Arginase inhibition prevents inflammation and remodeling in a guinea pig model of chronic obstructive pulmonary disease.

Airway inflammation and remodeling are major features of chronic obstructive pulmonary disease (COPD), whereas pulmonary hypertension is a common comorbidity associated with a poor disease prognosis. Recent studies in animal models have indicated that increased arginase activity contributes to features of asthma, including allergen-induced airway eosinophilia and mucus hypersecretion. Although cigarette smoke and lipopolysaccharide (LPS), major risk factors for COPD, may increase arginase expression, the role of arginase in COPD is unknown. This study aimed to investigate the role of arginase in pulmonary inflammation and remodeling using an animal model of COPD. Guinea pigs were instilled intranasally with LPS or saline twice weekly for 12 weeks and pretreated by inhalation of the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) or vehicle. Repeated LPS exposure increased lung arginase activity, resulting in increased l-ornithine/l-arginine and l-ornithine/l-citrulline ratios. Both ratios were reversed by ABH. ABH inhibited the LPS-induced increases in pulmonary IL-8, neutrophils, and goblet cells as well as airway fibrosis. Remarkably, LPS-induced right ventricular hypertrophy, indicative of pulmonary hypertension, was prevented by ABH. Strong correlations were found between arginase activity and inflammation, airway remodeling, and right ventricular hypertrophy. Increased arginase activity contributes to pulmonary inflammation, airway remodeling, and right ventricular hypertrophy in a guinea pig model of COPD, indicating therapeutic potential for arginase inhibitors in this disease.

Tiotropium inhibits pulmonary inflammation and remodelling in a guinea pig model of COPD.

Airway remodelling and emphysema are major structural abnormalities in chronic obstructive pulmonary disease (COPD). In addition, pulmonary vascular remodelling may occur and contribute to pulmonary hypertension, a comorbidity of COPD. Increased cholinergic activity in COPD contributes to airflow limitation and, possibly, to inflammation and airway remodelling. This study aimed to investigate the role of acetylcholine in pulmonary inflammation and remodelling using an animal model of COPD. To this aim, guinea pigs were instilled intranasally with lipopolysaccharide (LPS) twice weekly for 12 weeks and were treated, by inhalation, with the long-acting muscarinic receptor antagonist tiotropium. Repeated LPS exposure induced airway and parenchymal neutrophilia, and increased goblet cell numbers, lung hydroxyproline content, airway wall collagen and airspace size. Furthermore, LPS increased the number of muscularised microvessels in the adventitia of cartilaginous airways. Tiotropium abrogated the LPS-induced increase in neutrophils, goblet cells, collagen deposition and muscularised microvessels, but had no effect on emphysema. In conclusion, tiotropium inhibits remodelling of the airways as well as pulmonary inflammation in a guinea pig model of COPD, suggesting that endogenous acetylcholine plays a major role in the pathogenesis of this disease.

Increased arginase activity contributes to airway remodelling in chronic allergic asthma.

Airway remodelling, characterised by increased airway smooth muscle (ASM) mass, subepithelial fibrosis, goblet cell hyperplasia and mucus gland hypertrophy, is a feature of chronic asthma. Increased arginase activity could contribute to these features via increased formation of polyamines and l-proline downstream of the arginase product l-ornithine, and via reduced nitric oxide synthesis. Using the specific arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH), we studied the role of arginase in airway remodelling using a guinea pig model of chronic asthma. Ovalbumin-sensitised guinea pigs were treated with ABH or PBS via inhalation before each of 12 weekly allergen or saline challenges, and indices of arginase activity, and airway remodelling, inflammation and responsiveness were studied 24 h after the final challenge. Pulmonary arginase activity of repeatedly allergen-challenged guinea pigs was increased. Allergen challenge also increased ASM mass and maximal contraction of denuded tracheal rings, which were prevented by ABH. ABH also attenuated allergen-induced pulmonary hydroxyproline (fibrosis) and putrescine, mucus gland hypertrophy, goblet cell hyperplasia, airway eosinophilia and interleukin-13, whereas an increased l-ornithine/l-citrulline ratio in the lung was normalised. Moreover, allergen-induced hyperresponsiveness of perfused tracheae was fully abrogated by ABH. These findings demonstrate that arginase is prominently involved in allergen-induced airway remodelling, inflammation and hyperresponsiveness in chronic asthma.

