Angela C. D'Aprile

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.

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.

Influence of regional differences in ETA and ETB receptor subtype proportions on endothelin-1-induced contractions in porcine isolated trachea and bronchus

1 Quantitative autoradiographic studies were conducted to determine the distributions and densities of ETA and ETB binding site subtypes in porcine tracheal and bronchial smooth muscle. In addition, the roles of ETA and ETB receptors in endothelin‐1‐mediated contraction of these tissues were assessed. 2 Quantitative autoradiographic studies revealed that both ETA and ETB binding sites for [125I]‐ endothelin‐1 were present in both bronchial and tracheal airway smooth muscle. However, the proportions of these sites were markedly different at these two levels within the respiratory tract. In tracheal smooth muscle, the proportions of ETA and ETB sites were 30± 1% and 70±1% respectively, whereas in bronchial smooth muscle, these proportions were virtually reversed, being 73±2% and 32±8% respectively. 3 Endothelin‐1 induced concentration‐dependent contraction of porcine tracheal and bronchial airway smooth muscle. Endothelin‐1 had similar potency (concentration producing 30% of the maximum carbachol contraction, Cmax) in trachea (22 nM; 95% confidence limits (c.l.), 9–55 nM; n = 9) and bronchus (22 nM; c.l., 9–55 nM; n = 6). Endothelin‐1 also produced comparable maximal contractions in trachea (59±5% Cmax; n = 9) and bronchus (65 ± 4% Cmax, n = 6). 4 In trachea, endothelin‐1 induced contractions were not significantly inhibited by either the ETA receptor‐selective antagonist, BQ‐123 (3 μm) or the ETB receptor‐selective antagonist, BQ‐788 (1 μm). However, in the combined presence of BQ‐123 and BQ‐788, the concentration‐effect curve to endothelin‐ 1 was shifted to the right by 3.7 fold (n = 8; P = 0.01). 5 In bronchus, concentration‐effect curves to endothelin‐1 were shifted to the right by BQ‐123 (3 μm; 4.3 fold; P<0.05), but not by BQ‐788 (1 μm). In the presence of both antagonists, concentration‐effect curves to endothelin‐1 were shifted by at least 6.7 fold (n = 6; P = 0.01). 6 Sarafotoxin S6c induced contraction in both tissue types, although the maximum contraction was greater in trachea (53 ± 7% Cmax; n = 6) than in bronchus (21 ± 5% Cmax; n = 6). BQ‐788 (1 μm) markedly reduced sarafotoxin S6c potency in both trachea and bronchus (e.g. by 50 fold in trachea; c.l., 14–180; n = 6; P < 0.05). 7 These data demonstrate that the proportions of functional endothelin receptor subtypes mediating contraction of airway smooth muscle to endothelin‐1, vary significantly at different levels in the porcine respiratory tract.

Leukaemia inhibitory factor (LIF) upregulates excitatory non-adrenergic non-cholinergic and maintains cholinergic neural function in tracheal explants.

The effect of leukaemia inhibitory factor (LIF) in modulating cholinergic and sensory nerve function was examined using guinea‐pig tracheal explants. Specific LIF receptors (LIFR) were immunolocalized to both cholinergic and sensory nerves. Release of SP in culture was not influenced by LIF. Similarly, maximum contraction to carbachol (Cmax) was not influenced by LIF. After 3 h, maximum (Emax) eNANC‐induced contraction in controls was 32±2.5% of Cmax. In LIF‐treated preparations, Emax was enhanced to 50±4.5% Cmax (P<0.05). Cholinergic nerve‐induced contractions after 3 h incubation with LIF were similar to control. After 24 h, control Emax was 25±4.5% Cmax (58% smaller than Emax at 3 h). In contrast, in LIF‐treated preparations, Emax was 37±2.5% Cmax, (24% smaller than at 3 h, P<0.05). This did not appear to be due to the effect of LIF on muscarinic M2 receptor expression or function. Thus LIF appears to differentially influence the function of airway nerves and thus may provide an important link between the immune and neural systems.

Influence of endothelin-1(1-31) on smooth muscle tone and cholinergic nerve-mediated contraction in rat isolated trachea

Endothelin-1(1-21) (ET-1(1-21)) is a strong candidate as a significant mediator in asthma, in part because of its powerful spasmogenic actions and its ability to enhance cholinergic nerve-mediated contraction in human and animal airway smooth muscle. In the study reported here, we have demonstrated that [125I]ET-1(1-31) binds specifically to BQ-123-sensitive sites (presumably ET(A)-receptors) and to sarafotoxin S6c (S6c)-sensitive sites (presumably ET(B)-receptors) in rat tracheal and pulmonary airways, as well as in lung alveoli. These sites coexist in tracheal airway smooth muscle and in alveolar tissue in approximately equal proportions. ET-1(1-21) and ET-1(1-31) were equipotent and approximately equally active as spasmogens in rat tracheal smooth muscle. Importantly, both peptides were shown to potentiate cholinergic nerve-mediated rat tracheal contraction, although ET-1(1-31) was less active in this regard. These data are consistent with the idea that ET-1(1-31) could play a significant mediator role in obstructive airway diseases such as asthma.

