Arachidonylethanolamide, an arachidonic acid derivative in porcine brain, was identified in a screen for endogenous ligands for the cannabinoid receptor. The structure of this compound, which has been named "anandamide," was determined by mass spectrometry and nuclear magnetic resonance spectroscopy and was confirmed by synthesis. Anandamide inhibited the specific binding of a radiolabeled cannabinoid probe to synaptosomal membranes in a manner typical of competitive ligands and produced a concentration-dependent inhibition of the electrically evoked twitch response to the mouse vas deferens, a characteristic effect of psychotropic cannabinoids. These properties suggest that anandamide may function as a natural ligand for the cannabinoid receptor.

https://science.sciencemag.org/content/sci/258/5090/1946.full.pdf

Isolation and structure of a brain constituent that binds to the cannabinoid receptor

Marijuana and many of its constituent cannabinoids influence the central nervous system (CNS) in a complex and dose-dependent manner. Although CNS depression and analgesia are well documented effects of the cannabinoids, the mechanisms responsible for these and other cannabinoid-induced effects are not so far known. The hydrophobic nature of these substances has suggested that cannabinoids resemble anaesthetic agents in their action, that is, they nonspecifically disrupt cellular membranes. Recent evidence, however, has supported a mechanism involving a G protein-coupled receptor found in brain and neural cell lines, and which inhibits adenylate cyclase activity in a dose-dependent, stereoselective and pertussis toxin-sensitive manner. Also, the receptor is more responsive to psychoactive cannabinoids than to non-psychoactive cannabinoids. Here we report the cloning and expression of a complementary DNA that encodes a G protein-coupled receptor with all of these properties. Its messenger RNA is found in cell lines and regions of the brain that have cannabinoid receptors. These findings suggest that this protein is involved in cannabinoid-induced CNS effects (including alterations in mood and cognition) experienced by users of marijuana.

Structure of a cannabinoid receptor and functional expression of the cloned cDNA

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.

Drug addiction, dysregulation of reward, and allostasis.

The determination and characterization of a cannabinoid receptor from brain are reported. A biologically active bicyclic cannabinoid analgetic CP-55,940 was tritium-labeled to high specific activity. Conditions for binding to rat brain P2 membranes and synaptosomes were established. The pH optimum was between 7 and 8, and specific binding could be eliminated by heating the membranes to 60 degrees. Binding to the P2 membranes was linear within the range of 10 to 50 micrograms of protein/ml. Specific binding (defined as total binding displaced by 1 microM delta 9-tetrahydrocannabinol (delta 9-THC) or 100 nM desacetyllevonantradol) was saturable. The Kd determined from Scatchard analysis was 133 pM, and the Bmax for rat cortical P2 membranes was 1.85 pmol/mg of protein. The Hill coefficient for [3H]CP-55,940 approximated 1, indicating that, under the conditions of assay, a single class of binding sites was determined that did not exhibit cooperativity. The binding was rapid (kon approximately 2.6 x 10(-4) pM-1 min-1) and reversible (Koff approximately 0.016 min-1) and (koff' greater than 0.06 min-1). The two Kd values estimated from the kinetic constants approximately 55 pM and exceeded 200 pM, respectively. The binding of the agonist ligand [3H]CP-55,940 was decreased by the nonhydrolyzable GTP analog guanylylimidodiphosphate. The guanine nucleotide induced a more rapid dissociation of the ligand from the binding site, consistent with an allosteric regulation of the putative receptor by a G protein. The binding was also sensitive to MgCl2 and CaCl2. Binding of [3H]CP-55,940 was displaced by cannabinoid drugs in the following order of potency: CP-55,940 greater than or equal to desacetyllevonantradol greater than 11-OH-delta 9-THC = delta 9-THC greater than cannabinol. Cannabidiol and cannabigerol displaced [3H]CP-55,940 by less than 50% at 1 microM concentrations. The (-)-isomer of CP-55,940 displaced with 50-fold greater potency than the (+)-isomer. This pharmacology is comparable to both the inhibition of adenylate cyclase in vitro and the analgetic activity of these compounds in vivo. The criteria for a high affinity, stereoselective, pharmacologically distinct cannabinoid receptor in brain tissue have been fulfilled.

Determination and characterization of a cannabinoid receptor in rat brain.

[3H]CP 55,940, a radiolabeled synthetic cannabinoid, which is 10-100 times more potent in vivo than delta 9-tetrahydrocannabinol, was used to characterize and localize a specific cannabinoid receptor in brain sections. The potencies of a series of natural and synthetic cannabinoids as competitors of [3H]CP 55,940 binding correlated closely with their relative potencies in several biological assays, suggesting that the receptor characterized in our in vitro assay is the same receptor that mediates behavioral and pharmacological effects of cannabinoids, including human subjective experience. Autoradiography of cannabinoid receptors in brain sections from several mammalian species, including human, reveals a unique and conserved distribution; binding is most dense in outflow nuclei of the basal ganglia--the substantia nigra pars reticulata and globus pallidus--and in the hippocampus and cerebellum. Generally high densities in forebrain and cerebellum implicate roles for cannabinoids in cognition and movement. Sparse densities in lower brainstem areas controlling cardiovascular and respiratory functions may explain why high doses of delta 9-tetrahydrocannabinol are not lethal.

