How nerve signals may trigger the body’s own anandamide

The endogenous cannabinoid signalling system consists of G protein-coupled receptors, messengers - 2-arachidonoylglycerol (2-AG) and anandamide - and enzymatic machinery to synthesize and metabolize these messengers. Anandamide is physiologically important, but its synthesis is incompletely understood. N-Arachidonoyl-phosphatidylethanolamide phospholipase D (NAPE-PLD) synthesizes acylethanolamines, including anandamide, but how is NAPE-PLD activated? We used genetically encoded G protein-coupled receptor based (GRAB) sensors for endocannabinoids (eCBs) and immunohistochemistry to investigate. Human embryonic kidney 293 (HEK293) cells natively express Gq-coupled muscarinic M3 receptors. The muscarinic agonist oxotremorine-M stimulated the GRABeCB sensor only when HEK293 cells were cotransfected with NAPE-PLD. This signal was reduced by the NAPE-PLD inhibitor LEI401 and required both phospholipase C and internal calcium stores. Gq-coupled mGluR5 glutamate receptors effectively substitute for M3 receptors. Thus M3 receptors stimulate NAPE-PLD synthesis of acylethanolamines such as anandamide via Gq signalling pathways resembling those for 2-arachidonoylglycerol. Parasympathetic activation stimulates tearing and salivation via M3 receptors on myoepithelial cells. The cannabinoid signalling system may act as a feedback inhibitor to inhibit acetylcholine release, but the identity and source of the endogenous cannabinoid messenger are uncertain. NAPE-PLD and M3 proteins colocalize in myoepithelial cells; fatty acid amide hydrolase (FAAH) resides in glandular acinar cells. Oxotremorine-M stimulation of lacrimal or submandibular salivary glands co-cultured with HEK293-GRABeCB cells stimulated GRABeCB responses. This response was diminished by LEI401 and was also present in cells expressing GRABAEA, an anandamide-specific sensor, but not those expressing GRAB2AG. We conclude that muscarinic M3 receptors stimulate NAPE-PLD to produce anandamide in cell lines and exocrine glands. This means of stimulating anandamide synthesis may play many roles in the body. KEY POINTS: The cannabinoid signalling system plays important roles in the body and in doing so makes use of two endogenous messengers, 2-arachidonoylglycerol (2-AG) and anandamide. The regulation of anandamide synthesis is still poorly understood. This study demonstrates that muscarinic Gq-coupled GPCRs can stimulate anandamide production by activating the enzyme N-arachidonoyl-phosphatidylethanolamide phospholipase D (NAPE-PLD) and further shows that this occurs natively in salivary and lacrimal glands. Muscarinic Gq-coupled G protein-coupled receptor (GPCR) activation of NAPE-PLD to induce anandamide synthesis may play many roles in the body.