Endothelium-dependent relaxations mediated by inducible B1 and constitutive B2 kinin receptors in the bovine isolated coronary artery

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Abstract
1 Rings of bovine left anterior descending coronary artery (LAD) were contracted with the thromboxane A2-mimetic, U46619 (1–30 nM), to approximately 40% of their maximum contraction to 125 mM KCl Krebs solution (KPSSmax) for comparison of responses to the B1 and B2 kinin receptor agonists, des-Arg9-bradykinin (des-Arg9-BK) and bradykinin (BK), respectively. Relaxation responses were normalized as percentages of the initial U46619-induced contraction level, while contractile responses were expressed as percentages of KPSSmax. 2 After 6 h of in vitro incubation in Krebs solution at 37°C, des-Arg9-BK (pEC50, 8.00 ±0.08; maximum response (Rmax), 93.9 ±1.9%) and BK (pEC50, 9.75 ±0.07; Rmax, 100.1 ±0.7%) caused endothelium-dependent relaxations in precontracted rings of bovine LAD which were competitively and selectively antagonized by the B. receptor antagonist, des-Arg9-[Leu8]-BK (pA2, 6.27 ±0.11) and the B2 receptor antagonist Hoe-140 (pA2, 9.63 +0.14), respectively. 3 At 3 h of in vitro incubation, the sensitivity (pEC50, 7.45 ±0.10) and Rmax (84.6 ±3.3%) to des-Arg9-BK were significantly less than those obtained in the same tissues at 6 h (pEC50, 7.94 ± 0.06; Rmax, 91.4±2.5%), whereas endothelium-dependent relaxations to BK and ACh were unaffected by incubation time. 4 Relaxation responses to des-Arg9-BK, but not BK, at both 3 h and 6 h were significantly attenuated by the protein synthesis inhibitors, cycloheximide (30 and 100 μm) and actinomycin D (2 μm). 5 At 6 h, the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine (L-NOARG, 100 μm), caused a significant 2 fold decrease in pEC50 (9.58 ±0.03) but had no effect on Rmax for BK. For des-Arg9-BK, L-NOARG (100 μm) caused a marked and significant decrease in both the pEC50 and Rmax and revealed contractions to low concentrations of des-Arg9-BK. In both cases, L-NOARG inhibition was reversed in the presence of L-arginine (10 mM). 6 At 6 h, removal of the endothelium abolished relaxation responses to des-Arg9-BK and BK, and for des-Arg9-BK, but not BK, unmasked concentration-dependent contractions (pEC50, 7.57 ±0.09; Rmax, 83.4±9.1%). The sensitivity of contractions to des-Arg9-BK increased slightly from 3h (pEC50, 7.37 ±0.08) to 6 h (pEC50, 7.62 ±0.12) of in vitro incubation; however, there was a small but significant depression in the maximum response over this time (Rmax, 126.8 ±8.5% and 103.3 ±8.6% for 3 h and 6 h of incubation respectively). 7 In conclusion, the bovine LAD contains inducible B1 and constitutive B2 endothelial cell kinin receptors, both of which mediate endothelium-dependent relaxation partly via the release of NO. B1 receptors were also present on the smooth muscle layer of the bovine LAD.