Characterisation of the anti-adrenergic response to endocannabinoids in rat isolated right atria

William Ford, Nichola Underdown. Cardiff University, Cardiff, United Kingdom.

Previously we have reported that the endocannabinoid anandamide (AEA) increased chronotropy in rat naïve, spontaneously contracting, isolated right atria in an indomethacin-sensitive manner (Underdown et al, 2003). Given that cannabinoid receptors couple to Gi-proteins (see review by Pertwee, 2006), and Gi-protein activation is known to oppose responses involving Gs-protein activation (Morris et al., 1999), we investigated cannabinoid responses in rat isolated right atria where an increase in chronotropic tone had been stimulated by isoprenaline (ISO).

Right atria were removed from male Wistar rats (250-350g) which had been killed by an overdose of sodium pentobarbital followed by cervical dislocation, and mounted in 20ml organ baths containing Krebs bicarbonate solution warmed to 37oC and gassed with 5% CO2 in oxygen. A resting tension of 0.5 g was applied to the tissue, which was allowed to beat spontaneously and isometric tension recorded. ISO (0.01μM) was added to the tissue and the resultant increase in chronotropy allowed to stabilise then cumulative concentration response curves (CRC) to Tocrisolve 100 (agonist vehicle, n=4)), AEA (n=7), methanandamide (MeAEA, n=5), the CB1-receptor selective agonist, ACPA (arachidonoylcyclopropylamide, n=3, see Pertwee, 2006) or the CB2-receptor selective agonist, JWH 133 (n=4, see Pertwee, 2006)) were constructed (one CRC was constructed per tissue). CRCs to AEA were also constructed in the presence of DMSO (antagonist vehicle, n=4), the selective CB1-receptor antagonists, rimonabant (1 μM, n= 5), AM281 (1 μM, n=5, see Pertwee, 2006) or the selective CB2-receptor antagonist SR144528 (1 μM, n=5, see Pertwee, 2006). Data are reported as mean ± s.e.mean. Responses are reported as those obtained with 10 μM (the highest concentration used) of the agonists tested expressed as a percentage of the increase in chronotropy induced by 0.01μM ISO.

ISO induced positive chronotropy was significantly inhibited by AEA (79±9%), MeAEA (46±12%) and JWH 133 (51±13) in a concentration dependent manner, but not ACPA (21±7%). ISO-induced positive inotropy was still observed in response to anandamide treatment in the presence of DMSO, albeit of a reduced magnitude (38±15%). Negative chronotropy associated with AEA treatment in the presence of ISO was not significantly affected by rimonabant (66±16%) or AM251 (22±8) but was significantly potentiated by SR144528 (90±14%) compared to DMSO-treated controls.

In contrast to its effect on rat naïve isolated right atria, AEA acted as a negative chronotrope in the presence of ISO. Our results suggest that the mechanism of this response is not mediated by products of AEA catabolism or CB1-receptor activation. Although negative chronotropy was observed with the CB2-receptor selective ligand, JWH133, responses to AEA were potentiated by SR144528. Therefore, AEA-induced negative chronotropy in the presence of ISO cannot easily be explained by the generation of active catabolites of AEA breakdown, CB1- or CB2-receptor activation.

Pertwee (2006) Int J Obes, 30: S13-8

Morris et al (1999) Physiol Rev, 79(4): 1373-1430

Underdown et al (2003) PA2, 1(4): 161P