L-arginine deficiency causes airway hyperresponsiveness after the late asthmatic reaction.

Peroxynitrite has been shown to be crucially involved in airway hyperresponsiveness (AHR) after the late asthmatic reaction (LAR). Peroxynitrite production may result from simultaneous synthesis of nitric oxide (NO) and superoxide by inducible NO-synthase (iNOS) at low L-arginine concentrations. L-arginine availability to iNOS is regulated by its cellular uptake, which can be inhibited by eosinophil-derived polycations and by arginase, which competes with iNOS for the common substrate. Using a guinea pig model of allergic asthma, we investigated whether aberrant L-arginine homeostasis could underlie peroxynitrite-mediated AHR after the LAR. After the LAR, arginase activity in the airways and eosinophil peroxidase release from bronchoalveolar lavage cells were increased. These changes were associated with a 2.0-fold AHR to methacholine as measured in isolated perfused tracheal preparations. AHR was reduced by exogenous L-arginine administration. Moreover, both the arginase inhibitor N(omega)-hydroxy-nor-L-arginine (nor-NOHA) and the polycation antagonist heparin normalised airway responsiveness. These effects were reversed by the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME), indicating that both agents reduced AHR by restoring bronchodilating NO production. In conclusion, in allergen-challenged guinea pigs, the AHR after the LAR is caused by arginase- and polycation-induced attenuation of L-arginine availability to iNOS, which may switch the enzyme to simultaneous production of superoxide and NO, and, consequently, peroxynitrite.

Airway hyperresponsiveness in asthma: lessons from in vitro model systems and animal models.

Airway hyperresponsiveness (AHR) is a hallmark clinical symptom of asthma. At least two components of AHR have been identified: 1) baseline AHR, which is persistent and presumably caused by airway remodelling due to chronic recurrent airway inflammation; and 2) acute and variable AHR, which is associated with an episodic increase in airway inflammation due to environmental factors such as allergen exposure. Despite intensive research, the mechanisms underlying acute and chronic AHR are poorly understood. Owing to the complex variety of interactive processes that may be involved, in vitro model systems and animal models are indispensable to the unravelling of these mechanisms at the cellular and molecular level. The present paper focuses on a number of translational studies addressing the emerging central role of the airway smooth muscle cell, as a multicompetent cell involved in acute airway constriction as well as structural changes in the airways, in the pathophysiology of airway hyperresponsiveness.

Insulin induces airway smooth muscle contraction.

BACKGROUND AND PURPOSE: Recently, the use of inhaled insulin formulations for the treatment of type I and type II diabetes has been approved in Europe and in the United States. For regular use, it is critical that airway function remains unimpaired in response to insulin exposure. EXPERIMENTAL APPROACH: We investigated the effects of insulin on airway smooth muscle (ASM) contraction and contractile prostaglandin (PG) production, using guinea-pig open-ring tracheal smooth muscle preparations. KEY RESULTS: It was found that insulin (1 nM-1 microM) induced a concentration-dependent contraction that was insensitive to epithelium removal. These sustained contractions were susceptible to inhibitors of cyclooxygenase (indomethacin, 3 microM), Rho-kinase (Y-27632, 1 microM) and p42/44 MAP kinase (PD-98059, 30 microM and U-0126, 3 microM), but not of PI-3-kinase (LY-294002,10 microM). In addition, insulin significantly increased PGF(2alpha)-production which was inhibited by indomethacin, but not Y-27632. Moreover, the FP-receptor antagonist AL-8810 (10 microM) and the EP(1)-receptor antagonist AH-6809 (10 microM) strongly reduced insulin-induced contractions, supporting a pivotal role for contractile prostaglandins. CONCLUSIONS AND IMPLICATIONS: Collectively, the results show that insulin induces guinea-pig ASM contraction presumably through the production of contractile prostaglandins, which in turn are dependent on Rho-kinase for their contractile effects. The data suggest that administration of insulin as an aerosol could result in some acute adverse effects on ASM function.

