Lynette Fernandes

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.

EndothelinB (ETB) receptor‐activated potentiation of cholinergic nerve‐mediated contraction in human bronchus

In human isolated bronchial preparations, the endothelinB (ETB) receptor‐selective agonist, sarafotoxin S6c (Stx6c; 1 nM) increased nerve‐mediated contraction in response to electrical field stimulation (EFS) at 0.5‐1 Hz from 19±4% to 42±7% (n = 9). This effect was blocked in the presence of the ETB receptor‐selective antagonist, BQ‐788 (10 μm). These data are consistent with findings in some animal species that ET‐1 and related peptides have marked neuromodulatory influences on the cholinergic system. Furthermore, they provide additional support for the concept that ET‐1 may have a mediator role in bronchial obstruction in asthma.

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 μm), 1,1‐dimethyl‐4‐phenylpiperazinium (DMPP) (25 μm), calcium ionophore A23187 (0.5 μm), bradykinin (0.05‐0.5 μm), substance P (5 μm), 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 μm) the cyclo‐oxygenase/lipoxygenase inhibitor with radical scavenging properties or the cytochrome P‐450 inhibitors metyrapone (1 mm) and SKF 525A (25 μm). 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 μm), 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 β‐adrenoceptor antagonist, (−)−propranolol (1 μm), and the PAF‐receptor antagonist, WEB 2086 (50 μm), also failed to alter significantly EpDIF‐modulated relaxations. These data suggest that EpDIF is neither a stimulant of β‐adrenoceptors 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.

Rho kinase as a therapeutic target in the treatment of asthma and chronic obstructive pulmonary disease

Asthma is a complex inflammatory disease of the airways involving reversible bronchoconstriction. Chronic obstructive pulmonary disease is typified by inflammation and airflow limitation that has an irreversible component. There is now substantial evidence that Rho kinase is involved in many of the pathways that contribute to the pathologies associated with these respiratory diseases including bronchoconstriction, airway inflammation, airway remodelling, neuromodulation and exacerbations due to respiratory tract viral infection. Indeed the Rho kinase inhibitor Y-27632 causes bronchodilatation and reduces pulmonary eosinophilia trafficking and airways hyperresponsiveness. Furthermore, accumulating evidence suggests that inhibition of Rho kinase could have a major beneficial impact on symptoms and disease progression in asthma and COPD by modulating several other systems and processes. Thus, the Rho kinase pathway may indeed be a worthwhile therapeutic target in the treatment of asthma and chronic obstructive pulmonary disease.

Epithelium-derived inhibitory factor in human brochus

The potencies of histamine and methacholine were significantly increased by approximately 2- and 5-fold respectively in human non-diseased isolated bronchi on removal of the epithelium. In contrast, no increases in spasmogen potency were observed following epithelium removal in bronchi obtained from a sample of asthmatic human lung. The failure of epithelium removal to increase asthmatic bronchial sensitivity to histamine may have been due to a reduction in the release of an epithelium-derived inhibitory factor (EpDIF) resulting from disease-induced epithelial damage. A co-axial bioassay system in which endothelium-denuded rat aorta was used as the assay tissue was used to detect the release of a vasorelaxant EpDIF from human bronchial tissue. Histamine (100 microM) and methacholine (25 microM), in the presence of indomethacin (5 microM), reduced phenylephrine-induced tone in endothelium-denuded rat aorta in co-axial assemblies by 75 +/- 11 and 67 +/- 9% respectively. Removal of the bronchial epithelium abolished these responses, indicating that they were mediated by an EpDIF. It is possible that human airway smooth muscle is sensitive to this vasorelaxant EpDIF and that the absence of the source of this factor following epithelium removal caused the increases in sensitivity to spasmogens. Alternatively, the human bronchial epithelium may also release an EpDIF selective for airway smooth muscle.

Potent bronchodilation and reduced stiffness by relaxant stimuli under dynamic conditions

Airway relaxation in response to isoprenaline, sodium nitroprusside (SNP) and electrical field stimulation (EFS) was compared under static and dynamic conditions. The capacity of relaxants to reduce airway stiffness and, thus, potentially contribute to bronchodilation was also investigated. Relaxation responses were recorded in fluid filled bronchial segments from pigs under static conditions and during volume oscillations simulating tidal and twice tidal manoeuvres. Bronchodilation was assessed from the reduction in carbachol-induced lumen pressure, at isovolume points in pressure cycles produced by volume oscillation, and stiffness was assessed from cycle amplitudes. Under static conditions, all three inhibitory stimuli produced partial relaxation of the carbachol-induced contraction. Volume oscillation alone also reduced the contraction in an amplitude-dependent manner. However, maximum relaxation was observed when isoprenaline or SNP were combined with volume oscillation, virtually abolishing contraction at the highest drug concentrations. The proportional effects of isoprenaline and EFS were not different under static or oscillating conditions, whereas relaxation to SNP was slightly greater in oscillating airways. All three inhibitory stimuli also strongly reduced carbachol-induced airway stiffening. The current authors conclude that bronchoconstriction is strongly suppressed by combining the inhibitory stimulation of airway smooth muscle with cyclical mechanical strains. The capacity of airway smooth muscle relaxants to also reduce stiffness may further contribute to bronchodilation.

