Peter J. Henry

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.

Endothelin-1 (ET-1)-induced contraction in rat isolated trachea: involvement of ETA and ETB receptors and multiple signal transduction systems.

1 Quantitative autoradiographic, biochemical and functional studies were performed to investigate the endothelin receptor subtypes and signal transduction systems that mediate endothelin‐1 (ET‐1)‐induced contraction in rat isolated tracheal smooth muscle. 2 Specific binding of 0.5 nm [125I]‐ET‐1 to tracheal smooth muscle was inhibited by at least 40% in the presence of either the ETA receptor selective ligand BQ‐123 (1 μm) or the ETB receptor‐selective ligand sarafotoxin S6c (30 nm), indicating the presence of both ETA and ETB receptors in this tissue. 3 ET‐1 and sarafotoxin S6c were both potent spasmogens of rat isolated tracheal smooth muscle preparations. Sarafotoxin S6c‐induced contractions were unaffected in the presence of the ETA receptor antagonist BQ‐123 (10 μm), but were markedly attenuated in tissue previously exposed to 100 nm sarafotoxin S6c to induce ETB receptor desensitization. ET‐1‐induced contractions were, at most, only partially attenuated either by blocking the ETA receptor‐effector system (with 10 μm BQ‐123) or by desensitizing the ETB receptor‐effector system with sarafotoxin S6c. However, ET‐1‐induced contractions were markedly attenuated by blocking both receptor‐effector systems simultaneously. These findings suggest that ET‐1 could induce contraction by stimulating either ETA or ETB receptors. 4 ET‐1 (10 μm) induced a 7 fold increase in intracellular [3H]‐inositol phosphate accumulation over basal levels in rat isolated tracheal smooth muscle. In contrast, sarafotoxin S6c (2.5 μm) increased intracellular [3H]‐inositol phosphate accumulation by only 2 fold. ET‐1‐induced accumulation of [3H]‐inositol phosphates was abolished by 10 μm BQ‐123. 5 In Ca2+‐free Krebs bicarbonate solution, 100 nm ET‐1 induced a significantly larger contraction than that induced by 100 nm sarafotoxin S6c (46.6 ± 5.6% Cmax versus 8.8 ± 2.8% Cmax, n = 5–7). This presumed intracellular Ca2+‐dependent phase of contraction induced by ET‐1 was significantly inhibited by 10 μm BQ‐123 (7.5 ± 1.0% Cmax). Subsequent addition of 2.5 mm Ca2+ induced a second phase of contraction. The extracellular Ca2+‐dependent phase of contraction induced by ET‐1 was similar in magnitude to that induced by sarafotoxin S6c (63.6 ± 4.5% Cmax versus 58.0 ± 3.7% Cmax) and was not inhibited by BQ‐123. Sarafotoxin S6c‐induced contractions were not inhibited by the L‐type Ca2+‐channel antagonists, nicardipine or verapamil. 6 In summary, ETA and ETB receptors coexist in rat isolated tracheal smooth muscle and stimulation of both receptor subtypes contributes to ET‐1‐induced contraction in this tissue. However, stimulation of these receptor subtypes appears to induce contraction by activating different second messenger pathways; ETA receptor stimulation induces phosphoinositide turnover and subsequent release of intracellular Ca2+ whereas stimulation of ETB receptors facilitates the influx of extracellular Ca2+.

Role of PGE2 in protease-activated receptor-1, -2 and -4 mediated relaxation in the mouse isolated trachea

The potential mediator role of the prostanoid PGE2 in airway smooth muscle relaxations induced by peptidic and proteolytic activators of PAR‐1, PAR‐2, PAR‐3 and PAR‐4 was investigated in carbachol‐precontracted mouse isolated tracheal segments. The tethered ligand domain sequences of murine PAR‐1 (SFFLRN‐NH2), PAR‐2 (SLIGRL‐NH2) and PAR‐4 (GYPGKF‐NH2), but not PAR‐3 (SFNGGP‐NH2), induced smooth muscle relaxation that was abolished by the non‐selective cyclo‐oxygenase (COX) inhibitor, indomethacin. The relative order for mean peak relaxation was SLIGRL‐NH2>GYPGKF‐NH2 ∼amp; SFFLRN‐NH2>SFNGGP‐NH2. SFFLRN‐NH2, SLIGRL‐NH2 and GYPGKF‐NH2, but not SFNGGP‐NH2, induced significant PGE2 release that was abolished by indomethacin. Like that for relaxation, the relative order for mean PGE2 release was SLIGRL‐NH2>GYPGKF‐NH2>SFFLRN‐NH2>SFNGGP‐NH2. In dose‐response studies, SLIGRL‐NH2 induced concentration‐dependent increases in PGE2 release (EC50=20.4 μM) and smooth muscle relaxation (EC50=15.8 μM). The selective COX‐2 inhibitor, nimesulide, but not the COX‐1 inhibitor valeryl salicylate, significantly attenuated SLIGRL‐NH2‐induced smooth muscle relaxation and PGE2 release. Exogenously applied PGE2 induced potent smooth muscle relaxation (EC50=60.3 nM) that was inhibited by the mixed DP/EP1/EP2 prostanoid receptor antagonist, AH6809. SLIGRL‐NH2‐induced relaxation was also significantly inhibited by AH6809. In summary, the results of this study strongly suggest that PAR‐mediated relaxation in murine tracheal smooth muscle is dependent on the generation of the spasmolytic prostanoid, PGE2. PAR‐stimulated PGE2 release appears to be generated preferentially by COX‐2 rather than COX‐1, and induces relaxation via activation of the EP2 receptor.

