ACh-induced Bronchoconstriction Research Articles

The impact of long-acting muscarinic antagonists on mucus hypersecretion and cough in chronic obstructive pulmonary disease: a systematic review.

Patients suffering from chronic obstructive pulmonary disease (COPD) clinically manifest airway mucus hypersecretion as sputum expectoration and cough. Evidence accumulated in the past decade has shown that the cholinergic system not only regulates airway smooth muscle contraction but also the activity of inflammatory and airway epithelial cells, including goblet cells, and submucosal gland activity. Long-acting muscarinic antagonists (LAMAs) with the most favourable M3/M2 muscarinic acetylcholine (ACh) receptors residency properties are not only excellent bronchodilators but potentially also mucus-modifying agents, able to positively impact on mucus hypersecretion and cough. The aim of this systematic review was to investigate the impact of LAMAs on mucus hypersecretion and cough in COPD patients. The evidence confirmed that LAMAs, mainly tiotropium and aclidinium, improved sputum production and cough in moderate to severe COPD. Thus, LAMAs not only antagonise the ACh-induced bronchoconstriction of the airways but also appear to limit the production of mucus secreted in response to ACh by airway goblet cells and/or submucosal glands. Further clinical studies are necessary to evaluate the impact of LAMAs exclusively on sputum symptoms and cough as primary end-points and to investigate whether LAMAs have a modulatory action on the rheological properties of mucus.

Onset of diabetes modulates the airway smooth muscle reactivity of guinea pigs: role of epithelial mediators.

Background: Diabetes induces lung dysfunction, leading to alteration in the pulmonary functions. Our aim was to investigate whether the early stage of diabetes alters the epithelium-dependent bronchial responses and whether nitric oxide (NO), KATP channels and cyclooxygenase (COX) pathways contribute in this effect. Methods: Guinea pigs were treated with a single injection of streptozotocin (180 mg/kg, ip) for induction of diabetes. Airway conductivity was assessed by inhaled histamine, using a non-invasive body plethysmography. The contractile responses of tracheal rings induced by acetylcholine (ACh) and relaxant responses of precontracted rings, induced by isoproterenol (IP) were compared in the presence and absence of the epithelium. Effects of Nω-Nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor), glybenclamide (a KATP channel inhibitor) and indomethacin (a COX inhibitor) were also assessed in diabetic guinea pigs. Results: Early stage diabetes did not alter the airway conductivity. ACh-induced bronchoconstriction in epithelium intact tracheal rings was not affected by the onset of diabetes, however a reduction in the increased ACh responses due to epithelium removal, to L-NAME or to indomethacin was observed. The relaxation response to IP was impaired in trachea from guinea pigs in which diabetes had just developed. Early diabetes significantly reduced the IP response to glybenclamide and to indomethacin. Conclusion: Our results demonstrate that the early stage of diabetes, modulate the bronchial reactivity to both ACh and IP by disrupting the NO, KATP channels and COX pathways, without affecting the airway conductivity in guinea pigs.

Pharmacological Characterization of GSK573719 (Umeclidinium): A Novel, Long-Acting, Inhaled Antagonist of the Muscarinic Cholinergic Receptors for Treatment of Pulmonary Diseases

Activation of muscarinic subtype 3 (M3) muscarinic cholinergic receptors (mAChRs) increases airway tone, whereas its blockade improves lung function and quality of life in patients with pulmonary diseases. The present study evaluated the pharmacological properties of a novel mAChR antagonist, GSK573719 (4-[hydroxy(diphenyl)methyl]-1-{2-[(phenylmethyl)oxy]ethyl}-1-azoniabicyclo[2.2.2]octane; umeclidinium). The affinity (Ki) of GSK573719 for the cloned human M1-M5 mAChRs ranged from 0.05 to 0.16 nM. Dissociation of [(3)H]GSK573719 from the M3 mAChR was slower than that for the M2 mAChR [half-life (t1/2) values: 82 and 9 minutes, respectively]. In Chinese hamster ovary cells transfected with recombinant human M3 mAChRs, GSK573719 demonstrated picomolar potency (-log pA2 = 23.9 pM) in an acetylcholine (Ach)-mediated Ca(2+) mobilization assay. Concentration-response curves indicate competitive antagonism with partial reversibility after drug washout. Using isolated human bronchial strips, GSK573719 was also potent and showed competitive antagonism (-log pA2 = 316 pM) versus carbachol, and was slowly reversible in a concentration-dependent manner (1-100 nM). The time to 50% restoration of contraction at 10 nM was about 381 minutes (versus 413 minutes for tiotropium bromide). In mice, the ED50 value was 0.02 μg/mouse intranasally. In conscious guinea pigs, intratracheal administration of GSK573719 dose dependently blocked Ach-induced bronchoconstriction with long duration of action, and was comparable to tiotropium; 2.5 μg elicited 50% bronchoprotection for >24 hours. Thus, GSK573719 is a potent anticholinergic agent that demonstrates slow functional reversibility at the human M3 mAChR and long duration of action in animal models. This pharmacological profile translated into a 24-hour duration of bronchodilation in vivo, which suggested umeclidinium will be a once-daily inhaled treatment of pulmonary diseases.

