Airway epithelium-derived inhibitory factor.

Various bronchoactive agents can induce the release from the airway epithelium of an inhibitory substance that is able to relax certain tissues including rat aorta and possibly also airway smooth muscle. This substance, whose existence has recently been confirmed using a new bioassay system, is distinct from nitric oxide (EDRF) and is also known to be non-prostanoid in nature. Roy Goldie and colleagues describe the properties of this factor, and its potential clinical significance.

Endothelin and the respiratory system.

Pharmacological research involving the endothelin peptides has emphasized their activities in vascular systems, from both physiological and pathophysiological perspectives. However, the endothelins are known also to be synthesized and released from respiratory epithelial cells and to have potent effects in nonvascular components of the respiratory tract. Douglas Hay, Peter Henry and Roy Goldie summarize present understanding of the pharmacology of the endothelins in the respiratory system and assess the potential pathophysiological role in asthma.

Adrenoceptors in airway smooth muscle.

This review examines the roles and functional significance of alpha and beta-adrenoceptor subtypes in airway smooth muscle, with emphasis on human airway function and the influence of asthma. Specifically, we have examined the distribution of beta-adrenoceptors in lung and the influence of age, the epithelium, respiratory viruses and inflammation associated with asthma on airway smooth muscle beta-adrenoceptor function. Sites of action, beta 2-selectivity, efficacy and tolerance are also examined in relation to the use of beta 2-agonists in man. In addition, alpha-adrenoceptor function in airway smooth muscle has been reviewed, with some emphasis on comparing observations made in airway smooth muscle with those in animal models.

Beta 1-adrenoceptors mediate smooth muscle relaxation in mouse isolated trachea.

1. The relaxant effects to the beta-adrenoceptor agonists isoprenaline, adrenaline, noradrenaline, RO363, procaterol and fenoterol were investigated in carbachol-contracted mouse isolated tracheal preparations. 2. The order of potencies for those beta-adrenoceptor agonists that induced full relaxation of carbachol-contracted mouse tracheal preparations was isoprenaline greater than RO363 greater than noradrenaline = adrenaline greater than fenoterol. The EC50 value of isoprenaline for relaxation was 46 nM. The beta 1-adrenoceptor-selective agonist, RO363 was ten times more potent than the beta 2-adrenoceptor-selective agonist, fenoterol. The highly beta 2-adrenoceptor-selective agonist procaterol was a partial relaxant and induced only 28 +/- 4% relaxation. 3. Relaxations induced by noradrenaline and isoprenaline were not significantly affected by the neuronal uptake inhibitor, cocaine (10 microM) or by the extraneuronal uptake inhibitor, deoxycorticosterone acetate (25 microM) respectively. The alpha-adrenoceptor agonist methoxamine induced no observable elevation of mouse tracheal smooth muscle tone. 4. Schild plots for the beta-adrenoceptor antagonists, atenolol and betaxolol (beta 1-adrenoceptor-selective) and ICI 118,551 (beta 2-adrenoceptor-selective) were linear, with slope values approaching unity. Mean pA2 values derived for atenolol, betaxolol and ICI 118,551 for antagonism of beta-adrenoceptor-mediated relaxation were 7.1, 8.4 and 7.2, respectively. These data were independent of the use of isoprenaline or noradrenaline as the agonist. 5. These findings indicate that beta-adrenoceptor-mediated relaxations of mouse isolated trachea occur predominantly through activation of beta 1-adrenoceptors.

Potentiation by endothelin-1 of cholinergic nerve-mediated contractions in mouse trachea via activation of ETB receptors.

