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

Abstract

1. Mechanisms of protease-activated receptor-1 (PAR1)- 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 PAR1-activating peptide SFLLRN (0.1-10 μM) caused concentration- and endothelium-dependent relaxation. pEC50s (-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 (pEC50 2.3 ± 0.2; R(max) 94.1 ± 1.9%) and the PAR2-activating peptide SLIGRL (pEC50 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 μM) and the NO scavenger oxyhaemoglobin (20 μM), both separately and in combination. 4. In the presence of the L-type voltage-operated calcium channel (L-VOCC) inhibitor nifedipine (0.3 μM), K+ (67 mM) abolished the L-NOARG-resistant relaxations to thrombin, SFLLRN, trypsin and SLIGRL. However, nifedipine alone significantly (P < 0.05) reduced the pEC50 (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 μM) and diltiazem (3 μM). 6. Our results suggest heterogeneous mechanisms in the NO-independent retaliation to thrombin and peptide activators of PAR1 in the porcine coronary artery.