P.G. Knott

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.

Predominance of endothelinA (ETA) receptors in ovine airway smooth muscle and their mediation of ET-1-induced contraction

1 Autoradiographic studies were conducted to investigate the receptor subtypes for endothelin‐1 (ET‐1) that were present in the ovine respiratory tract. In addition, the receptor subtypes mediating contraction of airway smooth muscle and the possible involvement of extracellular Ca2+ and inositol phosphate generation in intracellular signal transduction were assessed. 2 Specific [125I]‐ET‐1 binding in ovine trachea increased in a time‐ and concentration‐dependent manner. Autoradiographic studies demonstrated that significant binding was associated with airway smooth muscle, although higher densities of specific binding were associated with submucosal glands and with cells immediately below the epithelial basement membrane (lamina propria). The ETA receptor‐selective antagonist, BQ 123 (1 μm), virtually abolished specific binding to airway smooth muscle. Quantitative analyses of autoradiographic data describing the time‐dependence of specific [125I]‐ET‐1 binding in ovine airway smooth muscle in the presence and absence of BQ 123 or sarafotoxin S6c, revealed a homogeneous population of ETA receptors. BQ 123 (1 μm) also abolished specific binding to structures associated with submucosal glands, whereas the ETB receptor selective agonist, sarafotoxin S6c (100 nm) had little effect on this binding, indicating the predominance of ETA receptors at these sites. In contrast, ETB receptors predominated in the lamina propria, since sarafotoxin S6c abolished specific binding in this tissue. 3 High levels of specific [125I]‐ET‐1 binding were also detected in the alveoli and in the walls of blood vessels and small airways in ovine peripheral lung. Specific binding associated with alveoli was reduced to similar extents by BQ 123 (1 μm; 54%) and sarafotoxin S6c (100 nm; 40%), suggesting the coexistence of both ETA and ETB receptors in approximately equal proportions in this tissue. In contrast, specific binding to blood vessels and to peripheral bronchial smooth muscle was abolished in the presence of BQ 123 (1 μm), but was unaffected by sarafotoxin S6c, indicating the presence of only ETA receptors at these sites. 4 ET‐1 caused concentration‐dependent contractions of ovine tracheal smooth muscle which were inhibited in the presence of BQ 123 (1 μm). ET‐1 also caused concentration‐dependent contraction of ovine lung parenchyma strips. In contrast, the ETB receptor‐selective agonists, sarafotoxin S6c and BQ 3020, were virtually inactive as spasmogens in both tracheal smooth muscle and lung strip preparations. Thus contraction was mediated by ETA receptors in ovine tracheal smooth muscle and this is consistent with binding and autoradiographic data demonstrating a homogeneous population of these binding sites in this tissue. Contraction of parenchymal lung strip preparations to ET‐1 was mediated via non‐ETB receptors, presumably ETA receptors, with contributions to this response perhaps coming from airway and vascular smooth muscle and from alveolar wall contractile cells. 5 ET‐1‐induced contraction of tracheal smooth muscle was not significantly altered in the presence of indomethacin (5 μm), indicating that cyclo‐oxygenase metabolites of arachidonic acid were not involved in this response. Contraction induced by ET‐1 was virtually abolished in Ca2+‐free medium containing 0.1 mm EGTA, indicating that this response was dependent upon the influx of extracellular Ca2+. Contraction was inhibited by about 50% in the presence of nicardipine (1 μm), indicating that a significant component of this response was mediated via the activation of L‐type Ca2+ channels. 6 ET‐1 caused poorly defined increases in the accumulation of intracellular inositol phosphates in ovine tracheal smooth muscle. The maximal response to ET‐1 was less than 20% of that to the cholinoceptor agonist, carbachol. Furthermore, sarafotoxin S6c was inactive. These data, when taken together with the results of autoradiographic and contraction studies, indicate that ovine airway smooth muscle contraction in response to ET‐1 is mediated via ETA receptors which are linked to the influx of extracellular Ca2+, partly through voltage‐dependent channels. ETB receptors also exist in the lamina propria of ovine trachea and in peripheral alveoli, perhaps residing in vascular endothelial cells.

Influence of parainfluenza-1 respiratory tract viral infection on endothelin receptor-effector systems in mouse and rat tracheal smooth muscle.

