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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-9␣,11␣-methanoepoxy PGF2␣) 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 concentrationdependent increases in PGE 2 levels and smooth muscle relaxation, although both effects were significantly greater in lower tracheal segments than in upper tracheal segments. SLIGRLinduced 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-␥ 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.

The principal aim of the study was to determine the influence of influenza A virus infection on capsaicin-induced relaxation responses in mouse isolated tracheal segments and clarify the underlying mechanisms. Anesthetized mice were intranasally inoculated with influenza A/PR-8/34 virus (VIRUS) or vehicle (SHAM), and 4 days later tracheal segments were harvested for isometric tension recording and biochemical and histologic analyses. Capsaicin induced dose-dependent relaxation responses in carbachol-contracted SHAM trachea (e.g., 10 M capsaicin produced 66 Ϯ 4% relaxation; n ϭ 11), which were significantly inhibited by capsazepine prostanoid (EP) 2 and EP 4 receptor antagonists, respectively], indicating that capsaicin-induced relaxation involved the TRPV1-mediated release of substance P (SP), activation of epithelial NK 1 receptors, and production of COX products capable of activating relaxant EP 2 /EP 4 receptors. Consistent with this postulate, capsaicin-induced relaxation was associated with the significant release of SP and prostaglandin E 2 (PGE 2 ) from mouse tracheal segments. As expected, influenza A virus infection was associated with widespread disruption of the tracheal epithelium. Tracheal segments from VIRUS mice responded weakly to capsaicin (7 Ϯ 3% relaxation) and were 25-fold less responsive to SP than tracheas from SHAM mice. In contrast, relaxation responses to exogenous PGE 2 and the ␤-adrenoceptor agonist isoprenaline were not inhibited in VIRUS trachea. Virus infection was associated with impaired capsaicin-induced release of PGE 2 , but the release of SP was not affected. In summary, influenza A virus infection profoundly inhibits capsaicin-and SP-induced relaxation responses, most likely by inhibiting the production of PGE 2 .

Proteinase-activated receptor 2 (PAR 2 ) is widely expressed in the respiratory tract and is an integral component of the host antimicrobial defense system. The principal aim of this study was to investigate the influence of a PAR 2 -activating peptide, SLIGRL, on influenza A virus (IAV)-induced pathogenesis in mice. Intranasal inoculation of BALB/c mice with influenza A/PR/8/34 virus caused time-dependent increases in the number of pulmonary leukocytes (recovered from bronchoalveolar lavage fluid), marked airway histopathology characterized by extensive epithelial cell damage, airway hyper-responsiveness to the bronchoconstrictor methacholine, and elevated levels of inflammatory chemokines (keratinocyte-derived chemokine and macrophage inflammatory protein 2) and cytokines (interferon-␥). It is noteworthy that these IAV-induced effects were dose-dependently attenuated in mice treated with a PAR 2 -activating peptide, SLIGRL, at the time of IAV inoculation. However, SLIGRL also inhibited IAV-induced increases in pulmonary leukocytes in PAR 2 -deficient mice, indicating these antiviral actions were not mediated by PAR 2 . The potency order obtained for a series of structural analogs of SLIGRL for anti-IAV activity (IGRL Ͼ SLIGRL Ͼ LSIGRL Ͼ2-furoyl-LIGRL) was also inconsistent with a PAR 2 -mediated effect. In further mechanistic studies, SLIGRL inhibited IAV-induced propagation in ex vivo perfused segments of trachea from wild-type or PAR 2 (Ϫ/Ϫ) mice, but did not inhibit viral attachment or replication in MadinDarby canine kidney cells and chorioallantoic membrane cells, which are established hosts for IAV. In summary, SLIGRL protected mice from IAV infection independently of PAR 2 and independently of direct inhibition of IAV attachment or replication, potentially through the activation of endogenous antiviral pathways within the mouse respiratory tract.

