A peptide corresponding to a proteinase-activated receptor 2 (PAR₂)-activating peptide with an N-terminal furoyl group modification, 2-furoyl-LIGRLO-NH₂, was assessed for PAR₂-dependent and -independent biological activities. 2-Furoyl-LIGRLO-NH₂ was equally effective to and 10 to 25 times more potent than SLIGRLNH₂ for increasing intracellular calcium in cultured human and rat PAR₂-expressing cells, respectively. In bioassays of tissue PAR₂ activity, measured as arterial vasodilation and hyperpolarization, 2-furoyl-LIGRLO-NH₂ was 10 to 300 times more potent than SLIGRL-NH₂. Unlike trans-cinnamoyl-LIGRLO-NH₂, 2-furoyl-LI-GRLO-NH₂ did not cause a prominent non-PAR₂-mediated contraction of murine femoral arteries. In conclusion, 2-furoyl-LI-GRLO-NH₂ represents the most potent and selective activator of PAR₂ in biological systems described to date.

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BACKGROUND AND PURPOSE In non-obese diabetic animals, protease-activated receptor-2 (PAR2) agonists are more effective vasodilators, which is attributed to increased COX-2 and endothelial NOS (eNOS) activities. Under conditions of diabetes and obesity, the effectiveness of PAR2 agonists is unknown. We compared the vasodilator responses of small calibre mesenteric arteries from obese diabetic B6.BKS(D)-Lepr^db/J (db/db) induced by PAR2-activating agonists 2-furoyl-LIGRLO-amide (2fly) and trypsin to those obtained in controls [C57BL/6J (C57)], and assessed the contributions of COX, NOS and calcium-activated potassium channels (K_Ca) to these responses. EXPERIMENTAL APPROACH Arteries mounted in wire myographs under isometric tension conditions were contracted submaximally by U46619 then exposed to vasodilators. mRNA and protein expression of PAR2, eNOS and soluble GC (sGC) were determined by real-time PCR and Western blots. KEY RESULTS ACh- and nitroprusside-induced relaxations were attenuated in db/db compared with C57. In contrast, 2fly- and trypsin-induced relaxations were largely retained in db/db. A NOS inhibitor partly inhibited ACh- and 2fly-induced relaxations in C57, but not those in db/db. Inhibitors of the COX-cAMP pathway (FR122044, SC560, NS398, SC58125, SQ22536, CAY10441) did not affect these relaxation responses in either strain. Charybdotoxin (BK_Ca, SK3.1 blocker), but not iberiotoxin (BK_Ca blocker), inhibited responses to the PAR2 agonists in db/db. In db/db protein levels of eNOS were higher, whereas those of sGC were lower than in C57. PAR2 mRNA expression in db/db was higher than in C57. CONCLUSIONS AND IMPLICATIONS PAR2-mediated vasodilatation is protected against the negative effects of obesity and diabetes in mice. In diabetic vascular dysfunction, preserved PAR2 vasodilatation was linked to activation of SK3.1.

We studied the actions of the proteinase-activated receptor-2-activating peptide (PAR2-AP)trans-cinnamoyl-LIGRLO-amide (tc-LI) in femoral (FA), renal, and small mesenteric (MA) arterial vessels from C57BL/6 [PAR2 (+/+)] and PAR2 (−/−) mice. The actions of tc-LI were compared with those of the parent PAR2-AP Ser-Leu-Ile-Gly-Arg-Leu-amide (SLIGRL-amide; SLI-NH₂). Either SLI-NH₂ or tc-LI (0.1–10 μM) induced relaxation of either 9,11-dideoxy-9α,11α-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U46619)- or cirazoline-precontracted FA from PAR2 (+/+) in endothelium-intact preparations but did not relax vessels from PAR2 (−/−) mice. This FA relaxation by SLI-NH₂ and by tc-LI was inhibited by 1) pretreatment with a combination ofl-N^G-nitroarginine methyl ester (l-NAME) and 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), 2) precontraction with 30 mM KCl, or 3) removal of the endothelium. In contrast, tc-LI caused anl-NAME/ODQ/indomethacin-resistant relaxation of MA from PAR2 (+/+) mice. In contrast with SLI-NH₂, tc-LI (>30 μM) contracted arteries from both PAR2 (−/−) and PAR2 (+/+) mice. Pretreatment of tissues with a combination of cyclopiazonic acid plus caffeine reduced significantly tc-LI-induced contractions, whereas nifedipine, CdCl₂, and Ca²⁺-free conditions did not. Inhibitors of vascular muscarinic, α₁-adrenergic, neurokinin, thromboxane A₂, histamine, angiotensin II, or endothelin-1 receptors failed to inhibit contractions by 50 μM tc-LI. At resting tension, SLI-NH₂ (>10 μM) contracted all arteries in an endothelium-independent manner but only from PAR2 (+/+) mice. We conclude that the endothelium-dependent vasodilation initiated by SLI-NH₂ and tc-LI, but not the endothelium-independent contraction initiated by tc-LI, are due to the activation of PAR2. Indeed, the data from PAR2 (−/−) mice indicate that tc-LI, in addition to activating PAR2, is an agonist of vascular smooth muscle contraction via a receptor different than PAR2.