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

An intense effects of steric hindrance on gas-phase protonation under chemical ionization (Cl) conditions, reflected by a very high site specificity in chemical ionization mass spectrometry (CIMS) of isomeric ethyl methyl esters of 2-t-butylmaleic and 2-t-butylsuccinic acids, provides new direct evidence of kinetic control in the protonation process.

Site specific gas-phase protonation of 2-t-butylmaleates and 2-t-butylsuccinates upon chemical ionization: stereochemical effects and kinetic control

The isobutane chemical ionization (CI) mass spectra of cis- and trans-1,4-di(alkoxymethyl)cyclohexanes, with a tertiary alkoxy group ROH and a primary group R'OH, are identical, and they exhibit exclusive elimination of the alcohol ROH originating from the tertiary alkoxyl. The high abundance of the [MH - ROH] + ion and the absence of [MH - R'OH] + and MH + ions is unexpected in the case of trans-diethers, and it suggests proton transfer from the primary alkoxyl OR' to the tertiary OR group prior to the elimination of ROH, despite the large distance between them in the trans configuration. Various isomerization pathways were explored in order to account for the similar behavior of the cis- and trans-isomers upon chemical ionization. The results show that the hydrogens at positions 1 and 4 are not involved in the elimination of ROH. Methyl substitution at positions 1 and 4 leads to the competitive elimination of ROH and R'OH, indicating suppression of the proton transfer. Methyl substitution at position 1 adjacent to the primary alkoxy group has a minor effect on the chemical ionization behavior of the diethers. On the other hand, methyl substitution at position 4, adjacent to the tertiary alkoxy group, suppresses the proton transfer, (i.e. both [MH - ROH] + and [MH - R'OH] + are abundant in the CI mass spectra of the trans-isomers), indicating an effect of steric hindrance. The results suggest that direct proton transfer via a strained proton-bound transition-state occurs, and methyl substitution at positions 1 and 4 can affect the relative stability of the transition-state structures involved. Similar behavior has been observed in the CI and collision-induced dissociation spectra of 1,4-hydroxy ester cyclohexanes.

Proton transfer via strained transition states in the elimination of alcohols from MH+ ions of stereoisomeric diethers and hydroxy esters upon chemical ionization and collision‐induced dissociation

Norbornene derivatives with one or two carbonyl-containing substituents at positions 5 and 6 (anhydrides, esters, amides, cyclic ketones) undergo an electron impact induced rerro-Diels-Alder fragmentation accompanied by the migration of one hydrogen atom (RDA + H) giving rise to the [dienophile + H]+ ions. Bicyclo[2.2.2]octene derivatives substituted at C(5) and C(6) by a ring with two carbonyl groups (anhydrides, imides, diketones) undergo an RDA fragmentation accompanied by the transfer of two hydrogen atoms (RDA + 2H). Bicyclo[2.2.2]octene 5,6-diesters undergo both the RDA + H and RDA + 2H fragmentations, and the relative abundance of the resulting [dienophile + H]+ and [dienophile + 2H]+˙ ions is strongly affected by configuration.

One and two hydrogen atom migrations in the retro‐diels‐alder fragmentation of norbornene and bicyclo [2.2.2] octene derivatives under electron impact

Synthesis of exo-bicyclo[2.2.2]oct-5-ene-2,3-dicarboxylic anhydrides by thermal isomerization of trans-diacids

Elimination of CO gives rise to prominent peaks in the mass spectra of cis-1,2,3,6-tetrahydrophthalic anhydride (Ia) and its 4,5-dimethyl analogue which are almost absent in the corresponding trans-isomers. The striking difference in the fragmentation of the isomers shows that practically no rearrangement in the molecular ion occurs prior to this fragmentation. In this case a high degree of stereospecificity is observed, although hydrogen migration is apparently not involved in the process.

Asher Mandelbaum

Papers

Site specific gas-phase protonation of 2-t-butylmaleates and 2-t-butylsuccinates upon chemical ionization: stereochemical effects and kinetic control

Proton transfer via strained transition states in the elimination of alcohols from MH+ ions of stereoisomeric diethers and hydroxy esters upon chemical ionization and collision‐induced dissociation

One and two hydrogen atom migrations in the retro‐diels‐alder fragmentation of norbornene and bicyclo [2.2.2] octene derivatives under electron impact

Synthesis of exo-bicyclo[2.2.2]oct-5-ene-2,3-dicarboxylic anhydrides by thermal isomerization of trans-diacids

Studies in mass spectrometry—XVI. The striking difference between the mass spectra of stereoisomeric cyclic anhydrides