Anandamide (N-arachidonoyl-ethanolamine) was recently identified as a brain arachidonate derivative that binds to and activates cannabinoid receptors, yet the mechanisms underlying formation, release and inactivation of this putative messenger molecule are still unclear. Here we report that anandamide is produced in and released from cultured brain neurons in a calcium ion-dependent manner when the neurons are stimulated with membrane-depolarizing agents. Anandamide formation occurs through phosphodiesterase-mediated cleavage of a novel phospholipid precursor, N-arachidonoyl-phosphatidylethanolamine. A similar mechanism also governs the formation of a family of anandamide congeners, whose possible roles in neuronal signalling remain unknown. Our results and those of others indicate therefore that multiple biochemical pathways may participate in anandamide formation in brain tissue. The life span of extracellular anandamide is limited by a rapid and selective process of cellular uptake, which is accompanied by hydrolytic degradation to ethanolamine and arachidonate. Our results thus strongly support the proposed role of anandamide as an endogenous neuronal messenger.

1 (7)-Cannabidiol (CBD) is a non-psychotropic component of Cannabis with possible therapeutic use as an anti-in¯ammatory drug. Little is known on the possible molecular targets of this compound. We investigated whether CBD and some of its derivatives interact with vanilloid receptor type 1 (VR1), the receptor for capsaicin, or with proteins that inactivate the endogenous cannabinoid, anandamide (AEA). 2 CBD and its enantiomer, (+)-CBD, together with seven analogues, obtained by exchanging the C-7 methyl group of CBD with a hydroxy-methyl or a carboxyl function and/or the C-5' pentyl group with a di-methyl-heptyl (DMH) group, were tested on: (a) VR1-mediated increase in cytosolic Ca 2+ concentrations in cells over-expressing human VR1; (b) [ 14 C]-AEA uptake by RBL-2H3 cells, which is facilitated by a selective membrane transporter; and (c) [ 14 C]-AEA hydrolysis by rat brain membranes, which is catalysed by the fatty acid amide hydrolase. 3 Both CBD and (+)-CBD, but not the other analogues, stimulated VR1 with EC 50 =3.2 ± 3.5 mM, and with a maximal e ect similar in e cacy to that of capsaicin, i.e. 67 ± 70% of the e ect obtained with ionomycin (4 mM). CBD (10 mM) desensitized VR1 to the action of capsaicin. The e ects of maximal doses of the two compounds were not additive. 4 (+)-5'-DMH-CBD and (+)-7-hydroxy-5'-DMH-CBD inhibited [ 14 C]-AEA uptake (IC 50 =10.0 and 7.0 mM); the (7)-enantiomers were slightly less active (IC 50 =14.0 and 12.5 mM). CBD and (+)-CBD were also active (IC 50 =22.0 and 17.0 mM). 5 CBD (IC 50 =27.5 mM), (+)-CBD (IC 50 =63.5 mM) and (7)-7-hydroxy-CBD (IC 50 =34 mM), but not the other analogues (IC 50 4100 mM), weakly inhibited [ 14 C]-AEA hydrolysis. 6 Only the (+)-isomers exhibited high a nity for CB 1 and/or CB 2 cannabinoid receptors. 7 These ®ndings suggest that VR1 receptors, or increased levels of endogenous AEA, might mediate some of the pharmacological e ects of CBD and its analogues. In view of the facile high yield synthesis, and the weak a nity for CB 1 and CB 2 receptors, (7)-5'-DMH-CBD represents a valuable candidate for further investigation as inhibitor of AEA uptake and a possible new therapeutic agent.

1 Endocannabinoids are implicated in appetite and body weight regulation. In rodents, anandamide stimulates eating by actions at central CB1 receptors, and hypothalamic endocannabinoids may be under the negative control of leptin. However, changes to brain endocannabinoid levels in direct relation to feeding or changing nutritional status have not been investigated. 2 We measured anandamide and 2-arachidonoyl glycerol (2-AG) levels in feeding-associated brain regions of rats, during fasting, feeding of a palatable food, or after satiation. Endocannabinoid levels were compared to those in rats fed ad libitum, at a point in their daily cycle when motivation to eat was absent. Fasting increased levels of anandamide and 2-AG in the limbic forebrain and, to a lesser extent, of 2-AG in the hypothalamus. By contrast, hypothalamic 2-AG declined as animals ate. No changes were detected in satiated rats. Endocannabinoid levels in the cerebellum, a control region not directly involved in the control of food intake, were una ected by any manipulation. 3 As 2-AG was most sensitive to variation during feeding, and to leptin regulation in a previous study, we examined the behavioural e ects of 2-AG when injected into the nucleus accumbens shell, a limbic forebrain area strongly linked to eating motivation. 2-AG potently, and dose-dependently, stimulated feeding. This e ect was attenuated by the CB1 receptor antagonist SR141716. 4 These ®ndings provide the ®rst direct evidence of altered brain levels of endocannabinoids, and of 2-AG in particular, during fasting and feeding. The nature of these e ects supports a role for endocannabinoids in the control of appetitive motivation.

The purpose of these studies was to support the hypothesis that an undiscovered cannabinoid receptor exists in brain. [(35)S]GTP gamma S binding was stimulated by anandamide and WIN55212-2 in brain membranes from both CB(1)(+/+) and CB(1)(-/-) mice. In contrast, a wide variety of other compounds that are known to activate CB(1) receptors, including CP55940, HU-210, and Delta(9)-tetrahydrocannabinol, failed to stimulate [(35)S]GTP gamma S binding in CB(1)(-/-) membranes. In CB(1)(-/-) membranes, SR141716A affected both basal and anandamide- or WIN55212-2-induced stimulation of [(35)S]GTP gamma S binding only at concentrations greater than 1 microM. In CB(1)(+/+) membranes, SR141716A inhibited only 84% of anandamide and 67% of WIN55212-2 stimulated [(35)S]GTP gamma S binding with an affinity appropriate for mediation by CB(1) receptors (K(B) approximately 0.5 nM). The remaining stimulation seemed to be inhibited with lower potency (IC(50) approximately 5 microM) similar to that seen in CB(1)(-/-) membranes or in the absence of agonist. Further experiments determined that the effects of anandamide and WIN55212-2 were not additive, but that the effect of mu opioid, adenosine A1, and cannabinoid ligands were additive. Finally, assays of different central nervous system (CNS) regions demonstrated significant activity of cannabinoids in CB(1)(-/-) membranes from brain stem, cortex, hippocampus, diencephalon, midbrain, and spinal cord, but not basal ganglia or cerebellum. Moreover, some of these same CNS regions also showed significant binding of [(3)H]WIN55212-2, but not [(3)H]CP55940. Thus anandamide and WIN55212-2 seemed to be active in CB(1)(-/-) mouse brain membranes via a common G protein-coupled receptor with a distinct CNS distribution, implying the existence of an unknown cannabinoid receptor subtype in brain.