Non‐bronchodilating mechanisms of tiotropium prevent airway hyperreactivity in a guinea‐pig model of allergic asthma

Asthma is characterized by reversible bronchoconstriction and airway hyperreactivity. Although M(3) muscarinic receptors mediate bronchoconstriction, non-selective muscarinic receptor antagonists are not currently recommended for chronic control of asthma. We tested whether selective blockade of M(3) receptors, at the time of antigen challenge, blocks subsequent development of airway hyperreactivity in antigen-challenged guinea-pigs. Ovalbumin-sensitized guinea-pigs were pretreated with 1 µg·kg(-1) of a kinetically selective M(3) receptor antagonist, tiotropium, or 1 mg·kg(-1) of a non-selective muscarinic receptor antagonist, atropine, and challenged with inhaled ovalbumin. Animals were anaesthetized, paralyzed, ventilated and vagotomized 24 h later. We measured vagally mediated bronchoconstriction and i.v. ACh-induced bronchoconstriction. Electrical stimulation of both vagus nerves induced frequency-dependent bronchoconstriction in sensitized animals that was significantly increased after antigen challenge. Antigen-induced hyperreactivity was completely blocked by tiotropium pretreatment but only partially blocked by atropine pretreatment. Surprisingly, although tiotropium blocked bronchoconstriction induced by i.v. ACh, it did not inhibit vagally-induced bronchoconstriction in sensitized controls, suggesting that tiotropium does not block hyperreactivity by blocking receptors for vagally released ACh. Rather, tiotropium may have worked through an anti-inflammatory mechanism, since it inhibited eosinophil accumulation in the lungs and around nerves. These data confirm that testing M(3) receptor blockade with exogenous ACh does not predict vagal blockade. Our data also suggest that selective blockade of M(3) receptors may be effective in asthma via mechanisms that are separate from inhibition of bronchoconstriction.

Bronchodilator Activity of (3R)-3-[[[(3-fluorophenyl)[(3,4,5-trifluorophenyl)methyl]amino] carbonyl]oxy]-1-[2-oxo-2-(2-thienyl)ethyl]-1-azoniabicyclo[2.2.2]octane bromide (CHF5407), a Potent, Long-Acting, and Selective Muscarinic M3 Receptor Antagonist

The novel quaternary ammonium salt (3R)-3-[[[(3-fluorophenyl)[(3,4,5-trifluorophenyl)methyl]amino]carbonyl]oxy]-1-[2-oxo-2-(2-thienyl)ethyl]-1-azoniabicyclo[2.2.2]octane bromide (CHF5407) showed subnanomolar affinities for human muscarinic M1 (hM1), M2 (hM2), and M3 (hM3) receptors and dissociated very slowly from hM3 receptors (t(½) = 166 min) with a large part of the receptorial complex (54%) remaining undissociated at 32 h from radioligand washout. In contrast, [(3)H]CHF5407 dissociated quickly from hM2 receptors (t(½) = 31 min), whereas [(3)H]tiotropium dissociated slowly from both hM3 (t(½) = 163 min) and hM2 receptor (t(½) = 297 min). In the guinea pig isolated trachea and human isolated bronchus, CHF5407 produced a potent (pIC(50) = 9.0-9.6) and long-lasting (up to 24 h) inhibition of M3 receptor-mediated contractile responses to carbachol. In the guinea pig electrically driven left atrium, the M2 receptor-mediated inhibitory response to carbachol was recovered more quickly in CHF5407-pretreated than in tiotropium-pretreated preparations. CHF5407, administered intratracheally to anesthetized guinea pigs, potently inhibited acetylcholine (Ach)-induced bronchoconstriction with an ED(50) value of 0.15 nmol/kg. The effect was sustained over a period of 24 h, with a residual 57% inhibition 48 h after antagonist administration at 1 nmol/kg. In conscious guinea pigs, inhaled CHF5407 inhibited Ach-induced bronchoconstriction for at least 24 h as did tiotropium at similar dosages. Cardiovascular parameters in anesthetized guinea pigs were not significantly changed by CHF5407, up to 100 nmol/kg i.v. and up to 1000 nmol/kg i.t. In conclusion, CHF5407 shows a prolonged antibronchospastic activity both in vitro and in vivo, caused by a very slow dissociation from M3 receptors. In contrast, CHF5407 is markedly short-acting at M2 receptors, a behavior not shared by tiotropium.

