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Leptin-regulated endocannabinoids are involved in maintaining food intake

Leptin is the primary signal through which the hypothalamus senses nutritional state and modulates food intake and energy balance. Leptin reduces food intake by upregulating anorexigenic (appetite-reducing) neuropeptides, such as α-melanocyte-stimulating hormone, and downregulating orexigenic (appetite-stimulating) factors, primarily neuropeptide Y. Genetic defects in anorexigenic signalling, such as mutations in the melanocortin-4 (ref. 5) or leptin receptors, cause obesity. However, alternative orexigenic pathways maintain food intake in mice deficient in neuropeptide Y. CB1 cannabinoid receptors and the endocannabinoids anandamide and 2-arachidonoyl glycerol are present in the hypothalamus, and marijuana and anandamide stimulate food intake. Here we show that following temporary food restriction, CB1 receptor knockout mice eat less than their wild-type littermates, and the CB1 antagonist SR141716A reduces food intake in wild-type but not knockout mice. Furthermore, defective leptin signalling is associated with elevated hypothalamic, but not cerebellar, levels of endocannabinoids in obese db/db and ob/ob mice and Zucker rats. Acute leptin treatment of normal rats and ob/ob mice reduces anandamide and 2-arachidonoyl glycerol in the hypothalamus. These findings indicate that endocannabinoids in the hypothalamus may tonically activate CB1 receptors to maintain food intake and form part of the neural circuitry regulated by leptin.

An endogenous capsaicin-like substance with high potency at recombinant and native vanilloid VR1 receptors

The vanilloid receptor VR1 is a nonselective cation channel that is most abundant in peripheral sensory fibers but also is found in several brain nuclei. VR1 is gated by protons, heat, and the pungent ingredient of “hot” chili peppers, capsaicin. To date, no endogenous compound with potency at this receptor comparable to that of capsaicin has been identified. Here we examined the hypothesis, based on previous structure-activity relationship studies and the availability of biosynthetic precursors, that N-arachidonoyl-dopamine (NADA) is an endogenous “capsaicin-like” substance in mammalian nervous tissues. We found that NADA occurs in nervous tissues, with the highest concentrations being found in the striatum, hippocampus, and cerebellum and the lowest concentrations in the dorsal root ganglion. We also gained evidence for the existence of two possible routes for NADA biosynthesis and mechanisms for its inactivation in rat brain. NADA activates both human and rat VR1 overexpressed in human embryonic kidney (HEK)293 cells, with potency (EC50 ≈ 50 nM) and efficacy similar to those of capsaicin. Furthermore, NADA potently activates native vanilloid receptors in neurons from rat dorsal root ganglion and hippocampus, thereby inducing the release of substance P and calcitonin gene-related peptide (CGRP) from dorsal spinal cord slices and enhancing hippocampal paired-pulse depression, respectively. Intradermal NADA also induces VR1-mediated thermal hyperalgesia (EC50 = 1.5 ± 0.3 μg). Our data demonstrate the existence of a brain substance similar to capsaicin not only with respect to its chemical structure but also to its potency at VR1 receptors.

Endocannabinoids control spasticity in a multiple sclerosis model

Spasticity is a complicating sign in multiple sclerosis that also develops in a model of chronic relapsing experimental autoimmune encephalomyelitis (CREAE) in mice. In areas associated with nerve damage, increased levels of the endocannabinoids, anandamide (arachidonoylethanolamide, AEA) and 2‐arachidonoyl glycerol (2‐AG), and of the AEA congener, palmitoylethanolamide (PEA), were detected here, whereas comparable levels of these compounds were found in normal and non‐spastic CREAE mice. While exogenously administered endocannabinoids and PEA ameliorate spasticity, selective inhibitors of endocannabinoid re‐uptake and hydrolysis—probably through the enhancement of endogenous levels of AEA, and, possibly, 2‐arachidonoyl glycerol—significantly ameliorated spasticity to an extent comparable with that observed previously with potent cannabinoid receptor agonists. These studies provide definitive evidence for the tonic control of spasticity by the endocannabinoid system and open new horizons to therapy of multiple sclerosis, and other neuromuscular diseases, based on agents modulating endocannabinoid levels and action, which exhibit little psychotropic activity.

Anandamide inhibits metabolism and physiological actions of 2-arachidonoylglycerol in the striatum

Of the endocannabinoids (eCBs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG) have received the most study. A functional interaction between these molecules has never been described. Using mouse brain slices, we found that stimulation of metabotropic glutamate 5 receptors by 3,5-dihydroxyphenylglycine (DHPG) depressed inhibitory transmission in the striatum through selective involvement of 2-AG metabolism and stimulation of presynaptic CB1 receptors. Elevation of AEA concentrations by pharmacological or genetic inhibition of AEA degradation reduced the levels, metabolism and physiological effects of 2-AG. Exogenous AEA and the stable AEA analog methanandamide inhibited basal and DHPG-stimulated 2-AG production, confirming that AEA is responsible for the downregulation of the other eCB. AEA is an endovanilloid substance, and the stimulation of transient receptor potential vanilloid 1 (TRPV1) channels mimicked the effects of endogenous AEA on 2-AG metabolism through a previously unknown glutathione-dependent pathway. Consistently, the interaction between AEA and 2-AG was lost after pharmacological and genetic inactivation of TRPV1 channels.

Cannabinoid CB1‐receptor mediated regulation of gastrointestinal motility in mice in a model of intestinal inflammation

We have studied the effect of cannabinoid agonists (CP 55,940 and cannabinol) on intestinal motility in a model of intestinal inflammation (induced by oral croton oil in mice) and measured cannabinoid receptor expression, endocannabinoids (anandamide and 2‐arachidonylglycerol) and anandamide amidohydrolase activity both in physiological and pathophysiological states. CP 55,940 (0.03 – 10 nmol mouse−1) and cannabinol (10 – 3000 nmol mouse−1) were more active in delaying intestinal motility in croton oil‐treated mice than in control mice. These inhibitory effects were counteracted by the selective cannabinoid CB1 receptor antagonist SR141716A (16 nmol mouse−1). SR141716A (1 – 300 nmol mouse−1), administered alone, increased intestinal motility to the same extent in both control and croton oil‐treated mice Croton oil‐induced intestinal inflammation was associated with an increased expression of CB1 receptor, an unprecedented example of up‐regulation of cannabinoid receptors during inflammation. High levels of anandamide and 2‐arachidonylglycerol were detected in the small intestine, although no differences were observed between control and croton oil‐treated mice; by contrast anandamide amidohydrolase activity increased 2 fold in the inflamed small intestine. It is concluded that inflammation of the gut increases the potency of cannabinoid agonists possibly by ‘up‐regulating’ CB1 receptor expression; in addition, endocannabinoids, whose turnover is increased in inflamed gut, might tonically inhibit intestinal motility.

Endocannabinoid signalling in the blood of patients with schizophrenia

AimTo test the hypothesis that schizophrenia might be associated with alterations of the endogenous cannabinoid system in human blood.ResultsBlood from 20 healthy volunteers and 12 patients with schizophrenia, 5 of which both before and after a successful antipsychotic treatment, was analysed for: 1) the amounts of the endocannabinoid anandamide; 2) the levels of cannabinoid CB1 and CB2 receptor mRNAs, and 3) the levels of the mRNA encoding the enzyme fatty acid amide hydrolase (FAAH), responsible for anandamide degradation.The amounts of anandamide were significantly higher in the blood of patients with acute schizophrenia than in healthy volunteers (7.79 ± 0.50 vs. 2.58 ± 0.28 pmol/ml). Clinical remission was accompanied by a significant decrease of the levels of anandamide (3.88 ± 0.72 pmol/ml) and of the mRNA transcripts for CB2 receptors and FAAH.ConclusionThese findings indicate that endocannabinoid signalling might be altered during the acute phase of schizophrenia not only in the central nervous system but also in the blood. These changes might be related to the several immunological alterations described in schizophrenia.

Exogenous anandamide protects rat brain against acute neuronal injury in vivo

The endocannabinoid anandamide [N-arachidonoylethanolamine (AEA)] is thought to function as an endogenous protective factor of the brain against acute neuronal damage. However, this has never been tested in an in vivo model of acute brain injury. Here, we show in a longitudinal pharmacological magnetic resonance imaging study that exogenously administered AEA dose-dependently reduced neuronal damage in neonatal rats injected intracerebrally with the Na+/K+-ATPase inhibitor ouabain. At 15 min after injury, AEA (10 mg/kg) administered 30 min before ouabain injection reduced the volume of cytotoxic edema by 43 ± 15% in a manner insensitive to the cannabinoid CB1receptor antagonist SR141716A. At 7 d after ouabain treatment, 64 ± 24% less neuronal damage was observed in AEA-treated (10 mg/kg) rats compared with control animals. Coadministration of SR141716A prevented the neuroprotective actions of AEA at this end point. In addition, (1) no increase in AEA and 2-arachidonoylglycerol levels was detected at 2, 8, or 24 hr after ouabain injection; (2) application of SR141716A alone did not increase the lesion volume at days 0 and 7; and (3) the AEA-uptake inhibitor, VDM11, did not affect the lesion volume. These data indicate that there was no endogenous endocannabinoid tone controlling the acute neuronal damage induced by ouabain. Although our data seem to question a possible role of the endogenous cannabinoid system in establishing a brain defense system in our model, AEA may be used as a structural template to develop neuroprotective agents.

Lipid rafts control signaling of type-1 cannabinoid receptors in neuronal cells: Implications for anandamide-induced apoptosis

Several G protein-coupled receptors function within lipid rafts plasma membrane microdomains, which may be important in limiting signal transduction. Here we show that treatment of rat C6 glioma cells with the raft disruptor methyl-β-cyclodextrin (MCD) doubles the binding efficiency (i.e. the ratio between maximum binding and dissociation constant) of type-1 cannabinoid receptors (CB1R), which belong to the rhodopsin family of G protein-coupled receptors. In parallel, activation of CB1R by the endogenous agonist anandamide (AEA) leads to ∼3-fold higher [35S]GTPγS binding in MCD-treated cells than in controls, and CB1R-dependent signaling via adenylate cyclase, and p42/p44 MAPK is almost doubled by MCD. Unlike CB1R, the other AEA-binding receptor TRPV1, the AEA synthetase NAPE-PLD, and the AEA hydrolase FAAH are not modulated by MCD, whereas the activity of the AEA membrane transporter (AMT) is reduced to ∼50% of the controls. We also show that MCD reduces dose-dependently AEA-induced apoptosis in C6 cells but not in human CHP100 neuroblastoma cells, which mirror the endocannabinoid system of C6 cells but are devoid of CB1R. MCD reduces also cytochrome c release from mitochondria of C6 cells, and this effect is CB1R-dependent and partly mediated by activation of p42/p44 MAPK. Altogether, the present data suggest that lipid rafts control CB1R binding and signaling, and that CB1R activation underlies the protective effect of MCD against apoptosis.

Characterization of the endocannabinoid system in boar spermatozoa and implications for sperm capacitation and acrosome reaction

Anandamide (AEA) is the endogenous ligand of cannabinoid (CB) receptors, and as such it plays several central and peripheral activities. Regulation of female fertility by AEA has attracted growing interest, yet a role for this endocannabinoid in controlling sperm function and male fertility in mammals has been scarcely investigated. In this study we report unprecedented evidence that boar sperm cells have the biochemical machinery to bind and degrade AEA, i.e. type-1 cannabinoid receptors (CB1R), vanilloid receptors (TRPV1), AEA-synthesizing phospholipase D (NAPE-PLD), AEA transporter (AMT) and AEA hydrolase (FAAH). We also show that the non-hydrolyzable AEA analogue methanandamide reduces sperm capacitation and, as a consequence, inhibits the process of acrosome reaction (AR) triggered by the zona pellucida, according to a cyclic AMP-dependent pathway triggered by CB1R activation. Furthermore, activation of TRPV1 receptors seems to play a role of stabilization of the plasma membranes in capacitated sperm, as demonstrated by the high incidence of spontaneous AR occurring during the cultural period when TRPV1 activity was antagonized by capsazepine. We show that sperm cells have a complete and efficient endocannabinoid system, and that activation of cannabinoid or vanilloid receptors controls, at different time-points, sperm functions required for fertilization. These observations open new perspectives on the understanding and treatment of male fertility problems.

Neuroprotection by the Endogenous Cannabinoid Anandamide and Arvanil against In Vivo Excitotoxicity in the Rat: Role of Vanilloid Receptors and Lipoxygenases

Type 1 vanilloid receptors (VR1) have been identified recently in the brain, in which they serve as yet primarily undetermined purposes. The endocannabinoid anandamide (AEA) and some of its oxidative metabolites are ligands for VR1, and AEA has been shown to afford protection against ouabain-induced in vivo excitotoxicity, in a manner that is only in part dependent on the type 1 cannabinoid (CB1) receptor. In the present study, we assessed whether VR1 is involved in neuroprotection by AEA and by arvanil, a hydrolysis-stable AEA analog that is a ligand for both VR1 and CB1. Furthermore, we assessed the putative involvement of lipoxygenase metabolites of AEA in conveying neuroprotection. Using HPLC and gas chromatography/mass spectroscopy, we demonstrated that rat brain and blood cells converted AEA into 12-hydroxy-N-arachidoylethanolamine (12-HAEA) and 15-hydroxy-N-arachidonoylethanolamine (15-HAEA) and that this conversion was blocked by addition of the lipoxygenase inhibitor nordihydroguaiaretic acid. Using magnetic resonance imaging we show the following: (1) pretreatment with the reduced 12-lipoxygenase metabolite of AEA, 12-HAEA, attenuated cytotoxic edema formation in a CB1 receptor-independent manner in the acute phase after intracranial injection of the Na+/K+-ATPase inhibitor ouabain; (2) the reduced 15-lipoxygenase metabolite, 15-HAEA, enhanced the neuroprotective effect of AEA in the acute phase; (3) modulation of VR1, as tested using arvanil, the VR1 agonist capsaicin, and the antagonist capsazepine, leads to neuroprotective effects in this model, and arvanil is a potent neuroprotectant, acting at both CB1 and VR1; and (4) the in vivo neuroprotective effects of AEA are mediated by CB1 but not by lipoxygenase metabolites or VR1.