Inhibition of airway hyper-responsiveness by TRPV1 antagonists (SB-705498 and PF-04065463) in the unanaesthetized, ovalbumin-sensitized guinea pig.

BACKGROUND AND PURPOSE Airway sensory nerves play a key role in respiratory cough, dyspnoea, airway hyper-responsiveness (AHR), all fundamental features of airway diseases [asthma and chronic obstructive pulmonary disease (COPD)]. Vagally mediated airway reflexes such as cough, bronchoconstriction and chest tightness originate from stimulation of airway sensory nerve endings. The transient receptor potential vanilloid 1 receptor (TRPV1) is present on peripheral terminals of airway sensory nerves and modulation of its activity represents a potential target for the pharmacological therapy of AHR in airway disease. EXPERIMENTAL APPROACH As guinea pig models can provide some of the essential features of asthma, including AHR, we have established the model with some classical pharmacological agents and examined the effect of the TRPV1 antagonists, SB-705498 and PF-04065463 on AHR to histamine evoked by ovalbumin (OA) in unanaesthetized sensitized guinea pigs restrained in a double chamber plethysmograph. Specific airway conductance (sGaw) derived from the airflow was calculated as a percentage of change from baseline. KEY RESULTS Cetirizine and salbutamol significantly inhibited OA-evoked bronchoconstriction [sGaw area under the curve (AUC): 70 and 78%, respectively]. Atropine, SB-705498 and PF-04065463 significantly inhibited OA-evoked AHR to histamine in unanaesthetized, OA-sensitized guinea pigs (sGaw AUC: 94%, 57% and 73%, respectively). Furthermore, this effect was not related to antagonism of histamine's activity. CONCLUSION AND IMPLICATIONS These data suggest that TRPV1 receptors located on airway sensory nerves are important in the development of AHR and that modulation of TRPV1-receptor activity represents a potential target for the pharmacological therapy of AHR in airway disease.

RSD931, a novel anti-tussive agent acting on airway sensory nerves.

1 The anti-tussive effects, of the local anaesthetic, lidocaine and carcainium chloride (RSD931) have been investigated in guinea-pigs and rabbits. 2 Pre-treatment of guinea-pigs with aerosols of lidocaine or RSD931 at 0.1, 1.0 and 10 mg ml(-1) reduced the number of citric acid-induced coughs by 9.3, 32.6 and 40.9% (P>0.05) for lidocaine and by 25.3% (P>0.05), 40.4% (P>0.05) and 97.6% (P<0.01) for RSD931, respectively and increased the latency to onset of cough at 10.0 mg ml(-1) only. In addition, RSD931 at 10 mg ml(-1) reduced citric acid-evoked cough responses in rabbits (with prior exposure to ozone at 3 p.p.m. for 1 h) from 22.1+/-5.1 to 2.7+/-0.9 coughs (P<0.01). 3 Acute pre-treatment of guinea-pigs with aerosols of lidocaine or RSD931 at 10.0 and 30.0 mg ml(-1) reduced the number of capsaicin-evoked coughs by 42.2 and 10.3% (P>0.05) (lidocaine) and by 25% (P>0.05) and 76.9% (P<0.01) (RSD931), respectively. Lidocaine had little effect on the latency of cough onset at either 10.0 or 30.0 mg ml(-1), however, RSD at 30.0 mg ml(-1) significantly (P<0.05) prolonged the latency of cough onset. 4 RSD931 (10.0 mg ml(-1)) significantly (P<0.05-<0.01) reduced the spontaneous and histamine-evoked discharges in Adelta-fibres originating from airway, rapidly adapting stretch receptors (RARs) without affecting histamine-evoked bronchoconstriction. Lidocaine at 10.0 mg ml(-1) also significantly (P<0.05) inhibited the spontaneous and histamine-induced discharges of RARs without affecting histamine-evoked bronchoconstriction. 5 Aerosols of RSD931 (10.0 mg ml(-1)) caused a transient, but significant (P<0.05), activation of pulmonary C-fibre endings 2.5 min after administration started. RSD931 had no significant (P>0.05) effects on discharges in bronchial C-fibres originating from bronchial C-fibre endings, capsaicin-evoked discharges of either pulmonary or bronchial C-fibre endings or on capsaicin-evoked bronchoconstriction. In contrast, lidocaine (10.0 mg ml(-1)) significantly (P<0.05) inhibited spontaneous and capsaicin-induced discharges in both pulmonary and bronchial C-fibres respectively. Lidocaine also significantly (P<0.05) reduced capsaicin-evoked bronchoconstriction. 6 These studies suggest that the anti-tussive actions of RSD931 are mediated via inhibition of discharges in Adelta-fibres originating from airway RARs. The mechanism of action of RSD931 is distinct from that of the local anaesthetic lidocaine and RSD931 may represent a novel class of anti-tussive agent.