Muscarinic receptors involved in airway vascular leakage induced by experimental gastro-oesophageal reflux

Gastro-oesophageal acid reflux may cause airway responses such as cough, bronchoconstriction and inflammation in asthmatic patients. Studies in humans or in animals have suggested that these responses involve cholinergic nerves. The purpose of this study was to investigate the role of the efferent vagal component on airway microvascular leakage induced by instillation of hydrochloric acid (HCl) into the oesophagus of guinea-pigs and the subtype of muscarinic receptors involved. Airway microvascular leakage induced by intra-oesophageal HCl instillation was abolished by bilateral vagotomy or by the nicotinic receptor antagonist, hexamethonium. HCl-induced leakage was inhibited by pretreatment with atropine, a non-specific muscarinic receptor antagonist, and also by pretreatment with either pirenzepine, a muscarinic M 1 receptor antagonist, or 4-DAMP, a muscarinic M 3 receptor antagonist. Pirenzepine was more potent than atropine and 4-DAMP. These antagonists were also studied on airway microvascular leakage or bronchoconstriction induced by intravenous administration of acetylcholine (ACh). Atropine, pirenzepine and 4-DAMP inhibited ACh-induced airway microvascular leakage with similar potencies . In sharp contrast, 4-DAMP and atropine were more potent inhibitors of ACh-induced bronchoconstriction than pirenzepine. Methoctramine, a muscarinic M 2 receptor antagonist, was ineffective in all experimental conditions. These results suggest that airway microvascular leakage caused by HCl intra-oesophageal instillation involves ACh release from vagus nerve terminals and that M 1 and M 3 receptors play a major role in cholinergic-mediated microvascular leakage, whereas M 3 receptors are mainly involved in ACh-induced bronchoconstriction.

Macrophages mediate organophosphorus pesticide‐induced airway hyperreactivity in guinea pigs

Organophosphorus pesticide (OP) exposure is implicated in human asthma. At doses that do not inhibit acetycholinesterase, OPs such as parathion (Pth) block inhibitory neuronal M2 receptor function, which increases acetylcholine (ACh) release and potentiates vagally‐induced bronchoconstriction in guinea pigs. We tested whether macrophages mediate Pth‐induced airway hyperreactivity. Guinea pig alveolar macrophages were depleted with liposome‐encapsulated dichloromethylene‐diphosphonate (clodronate; prepared by Nico van Rooijen, Amsterdam) given intranasally 1 and 3 days prior to 1.0 mg/kg Pth sc. 24 hrs after Pth administration, animals were anesthetized and ventilated. In non‐Pth treated animals, bronchoconstriction induced by electrical stimulation of both vagus nerves was not affected by clodronate or PBS liposomes. Pth significantly potentiated bronchoconstriction in animals receiving PBS liposomes but did not potentiate bronchoconstriction in those pre‐treated with clodronate. ACh‐induced bronchoconstriction was not affected by any treatment, indicating that smooth muscle responsiveness was not changed. Macrophages mediate Pth‐induced airway hyperreactivity possibly by the release of interferon‐ã which decreases neuronal M2 RNA expression.Funding: NIH ES014521, ES014601, HL55543, HL54659, HL071795

Protective effects of volatile agents against acetylcholine‐induced bronchoconstriction in isolated perfused rat lungs

