Kinase experiment:

Tissues are routinely contracted with 100 mM potassium chloride (KCl) to test their viability. Then, after re-equilibration for 20 min in fresh buffer, tissues are contracted with 1 μM of phenylephrine and a test concentration of 1 μM ACh is added and the presence or absence of a relaxant response is monitored to verify the presence or absence of an intact functional endothelium. The contractile response to phenylephrine is expressed as a percentage of the contractile response caused by 100 mM KCl (% KCl). Upon standardizing the preparation with the use of KCl and ACh, the effects of added SLIGRL-NH2, 2-furoyl-LIGRLO-NH2, LRGILS-NH2, LSIGRL-NH2 and 2-furoyl-OLRGIL-NH2 on the tension of the phenylephrine-contracted preparations (1 μM phenylephrine) is monitored for tissues with/without an intact endothelium and with/without adherent PVAT. Relaxation (%) is expressed as a percentage reduction of the plateau tension developed in the presence of phenylephrine. The effects of the inhibitors (L-NAME, ODQ, indomethacin, 4-aminopyridine, combined apamin + charybdotoxin, glibenclamide, genistein, H89 and catalase) are measured by treating the tissues with the inhibitors for 15 min before their contraction with 1 μM phenylephrine, then followed by the addition of test concentrations of SLIGRL-NH2, 2-furoyl-LIGRLO-NH2, LRGILS-NH2, LSIGRL-NH2 and 2-furoyl-OLRGIL-NH2. In most experiments evaluating a role for PAR2, SLIGRL-NH2 is used at a concentration of 20 μM to ensure selectivity for PAR2[2].

Protease-Activated Receptor-2 Activating Peptide is an agonist of Protease-Activated Receptor-2 (PAR-2).

Protease-Activated Receptor-2 Activating Peptide (SLIGRL-NH2) is an agonist of PAR-2 and MrgprC11[1]. Protease-Activated Receptor-2 Activating Peptide (SLIGRL-NH2) causes an L-NAME-inhibited relaxation. Based on SLIGRL-NH2 causing a concentration-dependent relaxation with an EC50 of 10 μM in endothelium-free preparations in the presence of perivascular adipose tissue (PVAT) , 20 μM is used as a suitable ‘test’ concentration of peptide in subsequent experiments designed to evaluate the effects of potential inhibitors of ADRF release/action. In the endothelium-free aorta preparations, SLIGRL-NH2 causes a concentration-dependent relaxation in preparations only in the presence of PVAT [+PVAT, -ENDO (endothelium)][2].

References:
[1]. Akiyama T, et al. Behavioral model of itch, alloknesis, pain and allodynia in the lower hindlimb and correlativeresponses of lumbar dorsal horn neurons in the mouse. Neuroscience. 2014 Apr 25;266:38-46.
[2]. Li Y, et al. Perivascular adipose tissue-derived relaxing factors: release by peptide agonists via proteinase-activated receptor-2 (PAR2) and non-PAR2 mechanisms. Br J Pharmacol. 2011 Dec;164(8):1990-2002.