The role of central 5-HT receptors in the bronchoconstriction evoked by inhaled capsaicin in anaesthetised guinea-pigs.

The effects of intracisternal (i.c) injections of the 5-HT1A receptor agonists, buspirone and 8-OH-DPAT, and the antagonists WAY-100635; and (-)-pindolol, the 5-HT1B/1D receptor agonist sumatriptan and antagonist GR127935, the 5-HT2 receptor agonist DOI and the antagonist cinanserin, the 5-HT3 receptor antagonist granisetron, the alpha-adrenoceptor agonist clonidine and the antagonist idazoxan, the D2 receptor antagonists (-)-sulpiride and the 5-HT uptake inhibitor fluoxetine on capsaicin-evoked increase in tracheal inflation pressure (bronchoconstriction) were investigated in alpha-chloralose anaesthetised, neuromuscularly blocked, artificially ventilated guinea-pigs. Buspirone, 8-OH-DPAT and fluoxetine significantly potentiated while WAY-100635 (-)-pindolol and sumatriptan attenuated the evoked bronchoconstriction when applied i.c. Granisetron attenuated the response when applied i.v. but not when given i.c. The 5-HT2, alpha2-adrenoceptor and D2 dopamine receptor ligands did not have any significant effect on the evoked bronchoconstriction. Pretreatment i.v. with WAY-100635 alone had no effect on the capsaicin-evoked bronchoconstriction but blocked the potentiating action of i.c. buspirone. The effects of sumatriptan could be completely blocked by pretreatment i.v. with GR127935. Only DOI, in the presence (i.v.) of the peripheral acting 5-HT2 receptor antagonist BW501C67, caused a significant increase in baseline tracheal inflation pressure. It is concluded that activation of central 5-HT1A and 5-HT1B/1D receptors have opposing roles, facilitation and inhibition respectively, on the reflex activation of bronchoconstrictor vagal preganglionic neurones.

The effect of 15-HPETE on airway responsiveness and pulmonary cell recruitment in rabbits.

1. In the present study we have investigated the effect of 15-hydroperoxyeicosatetraenoic acid (15-HPETE) and 15-hydroxyeicosatetraenoic acid (15-HETE) on airway responsiveness to inhaled histamine in rabbits in vivo. 2. 15-HPETE increased airway responsiveness to histamine 24 h after tracheal instillation and this was associated with a cellular infiltration consisting mainly of neutrophils, as measured by bronchoalveolar lavage. The airway hyperresponsiveness induced by 15-HPETE was still present 72 h after tracheal instillation of 15-HPETE, but had returned to baseline values one week post challenge. The number of neutrophils in bronchoalveolar lavage remained significantly elevated compared to pre-challenge levels. In contrast to 15-HPETE, the major metabolite 15-HETE, failed to alter airway hyperresponsiveness to histamine despite the recruitment of neutrophils into the lung, suggesting that the effect of 15-HPETE was not secondary to the generation of this metabolite nor dependent on the influx of neutrophils. 3. Both capsaicin and atropine but not the peripherally acting mu-opioid receptor agonist, BW443C (H-Tyr-D-Arg-Gly-Phe(4-NO2)-Pro-NH4), attenuated 15-HPETE-induced hyperresponsiveness. The increased cellular infiltration induced by 15-HPETE was only attenuated by capsaicin. 4. The results of the present study suggest that the release of 15-HPETE into the airways could contribute to sensitization of afferent nerve endings analogous to the hyperalgesia induced by this mediator in skin.