Bronchoactive properties of volatile agents against lung constriction are well established. The purpose of this study was to investigate the ability of halothane (Hal), isoflurane (Iso), sevoflurane (Sev) and desflurane (Des) to alter the lung mechanics in the absence of an airway tone and during acetylcholine (Ach)-induced bronchoconstriction. Low-frequency pulmonary impedance data (ZL) were collected from isolated, normo-perfused rat lungs under baseline conditions and following the injection of Ach (0.1 mg/kg) into the pulmonary artery. Measurements were performed without the administration of any anaesthetic agent in the first phase of the experiments and during inhalation without any volatile agent (control group, n = 6) or during inhalation of Hal (n = 6), Iso (n = 9), Sev (n = 6) or Des (n = 8) at 1 minimum alveolar concentration (MAC). The airway resistance (Raw) and parenchymal damping and elastance were estimated from the Z(L) data by model fitting. Under baseline conditions, the basic value of Raw was significantly decreased by Des (- 31.2 +/- 3.8%) and Sev (- 18.0 +/- 4.5%) administration, whereas Hal and Iso did not have a statistically significant effect on Raw (- 3.3 +/- 5.1% and - 8.6 +/- 2.4%, respectively). Moreover, all four inhalation anaesthetics prevented the increase in Raw following Ach administration, the findings ranging between - 14.3 +/- 11.4% for Hal and - 37.5 +/- 10.9% for Sev. Our results on a denervated isolated perfused lung model demonstrate the potential of Des and Sev to decrease the basal airway tone, whereas Iso and Hal are ineffective in this regard. All of these volatile agents markedly protect against Ach-induced bronchoconstriction.

Treatment of bronchial asthma with tianeptine

Although circulating catecholamines and free serotonin in the plasma (f-5-HT) were found to be increased during asthma attacks, only f-5-HT levels correlated positively with bronchoconstriction and clinical severity. Tianeptine, a drug that enhances serotonin uptake by platelets and serotonergic axons at the central nervous system (CNS), provoked an abrupt disappearance of asthma attacks. This fact explains why tianeptine has proven to be a powerful therapeutic tool in controlling asthma. Its success has been demonstrated not only in two double-blind placebo, cross-over trials, but through an open study lasting more than seven years that included over 25,000 asthmatic patients. In the present article, we discuss the peripheral and central nervous system mechanisms that may explain the therapeutic success of tianeptine. These are summarized below. F-5-HT is taken up by pulmonary endocrine cells (PNEC) located at the parasympathetic terminals. A presynaptic element, these cells release serotonin and potentiate acetylcholine (ACh)-induced contraction of bronchial muscle. This effect is mediated by 5-HT(3) and 5-HT(4) postsynaptic receptors located at the bronchial muscle. According to the above, the increased f-5-HT plasma level, triggered by both platelet aggregation and nocturnal and/or diurnal hyperparasympathetic activity, potentiates ACh-induced bronchoconstriction. The fact that serotonin released by medullary serotonergic axons stimulates the medullary vagal cardiorespiratory neurons obliges us to think that serotonin-induced CNS mechanisms are also involved. Furthermore, the finding that drugs that interfere with serotonin uptake, by both platelets and 5-HT-terminals, worsen asthma symptoms and are able to provoke asthma attacks gives additional support to the above peripheral and CNS mechanisms.

Chronic systemic administration of salmeterol to rats promotes pulmonary beta(2)-adrenoceptor desensitization and down-regulation of G(s alpha).

