To develop potent and metabolically stable agonists for protease-activated receptor-2 (PAR-2), we prepared 2-furoylated (2f) derivatives of native PAR-2-activating peptides, 2f-LIGKV-OH, 2f-LIGRL-OH, 2f-LIGKV-NH 2 , and 2f-LIGRL-NH 2 , and systematically evaluated their activity in PAR-2-responsive cell lines and tissues. In both HCT-15 cells and NCTC2544 cells overexpressing PAR-2, all furoylated peptides increased cytosolic Ca 2ϩ levels with a greater potency than the corresponding native peptides, although a similar maximum response was recorded. The absolute potency of each peptide was greater in NCTC2544, possibly due to a higher level of receptor expression. Furthermore, the difference in potency between the 2-furoylated peptides and the native peptides was enhanced when evaluated in the rat superior mesenteric artery and further increased when measuring PAR-2-mediated salivation in ddY mice in vivo. The potency of 2f-LIGRL-NH 2 , the most powerful peptide, relative to SLIGKV-OH, was about 100 in the cultured cell Ca 2ϩ signaling assays, 517 in the vasorelaxation assay, and 1100 in the salivation assay. Amastatin, an aminopeptidase inhibitor, augmented salivation caused by native peptides, but not furoylated peptides. The PAR-2-activating peptides, including the furoylated derivatives, also produced salivation in the wild-type C57BL/6 mice, but not the PAR-2-deficient mice. Our data thus demonstrate that substitution of the N-terminal serine with a furoyl group in native PAR-2-activating peptides dramatically enhances the agonistic activity and decreases degradation by aminopeptidase, leading to development of 2f-LIGRL-NH 2 , the most potent peptide. Furthermore, the data from PAR-2-deficient mice provide ultimate evidence for involvement of PAR-2 in salivation and the selective nature of the 2-furoylated peptides.

No abstract

No abstract

No abstract

1 Proteinase-activated receptor-2 (PAR 2 ), expressed in capsaicin-sensitive sensory neurons, plays a protective role in gastric mucosa. The present study evaluated gastric mucosal cytoprotective effect of 2-furoyl-LIGRL-NH 2 , a novel highly potent PAR 2 agonist, in ddY mice and in wild-type and PAR 2 -knockout mice of C57BL/6 background. 2 Gastric mucosal injury was created by oral administration of HCl/ethanol solution in the mice. The native PAR 2 -activating peptide SLIGRL-NH 2 , administered intraperitoneally (i.p.) at 0.3-1 mmol kg À1 in combination with amastatin, an aminopeptidase inhibitor, but not alone, revealed gastric mucosal protection in ddY mice, which was abolished by ablation of capsaicin-sensitive sensory neurons. 3 I.p. administration of 2-furoyl-LIGRL-NH 2 at 0.1 mmol kg À1 , without combined treatment with amastatin, exhibited gastric mucosal cytoprotective activity in ddY mice, the potency being much greater than SLIGRL-NH 2 in combination with amastatin. This effect was also inhibited by capsaicin pretreatment. 4 Oral administration of 2-furoyl-LIGRL-NH 2 at 0.003-0.03 mmol kg À1 also protected against gastric mucosal lesion in a capsaicin-reversible manner in ddY mice. 5 I.p. 2-furoyl-LIGRL-NH 2 at 0.1-0.3 mmol kg À1 caused prompt salivation in anesthetized mice, whereas its oral administration at 0.003-1 mmol kg À1 was incapable of eliciting salivation. 6 In wild-type, but not PAR 2 -knockout, mice of C57BL/6 background, i.p. administration of 2-furoyl-LIGRL-NH 2 caused gastric mucosal protection. 7 Thus, 2-furoyl-LIGRL-NH 2 is considered a potent and orally available gastric mucosal protective agent. Our data also substantiate a role for PAR 2 in gastric mucosal protection and the selective nature of 2-furoyl-LIGRL-NH 2 .