Involvement of central 5-HT1A receptors in the reflex activation of pulmonary vagal motoneurones by inhaled capsaicin in anaesthetized cats.

1. The aim of the present experiments was to determine whether 5-HT1A receptors play a role in the control of the reflex activation of pulmonary vagal motoneurones. This was carried out by investigating the effects of intracisternal injections (i.c.) of the 5-HT1A receptor ligands, 8-OH-DPAT (50 micrograms kg-1), buspirone (200 micrograms kg-1), WAY-100635 (100 micrograms kg-1), methiothepin (200 micrograms kg-1) and (-)-pindolol (100 micrograms kg-1) and the 5-HT2 receptor antagonist, cinanserin (200 micrograms kg-1), on the reflex bronchoconstriction evoked by inhaled capsaicin aerosol in alpha-chloralose anaesthetized, neuromuscularly blocked and artificially ventilated cats. Recordings were made of heart rate, blood pressure and upper tracheal pressure. 2. Central application of all the 5-HT1A receptor antagonists (methiothepin, WAY-100635 and (-)-pindolol) attenuated the reflex bronchoconstriction in the upper trachea. However, the same dose of WAY-100635 given i.v. had no effect on this reflex bronchoconstriction. The 5-HT1A receptor agonist, 8-OH-DPAT (50 micrograms kg-1) given i.c., potentiated the capsaicin-evoked reflex bronchoconstriction, whereas buspirone (200 micrograms kg-1) i.c. had no effect. The 5-HT2 receptor antagonist, cinanserin (200 micrograms kg-1) also had no effect. 3. It is concluded that the reflex excitation of pulmonary vagal motoneurones by inhaled capsaicin in alpha-chloralose anaesthetized cats involves the activation of central 5-HT1A receptors.

Effects of aerosol-applied capsaicin, histamine and prostaglandin E2 on airway sensory receptors of anaesthetized cats.

1. Capsaicin, prostaglandin E2 (PGE2) and histamine are potent stimuli for reflex coughing and bronchoconstriction in many species including man. We have studied the effects of solutions of capsaicin, PGE2 and histamine on airway sensory receptors when administered as inhaled aerosols to the lower respiratory tract in anaesthetized, paralysed and artificially ventilated cats. 2. Histamine, administered by aerosol (6 breaths of a 1 mg ml-1 solution) and intravenously (10 micrograms kg-1), caused an increase in the rate of discharge from rapidly adapting stretch receptors (RARs) and caused bronchoconstriction. 3. Six breaths of a capsaicin aerosol generated from solutions of 0.1 or 1 mg ml-1 stimulated six out of nine RARs tested. Bronchoconstriction occurred with and without RAR stimulation. The diluent for the capsaicin aerosol had no significant effect on pulmonary mechanics or rate of RAR discharge. 4. Administration of increasing concentrations (0.001-1 mg ml-1) of PGE2 aerosol given in six breaths (at 6 min intervals) caused a dose-dependent increase in the rate of discharge of eight RARs tested and caused bronchoconstriction. The diluent for the PGE2 aerosol had no effect on pulmonary mechanics or rate of RAR discharge. 5. Inhalation of aerosols of histamine (6 breaths of 1 mg ml-1 solution) and capsaicin (3 breaths of 0.1 mg ml-1 solution) stimulated all six lung C fibre endings studied (3 pulmonary and 3 bronchial). These aerosols of capsaicin and histamine also caused bronchoconstriction. 6. We conclude that solutions of capsaicin and PGE2, when delivered by aerosol to the airway epithelial surface, are not selective stimulants of C fibres. Both agents can stimulate RARs. Activation of some but not all RARs tested, by inhaled capsaicin, suggests that there are subpopulations of capsaicin-sensitive and -insensitive receptors. Stimulation of airway RARs by a range of pharmacologically active agents released by airway inflammation may contribute to reflex coughing and bronchoconstriction in man.