1. The aim of the present study was to examine the effects of chronic infusion of the long-acting agonist salmeterol on pulmonary beta(2)-adrenoceptor function in Sprague-Dawley rats in vivo and to elucidate the molecular basis of any altered state. 2. Systemic administration of rats with salmeterol for 7 days compromised the ability of salmeterol and prostaglandin E(2) (PGE(2)), given acutely by the intravenous route, to protect against ACh-induced bronchoconstriction when compared to rats treated identically with vehicle. 3. beta(1)- and beta(2)-adrenoceptor density was significantly reduced in lung membranes harvested from salmeterol-treated animals, which was associated with impaired salmeterol- and PGE(2)-induced cyclic AMP accumulation ex vivo. 4. Three variants of G(s alpha) that migrated as 42, 44 and 52 kDa peptides on SDS polyacrylamide gels were detected in lung membranes prepared from both groups of rats but the intensity of each isoform was markedly reduced in rats that received salmeterol. 5. The activity of cytosolic, but not membrane-associated, G-protein receptor-coupled kinase was elevated in the lung of salmeterol-treated rats when compared to vehicle-treated animals. 6. The ability of salmeterol, administered systemically, to protect the airways of untreated rats against ACh-induced bronchoconstriction was short-acting (t(off) approximately 45 min), which contrasts with its long-acting nature when given to asthmatic subjects by inhalation. 7. These results indicate that chronic treatment of rats with salmeterol results in heterologous desensitization of pulmonary G(s)-coupled receptors. In light of previous data obtained in rats treated chronically with salbutamol, we propose that a primary mechanism responsible for this effect is a reduction in membrane-associated G(s alpha). The short-acting nature of salmeterol, when administered systemically, and the reduction in beta-adrenoceptor number may be due to metabolism to a biologically-active, short-acting and non-selective beta-adrenoceptor agonist.

J-104129, a novel muscarinic M3 receptor antagonist with high selectivity for M3 over M2 receptors

A new class of 4-acetamidopiperidine derivatives has been synthesized and investigated for human muscarinic receptor subtype selectivity. Introduction of a hydrocarbon chain of appropriate length into the piperidine nitrogen of the racemic N-(piperidin-4-yl)-2-cyclobutyl-2-hydroxy-2-phenylacetamide platform conferred up to 70-fold selectivity for human muscarinic M3 receptors over M2 receptors. Subsequent synthetic derivatizations resulted in highly potent M3 receptor antagonists with selectivity greater than two orders of magnitude for M3 over M2 receptors, from which the analogue 4r was selected. Preparation of both enantiomers of 4r led to the identification of (2R)-N-[1-(4-methyl-3-pentenyl)piperidin-4-yl]-2-cyclopentyl-2-hydroxy-2-phenylacetamide (J-104129, (R)-4r), which exhibited 120-fold selectivity for M3 receptors (Ki=4.2 nM) over M2 receptors (Ki=490 nM). In isolated rat trachea, (R)-4r potently and specifically antagonized acetylcholine (ACh)-induced responses with a KB value of 3.3 nM. The highly subtype-selective profile was also seen in isolated rat tissue assays (50-fold) and in anesthetized rats (>250-fold). Oral administration of J-104129 ((R)-4r) antagonized ACh-induced bronchoconstriction with an ED50 value of 0.58 mg/kg in rats. Thus, J-104129 ((R)-4r) may effectively facilitate bronchodilation in the treatment of obstructive airway disease.

Low frequency-dependent mechanism of the M 2 receptor function on vagally mediated bronchoconstriction in rabbits in vivo

The present study was carried out to investigate whether there is the difference between low and high frequencies of vagal stimulation on the functional appearance of M 2 receptors in the rabbit. The animals were anesthetized, artificially ventilated and bilaterally vagotomized. Bilateral vagus nerve stimulation (5 to 30 Hz) for 30 sec caused bronchoconstriction (measured as an increase in R L and a decrease in Cdyn) in a frequency-dependent manner. The bronchoconstriction evoked by ACh injection (1 and 3 μg kg ) was dose-dependent. Although administration of methoctramine (50 and 300 μg kg ), a selective M 2 receptor antagonist, had no significant effect on ACh-induced bronchoconstriction, methoctramine dose-dependently augmented the R L and Cdyn responses to vagal stimulation at 5–15 Hz but did not potentiate bronchoconstrictive responses to the stimulation at 30 Hz. Administration of [D-Pro 2, D-Try 7,9]-SP (0.5 mg kg , a selective tachykinin receptor antagonist) that had no significant effect on the R L and Cdyn responses to vagal stimulation (5–15 Hz) attenuated the bronchoconstrictive response to the stimulation at 30 Hz. Conversely, thiorphan (2 mg kg , a neutral endopeptidase inhibitor) potentiated the bronchoconstriction evoked by vagal stimulation at 30 Hz only. These results suggest that M 2 receptors function as the inhibitory receptors in the bronchoconstrictive response to vagal stimulation at the lower frequencies (5–15 Hz), but that the M 2 receptor antagonism is diminished when vagal stimulation at a higher frequency (30 Hz) results in the release of SP from the lungs.