1. We have previously shown that endothelin-1-induced contraction of mouse isolated tracheal smooth muscle was mediated via both ETA and ETB receptors. In the current study, we have investigated endothelin-1-induced potentiation of cholinergic nerve-mediated contractions in mouse isolated trachea and have characterized pharmacologically the endothelin receptors mediating this response. 2. Electrical field stimulation (EFS; 70 V, 0.5 ms duration, 10s train, 0.1-60 Hz) of mouse isolated trachea caused frequency-dependent, monophasic contractions (magnitude of contraction of 60 Hz was 56 +/- 4% Cmax (n = 6), where Cmax is the contractile response to 10 microM carbachol). EFS-induced contractions were abolished by either 0.1 microM atropine or 3 microM tetrodotoxin, but were not affected by 1 microM hexamethonium, indicating that they were induced by stimulation of postganglionic cholinergic nerves. In contrast, contractions induced by exogenously applied acetylcholine were inhibited by atropine, but not by either tetrodotoxin or hexamethonium. 3. The ETB receptor-selective agonist, sarafotoxin S6c, caused marked concentration-dependent potentiation of EFS-induced contractions in mouse isolated tracheal segments. At 0.1 nM, sarafotoxin S6c exerted no direct contractile effect, but significantly increased a standard EFS-induced contraction of 20% Cmax by 8 +/- 2% Cmax (i.e. 1.4 fold, n = 5, P < 0.05). At higher concentrations, 10 nM sarafotoxin S6c induced a large, transient contraction (peak response of 74 +/- 2% Cmax at 10 min; 3 +/- 2% Cmax at 45 min) and enhanced the standard EFS-induced contraction by 30 +/- 4% Cmax (i.e. 2.5 fold, n = 5, P < 0.01). In contrast, 10 nM sarafotoxin S6c did not enhance contractile responses to exogenously applied acetylcholine(n = 6).4. Endothelin-1 also modulated EFS-induced contractions. At 0.1 nM, endothelin-1 exerted no direct contractile effect, but significantly increased the standard EFS-induced contraction of 20%Cmax, by 7 +/- 2%Cma, (i.e. 1.35 fold, n = 5, P<0.05). At 1 nM, endothelin-l induced a small, sustained contraction(16 +/- 3%Cmo) and increased the standard EFS-induced contraction by 19 +/- 2%Cmax (i.e. 1.95 fold,n = 5, P <0.01). Finally, 10 nM endothelin-1 induced a large, sustained contraction (98 +/- 8%Cma), but the EFS-induced contraction was significantly reduced from 20%Cmax to 6 +/- 4%Cmax (n = 6, P <0.05).In contrast, in the presence of 3 microM BQ-123 (ETA receptor-selective antagonist), 1O nM endothelin-1 induced a transient contraction mediated via ETB receptors (peak response of 59 +/- 10%Cmax at 10 min;8 +/- 2%Cmax at 45 min). Under these conditions, the standard EFS-induced contraction was increased by 26+/- l%Cmax (i.e. 2.3 fold, n = 6, P<0.01).5. The potentiation of EFS-induced contractions produced by 1 nM endothelin-1 was not mediated by ETA receptors, since 3 microM BQ-123 did not diminish this effect (n = 6). Furthermore, 1 nM endothelin-1 did not potentiate EFS-induced contractions in preparations in which the function of the ETB receptor effector system had been attenuated by desensitization (n = 6).6. In summary, endothelin-1 potentiates cholinergic nerve-mediated contractions in mouse isolated trachea, apparently by activating prejunctional ETB receptors. This neuronal pathway offers an additional mechanism through which endothelin-1 may elevate bronchomotor tone.

Metabolism of [3H]-(+/-)-isoprenaline by isolated atria and coronary arteries of the kitten.

1 Isolated coronary arteries of the kitten accumulated more unchanged isoprenaline and metabolized more amine than atria following incubation for 1 to 20 min with [3H]-(+/-)-isoprenaline (25 ng/ml or 5 micrograms/ml). 2 Cortisol (10 or 80 microM), U-0521 (120 microM) and oxytetracycline (100 microM) all reduced metabolite formation. 3 Cortisol inhibited 'Iso InfluxMin' (cellular isoprenaline accumulation plus total metabolite production). In contrast, it increased, decreased or did not alter accumulation of unmetabolized isoprenaline, depending upon the experimental conditions. 4 Isoprenaline accumulation was increased in atria and reduced in coronary arteries by U-0521, while oxytetracycline reduced accumulation in coronary arteries at the high amine concentration. 5 It is concluded that in atria, cortisol inhibits metabolism and has differential effects on a number of extraneuronal compartments which accumulate isoprenaline. Both cortisol and U-0521 appear to be extraneuronal uptake inhibitors and inhibitors of catechol-O-methyltransferase in coronary arteries. Oxytetracycline may have effects additional to inhibition of isoprenaline binding to connective tissue fibres.

Catecholamine transport in isolated lung parenchyma of pig.