Effect of BW443C81, a novel opioid, on non-cholinergic bronchoconstrictor responses and neurogenic plasma extravasation in the guinea pig.

The novel, peripherally acting opioid peptide, BW443C81, which attenuates airway sensory nerve impulses, was examined on non-cholinergic (NC) constrictor responses in vitro and in vivo and neurogenic plasma extravasation in vivo in guinea-pig airways. Non-cholinergic contractions of guinea pig isolated bronchi, evoked by electrical field stimulation, were concentration-dependently inhibited by BW443C81 and morphine (10 nmol/1-100 mumol/l). In anaesthetised, artificially ventilated guinea pigs, frequency-related NC bronchoconstrictor responses evoked by antidromic electrical stimulation of the vagus nerves were reduced by BW443C81 (100 micrograms/kg/min i.v. infusion) and morphine (1 mg/kg i.v.). Neurogenic plasma extravasation produced by bilateral electrical vagal nerve stimulation in spontaneously breathing, anaesthetised guinea pigs was also inhibited by BW443C81 (1 mg/kg i.v.). The inhibitory effects of BW443C81 were reversed/prevented by naloxone. BW443C81 inhibits NC bronchoconstrictor responses and neurogenic plasma extravasation in guinea pig airways, consistent with its previously described mu-opioid receptor-mediated inhibitory action on airway sensory nerve function.

Capsaicin-induced bronchoconstriction in the guinea-pig: contribution of vagal cholinergic reflexes, local axon reflexes and their modulation by BW443C81.

1 The objective of the study was to investigate the central vagal and local axon reflex components of bronchoconstrictor responses evoked by inhalation of capsaicin aerosol in anaesthetized guinea-pigs. This was accomplished by comparing the effects of bilateral vagotomy, atropine and the peripherally-acting polar enkephalin analogue, BW443C81, on bronchoconstrictor responses evoked by capsaicin. The effects of codeine were also determined. 2 Aerosols of capsaicin were generated from a 0.9 microgram ml-1 solution. Inhalation of capsaicin aerosol in 5, 10 and 15 breaths evoked dose-related bronchoconstrictor responses. The responses were immediate in onset and of extended duration. 3 Capsaicin-induced bronchoconstrictor responses were significantly inhibited following bilateral vagotomy or atropine (0.3 mg kg-1, i.v.) pretreatment by 46% +/- 14% (P less than 0.05) and 59% +/- 13% (P less than 0.01), respectively. 4 Administration of BW443C81 by intravenous infusion (3, 30 and 100 micrograms kg-1 min-1) caused a significant inhibition of capsaicin-induced bronchoconstrictor responses which achieved a greater maximum than either bilateral vagotomy or atropine. Codeine (100 micrograms kg-1 min-1, i.v.) did not significantly inhibit the bronchoconstrictor responses. 5 Inhibition of capsaicin-induced bronchoconstrictor responses by BW443C81 (30 micrograms kg-1 min-1, i.v.) was significantly (P less than 0.05) reduced by the peripherally-acting opioid antagonist N-methyl nalorphine (100 micrograms kg-1 min-1, i.v.). 6 These results show that capsaicin-induced bronchoconstrictor responses are mediated by at least two mechanisms, a vagal and/or cholinergic reflex pathway and a non-cholinergic pathway. BW443C81, but not codeine, significantly inhibited (P < 0.005) both mechanisms of capsaicin-induced bronchoconstriction probably by an action on peripheral opioid receptors located on vagal sensory nerves.

Peripheral opioid receptors and the cough reflex.