1 In this study we have compared the effects of parainfluenza‐1 respiratory tract viral infection on the density and function of ETA and ETB receptors in rat and mouse tracheal airway smooth muscle. 2 The bronchoconstrictor effect of inhaled methacholine was significantly enhanced in virus‐infected rats, at both 4 and 12 days post‐inoculation. That is, the concentration of methacholine causing an increase in resistance of 100% (PC100 methacholine) was significantly lower in virus‐infected animals at both 4 and 12 days post‐inoculation (n = 6–8; P < 0.05). 3 Total specific binding of [125I]‐endothelin‐1 and the relative proportions of ETA and ETB binding sites for [125I]‐endothelin‐1 were assessed in tracheal airway smooth muscle in parainfluenza‐1‐infected rats and mice at days 2, 4 and 12 post‐inoculation using the ligands BQ‐123 (1 μm; ETA receptor‐selective) and sarafotoxin S6c (100 nM; ETB receptor‐selective). Total specific binding in mice was significantly reduced at day 2 post‐inoculation (n = 5; P < 0.05) but not at days 4 and 12 post‐inoculation (n = 5). In control mice, the proportions of ETA and ETB binding sites were 53%:47% at day 2 and 43%:57% at day 4 and these were significantly altered by parainfluenza‐1 infection such that, the ratios were 81%:19% at day 2 and 89%:11% at day 4 (P < 0.05). By day 12 post‐inoculation, the proportion of ETA and ETB binding sites in tracheal smooth muscle from mice infected with parainfluenza‐1 was not significantly different from control. In rat tracheal airway smooth muscle, neither total specific binding nor the ETA and ETB binding site ratio (64%:36%) were significantly altered in virus‐inoculated rats at days 2, 4 or 12 post‐inoculation (n = 5). 4 Parainfluenza‐1 infection in mice had no effect on the sensitivity or maximal contractile effect of endothelin‐1 in tracheal smooth muscle at days 2, 4 or 12 post‐inoculation (n = 4). In contrast, contraction in response to the ETB receptor‐selective agonist sarafotoxin S6c was attenuated by 39% at day 2 and by 93% at day 4 post‐inoculation (P < 0.05). However, by day 12 post‐inoculation, contractions to sarafotoxin S6c were not significantly different between control and virus‐infected mice. In parainfluenza‐1‐infected rats, there were small but significant reductions in the sensitivity to carbachol, endothelin‐1 and sarafotoxin S6c whilst the maximal responses to the highest concentrations of these agonists were not significantly altered by virus infection (n = 8). 5 BQ‐123 (3 μm) had no significant effect on cumulative concentration‐effect curves to endothelin‐1 in tracheal preparations from control mice (n = 4) or parainfluenza‐1‐infected rats (n = 8). In contrast, in tissues taken from virus‐infected mice at day 4 post‐inoculation, BQ‐123 caused a marked 9.6 fold rightward shift in the concentration‐effect curve to endothelin‐1 (n = 4). 6 In summary, we have demonstrated that parainfluenza‐1 infection in mice transiently reduced the density of tracheal airway smooth muscle ETB receptors and this was reflected in reduced responsiveness to the ETB receptor‐selective agonist sarafotoxin S6c. In contrast, whilst parainfluenza‐1 infection in rats was associated with the pathological features and bronchial hyperresponsiveness common to respiratory tract viral infection, there was no selective down‐regulation of ETB receptor expression or functional activity. The reasons for these species differences are not clear, but may relate to differences in the airway inflammatory response to parainfluenza‐1 virus.

Influence of respiratory tract viral infection on endothelin-1-induced modulation of cholinergic nerve-mediated contractions in murine airway smooth muscle.

The effects of endothelin-1 (ET-1) and sarafotoxin S6c (S6c) on cholinergic contractions elicited by electrical field stimulation (EFS) were examined in mouse tracheal preparations from healthy animals and from animals infected with parainfluenza-1 (P-1) virus. S6c (an ETB-selective agonist) and ET-1 caused marked ETA and/or ETB receptor-mediated potentiation of EFS-induced contraction in tracheal tissue from both groups. Despite the fact that such infection is known to markedly alter ET receptor density and function in mouse tracheal smooth muscle, no evidence for modulated neuronal ET receptor function was obtained. The reason for this differential sensitivity of smooth muscle and neuronal ET receptors to P-1 infection is unknown.