Modulation of airway smooth muscle tone by protease activated receptor‐1,‐2,‐3 and ‐4 in trachea isolated from influenza A virus‐infected mice

Relaxant and contractile effects of the tethered ligand domain sequences of murine PAR‐1, PAR‐2, PAR‐3 and PAR‐4, and of the proteases thrombin and trypsin were examined in mouse isolated tracheal preparations. The epithelium‐ and cyclo‐oxygenase‐dependence of these effects and the potential modulatory effects of respiratory tract viral infection were also investigated. In carbachol‐contracted preparations, trypsin, thrombin, and the tethered ligand domain sequences of murine PAR‐1 (SFFLRN‐NH2), PAR‐2 (SLIGRL‐NH2) and PAR‐4 (GYPGKF‐NH2), but not PAR‐3 (SFNGGP‐NH2), induced transient, relaxant responses that were abolished by the cyclo‐oxygenase inhibitor indomethacin. Repeated administration of SFFLRN‐NH2, SLIGRL‐NH2 or GYPGKF‐NH2 (30 μM) was associated with markedly diminished relaxation responses (homologous desensitization), although there was no evidence of cross‐desensitization between these peptides. The tethered ligand domain sequences for PAR‐1 and PAR‐4 induced a rapid, transient contractile response that preceded the relaxant response. Contractions were not inhibited by indomethacin and were not induced by either thrombin or trypsin. Influenza A virus infection did not significantly affect the responses induced by either the proteases or peptides. Furthermore, epithelial disruption caused by mechanical rubbing had no significant effect on responses to these PAR activators in preparations from either virus‐ or sham‐infected mice. In summary, the proteases trypsin and thrombin, and peptide activators of PAR‐1, PAR‐2 and PAR‐4 induced relaxant responses of mouse isolated tracheal smooth muscle preparations, which were mediated by a prostanoid, probably PGE2. Interestingly, PAR‐mediated relaxations were not significantly diminished following acute damage to the epithelium caused by mechanical rubbing and/or the respiratory tract viral pathogen, influenza A.

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.

Stimulation of protease‐activated receptor‐2 inhibits airway eosinophilia, hyperresponsiveness and bronchoconstriction in a murine model of allergic inflammation

