N ‐Arachidonoyl‐phosphatidylethanolamide phospholipase D activation by muscarinic receptors

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 G q ‐coupled muscarinic M 3 receptors. The muscarinic agonist oxotremorine‐M stimulated the GRAB eCB 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. G q ‐coupled mGluR5 glutamate receptors effectively substitute for M 3 receptors. Thus M 3 receptors stimulate NAPE‐PLD synthesis of acylethanolamines such as anandamide via G q signalling pathways resembling those for 2‐arachidonoylglycerol. Parasympathetic activation stimulates tearing and salivation via M 3 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 M 3 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‐GRAB eCB cells stimulated GRAB eCB responses. This response was diminished by LEI401 and was also present in cells expressing GRAB AEA , an anandamide‐specific sensor, but not those expressing GRAB 2AG . We conclude that muscarinic M 3 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. image 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 G q ‐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 G q ‐coupled G protein‐coupled receptor (GPCR) activation of NAPE‐PLD to induce anandamide synthesis may play many roles in the body.