Stimulation of cannabinoid receptor agonist 2-arachidonylglycerol by chronic ethanol and its modulation by specific neuromodulators in cerebellar granule neurons

In an earlier study, we reported that chronic ethanol (EtOH) stimulates the formation of anandamide in human SK-N-SH cells. In the present study, we investigated the effect of chronic EtOH on the formation of yet another cannabinoid receptor (CB1) agonist, 2-arachidonylglycerol (2-AG), in cerebellar granule neurons (CGNs). The formation of-[H-3]AG without any stimulation was more pronounced in the older cultures than in younger cultures. Exposure of CGNs to EtOH led to a significant increase in the level of 2-[H-3]AG (P < 0.05). Incubation with the anandamidehydrolase inhibitor phenylmethylsulfonyl fluoride and EtOH did result in an additive increase in 2-[H-3]AG, but did not with E-6-(bromomethylene)tetrahydro-3-(1-naphthelenyl)-2H-pyran-2-one. The formation of 2-[H-3]AG was enhanced by ionomycin in both the control and EtOH-exposed CGNs, and the ionomycin-stimulated 2-[H-3]AG synthesis was inhibited by the intracellular chelating agent 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Further, glutamate increased the formation of 2-[H-3]AG only in control CGNs. MK-801 inhibited the EtOH-induced 2-[H-3]AG synthesis, suggesting the participation of intracellular Ca2+ in EtOH-induted 2-[H-3]AG synthesis. The dopamine receptor (D2) agonist did not modify the 2-AG synthesis in either the control or EtOH-exposed CGNs. However, the D2 receptor antagonist inhibited the EtOH-induced formation of 2-[(HT)-H-3]AG. The EtOH-induced 2-[H-3]AG formation was inhibited by SR141716A and pertussis toxin, suggesting the CB 1 receptor- and Gi/o-protein-mediated regulation of 2-AG. The observed increase in 2-AG level in CGNs is possibly a mechanism for neuronal adaptation to the continuous presence of EtOH. These findings indicate that some of the pharmacological actions of EtOH may involve alterations in the endocannabinoid signaling system. (C) 2000 Elsevier Science B.V. All eights reserved.