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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.

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Background and purpose: Proteinase-activated receptor 2 (PAR2) is a G-protein coupled receptor associated with many pathophysiological functions. To date, the development of PAR2 antagonists has been limited. Here, we identify a number of novel peptide-mimetic PAR2 antagonists and demonstrate inhibitory effects on PAR2-mediated intracellular signalling pathways and vascular responses. Experimental approach: The peptide-mimetic compound library based on the structures of PAR2 agonist peptides were screened for inhibition of PAR2-induced calcium mobilisation in human keratinocytes. Representative compounds were further evaluated by radioligand binding and inhibition of NFkB transcriptional activity and IL-8 production. The vascular effects of the antagonists were assessed using in vitro and in vivo models.

We investigated proteinase-activated receptor-2 (PAR 2 )-triggered signal transduction pathways causing increased prostaglandin E 2 (PGE 2 ) formation in human lung-derived A549 epithelial cells. The PAR 2 agonist, SLIGRL-NH 2 (Ser-Leu-Ile-Gly-Arg-Leu-amide), evoked immediate cytosolic Ca 2ϩ mobilization and delayed (0.5-3 h) PGE 2 formation. The PAR 2 -triggered PGE 2 formation was attenuated by inhibition of the following signal pathway enzymes: cyclooxygenases 1 and 2 (COX-1 and COX-2, respectively), cytosolic Ca 2ϩ -dependent phospholipase A 2 (cPLA 2 ), the mitogenactivated protein kinases (MAPKs), mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) and p38 MAPK, Src family tyrosine kinase, epidermal growth factor (EGF) receptor tyrosine kinase (EGFRK), and protein kinase C (PKC), but not by inhibition of matrix metalloproteinases. SLIGRL-NH 2 caused prompt (5 min) and transient ERK phosphorylation, blocked in part by inhibitors of PKC and tyrosine kinases but not by an EGFRK inhibitor. SLIGRL-NH 2 also evoked a relatively delayed (15 min) and persistent (30 min) phosphorylation of p38 MAPK, blocked by inhibitors of Src and EGFRK but not by inhibitors of COX-1 or COX-2. SLIGRL-NH 2 elicited a Src inhibitor-blocked prompt (5 min) and transient phosphorylation of the EGFRK. SLIGRL-NH 2 up-regulated COX-2 protein and/or mRNA levels that were blocked by inhibition of p38 MAPK, EGFRK, Src, and COX-2 but not MEK-ERK. SLIGRL-NH 2 also caused COX-1-dependent up-regulation of microsomal PGE synthase-1 (mPGES-1). We conclude that PAR 2 -triggered PGE 2 formation in A549 cells involves a coordinated up-regulation of COX-2 and mPGES-1 involving cPLA 2 , increased cytosolic Ca 2ϩ , PKC, Src, MEK-ERK, p38 MAPK, Src-mediated EGF receptor trans-activation, and also metabolic products of both COX-1 and COX-2.Proteinase-activated receptors (PARs), a family of G protein-coupled seven-trans-membrane domain receptors, consisting of PARs 1 to 4, are now known to mediate a variety of intracellular signaling and subsequent cellular events caused by specific extracellular proteinases (Hollenberg