Virodhamine and CP55,940 modulate cAMP production and IL-8 release in human bronchial epithelial cells.

BACKGROUND AND PURPOSE: We investigated expression of cannabinoid receptors and the effects of the endogenous cannabinoid virodhamine and the synthetic agonist CP55,940 on cAMP accumulation and interleukin-8 (IL-8) release in human bronchial epithelial cells. EXPERIMENTAL APPROACH: Human bronchial epithelial (16HBE14o(-)) cells were used. Total mRNA was isolated and cannabinoid receptor mRNAs were detected by RT-PCR. Expression of CB(1) and CB(2) receptor proteins was detected with Western blotting using receptor-specific antibodies. cAMP accumulation was measured by competitive radioligand binding assay. IL-8 release was measured by ELISA. KEY RESULTS: CB(1) and CB(2) receptor mRNAs and proteins were found. Both agonists concentration-dependently decreased forskolin-induced cAMP accumulation. This effect was inhibited by the CB(2) receptor antagonist SR144528, and was sensitive to Pertussis toxin (PTX), suggesting the involvement of CB(2) receptors and G(i/o)-proteins. Cell pretreatment with PTX unmasked a stimulatory component, which was blocked by the CB(1) receptor antagonist SR141716A. CB(2) receptor-mediated inhibition of cAMP production by virodhamine and CP55,940 was paralleled by inhibition of tumor necrosis factor-alpha (TNF-alpha) induced IL-8 release. This inhibition was insensitive to SR141716A. In the absence of agonist, SR144528 by itself reduced TNF-alpha induced IL-8 release. CONCLUSIONS AND IMPLICATIONS: Our results show for the first time that 16HBE14o(-) cells respond to virodhamine and CP55,940. CB(1) and CB(2) receptor subtypes mediated activation and inhibition of adenylyl cyclase, respectively. Stimulation of the dominant CB(2) receptor signalling pathway diminished cAMP accumulation and TNF-alpha-induced IL-8 release. These observations may imply that cannabinoids exert anti-inflammatory properties in airways by modulating cytokine release.

[The treatment of asthma: indications for a change in approach]. / Behandeling van astma: aanwijzingen voor een verandering in aanpak.

In both children and adults with persistent asthma, treatment with an inhaled corticosteroid (ICS) is recommended. Moreover, inhaled bronchodilating agents have a clear role to play. The minimum effective dose of an ICS in the individual patient can be determined either by starting with a low dosage of ICS and increasing the dosage gradually on the basis of the symptoms (the 'step-up' approach), or by starting with a high dosage and, if the results are good, decreasing it to the pointwhere adequate control is maintained (the 'step-down' approach). In a study of the step-up approach with the ICS fluticasone, with or without salmeterol as a long-acting beta2-agonist (LABA) in adult patients with asthma, the approach with salmeterol produced the best results, namely, good asthma control in 71% of the patients and total control in 41%. In a study involving both children and adults with asthma, good results were obtained from treatment with a relatively low maintenance dose of ICS (budesonide) combined with a LABA (formoterol), whereby patients were permitted to use additional inhalations of the combination ICS and LABA. How the different therapeutic concepts result in long-term control, what the side effects are in the long term, and whether, in addition to the clinical symptoms, laboratory findings are also important as a therapeutic criterion are all unknown.

Effects of vasoactive intestinal polypeptide on heart rate in relation to vagal cardioacceleration in conscious dogs.