Rhinovirus exacerbates house-dust-mite induced lung disease in adult mice.

Human rhinovirus is a key viral trigger for asthma exacerbations. To date, murine studies investigating rhinovirus-induced exacerbation of allergic airways disease have employed systemic sensitisation/intranasal challenge with ovalbumin. In this study, we combined human-rhinovirus infection with a clinically relevant mouse model of aero-allergen exposure using house-dust-mite in an attempt to more accurately understand the links between human-rhinovirus infection and exacerbations of asthma. Adult BALB/c mice were intranasally exposed to low-dose house-dust-mite (or vehicle) daily for 10 days. On day 9, mice were inoculated with human-rhinovirus-1B (or UV-inactivated human-rhinovirus-1B). Forty-eight hours after inoculation, we assessed bronchoalveolar cellular inflammation, levels of relevant cytokines/serum antibodies, lung function and responsiveness/sensitivity to methacholine. House-dust-mite exposure did not result in a classical TH2-driven response, but was more representative of noneosinophilic asthma. However, there were significant effects of house-dust-mite exposure on most of the parameters measured including increased cellular inflammation (primarily macrophages and neutrophils), increased total IgE and house-dust-mite-specific IgG1 and increased responsiveness/sensitivity to methacholine. There were limited effects of human-rhinovirus-1B infection alone, and the combination of the two insults resulted in additive increases in neutrophil levels and lung parenchymal responses to methacholine (tissue elastance). We conclude that acute rhinovirus infection exacerbates house-dust-mite-induced lung disease in adult mice. The similarity of our results using the naturally occurring allergen house-dust-mite, to previous studies using ovalbumin, suggests that the exacerbation of allergic airways disease by rhinovirus infection could act via multiple or conserved mechanisms.

β-Adrenoceptor desensitization in guinea-pig isolated trachea

Exposure to (−)-isoprenaline (25 μM, 1 h) caused a stereoselective, time and concentration-related decrease in smooth muscle β2-adrenoceptor function in guinea-pig trachea. Furthermore, tracheal relaxant responsiveness to the β-adrenoceptor agonists (±)-fenoterol and (−)-noradrenaline was reduced, while that to theophylline and nitroprusside was unaffected. Responsiveness to forskolin was marginally but significantly reduced. Indomethacin, a cyclooxygenase inhibitor and mepacrine, an inhibitor of phospholipid turnover, had no significant effect on the extent of isoprenaline-induced desensitization. Conversely, cortisol (25 μM) significantly reduced desensitization and enhanced the rate of spontaneous recovery of responsiveness to isoprenaline. Desensitization was not accompanied by a reduction in the density of β-adrenoceptors in the trachea, as assessed by binding and light microscopic autoradiography using [125I]iodocyanopindolol ([125I]CYP). Thus, desensitization was probably caused primarily by β-adrenoceptor/adenyl cyclase uncoupling. This model may be useful in investigations of the effect of glucocorticoids on the β-adrenoceptor dysfunction recognized in severe asthma.

Epithelial modulation of the relaxant activity of atriopeptides in rat and guinea-pig tracheal smooth muscle

Three peptide components of atrial natriuretic factor (ANF) caused relaxation of carbachol-contracted guinea-pig isolated tracheal smooth muscle. These were the 1-28, 5-28 and 5-27 peptide sequences (ANF(1-28), ANF-(5-28) and ANF-(5-27)). The peptides were 10-30 times more potent in epithelium-denuded than in epithelium-intact preparations. In the absence of airway epithelium, ANF-(1-28) was the most potent relaxant (mean pD2 = 7.40 +/- 0.08), with ANF-(5-27) and ANF-(5-28) 2-3 fold less potent. The neutral endopeptidase inhibitor phosphoramidon (1 microM) increased the potency of ANF-(5-27) in both epithelium-intact and epithelium-denuded guinea-pig tracheal rings. In contrast, removal of the epithelium from rat trachea, or pretreatment with phosphoramidon (1 microM) decreased relaxant responsiveness to ANF-(5-27). Thus, in rat trachea, epithelial endopeptidase may convert ANF-(5-27) to a more active relaxant peptide. Human bronchial preparations with or without epithelium, obtained from non-diseased lung samples and from a single sample of asthmatic lung, were virtually unresponsive to ANF-(5-27). Consistent with the spasmolytic effects of ANF in guinea-pig trachea, autoradiographic analysis revealed the presence of a sparse population of specific binding sites for [125I]ANF-(1-28) over both tracheal smooth muscle and epithelium. The present study shows that the relaxant effects of atriopeptins in rat and guinea-pig airway smooth muscle were modulated by the epithelium and the activity of neutral endopeptidase. However, marked species differences in airway smooth muscle responsiveness to ANF and in the modulatory role of the airway epithelium were evident.