Effects of isoenzyme selective phosphodiesterase inhibitors on bacterial lipopolysaccharide-induced bronchial hyperreactivity in guinea pigs

1. 1. Effects of cilostazol and vesnarinone (selective PDE III inhibitors), rolipram (selective PDE IV inhibitor) and theophylline (nonselective PDE inhibitor) on LPS-induced bronchial hyperreactivity were investigated in guinea pigs. 2. 2. Cilostazol (50 mg/kg PO), vesnarinone (100 mg/kg PO), rolipram (20 mg/kg PO), and theophylline (50 mg/kg PO), significantly inhibited bronchial hyperreactivity to acetylcholine (Ach) and TNF release into bronchoalveolar lavage fluid following LPS exposure. None of these compounds influenced neutrophil influx into bronchoalveolar lavage fluid. 3. 3. Rolipram and theophylline antagonized Ach-induced bronchoconstriction in normal guinea pigs.

Modulation of airway responsiveness to acetylcholine by nitric oxide in a rabbit model.

. Nitric oxide (NO) is an important mediator in the regulation of bronchial muscle tone and airway responsiveness. We investigated the influence of exogenous NO on airway responsiveness to acetylcholine aerosols (ACH) in normal and in hyperresponsive rabbits. White New Zealand rabbits were anesthetized, intubated, and breathed room air spontaneously. Responses of respiratory parameters in ACH challenge tests were measured. In group A the influence of NO on ACH infusion-induced airway constriction was measured. Airway responses to aerosols from 0.25 to 8.0% ACH solutions in saline were measured with 150 and 300 ppm NO inhalation (groups B and C) and compared with the same animals' responses without NO. Moreover, we examined the influence of NO synthase inhibition on airway responsiveness (group D) and the modulatory effect of NO in hyperresponsive animals (group E). 300 ppm NO inhalation significantly decreased the bronchoconstrictor response to intravenously administered ACH (group A). However, the baseline value of dynamic elastance (Edyn) was only marginally lower under the influence of 300 ppm NO. During inhalation of 150 or 300 ppm NO, responses to nebulized 2.0% and less ACH solutions remained nearly unaltered. Responses to aerosols of 4.0 and 8.0% diminished significantly (groups B and C). Following 40 min of aerosolized N-nitro-l-arginine-methyl ester (l-NAME) solution (a NO synthase inhibitor, 1.2 mM) inhalation, the response of Edyn to ACH increased significantly in group D. In group E, animals inhaled 500 mg/m3 ammonium persulfate (APS), an oxidant with various industrial applications, after the first ACH challenge test (0.2, 1.0, and 2.0% ACH). After 2 h of APS exposure, the ACH-induced broncho constriction was increased significantly in the challenge test. After another 2 h of APS inhalation, the airway responsiveness to ACH was tested under the influence of 300 ppm NO. NO significantly decreased the response to ACH to almost the same level as before APS exposure. The results indicate that responses to high ACH concentrations as well as an APS-induced increase in ACH responsiveness were effectively reduced by high concentrations of inhaled NO.

Abnormal modulation of cholinergic neurotransmission by endogenous nitric oxide in the bronchus of rats with hyperresponsiveness induced by allergen challenge.

The involvement of endogenous nitric oxide (NO) in bronchial cholinergic neurotransmission was compared between normal rats and airway hyperresponsive (AHR) rats. Male Wistar rats were sensitized and repeatedly challenged with dinitrophenylated (DNP)-Ascaris antigen. Twenty-four hours after the last antigenic challenge, enhancements of both the electrical field stimulation (EFS)-induced bronchoconstriction and acetylcholine (ACh) release were observed. NG-Monomethyl-L-arginine (L-NMMA; NO synthase inhibitor, 0.1 mM) augmented the EFS-induced bronchoconstriction and ACh release without affecting exogenously applied ACh-induced bronchoconstriction in normal rats. Interestingly, the augmentative effects of L-NMMA seen in normal rats were not manifested in AHR rats. Sodium nitroprusside inhibited the EFS-induced bronchoconstriction in a concentration-dependent manner; the inhibition was much larger than that of exogenously applied ACh-induced constriction in both normal and AHR rats. Furthermore, dibutyryl cGMP (3 mM) inhibited the EFS-induced bronchoconstriction with no effect on the ACh-induced bronchoconstriction in both normal and AHR rats. These findings suggest that endogenous NO may have a modulatory role in bronchial cholinergic neurotransmission in normal rats and that the augmented ACh release in the AHR rats may result from the defect of endogenous NO-induced modulation of cholinergic nerve transmission.

