Heterogeneous mechanisms of endothelium-dependent relaxation for thrombin and peptide activators of protease-activated receptor-1 in porcine isolated coronary artery

1 Mechanisms of protease-activated receptor-1 (PARI)- and PAR2-induced relaxation were investigated in pre-contracted porcine coronary artery ring preparations. 2 Thrombin (0.01-0.3 u ml(-1)) and the PARI-activating peptide SFLLRN (0.1-10 mu M) caused concentration- and endothelium-dependent relaxation. pEC(50)s (-log u ml(-1) for enzymes, -log M for peptides) and maximum relaxations (R-max, %) for thrombin were 1.8 +/- 0.1 and 93.5 +/- 2.8% respectively, and for SFLLRN 6.8 +/- 0.1 and 90.8 +/- 1.3%. Similar concentration- and endothelium-dependent relaxations occurred with trypsin (pEC(50) 2.3 +/- 0.2; R-max 94.1 +/- 1.9%) and the PAR2-activating peptide SLIGRL (pEC(50) 6.5 +/- 0.2; R-max 92.4 +/- 1.6%). 3 Relaxations to thrombin, SFLLRN, trypsin and SLIGRL were significantly inhibited (P<0.05) to similar extents by the nitric oxide (NO) synthase inhibitor N-G-nitro-L-arginine (L-NOARG; 100 mu M) and the NO scavenger oxyhaemoglobin (20 mu M), both separately and in combination. 4 In the presence of the L-type voltage-operated calcium channel (L-VOCC) inhibitor nifedipine (0.3 mu M), K+ (67 mM) abolished the L-NOARG-resistant relaxations to thrombin, SFLLRN, trypsin and SLIGRL. However, nifedipine alone significantly (P<0.05) reduced the pEC(50) (1.5 +/- 0.1) and R-max (77.5 +/- 7.0%) for thrombin but had no effect on relaxations to SFLLRN, trypsin or SLIGRL. Furthermore, L-NOARG-resistant relaxations to thrombin were abolished by nifedipine, whereas relaxations to SFLLRN, trypsin or SLIGRL were not further inhibited by combined treatment with nifedipine and L-NOARG, than they were with L-NOARG treatment alone. 5 Similar selective inhibition of the L-NOARG-resistant relaxation to thrombin, but not SFLLRN, occurred with verapamil (1 mu M) and diltiazem (3 mu M). 6 Our results suggest heterogeneous mechanisms in the NO-independent relaxation to thrombin and peptide activators of PAR1 in the porcine coronary artery.