1 Lung parenchyma strips of the pig incubated at 37 degrees C with [(3)H]-(-)-noradrenaline ([(3)H]-NA) or [(3)H]-(+/-)-isoprenaline ([(3)H]-Iso), accumulated radioactivity via saturable, high affinity uptake processes. Apparent saturation constants (K(m)) for [(3)H]-NA and [(3)H]-Iso were 1.34 x 10(-6) M and 1.63 x 10(-6) M respectively, while apparent transport maxima (V(max)) were 4.86 and 1.63 x 10(-9) mol min(-1) g(-1) respectively.2 Cellular accumulation of radioactivity from radiolabelled catecholamines was greatly reduced by lowering the temperature to 7 degrees C, pretreatment with ouabain (100 muM), phentolamine (15 muM) or phenoxybenzamine (80 muM). However, accumulation of radioactivity derived from ((3)H]-NA was inhibited selectively by cocaine (10 muM) and desipramine (1 muM), while normetanephrine (80 muM) and 3-O-methylisoprenaline (50 muM) caused much greater reductions in cellular radioactivity from [(3)H]-Iso than from ((3)H]-NA. Taken together with information from kinetic studies, the results indicate that these amines are transported by separate uptake processes.3 Cocaine (50 muM) which selectively reduced [(3)H]-NA transport, had no significant effect on the sensitivity (EC(50)) of isolated parenchyma lung strips of the pig to the contractile effects of cumulative concentrations of NA. The catechol-O-methyl transferase (COMT) inhibitor, U-0521 (60 muM), also failed to alter the potency of NA, while normetanephrine (80 muM) caused a 2 fold decrease in potency.4 Phentolamine (15 muM), which reduced the cellular accumulation of radioactivity derived from [(3)H]-Iso by 64%, caused a small potentiation of Iso-induced relaxations of porcine lung strips. Normetanephrine (80 muM) and 3-O-methylisoprenaline (50 muM), which also depressed the accumulation of cellular radioactivity from [(3)H]-Iso by > 50%, caused rightward shifts in Iso concentration-effect curves as a result of beta-adrenoceptor blockade. In sharp contrast, cortisol (80 muM) and U-0521 (60 muM), which caused smaller reductions in the cellular accumulation of radioactivity derived from [(3)H]-Iso, both caused an approximately 9 fold potentiation of responses to Iso in isolated lung strips.5 The results indicate that the major sites of uptake and metabolism of NA in porcine parenchyma strip are remote from alpha-adrenoceptors mediating NA-induced contraction. Similarly, some major sites of uptake of Iso are remote from beta-adrenoceptors mediating Iso-induced relaxation. However, beta-adrenoceptors are apparently in close proximity to a compartment containing COMT activity.

Pharmacological evaluation of a guinea-pig tracheal epithelium-derived inhibitory factor (EpDIF).

1. An epithelium-derived inhibitory factor (EpDIF) released by guinea-pig tracheal epithelium was evaluated in a co-axial bioassay system consisting of an epithelium-intact guinea-pig tracheal tube surrounding endothelium-denuded rat aortic strip. 2. Histamine and several muscarinic agonists induced concentration-dependent relaxation of phenylephrine-contracted rat aorta via the release of EpDIF. However, several other agonists did not induce the release of EpDIF from guinea-pig trachea. These included the nicotinic cholinoceptor agonists nicotine (25 microM), 1,1-dimethyl-4-phenylpiperazinium (DMPP) (25 microM), calcium ionophore A23187 (0.5 microM), bradykinin (0.05-0.5 microM), substance P (5 microM), platelet activating factor (PAF, 1-100 nM), the leukotrienes (LT) LTC4, LTD4 and LTE4 (0.1-10 nM) as well as hyperosmotic stimuli. 3. Prostaglandin E2 (PGE2) induced concentration-dependent contraction of endothelium-denuded rat aortic preparations, indicating that this prostanoid could not be EpDIF. Furthermore, relaxation to histamine and methacholine, mediated via EpDIF, was not significantly altered in the presence of phenidone (50 microM) the cyclo-oxygenase/lipoxygenase inhibitor with radical scavenging properties or the cytochrome P-450 inhibitors metyrapone (1 mM) and SKF 525A (25 microM). This suggests that EpDIF is neither a prostanoid nor a cytochrome P-450 metabolite of arachidonic acid. 4. The soluble guanylate cyclase inhibitor, methylene blue (50 microM), caused small but significant increases in the potencies of both histamine and methacholine in co-axial assemblies, indicating that EpDIF did not activate this enzyme and therefore was not NO or a related substance. The beta-adrenoceptor antagonist, (-)-propranolol (1 microM), and the PAF-receptor antagonist, WEB 2086 (50 microM), also failed to alter significantly EpDIF-modulated relaxations. These data suggest that EpDIF is neither a stimulant of fiadrenoceptors nor of PAF receptors. 5. The present study provides some evidence that this vascular smooth muscle-sensitive EpDIF may not be related to the putative EpDIF previously hypothesized to modulate directly spasmogen-induced airway smooth muscle tone.

Contractile properties of synthetic cationic polypeptides in guinea-pig isolated trachea.

