Anandamide-induced relaxation of sheep coronary arteries: the role of the vascular endothelium, arachidonic acid metabolites and potassium channels

Abstract

1. The effects of the endocannabinoid, anandamide, and its metabolically stable analogue, methanandamide, on induced tone were examined in sheep coronary artery rings in vitro. 2. In endothelium-intact rings precontracted to the thromboxane A(2) mimetic, U46619, anandamide (0.01 - 30 microM) induced slowly developing concentration-dependent relaxations (pEC(50) [negative log of EC(50)]=6.1+/-0.1; R(max) [maximum response]=81+/-4%). Endothelium denudation caused a 10 fold rightward shift of the anandamide concentration-relaxation curve without modifying R(max). Methanandamide was without effect on U46619-induced tone. 3. The anandamide-induced relaxation was unaffected by the cannabinoid receptor antagonist, SR 141716A (3 microM), the vanilloid receptor antagonist, capsazepine (3 and 10 microM) or the nitric oxide synthase inhibitor, L-NAME (100 microM). 4. The cyclo-oxygenase inhibitor, indomethacin (3 and 10 microM) and the anandamide amidohydrolase inhibitor, PMSF (70 and 200 microM), markedly attenuated the anandamide response. The anandamide transport inhibitor, AM 404 (10 and 30 microM), shifted the anandamide concentration-response curve to the right. 5. Precontraction of endothelium-intact rings with 25 mM KCl attenuated the anandamide-induced relaxations (R(max)=7+/-7%), as did K(+) channel blockade with tetraethylammonium (TEA; 3 microM) or iberiotoxin (100 nM). Blockade of small conductance, Ca(2+)-activated K(+) channels, delayed rectifier K(+) channels, K(ATP) channels or inward rectifier K(+) channels was without effect. 6. These data suggest that the relaxant effects of anandamide in sheep coronary arteries are mediated in part via the endothelium and result from the cellular uptake and conversion of anandamide to a vasodilatory prostanoid. This, in turn, causes vasorelaxation, in part, by opening potassium channels.

Figure 1

Concentration-response curves for anandamide and methanandamide in endothelium-intact sheep coronary arteries precontracted to U46619. (a) Comparison of the effects of anandamide (n=17), methanandamide (n=7) and vehicle (ethanol or Soya oil/water emulsion; n=7) on U46619-induced tone. (b) Effects of 30 min pretreatment with SR 141716A (3 μM) on relaxant responses to anandamide (n=5). (c) Effects of 30 min pretreatment with capsazepine (3 and 10 μM) on relaxant responses to anandamide (n=4 – 10). Data are presented as means and were fitted to curves according the procedure described in Methods. Vertical bars indicate s.e.mean.

Figure 2

Effect of endothelium denudation or pretreatment with L-NAME or indomethacin on anandamide-induced relaxation in sheep coronary arteries precontracted to U46619. (a) Effects of endothelium denudation on relaxant responses to anandamide (n=7). (b) Effects of 30 min pretreatment with L-NAME (100 μM) on relaxant responses to anandamide (n=5). (c) Effects of 30 min pretreatment with indomethacin (3 and 10 μM) on relaxant responses to anandamide (n=4 – 8). Data are presented as means and were fitted to curves according the procedure described in Methods. Vertical bars indicate s.e.mean.

Figure 3

Effect of PMSF and AM 404 on anandamide-induced relaxation in endothelium-intact sheep coronary arteries precontracted to U46619. (a) Effects of 30 min pretreatment with PMSF (70 and 200 μM) on relaxant responses to anandamide (n=5 – 11). (b) Effects of 30 min pretreatment with AM404 (10 and 30 μM) on relaxant responses to anandamide (n=4 – 13). Data are presented as means and were fitted to curves according the procedure described in Methods. Vertical bars indicate s.e.mean.

Figure 4

Effects of 17-ODYA and miconazole on anandamide-induced relaxation in endothelium-intact sheep coronary arteries precontracted to U46619. (a) Effects of 30 min pretreatment with 17-ODYA (5 and 10 μM) on relaxant responses to anandamide (n=4 – 10). (b) Effects of 30 min pretreatment with miconazole (1 and 10 μM) on relaxant responses to anandamide (n=6 – 12). Data are presented as means and were fitted to curves according the procedure described in Methods. Vertical bars indicate s.e.mean.

Figure 5

Effects of high K⁺ (25 mM) and K⁺ channel blockade with TEA and IBTX on anandamide-induced relaxation in endothelium-intact sheep coronary arteries. (a) Comparison of the relaxant responses to anandamide in arterial rings precontracted to 25 mM K⁺ and U46619, respectively (n=8). (b) Effects of 30 min pretreatment with TEA (1 and 3 mM) on relaxant responses to anandamide in arterial rings precontracted to U46619 (n=4 – 10). (c) Effects of 30 min pretreatment with IBTX (100 nM) on relaxant responses to anandamide in arterial rings precontracted to U46619 (n=8). Data are presented as means and were fitted to curves according the procedure described in Methods. Vertical bars indicate s.e.mean.