Roy G. Goldie

Influence of the epithelium on responsiveness of guinea‐pig isolated trachea to contractile and relaxant agonists

1 The potency (pD2) and maximal contractile effect (Emax) of histamine, acetylcholine, carbachol and K+ were assessed from cumulative concentration‐effect curves in guinea‐pig isolated tracheal ring preparations with and without an intact epithelium. 2 Estimates of Emax were not significantly different in epithelium‐denuded preparations compared with those measured in intact preparations; pD2 values for acetylcholine, carbachol and K+ were not significantly altered. In contrast, the potency of histamine was significantly increased by about 4 fold in preparations devoid of epithelial cells. 3 Estimates of potency and Emax were also determined for the smooth muscle relaxants isoprenaline, forskolin and theophylline (which increase intracellular cyclic AMP) and for nitroglycerin (which increases cyclic GMP) in both intact and epithelium‐stripped tracheal rings. The pD2 values for these relaxants were not significantly altered by the removal of the epithelium. However, with the exception of nitroglycerin, Emax values for these relaxants were significantly lower in stripped than in intact tracheal rings that had been maximally precontracted with carbachol. 4 The autoradiographic localisation of binding sites for the non‐selective β‐adrenoceptor ligand [125I]‐iodocyanopindolol (I‐CYP) showed that the epithelium of the guinea‐pig trachea had a 75 ± 16% greater density of β‐adrenoceptors than the smooth muscle. Removing the epithelium did not significantly alter either the density of smooth muscle binding sites or the affinity of I‐CYP binding. It was concluded that the reduced functional response of guinea‐pig trachea to isoprenaline was probably not due to smooth muscle β‐adrenoceptor dysfunction. 5 Results indicate that the epithelium plays an important role in the modulation of responsiveness of guinea‐pig trachea to histamine and relaxants that mediate their effects by selectively increasing intracellular cyclic AMP levels.

Endothelin‐1‐induced potentiation of human airway smooth muscle proliferation: an ETA receptor‐mediated phenomenon

1 . In this study the mitogenic effects in human cultured tracheal smooth muscle cells of endothelin‐1 (ET‐1), ET‐3, and sarafotoxin S6c (S6c), the ETB receptor‐selective agonist, were explored either alone or in combination with the potent mitogen, epidermal growth factor (EGF). 2 . In confluent, growth‐arrested human airway smooth, neither ET‐1 (0.01 nM‐1μm) nor ET‐3 (0.001 nM‐1 μm) or S6c (0.01 nM‐1 μm) induced cell proliferation, as assessed by [3H]‐thymidine incorporation. In contrast, EGF (1.6 pM − 16 nM) produced concentration‐dependent stimulation of DNA synthesis (EC50 of about 0.06 nM). The maximum increase of about 60 fold above control, elicited by 16nM EGF, was similar to that obtained with 10% foetal bovine serum (FBS). EGF (0.16‐16 nM) also produced a concentration‐dependent increase in cell counts, whereas ET‐1 (1 − 100 nM) was without effect on this index of mitogenesis. 3 . ET‐1 (1–100 nM) potentiated EGF‐induced proliferation of human tracheal smooth muscle cells. For example, ET‐1 (100 nM), which alone was without significant effect, increased by 3.0 to 3.5 fold the mitogenic influence of EGF (0.16 nM). The potentiating effect of ET‐1 on EGF‐induced proliferation was antagonized by BQ‐123 (3 μm), the ETA receptor antagonist, but was unaffected by the ETB receptor antagonist BQ‐788 (10 μm). 4 . Neither ET‐3 (1 − 100 nM) nor S6c (1 − 100 nM) influenced the mitogenic effects of EGF (0.16‐1.6 nM). 5 . [125I]‐ET‐1 binding studies revealed that on average the ratio of ETA to ETB receptors in human cultured tracheal smooth muscle cells was 35:65 (±3; n=4), confirming the predominance of the ETB receptor subtype in human airway smooth muscle. 6 . These data indicate that ET‐1 alone does not induce significant human airway smooth muscle cell proliferation. However, it potently potentiated mitogenesis induced by EGF, apparently via an ETA receptor‐mediated mechanism. These findings suggest that ET‐1, a mediator detected in increased amounts in patients with acute asthma, may potentiate the proliferative effects of mitogens and contribute to the airway smooth muscle hyperplasia associated with chronic severe asthma.

Effect of steroids on β‐adrenoceptor‐mediated relaxation of pig bronchus

1 Progesterone, testosterone (40 μm), cortisol and cortisol hemisuccinate (80 μm) 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 17β‐Oestradiol (40 μm) 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 μm) 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 μm) 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 μm) or by normetanephrine, 17β‐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.

Relationship between endothelin-1 binding site densities and constrictor activities in human and animal airway smooth muscle.