1. The synthetic polypeptides, poly-L-arginine, poly-L-lysine and poly-D-lysine contract guinea-pig isolated trachea in a concentration-dependent, epithelium-independent manner. Indomethacin augmented the contractile response to poly-L-arginine. 2. The contractile response to poly-L-arginine was not significantly inhibited by nicardipine, a selective L-type calcium channel blocker or by the histamine H1-receptor antagonist, mepyramine nor significantly augmented by the neutral endopeptidase inhibitor, phosphoramidon. 3. The contractile response to poly-L-arginine was inhibited in a concentration-dependent manner by prior incubation of guinea-pig tracheal rings with a number of anionic polypeptides including, low molecular weight heparin, poly-L-aspartic acid and bovine serum albumin. 4. In vitro capsaicin-induced desensitization failed to attenuate the contractile response to poly-L-arginine, suggesting little, if any role for sensory neuropeptides in the functional response in the guinea-pig. 5. Synthetic polypeptides induce an epithelium-independent, charge-dependent contraction of guinea-pig isolated trachea.

Poly-L-arginine-mediated release of acetylcholine from parasympathetic nerves in rat and guinea-pig airways.

1. The synthetic cationic polypeptide, poly-L-arginine (0.03-1 mg ml-1) induced concentration-dependent contraction of guinea-pig and rat isolated trachea. In guinea-pig isolated trachea, this response was attenuated in the presence of the muscarinic cholinoceptor antagonist, atropine (0.1 microM) and augmented by the acetylcholinesterase inhibitor, ecothiophate (0.1 microM). The neuronal sodium channel blocker, tetrodotoxin (3 microM) failed to alter the contractile response to poly-L-arginine and acetylcholine. 2. The contractile response to poly-L-arginine in rat isolated trachea was inhibited in the presence of atropine (0.1 microM) and the 5-hydroxytryptamine (5-HT) receptor antagonist, methysergide (1 microM). Treatment of rat tracheal preparations with capsaicin (100 microM) or tetrodotoxin (3 microM) failed to alter the contractile response to poly-L-arginine. In contrast, ecothiophate (0.1 microM) augmented the contractile response to poly-L-arginine in rat isolated trachea. 3. Electrical field stimulation (5 Hz, 2 min) of epithelium-denuded guinea-pig tracheal preparations preloaded with [3H]-choline resulted in a contractile response and the simultaneous efflux of radioactivity into the superfusate. Both these responses were abolished in the presence of tetrodotoxin (1.5 microM). Poly-L-arginine (1 mg ml-1) also increased the efflux of total radioactivity from epithelium-denuded guinea-pig isolated tracheal preparations preloaded with [3H]-choline, but this response was tetrodotoxin-insensitive. The negatively charged polyanion, heparin (1 mg ml-1) failed to increase significantly the efflux of radioactivity from epithelium-denuded preparations. 4.In conclusion, the synthetic cationic polypeptide, poly-L-arginine, caused contraction of guinea-pig isolated tracheal preparations via the release of acetylcholine from parasympathetic nerves. Similarly,poly-L-arginine-induced contraction of rat isolated trachea is secondary to the release of acetylcholine from parasympathetic nerves and/or the release of mast cell-derived 5-HT.

ETB but not ETA receptor-mediated contractions to endothelin-1 attenuated by respiratory tract viral infection in mouse airways.