The Impact of Respiratory Syncytial Virus Infection on Endothelin Receptor Function and Release in Sheep Bronchial Explants

We investigated the impact of respiratory syncytial virus (RSV) infection, an important asthma precipitant, on endothelin receptor function and release in sheep bronchial explants. RSV infection was confirmed using polymerase chain reaction and immunohistochemistry. Since sheep airway smooth muscle contains only endothelin-A receptors, sarafotoxin (Stx) S6c did not cause airway contraction. In contrast, sarafotoxin S6c (300 nM) caused contraction in RSV-infected bronchial explants (8 ± 3% carbachol Emax). However, we could not detect airway smooth muscle endothelin-B receptors in explants using autoradiography. RSV infection per se did not alter the release of immunoreactive endothelin from sheep bronchial explants (control = 11.6 ± 0.9 pg versus RSV = 12.1 ± 0.9 pg). Interestingly, dexamethasone (1 μM) alone increased endothelin release in both control (17.9 ± 2.0 pg) and RSV-infected tissue (18.3 ± 3.1 pg). The combined presence of protease-activated receptor-2 (PAR-2) ligand (100 μM) and dexamethasone (1 μM) also increased endothelin release from control tissue (17.3 ± 1.4 pg), but endothelin release was suppressed by PAR-2 ligand in RSV-infected tissue (10.3 ± 0.8 pg), probably because PAR-2 expression was increased by RSV. In summary, the novel expression of endothelin-B receptors triggered by RSV might be relevant to RSV-associated asthma. Furthermore, activation of airway PAR-2 may be protective in asthma where endothelin levels are elevated in part via endothelin release suppression.

Impact of parainfluenza-3 virus infection on endothelin receptor density and function in guinea pig airways

We examined the impact of parainfluenza-3 (P-3) respiratory tract viral infection on the density and function of endothelin (ET) receptor subtypes (ET(A) and ET(B)) in guinea pig tracheal smooth muscle. Total specific binding of [(125)I]ET-1 and the relative proportions of ET(A) and ET(B) binding sites for this ligand were assessed at day 0 (control) and at 2, 4, 8 and 16 days post-inoculation. At day 0, the proportions of ET(A) and ET(B) binding sites were 30% and 70% respectively. Total specific binding was significantly reduced at day 4 post-inoculation (32% reduction, n=8-12, P<0.05) and was largely due to a corresponding fall in ET(B) receptor density at this time point (38% reduction, n=8-12, P<0.05). The density of ET(A) receptors also fell significantly at day 8 post-inoculation (33% reduction, n=6-12, P<0.05). By day 16 post-inoculation, the densities of ET(A) and ET(B) receptors had recovered to control values. The ratio of ET(A):ET(B) receptor subtypes did not alter with P-3 infection. While P-3 infection reduced the density of tracheal smooth muscle ET(A) and ET(B) receptors, the contractile sensitivity and maximum response to carbachol and ET-1 was not altered in tissue from day 4 post-inoculation compared with the control. There seems to be a significant functional reserve for both receptor subtypes in this species that buffers the impact of P-3 infection on airway smooth muscle responsiveness to ET-1.

Enhanced ET(A) receptor-mediated contractile function is associated with reduced ET(A) receptor density in sheep bronchial explants maintained in culture.

We are interested in developing an airway explant culture system using sheep bronchi in which to establish respiratory viral infection and from which tissue can be used for functional, biochemical and immunohistochemical studies involving the endothelins (ETs). Freshly harvested sheep bronchial airway smooth muscle contains a homogeneous population of the ET(A) receptor. However, the potency of ET-1 and maximum contractile response of sheep bronchial explants to ET-1 increased with time in culture, despite these parameters remaining constant for carbachol in explants maintained for up to 48 h. The possibility that this was caused by changes in ET receptor density was assessed using light microscopic quantitative autoradiography. In view of the increased responsiveness to ET-1 in cultured explants, it was surprising to demonstrate a significant decrease in total ET receptor (59+/-6% compared with the initial value, n=4-5; P<0.01) and ET(A) receptor (51+/-2% compared with the initial value, n=4-5, P<0.01) density in sheep bronchial explants after 48 h. No ET(B) receptors were detected. Thus, the culture of sheep bronchial explants was associated with an increase in ET(A) receptor-mediated contractile function that was accompanied by a decrease in ET(A) receptor density. In addition, the structural integrity of the ciliated epithelium was preserved using this culture protocol, a feature that is critical to successful respiratory viral infection. The significant changes in ET receptor density and function in these bronchial explants must be carefully considered when assessing any effects of respiratory viral infection in this model.