/pdf/cannabinoid-receptor-localization-in-brain-k6t4ds8tqu.pdf

Cannabinoid receptor localization in brain

Although a receptor exists for cannabinoid drugs, it is uncertain which pharmacological actions this receptor mediates. This structure-activity relationship investigation was initiated to determine which effects might correspond to binding affinity for the cannabinoid receptor, as well as to explore the binding requirements of this site. The ability of nearly 60 cannabinoids to displace [3H]CP-55,940 [(-)-3-[2-hydroxy-4-(1,1-dimethylheptyl) phenyl]-4-[3-hydroxy propyl] cyclohexan-1-ol] was determined before establishing correlations between receptor affinity and in vivo pharmacological potency. Analysis of [3H]CP-55,940 binding indicated a Hill coefficient of 0.97, a Bmax of 499 pM (3.3 pmol/mg of protein) and an apparent Kd of 924 pM. Closer inspection indicated the binding assay exhibited "zone B" characteristics, and use of correction equations indicated a true Kd for CP-55,940 of 675 pM. The structure-activity relationship indicated the importance of side chain structure to high-affinity binding, with the most potent analogs (K1 < 10 nM) possessing either a dimethylheptyl side-chain, a similarly complex branched side chain or a halogen substituent at the 5' position. Comparative analysis of K1 values to in vivo potency in a mouse model indicated a high degree of correlation between parameters for the depression of spontaneous locomotor activity (r = 0.91) and for the production of antinociception (r = 0.90), hypothermia (r = 0.89) and catalepsy (r = 0.85). Similarly high correlations were demonstrated between binding affinity and in vivo potency in both the rat drug discrimination model (r = 0.81) and for psychotomimetic activity in humans (r = 0.88).(ABSTRACT TRUNCATED AT 250 WORDS)

Cannabinoid structure-activity relationships: correlation of receptor binding and in vivo activities.

Extensive structure-activity relationship studies have demonstrated that specific requirements within the cannabinoid structure are necessary to produce potent analgesia. A three-point association between the agonist and the receptor mediating analgesia consists of: 1) the C ring hydroxyl, 2) the phenolic A ring hydroxyl, and 3) the A ring alkyl hydrophobic side chain. Potent tricyclic and bicyclic structures were synthesized as "nonclassical" cannabinoid analgetics that conform to this agonist-receptor three-point interaction model. At the cellular level, centrally active cannabinoid drugs inhibit adenylate cyclase activity in a neuroblastoma cell line. The structure-activity relationship profile for inhibition of adenylate cyclase in vitro was consistent with this same three-point association of agonists with the receptor. A correlation exists between the potency of drugs to produce analgesia in vivo and to inhibit adenylate cyclase in vitro. Enantio- and stereoselectivity were exhibited by the nonclassical cannabinoid compounds for both the analgetic response and the ability to inhibit adenylate cyclase. The magnitude of the enantioselective response was equal for both the biochemical and physiological endpoints. Based on the parallels in structure-activity relationships and the enantioselective effects, it is postulated that the receptor that is associated with the regulation of adenylate cyclase in vitro may be the same receptor as that mediating analgesia in vivo. A conceptualization of the cannabinoid analgetic receptor is presented.

Nonclassical cannabinoid analgetics inhibit adenylate cyclase: development of a cannabinoid receptor model.

Cannabimimetic compounds, such as delta 9-tetrahydrocannabinol (delta 9-THC), evoke analgesia in addition to other behavioral responses in humans and animals. The cannabinoid receptor mediating this response has been characterized by its ability to bind the cannabinoid agonist [3H]CP-55,940 and to inhibit adenylyl cyclase via Gi. An investigation of structural requirements for antinociceptive activity of cannabinoid structures led to the development of a simple bicyclic cannabinoid agonist, CP-47,497, that possessed a spectrum of cannabinoid activities in animals that resembled that of delta 9-THC. The present investigation examines several series of CP-47,497 analogs for their binding affinity at the cannabinoid receptor and their ability to evoke analgesia in rodents. Analogs substituted at the C-3 alkyl side chain exhibited maximal affinity for the cannabinoid receptor with side chains of seven or eight carbons in length. Analgesic potency paralleled the receptor-binding affinity. The cyclohexyl ring was optimized as a six- or seven-membered ring structure for binding as well as analgesic activity. Cyclohexyl alkyl side chain extensions of up to four carbons in length had little influence on the affinity for the receptor or analgesic activity. Hydroxyalkyl side chains exhibited optimal binding affinity and antinociceptive activity at three or four carbon atoms in length; however, polar groups closer to the ring diminished binding to the receptor. The importance of the phenolic and cyclohexyl hydroxyl groups for binding affinity was demonstrated. In general, analgesic activity correlated well with the affinity of these analogs for the cannabinoid receptor. Exceptions could be explained by metabolic transformations likely to occur in vivo.

M R Johnson

Papers

Determination and characterization of a cannabinoid receptor in rat brain.

Cannabinoid receptor localization in brain

Cannabinoid structure-activity relationships: correlation of receptor binding and in vivo activities.

Nonclassical cannabinoid analgetics inhibit adenylate cyclase: development of a cannabinoid receptor model.

Structure-activity relationships for cannabinoid receptor-binding and analgesic activity: studies of bicyclic cannabinoid analogs