Protease-activated receptors (PARs) are widely expressed throughout the respiratory tract, and PAR 2 has been investigated as a potential drug target for inflammatory airway diseases. The primary focus of this study was to determine the extent to which PAR 2 -activating peptides modulate lipopolysaccharide (LPS)-induced airway neutrophilia in mice and establish the underlying mechanisms. Intranasal administration of LPS induced dose-and time-dependent increases in the number of neutrophils recovered from bronchoalveolar lavage (BAL) fluid of mice. Coadministration of the PAR 2 -activating peptide f-LIGRL inhibited LPS-induced neutrophilia at 3 and 6 h after inoculation. PAR 2 -mediated inhibition of LPS-induced neutrophilia was mimicked by prostaglandin E 2 (PGE 2 ) and butaprost[selective E-prostanoid (EP 2 ) receptor agonist], and blocked by parecoxib (cyclooxygenase 2 inhibitor) and 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH6809) (EP 1 /EP 2 receptor antagonist). PAR 2 -activating peptides also blunted early increases in the levels of the key neutrophil chemoattractants keratinocyte-derived chemokine and macrophage inflammatory protein 2 (MIP-2) in the BAL of LPS-exposed mice. However, neither PAR 2 -activating peptides nor PGE 2 inhibited LPSinduced generation of MIP-2 in cultures of primary murine alveolar macrophages In summary, PAR 2 -activating peptides and PGE 2 suppressed LPS-induced neutrophilia in murine airways, independently of an inhibitory action on MIP-2 generation by alveolar macrophages.

Airway sensory C-fibres express TRPA1 channels which have recently been identified as a key chemosensory receptor for acrolein, a toxic and highly prevalent component of smoke. TRPA1 likely plays an intermediary role in eliciting a range of effects induced by acrolein including cough and neurogenic inflammation. Currently, it is not known whether acrolein-induced activation of TRPA1 produces other airway effects including relaxation of mouse airway smooth muscle. The aims of this study were to examine the effects of acrolein on airway smooth muscle tone in mouse isolated trachea, and to characterise the cellular and molecular mechanisms underpinning the effects of acrolein. Isometric tension recording studies were conducted on mouse isolated tracheal segments to characterise acrolein-induced relaxation responses. Release of the relaxant PGE₂ was measured by EIA to examine its role in the response. Use of selective antagonists/inhibitors permitted pharmacological characterisation of the molecular and cellular mechanisms underlying this relaxation response. Acrolein induced dose-dependent relaxation responses in mouse isolated tracheal segments. Importantly, these relaxation responses were significantly inhibited by the TRPA1 antagonists AP-18 and HC-030031, an NK₁ receptor antagonist RP-67580, and the EP₂ receptor antagonist PF-04418948, whilst completely abolished by the non-selective COX inhibitor indomethacin. Acrolein also caused rapid PGE₂ release which was suppressed by HC-030031. In summary, acrolein induced a novel bronchodilator response in mouse airways. Pharmacologic studies indicate that acrolein-induced relaxation likely involves interplay between TRPA1-expressing airway sensory C-fibres, NK₁ receptor-expressing epithelial cells, and EP₂-receptor expressing airway smooth muscle cells.

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 Ͻ 0.05, n ϭ 6 mice per group). Treatment of allergic mice with an endothelin receptor antagonist, SB-217242 (30 mg ⅐ kg Ϫ1 ⅐ day Ϫ1 ), 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. eosinophils; allergic inflammation THERE IS A GROWING BODY of evidence from human and animal studies supporting a mediator role for endothelin-1 (ET-1) in several characteristic features of the allergic airway response, including airway inflammation and variable airflow obstruction. In allergic asthmatic patients, increased ET-1 immunoreactivity has been demonstrated in the airway epithelium of bronchial biopsies and elevated levels of ET-1 have been detected in bronchoalveolar lavage fluid. Consistent with these findings, several recent studies in rats have demonstrated that allergic inflammation induced by ovalbumin or Sephadex is associated with an early increase in expression of lung prepro-ET-1 mRNA, increased ET-1 immunoreactivity in bronchial epithelium, and elevated ET-1 peptide levels in lung tissue and bronchoalveolar lavage fluid (11,31,32).Animal studies using ET-1 antibodies and endothelin receptor antagonists point to endogenous ET-1 contributing to several processes involved in the allergic inflammatory response. For example, the endothelin receptor antagonist BQ-123 reduced the late, but not the early, antigen-induced reduction in airflow response in allergic sheep (26) and guinea pigs (38). The involvement of ET-1 in the late-phase allergic response, which is associated with the migration of inflammatory cells into the airways, is in accord with the recent findings from several independent groups that endothelin receptor antagonists inhibit the antigeninduced influx of eosinophils into the airways and bronchoalveolar lavage fluid (12,15,31).In addition to its effects on eosinophil recruitment into the airways, ET-1 has many other purported actions within the airways that may promote inflammation and airflow obstruction. For example, ET-1 stimulates the release of a range of proinflammator...

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