THE major active ingredient of marijuana, Δ9-tetrahydrocannabi-nol (Δ9-THC), has been used as a psychoactive agent for thousands of years. Marijuana, and Δ9-THC, also exert a wide range of other effects including analgesia, anti-inflammation, immunosuppression, anticonvulsion, alleviation of intraocular pressure in glaucoma, and attenuation of vomiting1. The clinical application of cannabinoids has, however, been limited by their psychoactive effects, and this has led to interest in the biochemical bases of their action. Progress stemmed initially from the synthesis of potent derivatives of δ9-THC4,5, and more recently from the cloning of a gene encoding a G-protein-coupled receptor for cannabinoids6. This receptor is expressed in the brain but not in the periphery, except for a low level in testes. It has been proposed that the non-psychoactive effects of cannabinoids are either mediated centrally or through direct interaction with other, non-receptor proteins1,7,8. Here we report the cloning of a receptor for cannabinoids that is not expressed in the brain but rather in macrophages in the marginal zone of spleen.

Molecular characterization of a peripheral receptor for cannabinoids

Identification of an endogenous 2-monoglyceride, present in canine gut, that binds to cannabinoid receptors.

Two types of cannabinoid receptor have been discovered so far, CB(1) (2.1: CBD:1:CB1:), cloned in 1990, and CB(2) (2.1:CBD:2:CB2:), cloned in 1993. Distinction between these receptors is based on differences in their predicted amino acid sequence, signaling mechanisms, tissue distribution, and sensitivity to certain potent agonists and antagonists that show marked selectivity for one or the other receptor type. Cannabinoid receptors CB(1) and CB(2) exhibit 48% amino acid sequence identity. Both receptor types are coupled through G proteins to adenylyl cyclase and mitogen-activated protein kinase. CB(1) receptors are also coupled through G proteins to several types of calcium and potassium channels. These receptors exist primarily on central and peripheral neurons, one of their functions being to inhibit neurotransmitter release. Indeed, endogenous CB(1) agonists probably serve as retrograde synaptic messengers. CB(2) receptors are present mainly on immune cells. Such cells also express CB(1) receptors, albeit to a lesser extent, with both receptor types exerting a broad spectrum of immune effects that includes modulation of cytokine release. Of several endogenous agonists for cannabinoid receptors identified thus far, the most notable are arachidonoylethanolamide, 2-arachidonoylglycerol, and 2-arachidonylglyceryl ether. It is unclear whether these eicosanoid molecules are the only, or primary, endogenous agonists. Hence, we consider it premature to rename cannabinoid receptors after an endogenous agonist as is recommended by the International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification. Although pharmacological evidence for the existence of additional types of cannabinoid receptor is emerging, other kinds of supporting evidence are still lacking.

https://pharmrev.aspetjournals.org/content/pharmrev/54/2/161.full.pdf

International Union of Pharmacology. XXVII. Classification of Cannabinoid Receptors

The endogenous cannabinoid receptor agonist anandamide is a powerful vasodilator of isolated vascular preparations, but its mechanism of action is unclear. Here we show that the vasodilator response to anandamide in isolated arteries is capsaicin-sensitive and accompanied by release of calcitonin-gene-related peptide (CGRP). The selective CGRP-receptor antagonist 8-37 CGRP, but not the cannabinoid CB1 receptor blocker SR141716A, inhibited the vasodilator effect of anandamide. Other endogenous (2-arachidonylglycerol, palmitylethanolamide) and synthetic (HU 210, WIN 55,212-2, CP 55,940) CB1 and CB2 receptor agonists could not mimic the action of anandamide. The selective 'vanilloid receptor' antagonist capsazepine inhibited anandamide-induced vasodilation and release of CGRP. In patch-clamp experiments on cells expressing the cloned vanilloid receptor (VR1), anandamide induced a capsazepine-sensitive current in whole cells and isolated membrane patches. Our results indicate that anandamide induces vasodilation by activating vanilloid receptors on perivascular sensory nerves and causing release of CGRP. The vanilloid receptor may thus be another molecular target for endogenous anandamide, besides cannabinoid receptors, in the nervous and cardiovascular systems.