OBJECTIVE: The vagal cardiac accelerator (VCA) system takes part in the nervous control of the heart rate. In the present study we tried to adduce evidence that vasoactive intestinal polypeptide (VIP) contributes to vagally induced cardioaccelerations. METHODS: The effect of VIP on heart rate and arterial blood pressure was investigated after unmasking the inherent VCA activity by blocking the sympathetic accelerator and vagal decelerator influences on heart rate in conscious dogs. RESULTS: Following intravenous administration of VIP (10 micrograms i.v.) the heart rate increased by 43.6 +/- 6.7 (28.1 +/- 4.7%), from 165.6 +/- 8.5 to 209.1 +/- 7.0 beats/min (P < 0.001) and the mean arterial blood pressure decreased by 47.5 +/- 3.2 (37.9 +/- 3.0%), from 126.6 +/- 2.6 to 79.1 +/- 4.9 mmHg (P < 0.001) (n = 11). After VCA activity was reflexly enhanced by alpha 1-adrenoceptor stimulation with methoxamine, VIP increased heart rate by 36.9 +/- 7.3 (21.5 +/- 4.6%), from 179.8 +/- 5.2 to 216.7 +/- 5.8 beats/min (P < 0.001) and decreased mean arterial pressure by 79.1 +/- 6.4 (46.7 +/- 3.5%), from 168.2 +/- 4.1 to 89.1 +/- 5.0 mmHg (P < 0.001). Hence, the VIP-induced tachycardia, expressed in relative values, shows a significant attenuation after the administration of methoxamine (P < 0.05). The increase in heart rate induced by VIP appeared to be inversely related to the prevailing VCA activity, both before (r = -0.744, P = 0.009) and after methoxamine (r = -0.689, P = 0.019). The VIP-induced tachycardia is certainly not reflexly induced by the fall in arterial pressure, because intracoronary administration of VIP (0.5 microgram i.c.) caused an appreciable increase in the heart rate by 63.7 +/- 13.0 (46.4 +/- 10.4%), from 143.0 +/- 8.1 to 208.7 +/- 12.0 beats/min (P < 0.005), whereas the mean arterial pressure only slightly changed (-7.7 +/- 2.0 mmHg) (P < 0.05) (n = 6). In addition, VIP (10 micrograms i.v.) also caused a tachycardia in vagotomized dogs with blocked beta-adrenergic and muscarinic receptors. The administration of the VIP antagonists [D-p-CI-Phe6, Leu17]-VIP (50-150 micrograms i.c.) and [Lys1, Pro2,5, Leu17]-VIP (20 micrograms i.c.) did not result in alterations in VCA activity nor did the VIP antagonists block the VCA reflex response to a rise in arterial pressure. However, none of the VIP antagonists reduced the VIP-induced tachycardia either. CONCLUSION: Vasoactive intestinal polypeptide is likely to play a part in the vagal cardiac accelerator system. However, conclusive evidence for its role as the terminal transmitter in the VCA pathway will have to wait for the availability of a specific cardiac VIP receptor antagonist.

Beta-adrenoceptor studies. 2. Effects of alkyl substitution on beta-adrenoceptor blocking, antiarrhythmic, and local anesthetic activities of 1,1'-(o-phenylenedioxy)bis(3-isopropylamino-2-propanol).

A series of bis(2-hydroxy-3-isopropylaminopropyl) ethers of nuclear-substituted catechols (1-7) has been synthesized and examined in vitro for beta-adrenoceptor blocking activity, antagonism of ouabain-induced arrhythmias, and local anesthetic activity. Both tracheal and right atrial beta-adrenoceptor blocking activity are markedly decreased by alkyl substitution in position 3 of parent catechol diether 1. Substitution in position 4 still lowers the affinity to cardiac arrhythmias and local anesthetic activity increases with introduction of alkyl substituents in the 3 as well as in the 4 position. In contrast with biological activities, the partition coefficient 1-octanol-phosphate buffer, pH 7.40, of 1 did not change significantly by 3- and 4-methyl substitution. Stepwise multiple regression analyses were performed using log P or pi values in combination with pKa(m), E8, or sigma. With cardiac beta-adrenoceptor blocking activity the optimal equation contained E8 and pi parameters, tracheal activity appeared to depend mainly on the E8 parameter, whereas for antiarrhythmic and local anesthetic activities the lipophilicity of the substituents appeared to be the determinant factor.