1. The current study investigated the effects of respiratory tract viral infection on the density of ETA and ETB receptors in murine tracheal smooth muscle and on the contractile response to endothelin-1 mediated by these receptors. 2. Quantitative autoradiographic studies using [125I]-endothelin-1 revealed that tracheal smooth muscle from control mice contained ETA and ETB receptors in the ratio of 42%:58% (+/- 4%, n = 10 mice), respectively. In contrast, tracheal smooth muscle obtained from mice 2 days post-inoculation with Influenza A/PR-8/34 virus contained 23 +/- 2% fewer receptors for [125I]-endothelin-1 (n = 10, P < 0.01). This reflected a selective reduction in ETB receptor density and a change in the ratio of ETA and ETB receptors to 77%:23% (+/- 5%, n = 10 mice), respectively. 3. The ETB receptor-selective agonist, sarafotoxin S6c, was a potent spasmogen of murine isolated tracheal smooth muscle and the EC50 for contraction was similar in preparations from control (3.6 nM [95% confidence limits, 2.7-4.8 nM], n = 16 preparations from 8 mice) and virus-inoculated mice (3.0 nM [2.4-3.7 nM], n = 16 preparations from 8 mice). However, the maximum contractions induced by sarafotoxin S6c (100 nM) in the preparations from virus-inoculated mice (37 +/- 5% Cmax, where 100% Cmax was the response to 10 microM carbachol) were significantly smaller than those from control mice (85 +/- 4% Cmax, P < 0.01). 4. Contractions induced by endothelin-1 in tracheal smooth muscle preparations obtained from virus inoculated mice (EC50 for contraction, 6.5 nM [95% confidence limits, 2.7-16 nM]; maximum contraction,112 +/- 5% Cm.; n = 4) were similar to those induced by endothelin-1 in control preparations (ECm9.3 nM (4.2-21); maximum contraction, 110 +/- 3% Cmax; n = 4). Endothelin-1-induced contractions in control preparations were only marginally inhibited by the ETA receptor-selective antagonist BQ-123 (in the presence of 3 micro M BQ-123; EC50 for contraction, 5.9 nM [4.1-8.5]; maximum contraction, 82 +/- 4%Cmax; n = 4). In contrast, 3 microM BQ-123 caused a 50 fold rightward shift (17-160, n =4) of the concentration-effect curve to endothlin-1 in preparations obtained from virus-inoculated mice (measured at the 30% Cmax level of contraction).5. Tracheal smooth muscle preparations exposed to 100 nM sarafotoxin S6c for 30 min (followed by a 30 min washout period) did not contract to subsequently administered sarafotoxin S6c (1-100 nM;n = 8), but contracted normally in response to endothelin-1 (EC50 6.5 nM (2.3-18); maximum contraction,109 +/- 2% Cmax; n = 4). Endothelin-l-induced contractions in these ETB receptor desensitized preparations were markedly inhibited by 3 microM BQ-123, irrespective of whether the preparations were obtained from control (63 fold shift (10-400) at the 30% Cma. level of contraction, n = 4) or virus inoculated mice (46 fold shift (18-120), n = 4).6. In summary, tracheal smooth muscle obtained from mice infected with a respiratory tract virus,Influenza A/PR-8/34 had a reduced density of ETB receptors and an attenuated ETB receptor-mediated contractile response to sarafotoxin S6c and endothelin-1. Virus-inoculation was also associated with a modest increase in tracheal smooth muscle ETA receptor density, although no significant change in ETA receptor-mediated contractile activity was seen. Thus, virus infection in murine airways produced profound alterations in endothelin receptor density, some of which were associated with changes in receptor-mediated contractile activity.

Alpha 1-adrenoceptor function and autoradiographic distribution in human asthmatic lung.

1. The autoradiographic distribution of alpha 1-adrenoceptors was investigated in non-diseased and asthmatic human lung by use of [3H]-prazosin (H-PZ). To validate binding and autoradiographic methods, H-PZ binding was also measured in rat heart. 2. Significant levels of specific H-PZ binding were detected in sections of rat heart. This binding was associated with a single class of non-interacting sites of high affinity (dissociation constant, Kd = 1.17 +/- 0.26 nM). The maximum binding capacity (Bmax) was 59.5 +/- 4.5 fmol mg-1 protein. 3. In sharp contrast, very low levels of specific H-PZ binding were found in both human nondiseased and asthmatic bronchus, although a high level of binding of [125I]-iodocyanopindolol (I-CYP, 50 pM) to beta-adrenoceptors was detected in these airways. Furthermore, very low levels of autoradiographic grains representing specific H-PZ binding were found in all airway structures in human non-diseased or asthmatic lung parenchyma. 4. Consistent with these data, the alpha-adrenoceptor agonist phenylephrine failed to induce significant increases in tone in bronchi isolated from either non-diseased or asthmatic human lung. Results indicate that asthma does not involve significant increases in airway alpha 1-adrenoceptor function.

Co-axial bioassay of a smooth muscle relaxant factor released from guinea-pig tracheal epithelium.