Vanilloid receptors on sensory nerves mediate the vasodilator action of anandamide

Endogenous neuromodulatory molecules are commonly coupled to specific metabolic enzymes to ensure rapid signal inactivation. Thus, acetylcholine is hydrolysed by acetylcholine esterase and tryptamine neurotransmitters like serotonin are degraded by monoamine oxidases. Previously, we reported the structure and sleep-inducing properties of cis-9-octadecenamide, a lipid isolated from the cerebrospinal fluid of sleep-deprived cats. cis-9-Octadecenamide, or oleamide, has since been shown to affect serotonergic systems and block gap-junction communication in glial cells (our unpublished results). We also identified a membrane-bound enzyme activity that hydrolyses oleamide to its inactive acid, oleic acid. We now report the mechanism-based isolation, cloning and expression of this enzyme activity, originally named oleamide hydrolase, from rat liver plasma membranes. We also show that oleamide hydrolase converts anandamide, a fatty-acid amide identified as the endogenous ligand for the cannabinoid receptor, to arachidonic acid, indicating that oleamide hydrolase may serve as the general inactivating enzyme for a growing family of bioactive signalling molecules, the fatty-acid amides. Therefore we will hereafter refer to oleamide hydrolase as fatty-acid amide hydrolase, in recognition of the plurality of fatty-acid amides that the enzyme can accept as substrates.

Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides

Stereochemical assignment by mass spectrometry : Double bond geometrical isomers of 3,5-dialkylcyclohexylidene acetic acid esters and 3-alkyl-2-alkenoates

The eilmination of methanol from the MD + ion of 2,3-cis-3-methoxytricyclo[6220 2,7 ]dodeca-9-ene, endo-2, upon chemical ionization (CI) gives rise to both [MD - MeOD] + and [MD - MeOH] + ions Only the [MD - MeOH] + ion is formed under collision-induced dissociation (CID) conditions This is in contrast with the behavior of the 2,3-trans-stereoisomer exo-2 and with saturated analogs which undergo exclusive elimination of MeOD The unusual elimination of MeOH from endo-2 indicates transfer of the external deuteron in the MD + ion from the oxygen atom to the interior of the organic moiety and a back transfer of a hydrogen from the organic moiety to the oxygen atom prior to the C-O bond dissociation step A deuterium labeling study showed that the hydrogen atom involved in this elimination process originates at position 3 (formal 1,1-elimination) These results suggest a multi-step mechanism for this unique stereospecific methanol elimination, initiated by a proton transfer from the methoxy group to the double bond followed by a 1,4-methoxyl migration from C-3 to C-10 The proposed mechanism finds support in the Cl and CID study of deuterium-labeled analogs which have a methoxy group at position 10

Stereospecific multi‐step elimination of methanol involving 1,4‐methoxyl migration from the MH+ ion of 2,3‐cis‐3‐methoxytricyclo[6.2.2.02,7]dodeca‐9‐ene

Organometallic compounds and their preparation

The structural features of arenemetal carbonyl π-complexes have been shown 2 to be analogous to the “sandwich” structure of metallocenes such as ferrocene. It appeared to us that by suitable choice of the arene moiety, as for example m -methoxybenzoic acid, the synthesis of the chromium tricarbonyl derivative, III, should yield a racemic mixture since III does not have any elements of symmetry. The presence of the carboxylic group in III allowed for the preparation of diastereoisomers with optically active bases, their separation by fractional crystallization and the preparation of the two enantiomers of III. This appears to be the first reported 3 separation of enantiomers of an arenechromium tricarbonyl derivative. Work currently in progress is aimed at an investigation of the conditions for the racemization compounds such as III as well as of symmetrically substituted naphthalenechromium tricarbonyl derivatives, IV. In compounds such as III racemization could take place,e.g. by intermolecular transfers, whereas in IV racemization could supposedly take place by an intramolecular transfer of the —Cr(CO) 3 moiety from one arene ring to the other.

Asher Mandelbaum

Papers

Stereochemical assignment by mass spectrometry : Double bond geometrical isomers of 3,5-dialkylcyclohexylidene acetic acid esters and 3-alkyl-2-alkenoates

Stereospecific multi‐step elimination of methanol involving 1,4‐methoxyl migration from the MH+ ion of 2,3‐cis‐3‐methoxytricyclo[6.2.2.02,7]dodeca‐9‐ene

Organometallic compounds and their preparation

Organometallic studies, vii the resolution on an arenechromium tricarbonyl racemic mixture

Polycyclic Studies. II. The Ultraviolet Spectra of Some Diels-Alder Adducts1