1 Endothelin‐1 (ET‐1) binding site densities and constrictor activities were compared in airway smooth muscle preparations of human, guinea‐pig, rat and mouse. 2 The mean contractile response to 0.3 μm ET‐1 (measured as the % maximum response to 10 μm carbachol, % Cmax ± s.e.mean) and the mean concentration of ET‐1 producing 30% (95% confidence limits) were respectively; 85.9 ± 5.4% and 3.4nm (2.4–5.0) for mouse trachea (n = 11), 88.8 ± 4.7% and 18.2 nm (11.2–25.2) for rat trachea (n = 6), 71.0 ± 7.1% and 35.2 nm (5.4–231) for human bronchus (n = 3), and 32.3 ± 3.0% and 241 nm (125–460) for guinea‐pig trachea (n = 6). 3 Light microscopic autoradiography revealed specific [125I]‐ET‐1 binding sites localized to the smooth muscle band, with very low levels of binding associated with cartilage, submucosal and epithelial cells. 4 Quantitative autoradiographic analyses of the concentration‐dependence of specific [125I]‐ET‐1 binding (0.1–2nm) to smooth muscle revealed similar dissociation constants but markedly different specific binding site densities for the various animal species. The order of densities of specific [125I]‐ET‐1 binding sites was rat trachea (69.0 ± 11.2 amol mm−2) > human bronchus (42.7 ± 17.5 amol mm−2) > mouse trachea (28.7 ± 2.6 amol mm−2) > guinea‐pig trachea (8.3 ± 1.8 amol mm−2). 5 A positive relationship between [125I]‐ET‐l binding site density and ET‐1 constrictor activity was observed in airway smooth muscle preparations from rat, human and guinea‐pig. The greater sensitivity of mouse trachea to the constrictor actions of ET‐1 was not dependent on the release of cyclo‐oxygenaseor epithelium‐derived constrictor substances, but may have been due to an inter‐species difference in the receptor‐effector system for ET‐1.

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 μm) and methacholine (25 μm) 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 μm and 2.7 μm respectively. Histamine‐induced relaxation was inhibited in the presence of mepyramine (2 μm) and responses to methacholine were inhibited by atropine (0.1 μm). 6 Methylene blue (50 μm) had no effect on such relaxant responses, indicating that EpDRF does not activate guanylate cyclase. Furthermore, the cyclo‐oxygenase inhibitor indomethacin (5 μm), the cyclo‐oxygenase/lipoxygenase inhibitor BW 755C (150 μm) and the leukotriene receptor antagonist FPL 55712 (10 μm) each failed significantly to alter EpDRF‐mediated relaxation of vascular smooth muscle suggesting that EpDRF is not a prostanoid. Platelet activating factor (Paf) 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.

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.

Endothelins in Health and Disease: an overview

1. There is an ever increasing volume of evidence implicating endothelin‐1 and its isoforms in a range of disease processes. These include asthma, pulmonary and essential systemic hypertension, cardiac failure and uterine dysfunction.

PHARMACOLOGICAL RESPONSES OF HUMAN AND PORCINE LUNG PARENCHYMA, BRONCHUS AND PULMONARY ARTERY

1 Responses of preparations of human and porcine isolated bronchus and pulmonary artery to carbachol (CCh), methacholine, histamine, 5‐hydroxytryptamine (5‐HT), (−)‐noradrenaline (NA), (−)adrenaline (Adr) and (±)‐isoprenaline (Iso) were compared with responses to the same agonists in isolated lung parenchyma strips. 2 All preparations from both human and porcine lung contracted in response to histamine and all, except preparations of porcine pulmonary artery, contracted in response to CCh. Human and porcine pulmonary artery and parenchyma strip contracted in response to NA while bronchial preparations invariably relaxed. Iso caused relaxation of human and porcine bronchus and parenchyma strip. Although 5‐HT was completely inactive in tissues isolated from pig lung, this amine was a powerful spasmogen in human pulmonary artery, relaxed human bronchus and caused variable responses in human parenchyma. 3 Results indicate that the pharmacological characteristics of human and porcine parenchyma strips may be explained in terms of responses of vascular or airways smooth muscle.

Endothelins and asthma

In the decade since endothelin-1 (ET-1) and related endogenous peptides were first identified as vascular endothelium-derived spasmogens, with potential pathophysiological roles in vascular diseases, there has been a significant accumulation of evidence pointing to mediator roles in obstructive respiratory diseases such as asthma. Critical pieces of evidence for this concept include the fact that ET-1 is an extremely potent spasmogen in human and animal airway smooth muscle and that it is synthesised in and released from the bronchial epithelium. Importantly, symptomatic asthma involves a marked enhancement of these processes, whereas asthmatics treated with anti-inflammatory glucocorticoids exhibit reductions in these previously elevated indices. Despite this profile, a causal link between ET-1 and asthma has not been definitively established. This review attempts to bring together some of the evidence suggesting the potential mediator roles for ET-1 in this disease.

Receptors for endothelin‐1 in asthmatic human peripheral lung

[125I]‐endothelin‐1 ([125I]‐ET‐1) binding was assessed by autoradiography in peripheral airway smooth muscle and alveolar wall tissue in human non‐asthmatic and asthmatic peripheral lung. Levels of specific binding to these structures were similar in both non‐asthmatic and asthmatic lung. The use of the receptor subtype‐selective ligands, BQ‐123 (ETA) and sarafotoxin S6c (ETB), demonstrated the existence of both ETA and ETB sites in airway smooth muscle and in alveoli. In airway smooth muscle from both sources, the great majority of sites were of the ETB subtype. Quantitative analyses of asthmatic and non‐asthmatic alveolar wall tissue demonstrated that 29–32% of specific [125I]‐ET‐1 binding was to ETA sites and 68–71% was to ETB sites. Thus, asthma was not associated with any significant alteration in the densities of ETA and ETB receptors in peripheral human lung.