Direct evidence for the atypical nature of functional beta-adrenoceptors in rat adipocytes.

1. The nature of the rat epididymal adipocyte beta-adrenoceptor was investigated by studying the effects of beta 1- and beta 2-selective antagonists on lipolysis induced by (-)-isoprenaline and the lipolytically selective agonist BRL 37344. 2. From 10 nM to 10 microM, the potent and highly selective beta 1-adrenoceptor antagonist CGP 20712A did not influence the concentration-response curve (CRC) of BRL 37344 whereas small but consistent shifts to the right of the (-)-isoprenaline-induced CRC were observed. Clear rightward shifts of the CRCs induced by both (-)-isoprenaline and BRL 37344 were produced only at 100 microM CGP 20712A with the corresponding pA2 values being 4.80 and 4.61, respectively. 3. When the beta 2-selective antagonist ICI 118,551 was used at 10 microM and higher, clear and concentration-dependent shifts to the right of the CRCs of both agonists were observed. The slopes of the Schild plots did not deviate significantly from unity, the pA2 values being 5.49 and 5.33 against (-)-isoprenaline and BRL 37344, respectively. 4. The results demonstrate that (-)-isoprenaline-induced lipolysis in rat white adipocytes is mediated predominantly by atypical beta-adrenoceptors, whereas the typical beta 1-adrenoceptors play a small, subordinate role. The lipolytically selective agonist BRL 37344 acts solely through atypical beta-adrenoceptors.

Relationship between lipolysis and cyclic AMP generation mediated by atypical beta-adrenoceptors in rat adipocytes.

1. The nature of the beta-adrenoceptor(s) mediating adenylyl cyclase activation in rat adipocyte ghosts by (-)-isoprenaline and the lipolytically selective beta-adrenoceptor agonist, BRL 37344, was investigated by use of the beta 1-selective antagonist, CGP 20712A. The results were compared with lipolysis in adipocytes. 2. While in lipolysis BRL 37344 was a full and 10 times more potent agonist than (-)-isoprenaline, in adenylyl cyclase activation similar pD2 values for both agonists were found. BRL 37344 was only a partial agonist on rat adipocyte adenylyl cyclase, with an intrinsic activity of 0.62. 3. With CGP 20712A small rightward shifts of the (-)-isoprenaline concentration-response curve (CRC) were observed at concentrations up to 10 microM, while at 100 microM and 1 mM clear rightward shifts occurred. The BRL 37344 CRC was not shifted with antagonist concentrations up to 10 microM. Only at 100 microM and 1 mM CGP 20712A were rightward shifts observed. 4. CGP 20712A concentrations of 10 microM and 100 microM depressed the maximum of the (-)-isoprenaline CRC to 89 and 60%, while the BRL 37344 CRCs retained the control maximum effect (62% of (-)-isoprenaline). Only at 1 mM CGP 20712A, was the CRC of BRL 37344 depressed, while the (-)-isoprenaline maximum was diminished further. 5. It was concluded that as with lipolysis, (-)-isoprenaline acts both through typical beta 1- and atypical beta 3-adrenoceptors for activation of adenylyl cyclase, while BRL 37344 acts solely through atypical beta 3-adrenoceptors. 6. The results also demonstrate that the relationship between adenosine 3':5'-cyclic monophosphate (cyclic AMP) and lipolysis is different for BRL 37344 and (-)-isoprenaline. Although the maximum activation of adenylyl cyclase by BRL 37344 is only 62% of that by (-)-isoprenaline, the distance between the lipolysis and adenylyl cyclase CRCs is much larger in the case of BRL 37344, indicating a larger transduction reserve for this agonist.

The beta-adrenoceptors mediating relaxation of rat oesophageal muscularis mucosae are predominantly of the beta 3-, but also of the beta 2-subtype.

