Arachidonylethanolamide (anandamide), a candidate endogenous cannabinoid ligand, has recently been isolated from porcine brain and displayed cannabinoid-like binding activity to synaptosomal membrane preparations and mimicked cannabinoid-induced inhibition of the twitch response in isolated murine vas deferens. In this study, anandamide and several congeners were evaluated as cannabinoid agonists by examining their ability to bind to the cloned cannabinoid receptor, inhibit forskolin-stimulated cAMP accumulation, inhibit N-type calcium channels, and stimulate one or more functional second messenger responses. Synthetic anandamide, and all but one congener, competed for [3H]CP55,940 binding to plasma membranes prepared from L cells expressing the rat cannabinoid receptor. The ability of anandamide to activate receptor-mediated signal transduction was evaluated in Chinese hamster ovary (CHO) cells expressing the human cannabinoid receptor (HCR, termed CHO-HCR cells) and compared to control CHO cells expressing the muscarinic m5 receptor (CHOm5 cells). Anandamide inhibited forskolin-stimulated cAMP accumulation in CHO-HCR cells, but not in CHOm5 cells, and this response was blocked with pertussis toxin. N-type calcium channels were inhibited by anandamide and several active congeners in N18 neuroblastoma cells. Anandamide stimulated arachidonic acid and intracellular calcium release in both CHOm5 and CHO-HCR cells and had no effect on the release of inositol phosphates or phosphatidylethanol, generated after activation of phospholipase C and D, respectively. Anandamide appears to exhibit the essential criteria required to be classified as a cannabinoid/anandamide receptor agonist and shares similar nonreceptor effects on arachidonic acid and intracellular calcium release as other cannabinoid agonists.Both the psychoactive and medicinal properties of marijuana have been known for centuries, but not until the last decade has a clear mechanism of action been ascribed to A9-tetrahydrocannabinol (THC), the active principle of marijuana. It is now known that THC and other more potent synthetic cannabinoid agonists bind to specific cannabinoid receptors and couple functionally to inhibit adenylate cyclase (1, 2) and inhibit N-type calcium channels via a pertussis toxin-sensitive guanine nucleotide binding protein (G protein) (3,4). The existence of the cannabinoid receptor was corroborated with the cloning of a cannabinoid receptor gene from both rat and human (5, 6). To date only a single cannabinoid receptor gene has been identified and its nucleotide sequence indicates that it belongs to the superfamily of G-protein-coupled receptors. Expression studies indicateThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.that the cloned receptor and the native receptor display similar binding and functional coupling to the inhibition of adenylate cyclase (5-7). The ...

Chronic inflammation activates the tryptophan-degrading enzyme IDO, which is well known to impair T cell proliferation. We have previously established that bacille Calmette-Guérin (BCG), an attenuated form of Mycobacterium bovis, is associated with persistent activation of IDO in the brain and chronic depressive-like behavior, but a causative role has not been established. In these experiments we used both pharmacologic and genetic approaches to test the hypothesis that IDO activation is responsible for the development of chronic depression that follows BCG infection. BCG induced TNF-α, IFN-γ, and IDO mRNA steady-state transcripts in the brain as well as the enzyme 3-hydroxyanthranilic acid oxygenase (3-HAO) that lies downstream of IDO and generates the neuroactive metabolite, quinolinic acid. Behaviors characteristic of depression were apparent 1 wk after BCG infection. Pretreatment with the competitive IDO inhibitor 1-methyltryptophan fully blocked BCG-induced depressive-like behaviors. Importantly, IDO-deficient mice were completely resistant to BCG-induced depressive-like behavior but responded normally to BCG induction of proinflammatory cytokines. These results are the first to prove that the BCG-induced persistent activation of IDO is accompanied by the induction of 3-hydroxyanthranilic acid oxygenase and that IDO is required as an initial step for the subsequent development of chronic depressive-like behavior.

Anandamide (arachidonylethanolamide) is a novel lipid neurotransmitter first isolated from porcine brain which has been shown to be a functional agonist for the cannabinoid CB1 and CB2 receptors. Anandamide has never been isolated from human brain or peripheral tissues and its role in human physiology has not been examined. Anandamide was measured by LC/MS/MS and was found in human and rat hippocampus (and human parahippocampal cortex), striatum, and cerebellum, brain areas known to express high levels of CB1 cannabinoid receptors. Significant levels of anandamide were also found in the thalamus which expresses low levels of CB1 receptors. Anandamide was also found in human and rat spleen which expresses high levels of the CB2 cannabinoid receptor. Small amounts of anandamide were also detected in human heart and rat skin. Only trace quantities were detected in pooled human serum, plasma, and CSF. The distribution of anandamide in human brain and spleen supports its potential role as an endogenous agonist in central and peripheral tissues. The low levels found in serum, plasma, and CSF suggest that it is metabolized in tissues where it is synthesized, and that its action is probably not hormonal in nature.Key words: Anandamide; Cannabis; Cannabinoid receptor; Marijuana porcine brain and found to be a lipid of novel structure [7]. Anandamide displayed specific binding to the CBI receptor and inhibited a prototypical twitch response in mouse vas deferens. Anandamide has also been shown to induce similar behavioral [8,9], pharmacological [10,11], and signal transduction effects [12] as classical cannabinoid agonists, but high concentrations were required to induce these effects. Levels of anandamide were first estimated to occur at 0.4 pmol/g (133 pg/g) in whole porcine brain [7], and recently quantitated in porcine and bovine brain at 173 pmol/g (60 ng/g) and 101 pmol/g (35 ng/g) respectively [13]. A recent study reports levels of anandamide in rat testis to be considerably lower (6 pmol/ g, 2.1 ng/g) [14]. However, anandamide has never been isolated from human tissue or fluids. Furthermore, levels of anandamide have not been measured in regions of rat brain or in tissues such as spleen where CB2 receptors have been shown to be expressed at high levels. Studies of anandamide distribution should help elucidate the physiologic role of anandamide as a cannabimimetic eicosanoid and possibly broader functions. In this study we report the isolation and quantitation of anandamide by liquid chromatography/mass spectrometry in various tissues and fluids from postmortem human and rat.

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The microtexture and elemental composition of the backing of electrical tapes have been shown to be highly discriminating. In this study, the organic composition of electrical tape was evaluated as a complementary means of distinguishing tape brands. The backing and adhesive of 72 rolls of electrical tape were analyzed via Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR FTIR) and discriminant analysis was used to classify all samples by brand. Generally, the accuracy for FTIR data (88-99%) was higher than that for elemental data (86-94%). FTIR spectra from the adhesive layer were the most discriminating. In separate studies, two fragments of blast-damaged tape were correctly assigned to their brand of origin and discriminant analysis was used to quantitatively associate or exclude tape samples from two bombing cases.

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