1. The ability of guinea-pig trachea to release an epithelium-derived relaxant factor (EpDRF) was assessed in a co-axial bioassay system. 2. Histamine (100 microM) and methacholine (25 microM) caused endothelium-dependent relaxation of rat isolated aorta, presumably via the release of endothelium-derived relaxant factor (EDRF). In contrast, endothelium-denuded rat aorta did not relax in response to these agents. 3. EDRF release was detected in response to methacholine in a co-axial bioassay system, consisting of intact rabbit aorta tube (EDRF donor) and endothelium-denuded rat aorta strip (assay preparation). These results indicated the transfer of EDRF from a donor to an assay preparation, thereby validating the co-axial bioassay method. 4. Substitution of endothelium-intact rabbit aorta tube by epithelium-intact guinea-pig tracheal tube tissue in co-axial assemblies, still allowed the assay preparation to relax in response to histamine or methacholine. Removal of the intact tracheal tube from the system, or removal of the epithelium from the donor tracheal tube in co-axial preparations, abolished such relaxant responses. These observations are consistent with histamine- or methacholine-induced release of an epithelium-derived relaxant factor (EpDRF) from the trachea. 5. In the co-axial assembly comprising intact guinea-pig trachea and endothelium-denuded rat aorta, histamine and methacholine produced concentration-dependent, EpDRF-induced aortic relaxation. Mean concentrations of histamine and methacholine producing 50% of the maximum relaxation (EC50) were 39.8 microM and 2.7 microM respectively. Histamine-induced relaxation was inhibited in the presence of mepyramine (2 microM) and responses to methacholine were inhibited by atropine (0.1 microM). 6. Methylene blue (50 microM) had no effect on such relaxant responses, indicating that EpDRF does not activate guanylate cyclase. Furthermore, the cyclo-oxygenase inhibitor indomethacin (5 microM), the cyclo-oxygenase/lipoxygenase inhibitor BW 755C (150 microM) and the leukotriene receptor antagonist FPL 55712 (10 microM) each failed significantly to alter EpDRF-mediated relaxation of vascular smooth muscle suggesting that EpDRF is not a prostanoid. Platelet activating factor (Pat) failed to cause relaxation of endothelium-denuded rat aorta, indicating that this mediator was also not EpDRF. 7. EpDRF was also released from human bronchial segments. 8. This study provides direct evidence for the release of an EpDRF from non-diseased airway tissue and further suggests that healthy airway reactivity to spasmogens is modulated by the release of an endogenous protective, spasmolytic substance. The bronchial hyperreactivity of asthma may be partly caused by attenuated production of such an inhibitory signal.

Distribution of beta 1- and beta 2-adrenoceptors in mouse trachea and lung: a quantitative autoradiographic study.

1. Binding and quantitative autoradiography were used to detect [125I]-iodocyanopindolol (I-CYP) associated with beta 1- and beta 2-adrenoceptors in mouse tracheal epithelium and airway smooth muscle as well as in lung parenchymal tissue. 2. Specific I-CYP binding to slide-mounted tissue sections of both trachea and parenchyma was of high affinity (KD = 49.0 pM, n = 3, trachea; KD = 118.9 pM, n = 3, parenchyma) and saturable, involving single populations of non-interacting binding sites (Hill coefficient nH = 1.00 +/- 0.02, trachea; nH = 0.99 +/- 0.03, parenchyma). 3. Direct measurement of tissue radioactivity also showed that specific I-CYP binding was competitively inhibited in the presence of the beta-adrenoceptor antagonists (-)-propranolol (non-selective), CGP 20712A (beta 1-selective) and ICI 118,551 (beta 2-selective). Analysis of the competition binding curves for the two selective antagonists revealed mixed populations of beta 1- and beta 2-adrenoceptors in the approximate proportions 33% and 67% respectively in mouse trachea and 28% and 72% respectively in mouse lung parenchyma. 4. Densities of autoradiographic grains derived from specific I-CYP binding to alveolar wall tissue and to tracheal epithelium and airway smooth muscle were quantified by a computer-assisted image analysis system, which allowed the construction of competition binding curves in the presence of the selective beta-adrenoceptor antagonists CGP 20712A and ICI 118,551. Analysis of these data demonstrated that in alveolar wall, beta 1- and beta 2-adrenoceptors co-existed in the proportions 18% and 82%, respectively. 5. Quantitative autoradiographic analyses also showed that beta 1- and beta 2-adrenoceptors were differentially distributed in tracheal epithelium and airway smooth muscle. The beta 2-adrenoceptor subtype accounted for 71% of all beta-adrenoceptors in epithelium. Conversely, beta l-adrenoceptors which mediate relaxant responses of mouse trachea to beta,-adrenoceptor agonists (Henry & Goldie, 1990), accounted for 69% of all beta-adrenoceptors in the airway smooth muscle.

Inhibition by endothelin-1 of cholinergic nerve-mediated acetylcholine release and contraction in sheep isolated trachea.

