Activation of protease-activated receptors (PARs)-1 and -2 promotes alpha-smooth muscle actin expression and release of cytokines from human lung fibroblasts.

Previous studies have shown that protease-activated receptors (PARs) play an important role in various physiological processes. In the present investigation, we determined the expression of PARs on human lung fibroblasts (HLF-1) and whether they were involved in cellular differentiation and pro-inflammatory cytokine and prostaglandin (PGE2) secretion. PAR-1, PAR-2, PAR-3, and PAR-4 were detected in fibroblasts using RT-PCR, immunocytochemistry, and flow cytometry. Increased expression of PAR-4, but not other PARs, was observed in fibroblasts stimulated with phorbol myristate acetate. The archetypical activators of PARs, namely, thrombin and trypsin, as well as PAR-1 and PAR-2 agonist peptides, stimulated transient increases in intracellular Ca(2+), and promoted increased α-smooth muscle actin expression. The proteolytic and peptidic PAR activators also stimulated the release of IL-6 and IL-8, as well as PGE2, with a rank order of potency of PAR-1 > PAR-2. The combined stimulation of PAR-1 and PAR-2 resulted in an additive release of both IL-6 and IL-8. In contrast, PAR-3 and PAR-4 agonist peptides, as well as all the PAR control peptides examined, were inactive. These results suggest an important role for PARs associated with fibroblasts in the modulation of inflammation and remodeling in the airway.

Altered expression and in vivo lung function of protease-activated receptors during influenza A virus infection in mice.

Protease-activated receptors (PARs) are widely distributed in human airways, and recent evidence indicates a role for PARs in the pathophysiology of inflammatory airway disease. To further investigate the role of PARs in airway disease, we determined the expression and function of PARs in a murine model of respiratory tract viral infection. PAR-1, PAR-2, PAR-3, and PAR-4 mRNA and protein were expressed in murine airways, and confocal microscopy revealed colocalization of PAR-2 and cyclooxygenase (COX)-2 immunostaining in basal tracheal epithelial cells. Elevated levels of PAR immunostaining, which was particularly striking for PAR-1 and PAR-2, were observed in the airways of influenza A/PR-8/34 virus-infected mice compared with sham-infected mice. Furthermore, increased PAR-1 and PAR-2 expression was associated with significant changes in in vivo lung function responses. PAR-1 agonist peptide potentiated methacholine-induced increases in airway resistance in anesthetized sham-infected mice (and in indomethacin-treated, virus-infected mice), but no such potentiation was observed in virus-infected mice. PAR-2 agonist peptide transiently inhibited methacholine-induced bronchoconstriction in sham-infected mice, and this effect was prolonged in virus-infected mice. These findings suggest that during viral infection, the upregulation of PARs in the airways is coupled to increased activation of COX and enhanced generation of bronchodilatory prostanoids.

Diethylenetriamine nitric oxide adduct relaxes precontracted mouse tracheal smooth muscle.

1. Inhaled diethylenetriamine nitric oxide adduct (DETA/NO) has been shown to be a selective pulmonary vasodilator in animal and human studies. The aims of the present study were to investigate the effect of DETA/NO on mouse precontracted isolated tracheal smooth muscle preparations and to determine the active component of this compound. 2. Mouse isolated tracheal smooth muscle rings were precontracted with carbachol (10-7 mol/L). Changes in isometric tension were recorded after cumulative addition of DETA (30-300 micromol/L; n = 6), DETA/NO (30-300 micromol/L; n = 9) or diluent control (n = 3). In addition, some preparations (n = 5) were pretreated with the soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]-oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ; 30 micromol/L) before precontraction and exposure to DETA/NO. 3. Addition of DETA/NO caused a concentration-dependent relaxation of tracheal smooth muscle at 100 and 300 micromol/L, with an EC25%R of 109 micromol/L (95% confidence interval 72.6-164 micromol/L). The nucleophile amine carrier DETA had no effect on isometric tension. However, the relaxant effect of DETA/NO was completely abolished by pretreatment with ODQ. 4. We conclude that DETA/NO induces a concentration-dependent relaxation of mouse carbachol-contracted isolated tracheal smooth muscle that is mediated by NO released from DETA/NO via the activation of sGC.

Role of protease-activated receptors in airway function: a target for therapeutic intervention?

Protease-activated receptors (PARs) are G-protein-coupled, seven transmembrane domain receptors that act as cellular enzyme sensors. These receptors are activated by the proteolytic cleavage at the amino terminus, enabling interaction between the newly formed "tethered ligand" and the second extracellular loop of the receptor to confer cellular signalling. PARs can also be activated by small peptides that mimic the tethered ligand. In the respiratory tract, PARs may be regulated by endogenous proteases, such as airway trypsin and mast cell tryptase, as well as exogenous proteases, including inhaled aeroallergens such as those from house dust mite faecal pellets. Immunoreactive PARs have been identified in multiple cell types of the respiratory tract, and PAR activation has been reported to stimulate cellular mitogenesis and to promote tissue inflammation. Activation of PARs concurrently stimulates the release of bronchorelaxant and anti-inflammatory mediators, which may serve to induce cytoprotection and to minimise tissue trauma associated with severe chronic airways inflammation. Furthermore, airway inflammatory responses are associated with increased epithelial PAR expression and elevated concentrations of PAR-activating, and PAR-inactivating, proteases in the extracellular space. On this basis, PARs are likely to play a regulatory role in airway homeostasis, and may participate in respiratory inflammatory disorders, such as asthma and chronic obstructive pulmonary disease. Further studies focussing on the effects of newly developed PAR agonists and antagonists in appropriate models of airway inflammation will permit better insight into the role of PARs in respiratory pathophysiology and their potential as therapeutic targets.