Multiple mechanisms of vascular smooth muscle relaxation by the activation of proteinase-activated receptor 2 in mouse mesenteric arterioles

1 Activation of PAR2 in second-order mesenteric arteriole (MA) rings from C57BL/6J, NOS3 (-/-) and PAR2 (-/-) mice was assessed for the contributions of NO, cyclo-oxygenases, guanylyl cyclase, adenylyl cyclase, and of K+ channel activation to vascular smooth muscle relaxation. 2 PAR2 agonist, SLIGRL-NH2 (0.1 to 30 muM), induced relaxation of cirazoline-precontracted MA from C57BL/6J and NOS3 (-/-), but not PAR2 (-/-) mice. Maximal relaxation (E-max) was partially reduced by a combination of L-N-G-nitroarginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and indomethacin. An ODQ/L-NAME/indomethacin resistant relaxation was also caused by trypsin (30 nM) in PAR2 (+/+), but not in PAR2 (-/-) mice. Relaxation was endothelium-dependent and inhibited by either 30 mm KCl-precontraction, or pretreatment with apamin, charybdotoxin, and their combination; iberiotoxin did not substitute for charybdotoxin nor did scyllatoxin substitute fully for apamin. 3 Tetraethylammonium (TEA), glibenclamide, tetrodotoxin, 17-octadecynoic acid, carboxy-2-phenyl-4,4,5,5,-tetramethyl-imidazoline-1-oxyl-3-oxide, SQ22536, carbenoxolone, arachidonyl trifluoromethyl ketone, 7-nitroindazole, N-(3-(aminomethyl)benzyl)acetamidine (1400W), N-(2-cyclohexyloxy-4-nitrophenyl)-methanesulfonamide (NS-398) and propanolol did not inhibit relaxation. 4-aminopyridine significantly increased the potency of SLIGRL-NH2. A combination of 30 muM BaCl2, and 10 muM ouabain signiftcantly reduced the potency for relaxation, and in the presence of L-NAME, ODQ and indomethacin, E-max was reduced. 4 We conclude PAR2-mediated relaxation of mouse MA utilizes multiple mechanisms that are both NO-cGMP-dependent, and -independent. The data are also consistent with a role for endothelium-dependent hyperpolarization of vascular smooth muscle that involves the activation of an apamin/charybdotoxin-sensitive K+ channel(s) and, in part, may be mediated by K+.