1 An emerging body of evidence indicates that PGE2 has a privileged anti‐inflammatory role within the airways. Stimulants of protease‐activated receptor‐2 (PAR2) inhibit airway smooth muscle tone in vitro and in vivo predominantly via cyclooxygenase (COX)‐dependent generation of prostaglandin E2 (PGE2). Thus, the current study tested the hypothesis that PAR2‐induced generation of PGE2 inhibits the development of allergic airways inflammation and hyperresponsiveness. 2 Bronchoalveolar lavage (BAL) fluid recovered from ovalbumin (OVA)‐sensitised and ‐challenged (allergic) mice contained elevated numbers of eosinophils, which peaked at 48 h postchallenge. Intranasal (i.n.) administration of a PAR2‐activating peptide (PAR2‐AP) SLIGRL (25 mg kg−1, at the time of OVA challenge) caused a 70% reduction in the numbers of BAL eosinophils (compared to the scrambled peptide LSIGRL, 25 mg kg−1). 3 Pretreatment of allergic mice with either indomethacin (1 mg kg−1, dual COX inhibitor) or nimesulide (3 mg kg−1, COX‐2‐selective inhibitor) blocked SLIGRL‐induced reductions in BAL eosinophils. 4 I.n. SLIGRL, but not LSIGRL, inhibited the development of antigen‐induced airways hyperresponsiveness. The inhibitory effect of SLIGRL was blocked by indomethacin. 5 Exposure of isolated tracheal preparations from allergic mice to 100 μM SLIGRL was associated with a 5.0‐fold increase in PGE2 levels (P<0.05, compared to 100 μM LSIGRL). SLIGRL induced similar increases in PGE2 levels in control mice (OVA‐sensitised, saline‐challenged). 6 I.n. administration of PGE2 (0.15 mg kg−1) to allergic mice significantly inhibited eosinophilia and airways hyperresponsiveness to methacholine. 7 In anaesthetised, ventilated allergic mice, SLIGRL (5 mg kg−1, i.v.) inhibited methacholine‐induced increases in airways resistance. Consistent with this bronchodilator effect, SLIGRL induced pronounced relaxation responses in isolated tracheal preparations obtained from allergic mice. LSIGRL did not inhibit bronchomotor tone in either of these in vivo or in vitro experiments. 8 In summary, a PAR2‐AP SLIGRL inhibited the development of airway eosinophilia and hyperresponsiveness in allergic mice through a COX‐dependent pathway involving COX‐2‐mediated generation of the anti‐inflammatory mediator PGE2. SLIGRL also displayed bronchodilator activity in allergic mice. These studies support the concept that PAR2 exerts predominantly bronchoprotective actions within allergic murine airways.

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.

Distribution of β1- and β2-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 β1‐ and β2‐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 β‐adrenoceptor antagonists (−)‐propranolol (non‐selective), CGP 20712A (β1‐selective) and ICI 118,551 (β2‐selective). Analysis of the competition binding curves for the two selective antagonists revealed mixed populations of β1‐ and β2‐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 β‐adrenoceptor antagonists CGP 20712A and ICI 118,551. Analysis of these data demonstrated that in alveolar wall, β1‐ and β2‐adrenoceptors co‐existed in the proportions 18% and 82%, respectively. 5 Quantitative autoradiographic analyses also showed that β1‐ and β2‐adrenoceptors were differentially distributed in tracheal epithelium and airway smooth muscle. The β2‐adrenoceptor subtype accounted for 71% of all β‐adrenoceptors in epithelium. Conversely, β1‐adrenoceptors which mediate relaxant responses of mouse trachea to β‐adrenoceptor agonists (Henry & Goldie, 1990), accounted for 69% of all β‐adrenoceptors in the airway smooth muscle.

The distribution and density of receptor subtypes for endothelin-1 in peripheral lung of the rat, guinea-pig and pig

1 Quantitative autoradiographic studies were conducted to determine the distributions and densities of endothelin‐A (ETA) and ETB receptor subtypes in peripheral lung alveolar wall tissue of the rat, guinea‐pig and pig, with a view to assessing the potential suitability of these tissues as models for investigations of ET receptor function in human alveolar tissue. 2 High levels of specific [125I]‐ET‐1 binding were detected in peripheral lung components from all three species tested. In mature porcine alveolar wall tissue, specific binding increased in a time‐dependent manner to a plateau, consistent with the previously described pseudo‐irreversible binding of this ligand to a finite population of specific binding sites. 3 [125I]‐ET‐1 was associated specifically with both ETA and ETB binding site subtypes in alveolar wall tissue of foetal pig lung as early as 36 days gestation, raising the possibility of a functional role for ET‐1 in lung development. In addition, both ETA and ETB binding site subtypes were detected in alevolar wall tissue and in peripheral airway smooth muscle of mature lung parenchyma from all three species. However, the binding subtype proportions differed in these tissues. For example, in porcine peripheral bronchial smooth muscle, ETA sites apparently predominated, whereas ETB sites constituted the major subtype detected in alveolar wall in this species. These data suggest significant shifts in ET receptor subtype expression at different levels in the respiratory tract. 4 ET binding site subtype proportions in the alveolar wall also differed markedly between species. In rat lung alveoli, ETA and ETB sites were detected in similar proportions (52±3% and 43±5% respectively). In contrast, in guinea‐pig peripheral lung, ETB binding sites clearly predominated, constituting approximately 80% of total specific binding, with ETA sites accounting for only 12%. Porcine alveolar wall tissue also contained a mixture of these ET receptor subtypes, with ETA and ETB binding comprising 23±3% and 65±1% respectively of the total population of specific binding sites detected. These latter proportions are similar to values previously obtained in human peripheral lung tissue, suggesting that porcine lung might be a useful model of the human peripheral lung in subsequent studies of the functions of these pulmonary ET receptor subtypes.