1. The relative roles of ETA and ETB receptor activation on cholinergic nerve-mediated contraction and acetylcholine (ACh) release were examined in sheep isolated tracheal smooth muscle. 2. Electrical field stimulation (EFS; 90 V, 0.5 ms duration, 1 Hz, 10 s train) applied to sheep isolated tracheal smooth muscle strips induced monophasic contractile responses that were abolished by either 1 microM tetrodotoxin or 0.1 microM atropine, but were insensitive to 10 microM hexamethonium and 100 microM L-NAME. Thus, EFS-induced contractions resulted from the spasmogenic actions of ACh released from parasympathetic, postganglionic nerves. 3. As expected, sheep isolated tracheal smooth muscle preparations did not contract in response to the ETB receptor-selective agonist, sarafotoxin S6c (0.1-100 nM). However, sarafotoxin S6c caused a concentration-dependent and transient inhibition of EFS-induced contractions. The inhibitory effect induced by a maximally effective concentration of sarafotoxin S6c (10 nM; 72.1 +/- 5.7%, n = 6) was abolished in the presence of the ETB receptor-selective antagonist BQ-788 (1 microM). Contractile responses to exogenously administered ACh (10 nM-0.3 mM) were not inhibited by sarafotoxin S6c (1 or 10 nM; n = 7). 4. In contrast to sarafotoxin S6c, endothelin-1 induced marked contractions in sheep isolated tracheal smooth muscle. These contractions were inhibited by BQ-123, consistent with an ETA receptor-mediated response. In the presence of BQ-123 (3 microM), endothelin-1 produced a concentration-dependent inhibition of EFS-induced contractions (30 nM endothelin-1, 68.9 +/- 10.2% inhibition, n = 5). These responses were inhibited by 1 microM BQ-788, indicative of an ETB receptor-mediated process. Endothelin-1 was about 3 fold less potent than sarafotoxin S6c. 5. EFS (90 V, 0.5 ms duration, 1 Hz, 15 min train) induced the release of endogenous ACh (1.94 +/- 0.28 pmol mg-1 tissue, n = 12), as assayed by h.p.l.c. with electrochemical detection. EFS-induced release of ACh was inhibited to a similar extent by 100 nM endothelin-1 (47 +/- 4%, n = 9) and 10 nM sarafotoxin S6c (46 +/- 9%, n = 3). These effects of endothelin-1 on ACh release were inhibited by 1 microM BQ-788 alone (n = 4), by BQ-788 in the presence of 3 microM BQ-123 (n = 4), but not by 3 microM BQ-123 alone (n = 5). 6. In summary, sheep isolated tracheal smooth muscle contains two anatomically and functionally distinct endothelin receptor populations. ETA receptors located on airway smooth muscle mediate contraction, whereas ETB receptors appear to exist on cholinergic nerves that innervate tracheal smooth muscle cells and mediate inhibition of ACh release. The inhibitory effect of ETB receptor stimulation on cholinergic neurotransmission is in stark contrast to the enhancing effects hitherto described in the airways.

Catecholamine uptake by isolated coronary arteries and atria of the kitten.

Kitten coronary arteries and atria incubated with [3H]-(-)-noradrenaline or [3H]-(+/-)-isoprenaline showed concentration- and temperature-dependent accumulation of amines (unchanged and/or as metabolites). In addition, coronary arteries incubated with [3H]-(+/-)-isoprenaline showed temperature-insensitive amine accumulation due to binding to connective tissue fibres. 2 With low concentrations of [3H]-(-)-noradrenaline (20 ng/ml) accumulation of radioactivity in both tissues was neuronal since it was reduced by desipramine, metaraminol or cocaine but not by the extraneuronal inhibitor, cortisol. 3 In coronary arteries incubated with [3H]-(+/-)-isoprenaline (5 microgram/ml) for 1 or 10 min, uptake was extraneuronal since it was inhibited by cortisol, 17beta-oestradiol or phenoxybenzamine. 4 Atria incubated with (+/-)-isoprenaline (5 to 1000 microgram/ml) showed non-neuronal, biphasic accumulation of amine. There was a high capacity, low affinity initial uptake process (apparent Km 136 micron) which was resistant to steroidal extraneuronal uptake inhibitors but which could be abolished by phenoxybenzamine. A slower uptake occurred after 2 min which was sensitive to steroidal and other extraneuronal uptake inhibitors. 5 Inhibition of uptake processes did not alter sensitivity of coronary arteries to dilator effects of catecholamines. However, experiments with extraneuronal uptake inhibitors were limited by the relaxant effects of cortisol and 17beta-oestradiol themselves. 6 In atria inhibition of neuronal uptake by desipramine or cocaine led to supersensitivity to (-)-noradrenaline but not to (-)-adrenaline or (+/-)-isoprenaline. Chronotropic responses to (-)-noradrenaline were increased five fold and inotropic responses three fold.

Effect of hypothermia on beta 1-adrenoceptor-mediated relaxation of pig bronchus.

