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Proteinase-activated receptor 2 (PAR2) 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 PAR2-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-γ). It is noteworthy that these IAV-induced effects were dose-dependently attenuated in mice treated with a PAR2-activating peptide, SLIGRL, at the time of IAV inoculation. However, SLIGRL also inhibited IAV-induced increases in pulmonary leukocytes in PAR2-deficient mice, indicating these antiviral actions were not mediated by PAR2. The potency order obtained for a series of structural analogs of SLIGRL for anti-IAV activity (IGRL > SLIGRL > LSIGRL >2-furoyl-LIGRL) was also inconsistent with a PAR2-mediated effect. In further mechanistic studies, SLIGRL inhibited IAV-induced propagation in ex vivo perfused segments of trachea from wild-type or PAR2(−/−) mice, but did not inhibit viral attachment or replication in Madin-Darby canine kidney cells and chorioallantoic membrane cells, which are established hosts for IAV. In summary, SLIGRL protected mice from IAV infection independently of PAR2and independently of direct inhibition of IAV attachment or replication, potentially through the activation of endogenous antiviral pathways within the mouse respiratory tract.

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 μM capsaicin produced 66 ± 4% relaxation; n= 11), which were significantly inhibited by capsazepine [transient receptor potential vanilloid type 1 (TRPV1) antagonist], (2S,3S)-3-{[3,5-bis(trifluoromethyl)phenyl]methoxy}-2-phenylpiperidine hydrochloride (L-733,060) [neurokinin 1 (NK1) receptor antagonist], indomethacin [cyclooxygenase (COX) inhibitor], and the combination of 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH6809) and 7-[5α-([1S,1α(Z)-biphenyl]-4-ylmethoxy)-2β-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoic acid, calcium salt, hydrate (AH23848) [E-prostanoid (EP)2and EP4receptor antagonists, respectively], indicating that capsaicin-induced relaxation involved the TRPV1-mediated release of substance P (SP), activation of epithelial NK1receptors, and production of COX products capable of activating relaxant EP2/EP4receptors. Consistent with this postulate, capsaicin-induced relaxation was associated with the significant release of SP and prostaglandin E2(PGE2) 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 ± 3% relaxation) and were 25-fold less responsive to SP than tracheas from SHAM mice. In contrast, relaxation responses to exogenous PGE2and the β-adrenoceptor agonist isoprenaline were not inhibited in VIRUS trachea. Virus infection was associated with impaired capsaicin-induced release of PGE2, 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 PGE2.

Protease-activated receptors (PARs) are widely expressed throughout the respiratory tract, and PAR2has been investigated as a potential drug target for inflammatory airway diseases. The primary focus of this study was to determine the extent to which PAR2-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 PAR2-activating peptide f-LIGRL inhibited LPS-induced neutrophilia at 3 and 6 h after inoculation. PAR2-mediated inhibition of LPS-induced neutrophilia was mimicked by prostaglandin E2(PGE2) and butaprost [selective E-prostanoid (EP2) receptor agonist], and blocked by parecoxib (cyclooxygenase 2 inhibitor) and 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH6809) (EP1/EP2receptor antagonist). PAR2-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 PAR2-activating peptides nor PGE2inhibited LPS-induced generation of MIP-2 in cultures of primary murine alveolar macrophages In summary, PAR2-activating peptides and PGE2suppressed LPS-induced neutrophilia in murine airways, independently of an inhibitory action on MIP-2 generation by alveolar macrophages.

Stimulants of protease-activated receptor-2 (PAR2), such as Ser-Leu-Ile-Gly-Arg-Leu-NH2(SLIGRL), cause airway smooth muscle relaxation via the release of the bronchodilatory prostanoid prostaglandin E2(PGE2). The principal aim of the current study was to determine whether compounds that inhibit PGE2reuptake by the prostaglandin transporter [bromocresol green and U46619 (9,11-dideoxy-9α,11α-methanoepoxy PGF2α) and PGE2metabolism by 15-hydroxyprostaglandin dehydrogenase (thiazolidenedione compounds rosiglitazone and ciglitazone) significantly enhanced the capacity of SLIGRL to elevate PGE2levels and produce relaxation in isolated segments of upper and lower mouse trachea. SLIGRL produced concentration-dependent increases in PGE2levels and smooth muscle relaxation, although both effects were significantly greater in lower tracheal segments than in upper tracheal segments. SLIGRL-induced increases in PGE2levels 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-γ antagonist. SLI-GRL-induced relaxation responses were also significantly enhanced by ciglitazone and rosiglitazone, whereas responses to isoprenaline, a PGE2-independent smooth muscle relaxant, were unaltered. Ciglitazone and rosiglitazone alone produced concentration-dependent increases in PGE2levels 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 PGE2levels 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 PGE2reuptake and metabolism significantly potentiate PAR2-mediated increases in PGE2levels and smooth muscle relaxation in murine-isolated airways.

Contraction of vascular and nonvascular smooth muscle induced by the endothelin/sarafotoxin family of peptides frequently does not readily fit into the current classification criteria for ETAand ETBreceptors, raising the possibility of additional atypical receptors. In the current study, isometric tension recording and radioligand binding techniques were used to characterize the ETAreceptor population in sheep isolated tracheal smooth muscle. Endothelin-1 and sarafotoxin S6b induced similar concentration-dependent contractions, although endothelin-1 was 2.6-fold more potent (P< .05, n= 15–18). The ETAreceptor-selective antagonists BQ-123 and FR139317 caused concentration-dependent inhibition of the contractions induced by endothelin-1 and sarafotoxin S6b, but both antagonists were significantly less potent in inhibiting contractions induced by endothelin-1 than sarafotoxin S6b. For example, 0.03 μM FR139317 shifted the endothelin-1 and sarafotoxin S6b concentration-effect curves to the right by 1.8- and 8.3-fold, respectively (P< .01, n= 6–8). Although the observed agonist dependence of antagonist potency may indicate the presence of atypical ETAreceptors, competition binding studies using 125I-endothelin-1 and 125-I-sarafotoxin S6b identified only a single population of BQ-123- and sarafotoxin S6b-sensitive ETAreceptors. Additional association-, dissociation-, and saturation-binding studies revealed that 125I-endothelin-1 binding to these ETAreceptors was pseudoirreversible, whereas 125I-sarafotoxin S6b binding was readily reversible. Thus, marked differences in the kinetic profiles of ETAreceptor binding to endothelin-1, sarafotoxin S6b, and BQ-123, rather than the existence of another ETAreceptor subtype, may explain the stark agonist dependence of antagonist potency observed in contractile studies.