Evidence of a novel site mediating anandamide-induced negative inotropic and coronary vasodilatator responses in rat isolated hearts

Abstract

1. Cannabinoids are known to cause coronary vasodilatation and reduce left ventricular developed pressure (LVDP) in isolated hearts although the identity of the receptor(s) mediating these responses is unknown. Our objective was to pharmacologically characterize cannabinoid receptors mediating cardiac responses to the endocannabinoid, anandamide. 2. Dose-response curves for coronary perfusion pressure (CPP) and LVDP were constructed to anandamide, R-(+)-methanandamide, palmitoylethanolamide (PEA) and JWH015 in isolated Langendorff-perfused rat hearts. Anandamide dose-response curves were also constructed in the presence of antagonists selective for CB(1), CB(2) or VR(1) receptors. 3. Anandamide and methanadamide significantly reduced CPP and LVDP but the selective CB(2) receptor agonists, PEA and JWH015 had no significant effect, compared with equivalent vehicle doses. 4. Single bolus additions of the selective CB(1)-receptor agonist, ACEA (5 nmol), decreased LVDP and CPP. When combined with JWH015 (5 nmol) these responses were not augmented. 5. Anandamide-mediated reductions in CPP were significantly blocked by the selective CB(1) receptor antagonists SR 141716A (1 microM) and AM251 (1 microM) and the selective CB(2) receptor antagonist SR 144528 (1 microM) but not by another selective CB(2) receptor antagonist AM630 (10 microM) nor the vanilloid VR(1) receptor antagonist capsazepine (10 microM). 6. SR 141716A, AM281 and SR 144528 significantly blocked negative inotropic responses to anandamide that were not significantly affected by AM251, AM630 and capsazepine. 7. One or more novel sites mediate negative inotropic and coronary vasodilatatory responses to anandamide. These sites can be distinguished from classical CB(1) and CB(2) receptors, as responses are sensitive to both SR 141716A and SR 144528.

Figure 1

Dose-response curves for the effects of anandamide (n=6), R-(+)-methanandamide (n=6), palmitoylethanloamine (PEA, n=6) and JWH015 (n=6) on (A) left ventricular developed pressure (LVDP) and (B) coronary perfusion pressure (CPP). The effect of equivalent amounts of vehicle to that given with the various agonists are also shown (n=7). * Indicates significant differences (P<0.05) between agonist dose-response curves and the effects of equivalent vehicle doses (ANOVA with repeated measures followed by Bonferroni's post hoc test). †Indicates significant differences (P<0.05) between agonist dose response curves and those of anandamide (ANOVA with repeated measures followed by Bonferroni's post hoc test).

Figure 2

Dose-response curves for the effect of anandamide (n=6) on left ventricular developed pressure (LVDP) in the presence of (A) 1 μM AM281 (n=6), 1 μM AM251 (n=6) and 1 μM SR 141716A (n=6) or (B) 1 μM SR 144528 (n=5) and 10 μM AM630 (n=4) or (C) 10 μM capsazepine (n=5). The effect of equivalent amounts of vehicle to that given with various doses of anandamide are also shown (n=7). *Indicates significant difference (P<0.05) between dose response curve and the effects of equivalent vehicle doses (ANOVA with repeated measures followed by Bonferroni's post hoc test). †Indicates significant differences (P<0.05) between agonist dose response curves and those of anandamide (ANOVA with repeated measures followed by Bonferroni's post hoc test).

Figure 3

Dose-response curves for the effect of anandamide (n=6) on coronary perfusion pressure (CPP) in the presence of (A) 1 μM AM281 (n=6), 1 μM AM251 (n=6) and 1 μM SR 141716A (n=6) or (B) 1 μM SR 144528 (n=5) and 10 μM AM630 (n=4) or (C) 10 μM capsazepine (n=5). The effect of equivalent amounts of vehicle to that given with various doses of anandamide are also shown (n=7). *Indicates significant difference (P<0.05) between dose response curve and the effects of equivalent vehicle doses (ANOVA with repeated measures followed by Bonferroni's post hoc test). †Indicates significant differences (P<0.05) between agonist dose response curves and those of anandamide (ANOVA with repeated measures followed by Bonferroni's post hoc test).

Figure 4

Effect of single doses of 5 nmol ACEA (n=6) or co-administration of 5 nmol ACEA with 5 nmol JWH015 (n=6) on (A) left ventricular developed pressure (LVDP) and (B) coronary perfusion pressure (CPP). *Indicates significant difference (P<0.05) between ethanol vehicle and ACEA or ACEA+JWH015 addition (ANOVA followed by Bonferroni's post hoc test).