RESUMO
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.
Assuntos
Amidas/farmacologia , Inibidores Enzimáticos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Músculo Liso Vascular/efeitos dos fármacos , Neurotransmissores/metabolismo , Sistema Nervoso Parassimpático/efeitos dos fármacos , Sistema Nervoso Parassimpático/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Piridinas/farmacologia , Animais , Carbacol/farmacologia , Relação Dose-Resposta a Droga , Cobaias , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos CBA , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/inervação , Parassimpatomiméticos/farmacologia , Traqueia/efeitos dos fármacos , Quinases Associadas a rhoRESUMO
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.
Assuntos
Antiporters/antagonistas & inibidores , Proteínas de Ligação a DNA/antagonistas & inibidores , Dinoprostona/metabolismo , Relaxamento Muscular/efeitos dos fármacos , Receptor PAR-2/fisiologia , Traqueia/fisiologia , Animais , Verde de Bromocresol/farmacologia , Dinoprostona/análise , Feminino , Hidroxiprostaglandina Desidrogenases/análise , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Oligopeptídeos/farmacologia , Transportadores de Ânions Orgânicos , PPAR gama/fisiologia , Rosiglitazona , Tiazolidinedionas/farmacologia , Traqueia/químicaRESUMO
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.
Assuntos
Brônquios/metabolismo , Contração Muscular , Músculo Liso/metabolismo , Receptor de Endotelina B/metabolismo , Infecções por Vírus Respiratório Sincicial/metabolismo , Animais , Brônquios/efeitos dos fármacos , Brônquios/fisiopatologia , Brônquios/virologia , Meios de Cultivo Condicionados/metabolismo , Dexametasona/metabolismo , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Endotelina-1/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Indometacina/farmacologia , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiopatologia , Músculo Liso/virologia , Oligopeptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Piperidinas/farmacologia , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/efeitos dos fármacos , Receptor PAR-2/metabolismo , Infecções por Vírus Respiratório Sincicial/fisiopatologia , Ovinos , Fatores de Tempo , Técnicas de Cultura de Tecidos , Venenos de Víboras/farmacologiaRESUMO
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 x kg(-1) x 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.