The cough reflex originates from stimulation of sensory nerve endings located in the upper and lower respiratory tract. Opiates are among the most potent and widely used drugs which inhibit the cough reflex, and it has been generally assumed that they act generally. However, opioid receptors have been identified on the sensory arm of the vagus nerve. Although the functional significance of these receptors is not clear, recent evidence suggests that their activation may be involved in the modulation of airway reflexes. This review briefly examines the evidence to support the hypothesis that opioid receptors on vagal sensory nerves may mediate peripheral opioid-induced antitussive activity.

Effects of codeine, morphine and a novel opioid pentapeptide BW443C, on cough, nociception and ventilation in the unanaesthetized guinea-pig.

1. Antitussive, antinociceptive and respiratory depressant effects of codeine, morphine and H.Tyr.D-Arg.Gly.Phe(4-NO2) Pro.NH2 (compound BW443C) were investigated in unanaesthetized guinea-pigs. Antagonism of the antitussive and antinociceptive effects was investigated by the use of nalorphine and N-methylnalorphine. Naloxone was used to antagonize respiratory depression. 2. Antitussive ED50s (with 95% confidence limits) for inhibition of cough induced by citric acid vapour were for codeine, morphine and BW443C respectively, 9.1(5.8-15), 1.3(0.7-2.4) and 1.2(0.6-2.6) mg kg-1 s.c. and 8.7(4.2-12), 1.6(1.2-1.9) and 0.67(0.002-3.3) mg kg-1, i.v. The antitussive effects of subcutaneous codeine (25 mg kg-1) morphine (8.1 mg kg-1) and BW443C (2.5 mg kg-1) were significantly antagonized by subcutaneous nalorphine (3.0 mg kg-1) and N-methylnalorphine (3.0 mg kg-1). 3. In the multiple toe-pinch test, the antinociceptive ED50s (with 95% confidence limits) of codeine and morphine were 18(16-22) and 2.3(0.4-4.3) mg kg-1, s.c., respectively. Compound BW443C was ineffective in doses of 2.5 and 10 mg kg-1 s.c., a result consistent with its lacking penetration into the CNS. Subcutaneous nalorphine (3.0 mg kg-1) antagonized the antinociceptive action of codeine (25 mg kg-1) and morphine (8.1 mg kg-1). In contrast, N-methylnalorphine (3.0 mg kg-1) had no significant effect on the antinociceptive action of codeine and morphine, suggesting lack of penetration of the CNS by N-methylnalorphine. 4. At doses near to the i.v. ED50 values for the antitussive activity, morphine (1.5mg kg- ', i.v.) and codeine (10mg kg-', i.v.) caused small but significant depressions of ventilation (7.0 +/- 2.3% and 16.5 +/- 8.4% respectively). Higher doses of morphine (10, 30 and 60mg kg- ', i.v.) caused further doserelated depression of ventilation (9.6 +/- 5.3%, 22.4 +/- 6.2% and 36.2 +/- 9.6% respectively) whereas codeine (30 and 60mg kg-' i.v.) caused stimulation of ventilation which was marked (191.3 +/- 43.9%) at 60 mg kg-'. 5. Compound BW443C in doses of 1 or 10mgkg-',i.v. (approximately equal to, and 10 times the EDo for antitussive activity) did not cause significant depression of ventilation. Only at higher doses of 30 and 60mg kg-', i.v. was there a significant decrease in minute volume (13.1 +/- 6.8% and 15.9 +/- 1.89% respectively). The depression of ventilation caused by either BW443C (60mg kg-', i.v.) or morphine (60mg kg-', i.v.) was prevented by pretreatment with naloxone (3mg kg-', i.v.) administered 15 min before morphine or BW443C. 6. These results in the guinea-pig support the hypothesis that the antitussive action of the opiates codeine and morphine and the opioid pentapeptide BW443C do not require penetration of these drugs into the CNS.

Modification of human airway smooth muscle reactivity by drugs that interfere with arachidonic acid metabolism.