Abnormal Modulation of Cholinergic Neurotransmission by Endogenous Nitric Oxide in the Bronchus of Rats with Hyperresponsiveness Induced by Allergen Challenge

The involvement of endogenous nitric oxide (NO) in bronchial cholinergic neurotransmission was compared between normal rats and airway hyperresponsive (AHR) rats. Male Wistar rats were sensitized and repeatedly challenged with dinitrophenylated (DNP)-Ascaris antigen. Twenty-four hours after the last antigenic challenge, enhancements of both the electrical field stimulation (EFS)-induced bronchoconstriction and acetylcholine (ACh) release were observed. NG-Monomethyl-L-arginine (L-NMMA; NO synthase inhibitor, 0.1 mM) augmented the EFS-induced bronchoconstriction and ACh release without affecting exogenously applied ACh-induced bronchoconstriction in normal rats. Interestingly, the augmentative effects of L-NMMA seen in normal rats were not manifested in AHR rats. Sodium nitroprusside inhibited the EFS-induced bronchoconstriction in a concentration-dependent manner; the inhibition was much larger than that of exogenously applied ACh-induced constriction in both normal and AHR rats. Furthermore, dibutyryl cGMP (3 mM) inhibited the EFS-induced bronchoconstriction with no effect on the ACh-induced bronchoconstriction in both normal and AHR rats. These findings suggest that endogenous NO may have a modulatory role in bronchial cholinergic neurotransmission in normal rats and that the augmented ACh release in the AHR rats may result from the defect of endogenous NO-induced modulation of cholinergic nerve transmission.Keywords: Airway hyperresponsiveness, Nitric oxide, Cholinergic neurotransmission, Nitrergic dysfunction, Dinitrophenylated (ONP)-Ascaris

The tachykinin NK2 receptor antagonist SR 48968 inhibits citric acid-induced airway hyperresponsiveness in guinea pigs.

Airway hyperresponsiveness is a main feature of asthma, and several lines of evidence suggest that tachykinins might be involved in the pathogenesis of airway hyperresponsiveness in rodents. We conducted a study designed to describe an original model of airway hyperresponsiveness induced by citric acid administered as aerosol to guinea pigs, and to investigate the effects of the nonpeptide neurokinin1 (NK1) and neurokinin2 (NK2)-receptor antagonists, SR 140333 and SR 48968, respectively, on the development of this airway hyperresponsiveness. Animals received thiorphan 1 mg/kg intraperitoneally and 30 min later were exposed to an aerosol of citric acid 0.4 M for 1 h. After 24 h, the animals were anesthetized and ventilated. Airway hyperresponsiveness was evidenced by significant shifts to the left of dose-response curves for intravenous acetylcholine (ACh) without a change in maximum responses to ACh. Exposure to citric acid induced an airway hyperresponsive that was abolished by chronic pretreatment with capsaicin (120 mg/kg, 5 d before citric acid exposure). SR 48968 1 mg/kg intraperitoneally, given once at 30 min before the citric acid exposure, inhibited airway hyperresponsiveness, whereas SR 140333 1 mg/kg or codeine 30 mg/kg given under similar conditions did not. The inhibition of airway hyperresponsiveness by SR 48968 did not result from functional antagonism, since SR 48968 did not affect ACh-induced bronchoconstriction, nor did it result from inhibition of tachykinin, which could have been released under the influence of ACh in hyperresponsive animals, since SR 48968 given after the exposure to aerosolized citric acid failed to inhibit airway hyperresponsiveness. In conclusion, these results show that inhaled citric acid can induce the development of an airway hyperresponsiveness in the guinea pig through a release of tachykinins, and also demonstrate that NK2-receptor stimulation plays a predominant role in the development of airway hyperresponsiveness.