1. beta-Adrenoceptor-mediated relaxation of rat oesophageal smooth muscle was investigated by studying the effects of beta 1- and beta 2-selective antagonists on the relaxation induced by (-)-isoprenaline, the beta 2-selective agonists fenoterol and clenbuterol and the beta 3-agonist, BRL 37344. 2. The highly beta 1-selective antagonist CGP 20721A did not antagonize (-)-isoprenaline- or BRL 37344-induced relaxations in concentrations up to 10 microM. Only at 100 microM of CGP 20712A were clear rightward shifts of the agonist concentration-response curves (CRCs) observed, with pA2 values of 4.70 and 4.97 against (-)-isoprenaline and BRL 37344, respectively. 3. ICI 118,551, a potent and selective beta 2-antagonist, at 100 nM caused moderate rightward shifts of the CRCs of (-)-isoprenaline, fenoterol and clenbuterol; with fenoterol and clenbuterol, this was accompanied by a clear steepening of the curve. Only at the highest concentration (100 microM ICI 118,551) did the shifts to the right further increase substantially. Resulting Schild-plots were clearly biphasic. BRL 37344-induced relaxations were only antagonized at 100 microM ICI 118,551, yielding a pA2 value of 5.48. 4. These results clearly demonstrate that the BRL 37344-induced relaxation of rat oesophageal muscularis mucosae is mediated solely through beta 3-adrenoceptors, whereas (-)-isoprenaline-, fenoterol- and clenbuterol-induced relaxations were shown to involve both beta 2- and, predominantly, beta 3-adrenoceptors.

Presynaptic muscarinic receptors inhibiting endogenous noradrenaline release in the portal vein of the freely moving rat.

1. In the portal vein of the freely moving unanaesthetized rat, the existence of presynaptically located inhibitory muscarinic receptors was investigated by use of the muscarinic agonist methacholine (MCh) 2. Infusion of MCh (0.3 micrograms min-1) did not significantly inhibit the endogenous noradrenaline (NA) overflow in portal plasma. However, after inducing high intra-synaptic concentrations of NA by blocking the presynaptic alpha 2-adrenoceptors with yohimbine (1 mg kg-1), MCh (0.3 microgram min-1) was able to reduce the yohimbine-induced enhanced NA overflow by 38%. 3. The MCh-induced inhibition was almost completely abolished after blockade of the presynaptic muscarinic receptors with atropine (0.6 mg kg-1). 4. During electrical stimulation of the portal vein nervous plexus the evoked NA overflow was strongly inhibited (95%) during MCh-infusion (0.3 microgram min-1). Again atropine (0.6 mg kg-1) was able to reverse this inhibition. 5. These results show the existence of presynaptic muscarinic receptors inhibiting endogenous NA overflow from the portal vein nervous plexus under conditions of enhanced sympathetic activity in the freely moving rat.

Noradrenaline-induced relaxation of rat oesophageal muscularis mucosae: mediation solely by innervated beta 3-adrenoceptors.

We investigated the effects of cocaine and corticosterone on the noradrenaline-induced relaxation of rat oesophageal smooth muscle in the absence and presence of the selective beta2-antagonist, ICI 111,551. It was found that the concentration-response curve (CRC) of noradrenaline was not shifted by ICI 118,551 at 1 microM, whereas a clear shift to the right was observed at 100 microM of the antagonist. In the presence of corticosterone (10 microM), CRC's were clearly shifted to the left; with cocaine (10 microM) additionally present, a further leftward shift was observed, indicating the involvement of both neuronal and extraneuronal uptake sites. It was concluded that the relaxation of rat oesophageal muscularis mucosae by noradrenaline is solely mediated by beta3-adrenoceptors which are sympathetically innervated.

Characterization of the muscarinic receptor subtype involved in phosphoinositide metabolism in bovine tracheal smooth muscle.