Histamine-induced contractions of small airways from human lung were substantially augmented by the cyclo-oxygenase inhibitor, indomethacin, whereas the contraction of larger airways was not. Mixed cyclo-oxygenase/lipoxygenase inhibitors of arachidonic acid metabolism did not modify histamine-induced contractions of smooth muscle from either small or large airways but completely reversed the augmentation of histamine responses produced by indomethacin on the smaller airways. The SRS-A (slow reacting substance of anaphylaxis) antagonist, FPL55712, did not affect the augmentation of histamine-induced contractions by indomethacin.

A possible role for lipoxygenase products as regulators of airway smooth muscle reactivity.

The size of guinea-pig isolated tracheal contractions induced by histamine was substantially augmented by pretreatment with the cyclo-oxygenase inhibitor, indomethacin. However, compounds which inhibit both the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism not only did not augment histamine-induced contractions of the guinea-pig trachea, but also completely reversed the increased reactivity to histamine produced by indomethacin. The SRS-A (slow reacting substance of anaphylaxis) antagonist, FPL55712, and the anti-allergic drug, cromoglycate, had no effect on the augmentation of histamine-induced contractions by indomethacin.

Enhancement of anaphylactic mediator release from guinea-pig perfused lungs by fatty acid hydroperoxides.

Fragments of chopped lung from indomethacin treated guinea-pigs had an anti-aggregating effect when added to human platelet rich plasma (PRP), probably due to the production of prostacyclin (PGI2) since the effect was inhibited by 15-hydroperoxy arachidonic acid (15-HPAA, 10 micrograms ml(-1)). Both 15-HPAA (1-20 micrograms ml(-1) min (-1)) and 13-hydroperoxy linoleic acid (13-HPLA, 20 micrograms ml(-1) min(-1)) caused a marked enhancement of the anaphylactic release of histamine, slow-reacting substance of anaphylaxis (SRS-A) and rabbit aorta contracting substance (RCS) from guinea-pig isolated perfused lungs. This enhancement was not reversed by the concomitant infusion of either PGI2 (5 micrograms ml(-1) min (-1)) or 6-oxo-prostaglandin F1alpha (6-oxo-PGF1alpha, 5 micrograms ml(-1) min(-1)). Anaphylactic release of histamine and SRS-A from guinea-pig perfused lungs was not inhibited by PGI2 (10 ng - 10 microgram ml(-1) min(-1)) but was inhibited by PGE2 (5 and 10 micrograms ml(-1) min (-1)). Antiserum raised to 5,6-dihydro prostacyclin (PGI1) in rabbits, which also binds PGI2, had no effect on the release of anaphylactic mediators. The fatty acid hydroperoxides may enhance mediator release either indirectly by augmenting thromboxane production or by a direct effect on sensitized cells. Further experiments to distinguish between these alternatives are described in the accompanying paper (27).

The mechanism of enhancement by fatty acid hydroperoxides of anaphylactic mediator release.

Indomethacin augmented the release of histamine and SRS-A but abolished synthesis of TxB2. Compound CLI that inhibited both cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism did not augment release of anaphylactic mediators. 13-HPLA enhanced mediator release from lungs in which arachidonic acid metabolism was blocked by compount CLI. Thus, it is concluded that 13-HPLA enhances mediator release not by altering the balance of arachidonic acid metabolites, e.g. by inhibiting synthesis of prostacyclin, but by a direct effect on lung mast cells. A corollary to this conclusion is that the fatty acid hydroperoxide (HPETE) formed by lipoxygenase from arachidonic acid may also augment the release of anaphylactic mediators. Thus, the enhancement of mediator release by indomethacin may be attributed to increased synthesis of HPETE following inhibition of cyclo-oxygenase.

Separation of IgE from IgG subclasses using staphylococcal protein A.

When human serum was passed through a column of insolubilised protein A, IgE was not removed, but IgG sublasses 1,2 and 4 were retained. Protein A did not show a similar selectivity in its interaction with rat immunoglobulins. The separation of human IgE from the bulk of the IgG was used to examine the effect of IgG on IgE-mediated sensitization of lung tissue for histamine reslease. Removal of IgG led to a significant increase in histamine release, suggesting that homocytotropic IgG competes with IgE, for binding sites on mast cells.