Acetylcholine‐induced bronchoconstriction modified by noise exposure in normal but not in sensitized guinea‐pigs

1. The acute (6h) exposure of guinea-pigs to white noise (110 dB) as a stress stimulus, reduced bronchial reactivity to acetylcholine (Ach) (3-1000 micrograms kg-1 i.v.) in anaesthetized animals. 2. The hyporesponsiveness to Ach in stressed animals was not confirmed in vitro on tracheal preparations (Ach 1 x 10(-9)-1 x 10(-4) g ml-1) and disappeared in vivo when the animals were sensitized with ovalbumin (OA, 100 mg kg-1 i.p. + 100 mg kg-1 s.c.). The hyporesponsiveness was also absent in ovalbumin sensitized guinea-pigs exposed to an aerosol of ovalbumin 60 min before testing with Ach. 3. In non-sensitized guinea-pigs, pretreatment with butoxamine (1 mg kg-1 i.v.) or with theophylline (25 mg kg-1 i.v.), completely abolished the effect of noise-exposure. In contrast, pretreatment with L-NG-nitro-arginine methyl ester (L-NAME, 10 mg kg-1 i.v.), alpha-chymotrypsin (2 U kg-1 i.v.) or with enprofylline (10 mg kg-1 i.v.), did not affect it. 4. In conclusion, our experiments reveal inhibitory mechanisms upon Ach-induced bronchoconstriction activated by a stress stimulus and this is absent in sensitized animals. These mechanisms seem to be linked to the adrenergic beta 2-receptors and a role for the purinergic system (via A-receptors) may also be present.

Effects of Methylecgonidine on acetylcholine-induced bronchoconstriction and indicators of lung injury in guinea pigs

The fumarate salt of methylecgonidine (MEG; anhydroecgonine methylester), a pyrolysis product of cocaine, has previously been shown to antagonize contractions of guinea pig isolated trachea induced by acetylcholine (ACh) and other spasmogenics. We determined the effects of MEG fumarate on ACh-induced bronchoconstriction in vivo. Specific airway conductance (SGaw) was measured in guinea pigs receiving 30–300 mg/kg s.c. MEG fumarate and exposed one hour later to nebulized ACh (0.2–3.2%; by inhalation). MEG fumarate did not induce any changes in SGaw; neither did it antagonize dose-dependent decreases in SGaw induced by ACh. However, tremors, salivation, startle and increased numbers of fecal boli were observed after MEG administration. Thus, unlike antagonism of ACh-induced contractions of guinea pig isolated trachea observed in vitro, MEG fumarate does not antagonize ACh-induced bronchoconstriction in vivo, even at doses which induced changes in grossly-observable behavior. Inhalation of a condensation aerosol of MEG base induced lung damage as evidenced by the presence of blood and higher levels of protein and lactate dehydrogenase in the lung lavage fluid of MEG-treated animals than of control animals. Aerosols of MEG fumarate, on the other hand, did not induce lung damage when inhaled. These results extend previous observations that MEG base may contribute to detrimental pulmonary effects of crack smoking.

Endogenous nitric oxide influences acetylcholine-induced bronchovascular dilation in sheep.

To test whether endogenous endothelial nitric oxide (NO) influences baseline bronchial vascular tone and mediates acetylcholine (ACh)-induced bronchial vascular dilation and/or modulates bronchoconstriction in ovine airways, we studied anesthetized ventilated open-chest sheep and measured bronchial blood flow (Qbr) and pulmonary resistance (RL). In six sheep we measured the response of Qbr and RL to the dose of ACh required to produce 50% of the maximal increase in Qbr at baseline during infusion of the NO synthase inhibitor NG-nitro-L-arginine (L-NNA; 10(-2) M). Infusion of L-NNA decreased both the baseline Qbr (28 +/- 13 to 8 +/- 2 ml/min, P < 0.01) and the change in Qbr (delta Qbr) from the baseline value (84 +/- 42 to 33 +/- 18 ml/min, P < 0.05). There was no difference in baseline RL or in the response of RL to ACh at any time. In another six sheep, phenylephrine (5 x 10(-6) to 5 x 10(-7) M) decreased baseline Qbr (22 +/- 6 to 10 +/- 3 ml/min, P < 0.05) but not delta Qbr (62 +/- 13 to 66 +/- 21 ml/min, not significant). Infusion of L-NNA in these sheep decreased the baseline Qbr to a similar extent (11 +/- 5 ml/min) and also decreased delta Qbr (42 +/- 16 ml/min, P < 0.05). We conclude that endogenous endothelial NO influences baseline vascular tone and ACh-induced vasodilation of the ovine bronchial vasculature but has no effect on baseline RL or ACh-induced bronchoconstriction.