1. The muscarinic receptor subtype involved in the methacholine-induced enhancement of phosphoinositide metabolism in bovine tracheal smooth muscle was identified by using the M2-selective antagonist AF-DX 116 and the M3-selective antagonist 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) methobromide, in addition to the M1-selective antagonist pirenzepine, in a classical Schild analysis. 2. All the antagonists shifted the methacholine dose-response curve to the right in a parallel and concentration-dependent fashion, yielding Schild plots with slopes not significantly different from unity. The pA2 values (6.94, 6.32 and 8.54 for pirenzepine, AF-DX 116 and 4-DAMP methobromide respectively) indicate that it is the M3 (smooth muscle/glandular), but not the M2 (cardiac) muscarinic receptor subtype, present in this tissue, that mediates phosphoinositide turnover, in accordance with our previous contractile studies. 3. The results provide additional evidence for the involvement of phosphoinositide turnover in the pharmacomechanical coupling between muscarinic receptor stimulation and contraction in (bovine tracheal) smooth muscle.

Sustained prejunctional facilitation of noradrenergic neurotransmission by adrenaline as a co-transmitter in the portal vein of freely moving rats.

1. The duration of the facilitatory effect of adrenaline on the electrically evoked overflow of noradrenaline was studied in the portal vein of permanently adreno-demedullated freely moving rats. 2. Rats were infused with adrenaline (20 or 100 ng min-1) for 2 h. After an interval of 1 h, when plasma adrenaline had returned to undetectable levels, electrical stimulation resulted in an enhanced catecholamine overflow amounting to 219% (noradrenaline) and 241% (noradrenaline plus adrenaline) of control (saline infusion), respectively. 3. When stimulation was applied again, in the same animal, at 24, 48 and 72 h after the first stimulation episode, the evoked noradrenaline overflow was 150, 111 and 102% (after 20 ng ml-1 adrenaline) and 158, 134 and 105% (after 100 ng min-1 adrenaline) of control. 4. The beta 2-adrenoceptor antagonist, ICI 118,551 (0.3 mg kg-1), blocked the facilitatory effect obtained after the 100 ng min-1 adrenaline infusion on all days. 5. The results show that adrenaline, after being taken up by and released from sympathetic nerve terminals, is able to facilitate the evoked noradrenaline overflow through activation of prejunctional beta 2-adrenoceptors for at least 48 h after administration.

Role of nitric oxide and superoxide in allergen-induced airway hyperreactivity after the late asthmatic reaction in guinea-pigs.

1. In the present study, the roles of nitric oxide (NO) and superoxide anions (O2(-)) in allergen-induced airway hyperreactivity (AHR) after the late asthmatic reaction (LAR) were investigated ex vivo, by examining the effects of the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) and superoxide dismutase (SOD) on the responsiveness to methacholine of isolated perfused guinea-pig tracheae from unchallenged (control) animals and from animals 24 h after ovalbumin challenge. 2. At 24 h after allergen challenge, the animals developed AHR in vivo, as indicated by a mean 2.63 +/- 0.54 fold (P < 0.05) increase in sensitivity to histamine inhalation. 3. Compared to unchallenged controls, tracheal preparations from the ovalbumin-challenged guinea-pigs displayed a significant 1.8 fold (P < 0.01) increase in the maximal response (E(max)) to methacholine, both after intraluminal (IL) and extraluminal (EL) administration of the agonist. No changes were observed in the sensitivity (pEC(50)) to the agonist. Consequently, the DeltapEC(50) (EL-IL), as a measure of epithelial integrity, was unchanged. 4. In the presence of L-NAME (100 microM, IL), tracheae from control guinea-pigs showed a 1.6 fold (P < 0.05) increase in the E(max) of IL methacholine. By contrast, the E(max) of IL methacholine was significantly decreased in the presence of 100 u ml(-1) EL SOD (54% of control, P < 0.01). 5. Remarkably, the increased responsiveness to IL methacholine at 24 h after allergen challenge was reversed by L-NAME to control (P < 0.01), and a similar effect was observed with SOD (P < 0.01). 6. The results indicate that both NO and O2(-) are involved in the tracheal hyperreactivity to methacholine after the LAR, possibly by promoting airway smooth muscle contraction through the formation of peroxynitrite.

