Effect of ONO-1078, a leukotriene antagonist, on capsaicin- and substance P-induced bronchoconstriction and airway microvascular leakage in guinea pigs.

AIM: To study the effect of 4-oxo-8-[p-(4-phenylbutyloxy) benzoylamino]-2-(tetrazol-5-yl)-4H-1-benzopyran hemihydrate (ONO-1078), a specific leukotriene antagonist, on capsaicin (Cap)-sensitive sensory nerve functions in the airways, and clarify the modulating roles of endogenous peptido-leukotrienes. METHODS: Changes in intrapulmonary pressure (IPP), Evans blue extravasation in airways, and contraction of bronchial smooth muscles of guinea pigs induced by Cap, substance P (SP) and leukotriene C4 (LTC4) were observed. RESULTS: Cap (0.05 mg.kg-1, i.v.), SP (1 microgram.kg-1, i.v.) and LTC4 (0.5 microgram.kg-1, i.v.) enhanced IPP, and Evans blue extravasation in bronchi and intrapulmonary airways. ONO-1078 0.03 mg.kg-1, i.v. completely blocked the responses to LTC4, attenuated those to Cap, but had no effect to SP. In isolated bronchial smooth muscles, ONO-1078 (1 mumol.L-1) inhibited the contractile response to Cap, but not to SP. CONCLUSION: ONO-1078 partly inhibits Cap-sensitive sensory nerve actions in airways, but has no direct effect on SP, a sensory neuropeptide.

Effects of ONO-1078, a leukotriene antagonist, on cardiovascular responses induced by vagal stimulation, capsaicin, and substance P in guinea pigs.

AIM: To determine the role of ONO-1078, 4-oxo-8 -[p-(4-phenylbutyloxy) benzoylamino]- 2-(tetrazol-5-yl) -4H-1-benzopyran hemihydrate, in cardiovascular responses induced by vagal stimulation, capsaicin, and substance P. METHODS: Evans blue extravasation in the atrium and ventricle, and mean arterial pressure (MAP) were observed. RESULTS: Electric stimulation of vagus (ESV, 10 Hz, 5 ms, 2 or 10 V, for 90 s) increased Evans blue extravasation in the hearts of atropine (1 mg.kg-1, i.v.)-pretreated guinea pigs. Capsaicin (0.05 mg.kg-1, i.v.) and substance P (1 microgram.kg-1, i.v.) enhanced the dye extravasation and elicited a drop in MAP. ONO-1078 (0.03 and 0.1 mg.kg-1, i.v.) inhibited ESV-induced response, especially at stimulation of 2 V. ONO-1078 (0.03 mg.kg-1) attenuated capsaicin-induced cardiac microvascular leakage and hypotensive response, but failed to inhibit substance P-induced responses. CONCLUSION: ONO-1078 can modulate the cardiovascular responses in neurogenic inflammation, possibly mediated by inhibiting sensory neuropeptide release.

[Progress in the study of cyclic nucleotide phosphodiesterases].

There are five families of cyclic nucleotide phosphodiesterase isozymes, which have a common structure, but have different biochemical characteristics, physiologic functions and regulatory mechanisms. Various selective phosphodiesterase inhibitors may have potential uses for antiasthmatic, inotropic, vasodilative, antithrombotic and antidepressant drugs.

Inhibitory effect of tetrandrine on C fiber activation-induced contractions in trachea and bronchus of guinea pig.

AIM: To study the inhibitory effects of tetrandrine (Tet) on an activation of the sensory nervous C fibers in isolated trachea and bronchus of guinea pig. METHOD: The electric field stimulation induced a phase II contraction of the preparations, which was due to an activation of the C fifers. The effects of Tet on phase II contraction were analyzed. RESULTS: Tet 0.3-30 mumol.L(-1) inhibited phase II contractions in a concentration-dependent manner. That phase II contractions inhibited by Tet 1 mumol.L(-1) were 40 +/- 38% and 75 +/- 22% of control in tracheae and bronchi respectively. After pretreatment with chlorphenamine or atropine phase II contraction were still reduced by Tet 1 mumol.L(-1), inhibitory rates being 70 +/- 16% and 64 +/- 16% of control, respectively. The contractile responses of the preparations to exogenous substance P, however, were unaffected by treatment with Tet 1 mumol.L(-1). CONCLUSION: Tet 1 mumol.L(-1) inhibits phase II contraction, related to the inhibition of local release of neuropeptides from C fibers of guinea pig airway.

