Proteinase-activated receptor-1 (PAR1) and PAR2 mediate relaxation of guinea pig internal anal sphincter

Activation of proteinase-activated receptor-1 (PAR₁) and PAR₂ stimulates contraction of the rat but relaxation of the guinea pig colon. The aim of the present study was to investigate PAR effects on internal anal sphincter (IAS) motility. We measured relaxation of isolated muscle strips from the guinea pig IAS caused by PAR agonists using isometric transducers. Reverse transcription polymerase chain reaction (RT-PCR) was performed to determine the existence of PAR. In the IAS, thrombin and PAR₁ peptide agonists TFLLR-NH₂ and SFLLRN-NH₂ evoked moderate to marked relaxation in a concentration-dependent manner. In addition, trypsin and PAR₂ peptide agonists 2-furoyl-LIGRLO-NH₂, SLIGRL-NH₂ and SLIGKV-NH₂ produced relaxation. In contrast, both PAR₁ and PAR₂ inactive control peptides did not elicit relaxation. Furthermore, the selective PAR₁ antagonist vorapaxar and PAR₂ antagonist GB 83 specifically inhibited thrombin and trypsin-induced relaxations, respectively. RT-PCR revealed the presence of PAR₁ and PAR₂ in the IAS. This indicates that PAR₁ and PAR₂ mediate the IAS relaxation. The relaxant responses of TFLLR-NH₂ and trypsin were attenuated by N(omega)-Nitro-L-arginine (L-NNA), indicating involvement of NO. These responses were not affected by tetrodotoxin, implying that the PAR effects are not neurally mediated. On the other hand, PAR₄ agonists GYPGKF-NH₂, GYPGQV-NH₂ and AYPGKF-NH₂ did not cause relaxation or contraction, suggesting that PAR₄ is not involved in the sphincter motility. Taken together, these results demonstrate that both PAR₁ and PAR₂ mediate relaxation of the guinea pig IAS through the NO pathway. PAR₁ and PAR₂ may regulate IAS tone and might be potential therapeutic targets for anal motility disorders.