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This study examined the effects of the selective Rho-kinase inhibitor Y-27632 [(+)-(R)-trans-4-(1-aminoethyl)-(4-pyridyl)cyclohexanecarboxamide dihydrochloride]) on cholinergic nerve-mediated contraction and neurotransmitter release in murine and guinea-pig isolated tracheal preparations. In tracheal preparations obtained from both species, Y-27632 shifted carbachol concentration-effect curves to the right and reduced the maximal contractile response. Repeated electrical field stimulation (EFS) evoked transient, consistent and reproducible contractions in murine and guinea-pig tracheal preparations. Y-27632 inhibited these cholinergic nerve-mediated contractions in a concentration-dependent manner. EFS (0.1-30 Hz) elicited frequency-dependent cholinergic nerve-mediated contractile responses. In murine tracheal preparations, Y-27632 (3 microM and 10 microM) shifted frequency-response curves to EFS to the right by 5.5 and 13.0 fold respectively and markedly reduced the maximal contractile response. In murine and guinea-pig tracheal preparations loaded with [(3)H]-choline, Y-27632 (10 microM) significantly increased the EFS-induced outflow of radioactivity from airway cholinergic nerves by 27% and 54% respectively. Thus, Y-27632 inhibited both carbachol-induced and cholinergic nerve-mediated contractile responses. Conversely, Y-27632 increased neurotransmitter release from airway cholinergic nerves. However, since antagonism of acetylcholine-induced contraction by Y-27632 overwhelmed the increased neurotransmitter release, the overall effect of this Rho-kinase inhibitor was to inhibit cholinergic nerve-mediated contraction.

Stimulants of protease-activated receptor-2 (PAR(2)), such as Ser-Leu-Ile-Gly-Arg-Leu-NH(2) (SLIGRL), cause airway smooth muscle relaxation via the release of the bronchodilatory prostanoid prostaglandin E(2) (PGE(2)). The principal aim of the current study was to determine whether compounds that inhibit PGE(2) reuptake by the prostaglandin transporter [bromocresol green and U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy PGF2alpha) and PGE(2) metabolism by 15-hydroxyprostaglandin dehydrogenase (thiazolidenedione compounds rosiglitazone and ciglitazone) significantly enhanced the capacity of SLIGRL to elevate PGE(2) levels and produce relaxation in isolated segments of upper and lower mouse trachea. SLIGRL produced concentration-dependent increases in PGE(2) levels and smooth muscle relaxation, although both effects were significantly greater in lower tracheal segments than in upper tracheal segments. SLIGRL-induced increases in PGE(2) levels were significantly enhanced in the presence of ciglitazone and rosiglitazone, and these effects were not inhibited by GW9662 (2-chloro-5-nitrobenzanilide), a peroxisome proliferator-activated receptor-gamma antagonist. SLI-GRL-induced relaxation responses were also significantly enhanced by ciglitazone and rosiglitazone, whereas responses to isoprenaline, a PGE(2)-independent smooth muscle relaxant, were unaltered. Ciglitazone and rosiglitazone alone produced concentration-dependent increases in PGE(2) levels and smooth muscle relaxation, and these responses were inhibited by indomethacin, a cyclooxygenase inhibitor. Bromocresol green, an inhibitor of prostaglandin transport, significantly enhanced SLIGRL-induced increases in PGE(2) levels and relaxation. Immunohistochemical staining for 15-hydroxyprostaglandin dehydrogenase was relatively intense over airway smooth muscle, as was staining for the prostaglandin transporter over both airway smooth muscle and epithelium. In summary, inhibitors of PGE(2) reuptake and metabolism significantly potentiate PAR(2)-mediated increases in PGE(2) levels and smooth muscle relaxation in murine-isolated airways.

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.