[Inhibition of chemically induced microvascular leakage by ONO-1078, a leukotriene antagonist, in rat skin].

This study is to determine whether ONO-1078, a potent leukotriene antagonist, influences chemically induced rat skin microvascular leakage which is considered to be, at least in part, due to stimulation of sensory nerve ending and release of sensory neuropeptides. Evans blue dye was used as a tracer for plasma leakage. Intradermal injections of chemical stimuli, histamine (10 micrograms), capsaicin (10 micrograms) and formalin (0.5 mg), evoked Evans blue dye extravasation in rat skin. Intraperitoneal ONO-1078 dose-dependently inhibited the dye extravasation induced by these stimuli, with ID5v values of 1.98 mg.kg-1 for histamine, 1.78 mg.kg-1 for capsaicin, and 2.23 mg.kg-1 for formalin. In contrast to chlorpheniramine, a H1 receptor antagonist, the inhibitory effect of ONO-1078 was weaker on histamine, but more potent on capsaicin and formalin. The inhibitory effect of dexamethasone was more potent than that of ONO-1078 on these stimuli. On the other hand, ONO-1078 inhibited the dye extravasation induced by leukotriene D4 (0.05 micrograms), but showed no effect on those induced by substance P (0.5 micrograms, a sensory neuropeptide), a larger dose of histamine (100 micrograms), and bradykinin (1 microgram). These results suggest that inhibition of chemically induced skin microvascular leakage by ONO-1078 may be mediated by inhibiting the release of sensory neuropeptides from capsaicin-sensitive sensory fibers.

Mediator difference in contractions between trachea and bronchus of guinea pig induced by stimulation of C-fibers in vitro.

In trachea and bronchus of guinea pig in vitro, electric field stimulation (EFS) induced a rapid contraction (phase I) followed by a long-lasting contraction (phase II). The pretreatments of chlorphenamine (Chl) 1 mumol.L-1 and disodium cromoglycate (Cro) 10 mumol.L-1 reduced the tracheal contraction of phase II from 49 +/- 23 mg and 34 +/- 18 mg to 27 +/- 21 and 18 +/- 12 mg, respectively. The contractile responses of the tracheae to increasing concentrations of substance P (SP) 0.1-3.0 mumol.L-1 were reduced by the pretreatment of Cro 10 mumol.L-1 (P < 0.01). On the contrary, the contractile responses of the bronchi were not inhibited by Cro or Chl but were inhibited by pretreatment of atropine 1 mumol.L-1 from 61 +/- 36 mg to 36 +/- 15 mg. These results show that there are different mechanisms in the EFS-induced contractions between the trachea and bronchus; that different mediators amplify the phase II contractions.

[Releases of bradykinin and substance P by heating hind paw of rat].

Contribution of kallikrein-kinin system to heat-induced substance P (SP) release into the periphery was studied by using plasma kininogens-deficient strain Brown Norway Katholiek (B/N-Ka) and normal strain Brown Norway Kitasato (B/N-Ki) rats. Bradykinin (BK) and SP levels in the sc perfusates of the hind instep were measured by radioimmunoassay. In B/N-Ki rat, immersion of hind paw into hot water (47 degrees C) for 20 min led to an increase of BK (43 +/- s 34 fmol.min-1) and SP (11.1 +/- 9.7 fmol.min-1) in the perfusate, whereas those in B/N-Ka rat (BK 1.3 +/- 1.0 fmol.min-1 (P < 0.01), SP 5.5 +/- 3.5 fmol.min-1 (P < 0.05)) were remarkably less. Heat-induced extravasation (leakage of Evans blue) in B/N-Ka rat was also less than that in B/N-Ki rat (P < 0.05). Results suggest that kallikrein-kinin system is involved in the release of SP into the periphery, ie, BK released into the extravascular space by noxious heat stimulation intervenes in SP release.

