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Within the airways, endothelin-1 (ET-1) can exert a range of prominent effects, including airway smooth muscle contraction, bronchial obstruction, airway wall edema, and airway remodeling. ET-1 also possesses proinflammatory properties and contributes to the late-phase response in allergic airways. However, there is no direct evidence for the contribution of endogenous ET-1 to airway hyperresponsiveness in allergic airways. Allergic inflammation induced in mice by sensitization and challenge with the house dust mite allergen Der P1 was associated with elevated levels of ET-1 within the lung, increased numbers of eosinophils within bronchoalveolar lavage fluid and tissue sections, and development of airway hyperresponsiveness to methacholine ( P < 0.05, n = 6 mice per group). Treatment of allergic mice with an endothelin receptor antagonist, SB-217242 (30 mg · kg−1· day−1), during allergen challenge markedly inhibited airway eosinophilia (bronchoalveolar lavage fluid and tissue) and development of airway hyperresponsiveness. These findings provide direct evidence for a mediator role for ET-1 in development of airway hyperresponsiveness and airway eosinophilia in Der P1-sensitized mice after antigen challenge.

1. The effects of endothelin-1 (ET-1) and of the muscarinic cholinoceptor agonist, carbachol, on [3H]-inositol phosphate ([3H]-InsP) accumulation and smooth muscle contraction were determined in rat isolated tracheal tissue. 2. ET-1 (1 microM) and carbachol (10 microM) induced significant accumulation of [3H]-InsPs in myo-[2-3H]-inositol-loaded rat tracheal segments. Several components of the tracheal wall including the airway smooth muscle band, the cartilaginous region and the intercartilaginous region generated significant levels of [3H]-InsPs in response to ET-1 and carbachol. Following stimulation with ET-1, a greater proportion of tracheal [3H]-InsPs were generated in the intercartilaginous region (49%) than in either the airway smooth muscle band (25%) or cartilaginous region (26%). However, when the respective weights of these regions is taken into account, ET-1-induced accumulation of [3H]-InsPs was greatest in the airway smooth muscle band. The tracheal epithelium did not appear to generate [3H]-InsPs in response to ET-1 or modulate either basal or ET-1-induced accumulation of [3H]-InsPs in rat tracheal segments. 3. In the rat tracheal smooth muscle band, ET-1 caused a time- and concentration-dependent accumulation of [3H]-InsPs. Concentrations of ET-1 as low as 10 nM produced significant accumulation of [3H]-InsPs (1.23 +/- 0.10 fold increase above basal levels of 295 +/- 2 d.p.m. mg-1 wet wt., n = 3 experiments). At 10 microM, the highest concentration ?tsed, ET-1 produced similar levels of [3H]-InsP accumulation (7.03 +/- 0.55 fold above basal levels, t = 5) to that produced by a maximally effective concentration of carbachol (10 microM; 7.97 +/- 0.31 fold increase above basal levels, n = 4). ET-1-induced accumulation of [3H]-InsPs was not significantly affected by indomethacin (5 microM), nordihydroguaiaretic acid (NDGA, 10 microM), WEB 2086 (10 microM) or phosphoramidon (10 microM).4. ET-1 also produced concentration-dependent contractions of epithelium-denuded rat tracheal ring preparations. The mean concentration of ET-1 producing 50% of the maximum contractile response to carbachol (EC50) was 31 nm (95% confidence limits, 20-49 nM, n = 12). The presence of an intact tracheal epithelium, indomethacin (5 microM), WEB 2086 (10 microM) and phosphoramidon (10 microM) had no significant effect on the mean EC50 for ET-1-induced contraction (n = 5). In contrast, NDGA (10 microM) inhibited ET-1- induced contractions (4.0 fold increase in mean EC50, P < 0.001, n = 5). However, this effect of NDGA did not appear to be related to inhibition of leukotriene synthesis via lipoxygenase since the leukotriene antagonist SKF 104353 did not affect ET-1-induced contractions (n = 5) and moreover, leukotriene C4 and leukotriene D4 did not contract rat isolated tracheal smooth muscle preparations (n = 4).5. The threshold concentrations of ET-1 that produced increases in smooth muscle contraction and [3H]-InsPs accumulation were similar, although the EC50 for [3H]-InsP accumulation was 2.9 fold greater than that for smooth muscle contraction. For carbachol, the EC50 for [3H]-InsP accumulation (mean ECQO = 5.0 microM, 1.2-21 microM, n = 4) was 25 fold greater than that for smooth muscle contraction(mean EC50 = 0.20 miicroM, 0.17-0.24 microM, n = 12).6. It seems likely that ET-1 has a direct effect on InsP generation in rat tracheal smooth muscle and that this is largely responsible for the spasmogenic actions of this peptide.

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 microM) 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 microM) and dexamethasone (1 microM) 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.

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 alveolar 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.

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 microM ET-1 (measured as the % maximum response to 10 microM carbachol, % Cmax +/- s.e.mean) and the mean concentration of ET-1 producing 30% Cmax (95% confidence limits) were respectively; 85.9 +/- 5.4% and 3.4 nM (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 (0.1-2 nM) 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) greater than human bronchus (42.7 +/- 17.5 amol mm-2) greater than mouse trachea (28.7 +/- 2.6 amol mm-2) greater than guinea-pig trachea (8.3 +/- 1.8 amol mm-2). 5. A positive relationship between [125I]-ET-1 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.

The effects of N-acetylcysteine (NAC), 2-mercaptopropionylglycine (MPG) and dithiothreitol (DTT) on the metabolism and toxicity of acetaminophen (APAP) were examined in isolated mouse hepatocytes maintained in primary culture on collagen-coated dishes. Both NAC and MPG increased the formation of the glutathione and sulfate conjugates of APAP and decreased the covalent binding of the APAP reactive metabolite to cellular protein. DTT did not increase APAP metabolism but did decrease covalent binding. NAC, MPG and DTT decreased plasma membrane damage, as measured by leakage of lactate dehydrogenase from hepatocytes, during a 4-h incubation in 5.0 mM APAP. NAC, MPG and DTT also reduced the APAP-induced fall in glutathione levels in these cells. In other experiments, hepatocytes were exposed to 5.0 mM APAP for 1 h and then incubated during a post-exposure period in APAP-free medium. Damage increased during this post-exposure incubation. Addition of DTT, but not NAC or MPG, after APAP exposure protected the hepatocytes from plasma membrane damage during the post-exposure period. These results indicate that NAC and MPG exert their protective effects by their action on the reactive metabolite of APAP. As well as its effect in reducing the formation of the reactive metabolite, DTT has a potent protective effect against the toxic processes initiated by the APAP reactive metabolite.