Received for publication October 10, 2003.
Revised February 10, 2004.
Accepted for publication February 19, 2004.

Potent and Metabolically Stable Agonists for Protease-Activated Receptor-2: Evaluation of Activity in Multiple Assay Systems In Vitro and In Vivo

Abstract

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- NH₂ and 2f-LIGRL-NH₂, 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 Ca²⁺ 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- NH₂, the most powerful peptide, relative to SLIGKV-OH, was about 100 in the cultured cell Ca²⁺ 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 demonstrates 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-NH₂, the most potent peptide. Further, the data from PAR-2- deficient mice provides ultimate evidence for involvement of PAR-2 in salivation and the selective nature of the 2- furoylated peptides.

Key words: PAR-2, calcium signal, furoylation, receptor-activating peptide, salivation, vasorelaxation

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