[Effect of a leukotriene antagonist, ONO-1078, on electric stimulation of vagus (ESV)-induced bronchoconstriction and microvascular leakage in guinea pigs].

In atropine-pretreated guinea pigs, electric stimulation of vagus (ESV, 10 Hz, 5 ms, 2 V or 10 V, for 90 s) increased intrapulmonary pressure (IPP), and Evans blue extravasation in trachea, main bronchi, peripheral and distal intrapulmonary airways in a voltage-dependent manner. ONO-1078, a noval leukotriene antagonist, (0.03 and 0.1 mg.kg-1, iv) showed no remarkable inhibiting effect on ESV-induced increase of IPP. However, the agent significantly inhibited ESV-induced increase of Evans blue extravasation in the airways, especially in lower potency of stimulation (2 V). The results suggest that leukotrienes may be involved in airway microvascular leakage in response to neurogenic inflammation.

Vascular permeability increased by histamine aerosol, capsaicin, and electric stimulation of vagus nerves in guinea pigs.

The present study was to clarify the tissue differences in vascular permeability enhanced by 0.1% histamine aerosol, capsaicin (50 micrograms.kg-1, iv), and electric stimulation of vagus nerves (ESV, 12 V, 5 ms, 16 Hz, for 90 s) in different parts of airways, heart, esophagus, ileum, kidney, and liver of guinea pigs using Evans blue (30 mg.kg-1, iv) as a tracer. All the treatments significantly increased the dye extravasation in trachea, main bronchi, proximal and distal intrapulmonary airways (by 64-722%), as well as in the heart (by 89-126%), most remarkable for capsaicin on trachea and main bronchi. Capsaicin also markedly increased the dye content in esophagus. These results demonstrate that neurogenic inflammation induced by capsaicin and ESV primarily increases vascular permeability in the respiratory tract and heart.

[Effects of ketotifen on human neutrophil respiratory burst and intracellular free calcium].

The stimulatory effect of FMLP 5 nmol.L-1, OAG 25 nmol.L-1 and calcimycin 10 nmol.L-1 on luminol-dependent chemiluminescence (CL) was observed in human neutrophils (Neu). The rest level of Neu intracellular free calcium ([Ca]i) measured by Ca(2+)-sensitive probe Quin 2/AM was calculated to be 200 +/- s 19 nmol.L-1. By the addition of FMLP 0.1 nmol.L-1 or calcimycin 0.5 nmol.L-1, the peak [Ca]i increased to 769 +/- 104 nmol.L-1 and 953 +/- 53 nmol.L-1, respectively. Ketotifen (50-300 mumol.L-1) inhibited Neu CL in a dose-dependent manner with activator FMLP. Inhibition was also seen when Neu CL was activated by calcimycin and OAG, respectively. However, ketotifen did not inhibit Neu [Ca]i increment activated by FMLP and calcimycin.

[Effects of ketotifen on rabbit platelet aggregation and platelet activating factor formation from rat neutrophils].

The effects of ketotifen (Ket) on rabbit platelet aggregation induced by platelet activating factor (PAF), ADP and arachidonic acid (AA) and PAF formation from A-23187-stimulated rat neutrophils in vitro were studied. PAF (15-100 pmol/L) induced rabbit platelet aggregations, with an EC50 of 33 pmol/L. Ket shifted the PAF dose-dependent platelet aggregation curve to the right in a parallel fashion with no depression of the maximal response and reversed the secondary aggregation phase, suggesting that Ket had competitive antagonistic activity against PAF-induced platelet aggregation. It also showed inhibitory effects on platelet aggregations induced by ADP 10 mumol/L and AA 50 mumol/L, the IC50 were 94.5 and 143.5 mumol/L respectively. However, it failed to influence PAF formation from rat neutrophils stimulated by A-23187 2.5 mumol/L in vitro. The inhibitory effects of Ket on platelet activation, particularly PAF-induced platelet aggregation, may contribute to its anti-asthmatic properties.

