Anandamide, an endogenous cannabimimetic eicosanoid, binds to the cloned human cannabinoid receptor and stimulates receptor-mediated signal transduction.

Abstract

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 ...

Analysis of anandamide, an endogenous cannabinoid substance, and of other natural N-acylethanolamines

“…catalepsy, hypothermia and analgesia) (3, and, for review, 19). Anandamide was also shown to activate transfected cannabinoid receptors in Chinese hamster ovary cells, thereby triggering cannabinoidassociated transmembrane signalling events, thus further substantiating its role as an endogenous cannabinoid receptor ligand (4,5, and, for review, 6).…”

“…The possibility that, in addition to anandamide, other NAEs, with either saturated or polyunsaturated acyl moieties, are produced by mammalian neurons and participate in neuronal signalling has been strongly suggested by recent investigations. In these studies, several additional NAEs have been shown, on one hand, to exert cannabinoid-like activities similar to those of anandamide (5,15) and, on the other, to be produced by intact neurons challenged with various physiological stimuli (16) and to be present in brain tissue (15). Therefore, it can be foreseen that an ever increasing number of efforts will be aimed, in the next years, at fully understanding the physiological role of this important class of lipids, as well as the molecular mechanisms by which their synthesis and inactivation are regulated.…”

Cannabinoids and neuroinflammation

“…Anandamide induces AA release from J774 mouse macrophages, and this is blocked by pertussis toxin, an inhibitor of G i/o proteins . However, anandamide also induces AA release in cells that do not express CB 1 or CB 2 receptors (Felder et al, 1993;Felder et al, 1995). THC induces AA release from RAW 264.7 mouse macrophages, and this is likely mediated by the CB 2 receptor (Hunter & Burstein, 1997).…”

GC/MS Analysis of Anandamide and Quantification of N‐Arachidonoylphosphatidylethanolamides in Various Brain Regions, Spinal Cord, Testis, and Spleen of the Rat

“…NAE displayed similar in vivo and in vitro effects as a cannabinoid receptor agonist acting through the CB1 cannabinoid receptor (for review, see Felder and Glass, 1998). NAE induces antinociception, hypomotility, hypothermia, and catalepsy when injected into mice, and, in vitro, it is capable of inhibiting forskolininduced accumulation of intracellular cyclic AMP and Ntype Ca2+ channels in several types of cells (Felder et al, 1993;Vogel et al, 1993).…”