IP(3)R-mediated Ca(2+) release is modulated by anandamide in isolated cardiac nuclei

Cannabinoids (CBs) are known to alter coronary vascular tone and cardiac performance. They also exhibit cardioprotective properties, particularly in their ability to limit the damage produced by ischaemia reperfusion injury. The mechanisms underlying these effects are unknown. Here we investigate the intracellular localisation of CB receptors in the heart and examine whether they may modulate localised nuclear Ca²⁺ release. In isolated cardiac nuclear preparations, expression of both the inositol 1,4,5-trisphosphate receptor type 2 (IP₃R) and CB receptors (CB₁R and CB₂R) was demonstrated by immunoblotting. Both receptors localised to the nucleus and purity of the nuclear preparations was confirmed by co-expression of the nuclear marker protein nucleolin but absence of cytoplasmic actin. To measure effects of IP₃R and CBR agonists on nuclear Ca²⁺ release, isolated nuclei were loaded with Fluo5N-AM. This dye accumulates in the nuclear envelope. Isolated nuclei responded to IP₃ with rapid and transient Ca²⁺ release from the nuclear envelope. Anandamide inhibited this IP₃-mediated release. Preincubation of nuclear preparations with either the CB₁R antagonist (AM251) or the CB₂R antagonist (AM630) reversed anandamide-mediated inhibition to 80% and 60% of control values respectively. When nuclei were pre-treated with both CBR antagonists, anandamide-mediated inhibition of IP₃-induced Ca²⁺ release was completely reversed. These results are the first to demonstrate the existence of cardiac nuclear CB receptors. They are also the first to show that anandamide can negatively modulate IP₃-mediated nuclear Ca²⁺ release. As such, this provides evidence for a novel key mechanism underlying the action of CBs and CBRs in the heart.