[Effects of 7 drugs on cutaneous blood flow evoked by electric stimulation of rat sciatic nerve].

The non-invasive technique of laser-Doppler flowmeter (LDF) was used to measure the change of cutaneous blood flow evoked by electric stimulation of rat sciatic nerve. The sciatic nerve was cut centrally and placed on bipolar electrodes. Drug were infused in a carotid artery. Electric stimulation of the sciatic nerve containing sensory afferent fibers caused an increase in cutaneous blood flow. This increase was not modified by the ia infusion of adrenergic blocking agents (phentolamine, 0.1 mg/kg and propranolol, 0.5 mg/kg), anti-muscarinic agent (atropine 0.5 mg/kg), anti-histamines (mepyramine, 10 mg/kg and cimetidine, 10 mg/kg) and 5-HT antagonist (methysergide, 0.5 mg/kg). Pretreatment with capsaicin (50 mg/kg, sc) in the newborn rat or the ia infusion of spantide (1, 2 mumol/kg) significantly inhibited the the stimulation-induced increase of the blood flow. These results suggest that substance P released from the peripheral endings of sensory nerve may be involved in vasodilation following electric stimulation of the sciatic nerve in rat.

[Effects of neutrophils on histamine release from mast cells].

To determine whether neutrophils (NP) contribute to immediate anaphylaxis, the effects of NP on histamine release from mast cells (MC) were studied and changes in the NP morphology were investigated. When rat peritoneal MC and pleural NP collected from actively sensitized rats were mixed and challenged with antigen (trichosanthin), histamine release from MC was significantly increased. This promotive effect of NP on antigen-induced histamine release from MC was dosage-dependent. The observation of scanning and transmission electron microscopy showed that NP were activated and secretion was resulted. It is suggested that NP may be activated in immediate anaphylaxis and secrete some mediators which could promote histamine release from MC. Quercetin may inhibit the promotive effect of the NP on histamine release and the secretion of the NP while ketotifen and isoproterenol have no influence on this promotive effect.

[Effects of clonidine and norepinephrine on rabbit pulmonary artery strips with or without endothelium].

Relaxing responses of strips of rabbit pulmonary artery (RPA) with endothelium (+E) to norepinephrine (NE) during sustained contraction with KCl 20 mmol/L in the presence of propranolol (Pro) 10 mumol/L and prazosin (Pra) 1 mumol/L were more sensitive than those without endothelium (-E) to NE. These responses were inhibited by yohimbine (Yoh) 1 mumol/L. However, the relaxing responses of the strips to clonidine (Clo) were not different between RPA strips +E and -E in the presence of Pro+ Pra or Pro + Pra + Yoh 1 mumol/L. Relaxing responses of RPA strips -E precontracted by phenylephrine (PE) 1 mumol/L to Pra and Clo were greater than that of those precontracted by KCl 20 mmol/L. The relaxing responses of these strips precontracted by PE to Pra were larger than those precontracted by PE mumol/L; but that of those precontracted by PE and Clo to Yoh were not different. The results suggest that integrity of the endothelium is an important factor in the relaxing responses of RPA strips to NE. The relaxing effect of Clo on RPA strips precontracted by KCl 20 mmol/L may be due to alpha 1-adrenoceptor blockade on smooth muscle cells of the RPA strips.

[Antianaphylactic components of Asarum forbsii Maxin].

From the ethyl acetate extract of Asarum forbesii Maxin, four new constituents, asarumin A(I), B(II), C(III) and D(IV), were isolated along with elemicin (V), trans-asarone(VI) and linoleic acid(VII). The structures of the new compounds were elucidated as methyl 3 S-benzoyloxy-2 S-hydroxy-2-isopropylbutyrate for I, methyl 2 R-benzoyloxyisopentanoate for II, methyl 2 R-trans-cinnamoyloxyisopentanoate for III and methyl 2 R-piperonyloyloxyisopentanoate for IV. Compounds I, II, III and VII showed weak inhibition of PCA in rats, but the other compounds were inactive.

[Effects of tetrandrine on cytoplasmic free calcium in rat neutrophils].