The relaxant potencies of (+/-)-isoprenaline and of (-)-noradrenaline (NA) in the pig isolated bronchus were increased 5.4 and 3.1 fold respectively by lowering the organ bath temperature from 37 degrees C to 27 degrees C, whereas the potencies of the non-catecholamine beta-adrenoceptor agonists fenoterol and orciprenaline were not significantly changed. At 37 degrees C, the catechol-O-methyl transferase (COMT) inhibitor U-0521 (30 microM), caused a 7.2 fold increase in the potency of isoprenaline but had no effect on the potency of fenoterol. At 27 degrees C the potency of isoprenaline was similar in the absence or presence of U-0521 (30 microM). Furthermore, in bronchi where extraneuronal uptake was inhibited by phenoxybenzamine, the potency of NA was not significantly altered by reducing bathing temperature from 37 degrees C to 27 degrees C. These results suggest that the hypothermic potentiation of isoprenaline in pig bronchus resulted from inhibition of COMT or of access to COMT, rather than from sensitization of beta 1-adrenoceptors.

Effect of steroids on beta-adrenoceptor-mediated relaxation of pig bronchus.

1--Progesterone, testosterone (40 microM), cortisol and cortisol hemisuccinate (80 microM) caused 6-8 fold potentiations of (+/-)-isoprenaline (Iso)-induced relaxations of pig bronchus while several other steroids caused smaller potentiations or had no effect. 2--17 beta-Oestradiol (40 microM) increased the potency of Iso, (-)-adrenaline (Adr) and (-)-noradrenaline (NA) by 10.6, 2.3 and 2.6 fold respectively but had no significant effect on the potency of fenoterol (Fen). 3--Inhibition of catechol-O-methyl transferase (COMT) with U-0521 (30 microM) caused a 6 fold increase in the potency of Iso but failed to alter the potency of Adr, NA or Fen. The extraneuronal uptake inhibitor normetanephrine (50 microM) caused significant 2 fold increases in the potency of Iso and Adr but did not potentiate the responses to NA or Fen. 4--In preparations where the potency of Iso had already been increased by U-0521 (30 microM) or by normetanephrine, 17 beta-oestradiol produced no significant further increase in potency. These results indicate that steroid-induced increases in the potency of catecholamines in pig bronchus can be explained in terms of inhibition of COMT or extraneuronal uptake or both.

Classification of beta-adrenoceptors in isolated bronchus of the pig.

1 (+/-)-Isoprenaline (Iso), (-)-adrenaline (Ad), (-)-noradrenaline (NA), the beta 2-selective adrenoceptor agonist (+/-)-fenoterol (Fen) and the beta 1-selective adrenoceptor agonist (+/-)-RO363 caused concentration-dependent relaxation of preparations of pig bronchus pre-contracted with carbachol 40-ng/ml (0.22 microM). Iso, Ad, NA and Fen caused complete relaxation of carbachol-induced tone, but RO363 caused relaxation equivalent to only 59% of the maximal response to Iso. 2 When relaxation responses to these amines were plotted as a % of their maximal effects, comparison of EC50 values showed that the order of potency was RO363 greater than Iso greater than NA greater than Fen greater than Ad (14.4:4.6:1:0.4:0.3). 3 pA2 values determined for the beta-adrenoceptor antagonists propranolol (non-selective) and atenolol (beta 1-selective), or the partial agonist salbutamol (beta 2-selective) using Iso as agonist were 8.3, 7.3 and 4.4 respectively. The pA2 value for atenolol using RO363 as the agonist was 7.6. 4 These results indicate that porcine bronchus contains a homogeneous population of beta 1-adrenoceptors.

Age and region-dependent contraction to alpha-adrenoceptor agonists in rat and guinea-pig isolated trachea.

1. The influence of age and of region on alpha-adrenoceptor-mediated contraction to (-)-adrenaline and (-)-noradrenaline was examined in rat (4-136 weeks) and guinea-pig (2-156 weeks) isolated tracheal ring preparations with particular emphasis on the early (up to 12 weeks) maturation phase. 2. In rat tracheal rings, significant regional variation was observed with respect to maximal (-)-adrenaline-induced contraction, such that the greatest activity was seen in ring preparations from the laryngeal end of the trachea. Tracheal rings from the carinal end responded very poorly or were unresponsive to (-)-adrenaline, depending on animal age. These regional differences were seen across the age range. The potencies of (-)-adrenaline and (-)-noradrenaline remained unchanged with respect to animal age, but the maximum contractile tension that developed in response to these agonists increased with increasing animal age in all regions of the trachea. 3. In guinea-pig isolated tracheal tissue, maximum contractile responses (Emax) to (-)-adrenaline and (-)-noradrenaline remained unchanged with increasing animal age. In addition, there was no evidence for a region-dependence in the responsiveness of tracheal tissue to alpha-adrenoceptor-mediated contraction in this species. 4. In both guinea-pig and rat isolated tracheal tissue, alpha-adrenoceptor-mediated contraction appeared to involve the activation of alpha1-adrenoceptors.