No abstract

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.