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

Stereospecific Retro-Diels–Alder fragmentation in negative-ion mass spectrometry

The electron impact mass spectra of isomeric methyl ethym and ethyl methyl halosuccinates (X=Cl and Br) are surprisingly different. Only the isomers with the ethyl group remote from the halogen give rise to [M―X] + ions. A low-energy collision-induced dissociation study of deuterium-labelled analogues of the former isomers indicates that the [M―X] + ions are mixtures of protonated methyl ethyl maleate (major component, >85%) and fumarate, and the loss of the halogen atom is a multi-step process including at least two specific hydrogen transfers. Migration of a β-hydrogen atom to the carbonyl oxygen within the ethoxycarbonyl group produces a primary radical site in a distonic intermediate which, by subsequent abstraction of a hydrogen atom from C(3), triggers the ejection of X from C(2) with concomitant double fond formation. Whereas in the other isomer an [M-X] + ion is absent or negligible, a characteristic double loss of C 2 H 4 and CO 2 is observed

Behaviour of isomeric methyl ethyl and ethyl methyl halosuccinates under electron impact. Elimination of a halogen atom by a multi‐step mechanism

On the elimination of methanol from stereoisomeric arylcyclohexyl methyl ethers under electron impact

The elimination of HCN from the molecular ion of o-nitrobenzyl cyanide under electron impact is shown by deuterium labelling to take place with the abstraction of one of the benzylic hydrogens. This is the first case of a specific 1,1-elimination. o-Nitrobenzyl chloride and o-nitrobenzyl alcohol do not undergo similar eliminations.

Formal 1,1-elimination of hydrogen cyanide from o-nitrobenzyl cyanide under electron impact

Retro-Diels–Alder (RDA) fragmentation of cis- and trans-2,3-diethoxycarbonyl-5,6,7,8-dibenzo-bicyclo[2.2.2]octanes under isobutane and methane chemical ionization conditions is highly stereospecific, giving rise to protonated diethyl maleate and fumarate, respectively. This behaviour indicates a single-step concerted mechanism, analogous to the ground-state RDA process that occurs in neutral molecules in the condensed phase. The analogous dissociation is partially stereospecific in stereoisomeric endo-,exo- and trans-2,3-diethoxycarbonylbicyclo[2.2.1]heptanes and non-stereospecific in endo-,exo- and trans-2,3-diethoxycarbonyl-5,6-benzobicyclo[2.2.2]octanes, indicating the involvement of a stepwise mechanism in the latter two systems. The different behaviour of the above systems is explained in terms of the energy of the RDA fragmentation. The differentiation and quantitative estimates of protonated diethyl maleate and fumarate were obtained from collision-induced dissociation measurements.

Asher Mandelbaum

Papers

Stereospecific Retro-Diels–Alder fragmentation in negative-ion mass spectrometry

Behaviour of isomeric methyl ethyl and ethyl methyl halosuccinates under electron impact. Elimination of a halogen atom by a multi‐step mechanism

On the elimination of methanol from stereoisomeric arylcyclohexyl methyl ethers under electron impact

Formal 1,1-elimination of hydrogen cyanide from o-nitrobenzyl cyanide under electron impact

Stereospecificity and Concertedness of Retro‐Diels–Alder Fragmentation in Some Diester Systems Upon Chemical Ionization