No evidence for a role of muscarinic M2 receptors in functional antagonism in bovine trachea.

1. The functional antagonism between methacholine- or histamine-induced contraction and beta-adrenoceptor-mediated relaxation was evaluated in bovine tracheal smooth muscle in vitro. In addition, the putative contribution of muscarinic M2 receptors mediating inhibition of beta-adrenoceptor-induced biochemical responses to this functional antagonism was investigated with the selective muscarinic antagonists, pirenzepine (M1 over M2), AF-DX 116 and gallamine (M2 over M3), and hexahydrosiladiphenidol (M3 over M2). 2. By use of isotonic tension measurement, contractions were induced with various concentrations of methacholine or histamine, and isoprenaline concentration-relaxation curves were obtained in the absence or presence of the muscarinic antagonists. Antagonist concentrations were chosen so as to produce selective blockade of M2 receptors (AF-DX 116 0.1 microM, gallamine 30 microM), or half-maximal blockade of M3 receptors (pirenzepine 0.1 microM, AF-DX 116 0.5 microM, hexahydrosiladiphenidol 0.03 microM). Since these latter antagonist concentrations mimicked KB values towards bovine tracheal smooth muscle M3 receptors, antagonist-induced decreases in contractile tone were compensated for by doubling the agonist concentration. 3. It was found that isoprenaline-induced relaxation of bovine tracheal smooth muscle preparations was dependent on the nature and the concentration of the contractile agonist used. Thus, isoprenaline pD2 (-log EC50) values were decreased 3.7 log units as a result of increasing cholinergic tone from 22 to 106%, and 2.4 log units by increasing histamine tone over a similar range. Furthermore, maximal relaxability of cholinergic tone decreased gradually from 100% at low to only 1.3% at supramaximal contraction levels, whereas with histamine almost complete relaxation was maintained at all concentrations applied. As a result, isoprenaline relaxation was clearly hampered with methacholine compared to histamine at equal levels of contractile tone.4. In the presence of gallamine, isoprenaline relaxation was facilitated for most concentrations of methacholine, and for all concentrations of histamine. These changes could be explained by the decreased contraction levels for both contractile agonists in the presence of gallamine.5. Isoprenaline-induced relaxation of cholinergic contraction was also facilitated by AF-DX 116 as well as by pirenzepine and hexahydrosiladiphenidol, and these (small) changes were again related to the(small) decreases in cholinergic contraction levels that were present in these experiments despite the additional administration of the agonist to readjust contractile tone. Similarly, changes in isoprenaline relaxation of histamine-induced tone could be explained by different contraction levels.6. These results can be explained by the sole involvement of muscarinic M3 receptors, and provide no evidence for a role of muscarinic M2 receptors in functional antagonism in bovine trachea. Furthermore,they stress the importance of taking into account non-cholinergic controls as well as contraction levels in these experiments.

Deficiency of nitric oxide in polycation-induced airway hyperreactivity.

Using a perfused guinea-pig tracheal tube preparation, we investigated the role of endogenous nitric oxide (NO) in polycation-induced airway hyperreactivity (AHR) to methacholine. Intraluminal (IL) administration of the NO synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME; 100 microM) caused a 1.8 fold increase in the maximal contractile response (Emax) to IL methacholine compared to control, without an effect on the pEC50 (-log10 EC50). The polycation poly-L-arginine (100 microg ml(-1), IL) similarly enhanced the Emax for methacholine; however, the pEC50 value was also increased, by one log10 unit. L-NAME had no effect on the enhanced methacholine response of poly-L-arginine-treated airways, while the enhanced agonist response was completely normalized by the polyanion heparin (25 u ml(-1), IL). In addition, the effect of L-NAME was fully restored in the poly-L-arginine plus heparin treated airways. The results indicate that, in addition to enhanced epithelial permeability, a deficiency of endogenous NO contributes to polycation-induced AHR. The latter finding may represent a novel mechanism of AHR induced by eosinophil-derived cationic proteins in allergic asthma.