Neutrophils (NP) cytoplasmic free Ca2+ concentrations ([Ca2+]i) under several conditions were measured with Quin 2 in rats. The process of loading Quin 2-AM into the cells followed by its hydrolysis was assured by monitoring the shift of fluorescent emission spectrum of Quin 2-AM to that of Quin 2. The resting [Ca2+]i of NP in Ca2(+)-containing and Ca2(+)-free solutions were 186 +/- 45 and 46 +/- 16 nmol/L, respectively, indicating extracellular calcium concentration ([Ca2+]0) plays an important role on [Ca2+]i. Among agents tested, prostaglandin E2 (PGE2) 100 nmol/L did not change [Ca2+]i significantly. Calcimycin 25 mumol/L, leukotriene B4(LTB4) 60 nmol/L and platelet activating factor (PAF) 10 nmol/L increased [Ca2+]i of NP in Ca2(+)-containing solution from 185 +/- 54, 175 +/- 36 and 188 +/- 54 to 814 +/- 67, 577 +/- 229 and 540 +/- 174 nmol/L, respectively. But, compound 48/80 3.2 micrograms/ml, LTB4 300 nmol/L, PAF 10 nmol/L and PAF 5 nmol/L plus LTB4 150 nmol/L did not change significantly [Ca2+]i of NP in Ca2(+)-free solution, indicating that these agonists can not release intracellularly stored Ca2+ or no stored Ca2+ in NP is available. The rises of [Ca2+]i produced by calcimycin, PAF and LTB4 were markedly inhibited by tetrandrine (Tet) 65 mumol/L. These results, as a whole, show that Tet inhibits the rises of [Ca2+]i of NP induced by calcimycin, PAF and LTB4 via decreasing Ca2+ influx. The Ca2+ antagonism in this case may be related to its anti-allergic actions.

[Interaction between bradykinin and enkephalins in rat dental pulp].

The content of bradykinin (BK)-like peptides in rat dental pulp was significantly increased 1, 6 and 24 h after cavity formation at the neck of incisor. We have reported that enkephalin (EK)-like peptides in rat dental pulp were increased by cavity formation or BK. In the present study, the mechanism of the production of EK enhanced by BK was investigated using benzoyl-L-arginine-2-naphthylamide (BANA), a synthetic substrate. BK and its products cleft by carboxypeptidase B, des-Arg9-BK and arginine (Arg), activated the degradation of BANA. It is suggested that these substances may enhance the processing of enkephalins from precursor proteins. The activating effects were inhibited by EGTA. The BANA-degrading enzymes in lysosomal fraction were activated by BK, des-Arg9-BK and Arg, but the enzymes in supernatant were activated by Arg only. On the other hand, morphine and met-EK inhibited the production of BK-like peptides by trypsin from plasma kininogen. It is suggested that BK is cleft by carboxypeptidase B in pulp cell to des-Arg9-BK and Arg, which activate the lysosomal or soluble EK processing enzymes, and then the produced EK inhibits the production of BK from plasma kininogens in the pulp.

[Effect of tolmetin on responsiveness of isolated guinea pig tracheal smooth muscles].

Tolmetin directly relaxed the isolated guinea pig tracheal smooth muscles, increased the muscle responsiveness to histamine or carbachol and enhanced the tracheal Schultz-Dale reaction. Prostaglandin E2 synthesized by cyclooxygenase was transformed into prostaglandin B2 (PGB2) with alkali, PGB2 and the lipoxygenase metabolite leukotriene B4 (LTB4) were measured by high-performance liquid chromatography, whereas slow-reacting substance of allergy (SRS-A) was determined by bioassay. Tolmetin showed inhibitory effects on prostaglandin E2 synthesis, with the IC50 being 30 mumol/L, but no effect on LTB4 synthesis (up to 100 mumol/L). SRS-A release was potentiated by tolmetin at 10 and 50 mumol/L. The effects of tolmetin on arachidonic acid metabolism may be responsible for its effects on tracheal smooth muscles. It is suggested that asthma attacks may be induced or enhanced by tolmetin in patients with asthma.