment with either propranolol (1 mg/kg, i.v.), L-NAME (30 mg/kg, i.v.) or indomethacin (5 mg/kg, i.v.) completely blocked the inhibition of the methacholine-induced bronchoconstriction by bradykinin. The inhibition of the methacholine-induced bronchoconstriction after bradykinin was not affected by the NK1 receptor antagonist RP 67580 (17.5 microg/kg, i.v.). In conclusion, the results of this study demonstrate that bradykinin causes a dose-dependent inhibition of the methacholine-induced bronchoconstriction in vivo in mice. This response is B2 receptor-mediated and at least involves the activation of beta-adrenoceptors and the synthesis of nitric oxide and cyclo-oxygenase products.