Role of PGE(2) in protease-activated receptor-1, -2 and -4 mediated relaxation in the mouse isolated trachea

Abstract

1. The potential mediator role of the prostanoid PGE(2) in airway smooth muscle relaxations induced by peptidic and proteolytic activators of PAR-1, PAR-2, PAR-3 and PAR-4 was investigated in carbachol-precontracted mouse isolated tracheal segments. 2. The tethered ligand domain sequences of murine PAR-1 (SFFLRN-NH(2)), PAR-2 (SLIGRL-NH(2)) and PAR-4 (GYPGKF-NH(2)), but not PAR-3 (SFNGGP-NH(2)), induced smooth muscle relaxation that was abolished by the non-selective cyclo-oxygenase (COX) inhibitor, indomethacin. The relative order for mean peak relaxation was SLIGRL-NH(2)>GYPGKF-NH(2) approximately amp; SFFLRN-NH(2)>SFNGGP-NH(2). 3. SFFLRN-NH(2), SLIGRL-NH(2) and GYPGKF-NH(2), but not SFNGGP-NH(2), induced significant PGE(2) release that was abolished by indomethacin. Like that for relaxation, the relative order for mean PGE(2) release was SLIGRL-NH(2)>GYPGKF-NH(2)>SFFLRN-NH(2)>SFNGGP-NH(2). 4. In dose-response studies, SLIGRL-NH(2) induced concentration-dependent increases in PGE(2) release (EC(50)=20.4 microM) and smooth muscle relaxation (EC(50)=15.8 microM). 5. The selective COX-2 inhibitor, nimesulide, but not the COX-1 inhibitor valeryl salicylate, significantly attenuated SLIGRL-NH(2)-induced smooth muscle relaxation and PGE(2) release. 6. Exogenously applied PGE(2) induced potent smooth muscle relaxation (EC(50)=60.3 nM) that was inhibited by the mixed DP/EP(1)/EP(2) prostanoid receptor antagonist, AH6809. SLIGRL-NH(2)-induced relaxation was also significantly inhibited by AH6809. 7. In summary, the results of this study strongly suggest that PAR-mediated relaxation in murine tracheal smooth muscle is dependent on the generation of the spasmolytic prostanoid, PGE(2). PAR-stimulated PGE(2) release appears to be generated preferentially by COX-2 rather than COX-1, and induces relaxation via activation of the EP(2) receptor.

Figure 1

Representative tension-recording traces of PAR-induced relaxation on the mouse isolated trachea precontracted to 60% C_max with carbachol (1 μM). (a) Relaxations induced by a scrambled PAR peptide (in this case, 100 μM LSIGRL-NH₂), and then by the corresponding active PAR peptide (100 μM SLIGRL-NH₂). Supernatants were collected for PGE₂ quantification 10 min after addition of each peptide. (b) Relaxations induced by an enzymatic PAR activator (in this example, 100 U ml⁻¹ trypsin). (c) Unstimulated preparations were used as appropriate time controls to determine basal PGE₂ release.

Figure 2

Relaxant effects of 100 μM PAR-1, -2, -3 and -4 peptides on carbachol-precontracted mouse trachea in the absence or presence of 3 μM indomethacin (indo). Data are presented as mean±s.e.mean (n=4 – 5).

Figure 3

Effects of 100 μM PAR-1, -2, -3 and -4 peptides on PGE₂ release from carbachol-precontracted mouse trachea in the absence or presence of 3 μM indomethacin (indo). Data are presented as mean±s.e.mean (n=4 – 9).

Figure 4

(a) Concentration-response curve of SLIGRL-NH₂-induced smooth muscle relaxation and PGE₂ release from precontracted mouse isolated trachea (n=4 – 6). (b,c) Effects of 1 mM valeryl salicylate (vs), 1 μM nimesulide (nim), or 3 μM indomethacin (indo) on SLIGRL-NH₂-induced smooth muscle relaxation and PGE₂ release (n=5 – 7).

Figure 5

(a) Effect of 3 μM AH6809 on the cumulative concentration-response relationship generated by exogenous PGE₂ in the precontracted mouse isolated trachea (n=5 – 10). (b,c) Effect of 3 μM AH6809 (AH) on the single concentration addition of PGE₂ (30 nM) or SLIGRL-NH₂ (30 μM) in the precontracted mouse isolated trachea (n=6 – 13).