Brain regional cannabinoid CB 1 receptor signalling and alternative enzymatic pathways for 2-arachidonoylglycerol generation in brain sections of diacylglycerol lipase deficient mice Niina Aaltonen a,⇑ , Casandra Riera Ribas a , Marko Lehtonen a , Juha R. Savinainen b , Jarmo T. Laitinen b a School of Pharmacy, University of Eastern Finland, P.O. Box 1627, 70211 Kuopio, Finland b School of Medicine, Institute of Biomedicine/Physiology, University of Eastern Finland, P.O. Box 1627, 70211 Kuopio, Finland article info Article history: Received 22 March 2013 Received in revised form 19 July 2013 Accepted 26 August 2013 Available online 3 September 2013 Keywords: 2-Arachidonoylglycerol [ 35 S]GTPcS autoradiography Cannabinoid CB 1 receptor Diacylglycerol lipase-knockout Methylarachidonoylfluorophosphonate Tetrahydrolipstatin abstract Endocannabinoids are the endogenous ligands of the G protein-coupled cannabinoid receptors. The prin- cipal brain endocannabinoid, 2-arachidonoylglycerol (2-AG), is enzymatically produced by postsynaptic neurons and then activates presynaptic CB 1 receptors in a retrograde manner. The primary pathway for 2- AG generation is believed to be conversion from the diacylglycerols (DAGs) by two sn-1-specific lipases, DAGLa and DAGLb. Previous studies with DAGL-deficient mice indicated that DAGLa is the major enzyme needed for retrograde synaptic 2-AG signalling. The current study investigated whether the CB 1 receptor- mediated G i/o protein activity is altered in brain cryosections of DAGL-deficient mice when compared to wild-type mice and whether the sn-1-specific DAGLs are able to generate 2-AG in brain cryosections. Functional autoradiography indicated that brain regional CB 1 receptor-G i/o -activity largely remained unaltered in DAGLa-knockout and DAGLb-knockout mice when compared to wild-type littermates. Fol- lowing comprehensive pharmacological blockade of 2-AG hydrolysis, brain sections generated sufficient amounts of 2-AG to activate CB 1 receptors throughout the regions endowed with these receptors. As demonstrated by LC/MS/MS, this pool of 2-AG was generated via tetrahydrolipstatin-sensitive enzymatic pathways distinct from DAGLa or DAGLb. We conclude that in addition to the sn-1-specific DAGLs, addi- tional 2-AG generating enzymatic pathways are active in brain sections. Ó 2013 Elsevier B.V. All rights reserved. 1. Introduction Endocannabinoids (eCBs) are the body’s natural agonists for cannabinoid receptors (G protein-coupled CB 1 and CB 2 ) that also recognize D 9 -tetrahydrocannabinol (D 9 -THC), the psychoactive component of marijuana. The CB 1 receptor is highly expressed and widely distributed in the brain (Herkenham et al., 1991) and is primarily responsible for the neural actions of both exogenous cannabinoids and endocannabinoids. Unlike classical water soluble neurotransmitters, hydrophobic eCBs are not stored in synaptic vesicles, instead, they are produced and released locally by postsynaptic neurons only when needed, representing ‘‘on demand’’ production. After production, eCBs activate presynaptic CB 1 receptors in a retrograde manner, resulting in a reduction of neurotransmitter release at various synapses (Kano et al., 2009). Endocannabinoid signalling plays a role in several important pro- cesses during neuronal development (Argaw et al., 2011; Berghuis et al., 2007; Keimpema et al., 2010) as well as in adult neurogenesis (Goncalves et al., 2008). Two principal endocannabinoids, N-arachidonoylethanolamide (anandamide, AEA) and 2-arachidonoylglycerol (2-AG) have been characterized in mammals (Devane et al., 1992; Mechoulam et al., 1995; Sugiura et al., 1995). In the nervous system, 2-AG is produced by enzymatic pathways from phospholipid precursors. The synthesis is triggered by the increase in intracellular calcium concentrations induced by cell depolarization or stimulation of G q /G 11 protein-coupled receptors (Kano et al., 2009). The primary precursors for 2-AG are believed to be the diacylglycerols (DAGs) generated from phosphatidylinositols by the action of phospholipase C (PLCb; EC 3.1.4.11) or from phosphatidic acid (PA) by PA phosphohydrolase (Bisogno, 2008; Sugiura et al., 2006). DAGs are further hydrolyzed by sn-1-specific lipase (DAGL; 0928-0987/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejps.2013.08.035 Abbreviations: 2-AG, 2-arachidonoylglycerol; AEA, N-arachidonoylethanola- mide/anandamide; BSA, bovine serum albumin; DAGL, diacylglycerol lipase; eCB, endocannabinoid; FAAH, fatty acid amide hydrolase; KO, knockout; LC/MS/MS, liquid chromatography/tandem mass spectrometry; MAFP, meth- ylarachidonoylfluorophosphonate; MAGL, monoacylglycerol lipase; PLA, phospho- lipase A; PLC, phospholipase C; THL, tetrahydrolipstatin; WT, wild-type. ⇑ Corresponding author. Tel.: +358 40 3552418; fax. +358 17 162424. E-mail addresses: Niina.Aaltonen@uef.fi (N. Aaltonen), mcasandrariera@gmail.- com (C. Riera Ribas), Marko.Lehtonen@uef.fi (M. Lehtonen), Juha.Savinainen@uef.fi (J.R. Savinainen), Jarmo.Laitinen@uef.fi (J.T. Laitinen). European Journal of Pharmaceutical Sciences 51 (2014) 87–95 Contents lists available at ScienceDirect European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps