Abstract:
Endocannabinoids are endogenous agonists for the two types of cannabinoid receptors identified to date, the CB1 and CB2 receptors. CB1 receptors and tissue concentrations of endocannabinoids sufficient to activate them are present in the brain structures controlling energy intake (i.e., the hypothalamus, nucleus accumbens and nodose ganglion), as well as in all peripheral organs mostly involved in the regulation of energy homeostasis (i.e., the duodenum, small and large intestine, adipose tissue, skeletal muscle, pancreas and liver). Several peripheral neuropeptides and hormones involved in energy balance, and type of diet, regulate endocannabinoid levels, whereas endocannabinoids, in turn, regulate the expression and release of hypothalamic mediators involved in nutrient intake. Dysregulation of the endocannabinoid system might contribute to the development of eating disorders and explain why CB1 receptor blockers are efficacious at reducing not only food intake but also the metabolic consequences of visceral adiposity and obesity.

Abstract:
BACKGROUND AND AIMS: Diverticulosis is a common disease of not completely defined pathogenesis. Motor abnormalities of the intestinal wall have been frequently described but very little is known about their mechanisms. We investigated in vitro the neural response of colonic longitudinal muscle strips from patients undergoing surgery for complicated diverticular disease (diverticulitis). METHODS: The neural contractile response to electrical field stimulation of longitudinal muscle strips from the colon of patients undergoing surgery for colonic cancer or diverticulitis was challenged by different receptor agonists and antagonists. RESULTS: Contractions of colonic strips from healthy controls and diverticulitis specimens were abolished by atropine. The beta adrenergic agonist (-) isoprenaline and the tachykinin NK1 receptor antagonist SR140333 had similar potency in reducing the electrical twitch response in controls and diseased tissues, while the cannabinoid receptor agonist (+)WIN 55,212-2 was 100 times more potent in inhibiting contractions in controls (IC50 42 nmol/l) than in diverticulitis strips. SR141716, a selective antagonist of the cannabinoid CB1 receptor, had no intrinsic activity in control preparations but potentiated the neural twitch in diseased tissues by up to 196% in a concentration dependent manner. SR141716 inhibited (+)WIN 55,212-2 induced relaxation in control strips but had no efficacy on (+)WIN 55,212-2 responses in strips from diverticular disease patients. Colonic levels of the endogenous ligand of cannabinoid and vanilloid TRPV1 receptors anandamide were more than twice those of control tissues (54 v 27 pmol/g tissue). The axonal conduction blocker tetrodotoxin had opposite effects in the two preparations, completely inhibiting the contractions of control strips but potentiating those in diverticular preparations, an effect selectively inhibited by SR140333. CONCLUSIONS: Neural control of colon motility is profoundly altered in patients with diverticulitis. Their raised levels of anandamide, apparent desensitisation of the presynaptic neural cannabinoid CB1 receptor, and the SR141716 induced intrinsic response, suggest that endocannabinoids may be involved in the pathophysiology of complications of colonic diverticular disease.

Abstract:
We investigated the involvement of endocannabinoids in the control of neuronal damage and memory retention loss in rodents treated with the beta-amyloid peptide (1-42) (BAP). Twelve days after stereotaxic injection of BAP into the rat cortex, and concomitant with the appearance in the hippocampus of markers of neuronal damage, 2-arachidonoyl glycerol, but not anandamide, levels were enhanced in the hippocampus. VDM-11 (5 mg/kg, i.p.), an inhibitor of endocannabinoid cellular reuptake, significantly enhanced rat hippocampal and mouse brain endocannabinoid levels when administered sub-chronically starting either 3 or 7 days after BAP injection and until the 12-14th day. VDM-11 concomitantly reversed hippocampal damage in rats, and loss of memory retention in the passive avoidance test in mice, but only when administered from the 3rd day after BAP injection. We suggest that early, as opposed to late, pharmacological enhancement of brain endocannabinoid levels might protect against beta-amyloid neurotoxicity and its consequences.

Abstract:
Endocannabinoids were first defined in 1995 as 'endogenous substances capable of binding to and functionally activating the cannabinoid receptors'. To date, two well-established endocannabinoids, N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG), as well as a few other putative ligands, all derived from long-chain polyunsaturated fatty acids, have been identified in animal tissues. The biosynthetic and metabolic pathways for anandamide and 2-AG have been elucidated, and most of the enzymes therein involved have been cloned. We now know that CB1 receptors, and endocannabinoids in tissue concentrations sufficient to activate them, are more widely distributed than originally thought, and are found in brain and peripheral organs involved in the control of energy intake and processing, including the hypothalamus, nucleus accumbens, brainstem, vagus nerve, gastrointestinal tract, adipose tissue and liver. Endocannabinoid biosynthetic and inactivating pathways are under the regulation of neuropeptides and hormones involved in energy homeostasis, and endocannabinoid levels are directly affected by the diet. Endocannabinoids, in turn, regulate the expression and action of mediators involved in nutrient intake and processing. These cross-talks are at the basis of the proposed role of endocannabinoid signalling in the control of food intake, from invertebrates to lower vertebrates and mammals, and their perturbation appears to contribute to the development of eating disorders.

Abstract:
Endocannabinoid system evolution was estimated by searching for functional orthologs in the genomes of twelve phylogenetically diverse organisms: Homo sapiens, Mus musculus, Takifugu rubripes, Ciona intestinalis, Caenorhabditis elegans, Drosophila melanogaster, Saccharomyces cerevisiae, Arabidopsis thaliana, Plasmodium falciparum, Tetrahymena thermophila, Archaeoglobus fulgidus, and Mycobacterium tuberculosis. Sequences similar to human endocannabinoid exon sequences were derived from filtered BLAST searches, and subjected to phylogenetic testing with ClustalX and tree building programs. Monophyletic clades that agreed with broader phylogenetic evidence (i.e., gene trees displaying topographical congruence with species trees) were considered orthologs. The capacity of orthologs to function as endocannabinoid proteins was predicted with pattern profilers (Pfam, Prosite, TMHMM, and pSORT), and by examining queried sequences for amino acid motifs known to serve critical roles in endocannabinoid protein function (obtained from a database of site-directed mutagenesis studies). This novel transfer of functional information onto gene trees enabled us to better predict the functional origins of the endocannabinoid system. Within this limited number of twelve organisms, the endocannabinoid genes exhibited heterogeneous evolutionary trajectories, with functional orthologs limited to mammals (TRPV1 and GPR55), or vertebrates (CB2 and DAGLbeta), or chordates (MAGL and COX2), or animals (DAGLalpha and CB1-like receptors), or opisthokonta (animals and fungi, NAPE-PLD), or eukaryotes (FAAH). Our methods identified fewer orthologs than did automated annotation systems, such as HomoloGene. Phylogenetic profiles, nonorthologous gene displacement, functional convergence, and coevolution are discussed.

Abstract:
Endocannabinoids are endogenous substances capable of binding to and functionally activating the two types of cannabinoid receptors identified to date, the cannabinoid receptors type 1 and 2 (CB1 and CB2 receptors). The anabolic and catabolic pathways for the two most studied endocannabinoids, N-arachidonoylethanolamine (anandamide) and 2-arachidonoyl glycerol (2- AG), have been elucidated, thus leading to the molecular characterization of some of their catabolic and biosynthetic enzymes. These proteins are hydrolytic enzymes that also recognize as substrates other congeners of anandamide and 2-AG or of their biosynthetic precursors, respectively. CB1 receptor and tissue concentrations of endocannabinoids sufficient to activate them are more widely distributed than originally believed, and are present in all brain and peripheral organs involved in the control of energy homeostasis, including the hypothalamus, nucleus accumbens, brainstem, vagus nerve, gastrointestinal trad, adipose tissue, muscle and liver. Hypothalamic and peripheral neuropeptides and hormones involved in energy balance, as well as the type of diet, regulate endocannabinoid biosynthetic and inactivating pathways, whereas endocannabinoids, in turn, regulate the expression and action of mediators involved in nutrient intake and processing. The perturbation of these cross-talks might contribute to the development of eating disorders.

Abstract:
BACKGROUND & AIMS: Fatty acid amide hydrolase (FAAH) catalyzes the hydrolysis both of the endocannabinoids (which are known to inhibit intestinal motility) and other bioactive amides (palmitoylethanolamide, oleamide, and oleoylethanolamide), which might affect intestinal motility. The physiologic role of FAAH in the gut is largely unexplored. In the present study, we evaluated the possible role of FAAH in regulating intestinal motility in mice in vivo. METHODS: Motility was measured by evaluating the distribution of a fluorescent marker along the small intestine; FAAH messenger RNA (mRNA) levels were analyzed by reverse-transcription polymerase chain reaction (RT-PCR); endocannabinoid levels were measured by isotope-dilution, liquid chromatography, mass spectrometry. RESULTS: Motility was inhibited by N-arachidonoylserotonin (AA-5-HT) and palmitoylisopropylamide, 2 selective FAAH inhibitors, as well as by the FAAH substrates palmitoylethanolamide, oleamide, and oleoylethanolamide. The effect of AA-5-HT was reduced by the CB1 receptor antagonist rimonabant and by CB1 deficiency in mice but not by the vanilloid receptor antagonist 5'-iodoresiniferatoxin. In FAAH-deficient mice, pharmacologic blockade of FAAH did not affect intestinal motility. FAAH mRNA was detected in different regions of the intestinal tract. CONCLUSIONS: We conclude that FAAH is a physiologic regulator of intestinal motility and a potential target for the development of drugs capable of reducing intestinal motility.

Abstract:
The endocannabinoid system, consisting of two cannabinoid receptors (CB1 and CB2) and the endogenous ligands anandamide (arachidonoylethanolamide (AEA)) and 2-arachidonoylglycerol (2-AG), has been shown to control food intake in both animals and humans, modulating either rewarding or quantitative aspects of the eating behavior. Moreover, hypothalamic endocannabinoids seem to be part of neural circuitry involved in the modulating effects of leptin on energy homeostasis. Therefore, alterations of the endocannabinoid system could be involved in the pathophysiology of eating disorders, where a deranged leptin signalling has been also reported. In order to verify this hypothesis, we measured plasma levels of AEA, 2-AG, and leptin in 15 women with anorexia nervosa (AN), 12 women with bulimia nervosa (BN), 11 women with binge-eating disorder (BED), and 15 healthy women. Plasma levels of AEA resulted significantly enhanced in both anorexic and BED women, but not in bulimic patients. No significant change occurred in the plasma levels of 2-AG in all the patients' groups. Moreover, circulating AEA levels were significantly and inversely correlated with plasma leptin concentrations in both healthy controls and anorexic women. These findings show for the first time a derangement in the production of the endogenous cannabinoid AEA in drug-free symptomatic women with AN or with BED. Although the pathophysiological significance of this alteration awaits further studies to be clarified, it suggests a possible involvement of AEA in the mediation of the rewarding aspects of the aberrant eating behaviors occurring in AN and BED.

Abstract:
A cannabinoid receptor orthologue (CiCBR) has been described in the sea squirt Ciona intestinalis. Here we report that CiCBR mRNA expression is highest in cerebral ganglion, branchial pharynx, heart and testis of C. intestinalis, and that this organism also contains cannabinoid receptor ligands and some of the enzymes for ligand biosynthesis and inactivation. Using liquid chromatography-mass spectrometry, the endocannabinoid anandamide was found in all tissues analysed (0.063-5.423 pmol/mg of lipid extract), with the highest concentrations being found in brain and heart. The endocannabinoid 2-arachidonoylglycerol (2-AG) was fivefold more abundant than anandamide, and was most abundant in stomach and intestine and least abundant in heart and ovaries (2.677-50.607 pmol/mg of lipid extract). Using phylogenomic analysis, we identified orthologues of several endocannabinoid synthesizing and degrading enzymes. In particular, we identified and partly sequenced a fatty acid amide hydrolase (FAAH) orthologue, showing 44% identity with human FAAH and containing nearly all the amino acids necessary for a functional FAAH enzyme. Ciona intestinalis also contained specific binding sites for cannabinoid receptor ligands, and an amidase enzyme with pH-dependency and subcellular/tissue distribution similar to mammalian FAAHs. Finally, a typical C. intestinalis behavioural response, siphon reopening after closure induced by mechanical stimulation, was inhibited by the cannabinoid receptor agonist HU-210, and this effect was significantly attenuated by mammalian cannabinoid receptor antagonists.

Abstract:
Cannabinoid CB(1) receptors are involved in ocular physiology and may regulate intraocular pressure (IOP). However, endocannabinoid levels in human ocular tissues of cornea, iris, ciliary body, retina, and choroid from normal and glaucomatous donors have not been investigated. Anandamide (N-arachidonoylethanolamine; AEA), 2-arachidonoylglycerol (2-AG), and the anandamide congener, palmitoylethanolamide (PEA), were detected in all the human tissues examined. In eyes from patients with glaucoma, significantly decreased 2-AG and PEA levels were detected in the ciliary body, an important tissue in the regulation of IOP. The findings suggest that these endogenous compounds may have a role in this disease, particularly with respect to regulation of IOP.