The role of endothelin B (ETB) receptors in mediating ET ligand-induced contractions in mouse trachea was examined in ETB receptor knockout animals. Autoradiographic binding studies, using [125I]-ET-1, confirmed the presence of ETA receptors in tracheal and bronchial airway smooth muscle from wild-type (+/+) and homozygous recessive (−/−) ETB receptor knockout mice. In contrast, ETB receptors were not detected in airway tissues from (−/−) mice. In tracheae from (+/+) mice, the rank order of potencies of the ET ligands was sarafotoxin (Stx) S6c>ET-1>ET-3; Stx S6c had a lower efficacy than ET-1 or ET-3. In tissues from (−/−) mice there was no response to Stx S6c (up to 0.1 μM), whereas the maximum responses and potencies of ET-1 and ET-3 were similar to those in (+/+) tracheae. ET-3 concentration-response curve was biphasic in (+/+) tissues (via ETA and ETB receptor activation), and monophasic in (−/−) preparations (via stimulation of only ETA receptors). In (+/+) preparations SB 234551 (1 nM), an ETA receptor-selective antagonist, inhibited the secondary phase, but not the first phase, of the ET-3 concentration-response curve, whereas A192621 (100 nM), an ETB receptor-selective antagonist, had the opposite effect. In (−/−) tissues SB 234551 (1 nM), but not A192621 (100 nM), produced a rightward shift in ET-3 concentration-response curves. The results confirm the significant influence of both ETA and ETB receptors in mediating ET-1-induced contractions in mouse trachea. Furthermore, the data do not support the hypothesis of atypical ETB receptors. In this preparation ET-3 is not an ETB receptor-selective ligand, producing contractions via activation of both ETA and ETB receptors. British Journal of Pharmacology (2001) 132, 1905–1915; doi:10.1038/sj.bjp.0703957

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 have examined some of the binding characteristics and the autoradiographic distribution of binding sites for Na 125 I (INa) in airway tissue from the guinea-pig, monkey, pig, rat, mouse and from man. Basal INa (100 pM) binding levels were extremely low. However, in the presence of ascorbic acid (10 μM) or dithiothreitol (10 μM), INa binding was markedly increased in guinea-pig trachea, with lesser increases detected in monkey and rat trachea and in monkey and human bronchus. In guinea-pig trachea, ascorbic acid-induced INa binding was not saturable within the concentration range 100–620 pM and could not be reduced by washout. Autoradiography revealed that in central airways, INa binding was localized at or near the interface of the airway epithelium and submucosa in small clusters, apparently involving one or two cells per focus. The physiological significance of these binding sites is yet to be established, although they may be involved in intracellular iodine storage.

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. 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 microM), 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 microM) 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 MicroM; 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 MicroM), 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 MicroM). 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 MicroM), 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 MicroM), 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.

Administration of alpha-bungarotoxin and other curare-like drugs during embryogenesis arrests motoneuron death which normally occurs in the spinal cord from day 6 to day 10 of embryogenesis. The accepted explanation is that such motoneuron rescue is mediated by inhibition of neuromuscular transmission following the blockade of nicotinic cholinoceptors at the neuromuscular junction. In this study we investigated a further possibility, namely that motoneuron rescue might also involve the blockade of alpha-bungarotoxin-sensitive sites within the spinal cord. The kinetic profile of [125I]alpha-bungarotoxin binding was examined in the brachial and lumbar regions of chick spinal cord at embryonic day 15. Binding was specific and apparently saturable within the range 1-34 nM reaching a maximum after 45 min. Specific binding involved a single class of non-interacting sites with a KD of 8.0 nM and a Bmax of 106 +/- 12 fmol/mg of protein. Nicotine displaced specific [125I]alpha-bungarotoxin binding in a concentration-dependent manner. Furthermore, specific binding dissociated slowly in the absence of nicotine. Autoradiographs localizing [125I]alpha-bungarotoxin binding in embryonic spinal cord revealed that, at embryonic day 15, specific toxin binding sites could be detected throughout the gray matter. In contrast, at embryonic day 6, the ventral horn contained the majority of specific binding sites. Exogenously administered [125I]alpha-bungarotoxin reached and bound to nicotine-sensitive sites in the spinal cord at embryonic day 7. To conclude, these data demonstrate that central nicotine-sensitive sites which bind [125I]alpha-bungarotoxin in a saturable and specific manner were present at the beginning of the critical motoneuron death phase of neurogenesis and that they were accessible to exogenously administered toxin. It is proposed that the [125I]alpha-bungarotoxin binding characterized here is to a class of putative alpha-bungarotoxin-sensitive nicotinic cholinoceptors. These studies raise the possibility that alpha-bungarotoxin blockade of such putative nicotinic cholinoceptors within the spinal cord may contribute to toxin-induced arrest of naturally occurring motoneuron death.

The present study further characterizes ascorbic acid-dependent 125 I − ion binding in guinea-pig trachea. Binding of 125 I − ion in the presence of ascorbic acid was detected at the epithelial/submucosal interface, apparently involving individual cells containing peroxidase enzyme. A similar binding pattern was observed when hydrogen peroxide was substituted for ascorbic acid. Binding could be inhibited by the addition of the H 2 O 2 degrading enzyme catalase or the peroxidase inhibitor thiourea. Results demonstrated that this binding was dependent upon the addition of, or endogenous production of hydrogen peroxide and its metabolism via airway eosinophil peroxidase. The relevance of this anomalous iodine binding phenomenon to radioiodinated ligand binding studies is discussed.

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.