Serotonin 5‐HT 2A Receptor‐Coupled 2‐Arachidonoylglycerol (2‐AG) Release

Abstract

Serotonin 5-HT2A receptor (5-HT2AR) activation is believed to mediate the altered states of consciousness produced by hallucinogens, and is probably critical for normal cognitive functioning. Furthermore, the atypical antipsychotics possess antagonist or inverse agonist activity at this receptor. Although many studies have demonstrated that the 5-HT2AR is coupled to the release of arachidonic acid (AA) derivatives, few have confirmed the identity of these derivatives. There are many examples of G-protein-coupled receptor-mediated endocannabinoid release, and because endocannabinoids are AA derivatives, we hypothesized that 5-HT2AR stimulation would lead to endocannabinoid release. NIH3T3 cells expressing the rat 5-HT2AR were first incubated with [3H]-AA for 24 h. Following stimulation with agonists, supernatant was extracted and lipids were separated by thin layer chromatography. We found that the endocannabinoid 2-AG was produced after 5-HT2AR stimulation. The release of 2-AG was dependent on both phosphatidylinositol (PI)-specific phospholipase C (PLC) and diacylglycerol lipase (DGL) activation but not on phospholipase D (PLD) or phosphatidylcholine-specific-PLC activation. Staurosporine, an inhibitor of protein kinase C, as well as the calcium ionophore, A23187, potentiated 5-HT2AR-dependent 2-AG release. BAPTA-AM, a calcium chelator, failed to inhibit 5-HT2AR-dependent 2-AG release, supporting a calcium-independent mechanism of action. In conclusion, we are the first to conclusively demonstrate hallucinogen-induced endocannabinoid release mediated by 5-HT2AR-dependent PI-PLC and DGL activation. This research was supported by NIH grant DA02189 from NIDA.