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 ionotropic glutamate receptors are ligand-gated ion channels that mediate the vast majority of excitatory neurotransmission in the brain. The cloning of cDNAs encoding glutamate receptor subunits, which occurred mainly between 1989 and 1992 ([Hollmann and Heinemann, 1994][1]), stimulated this

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The glutamate receptor ion channels

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Inositol trisphosphate and diacylglycerol as second messengers.

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.

Drugs of abuse: anatomy, pharmacology and function of reward pathways

gamma-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in mammalian brain, is believed to act by increasing membrane conductance of chloride ions. In this study it was found that GABA agonists increased the uptake of chloride-36 by cell-free membrane preparations from mouse brain. This influx was rapid (less than 5 seconds), and 13 micromolar GABA produced a half-maximal effect. The GABA antagonists (bicuculline and picrotoxin) blocked the effect of GABA, whereas pentobarbital enhanced the action. This may be the first demonstration of functional coupling among GABA and barbiturate receptors and chloride channels in isolated membranes. The technique should facilitate biochemical and pharmacological studies of GABA receptor-effector coupling.

https://science.sciencemag.org/content/sci/228/4703/1108.full.pdf

Functional coupling of γ-aminobutyric acid receptors to chloride channels in brain membranes

Calcium/phospholipid-dependent protein kinase (protein kinase C, PKC) has been suggested to play a role in the sensitivity of gamma-aminobutyrate type A (GABAA) receptors to ethanol. We tested a line of null mutant mice that lacks the gamma isoform of PKC (PKC gamma) to determine the role of this brain-specific isoenzyme in ethanol sensitivity. We found that the mutation reduced the amount of PKC gamma immunoreactivity in cerebellum to undetectable levels without altering the levels of the alpha, beta I, or beta II isoforms of PKC. The mutant mice display reduced sensitivity to the effects of ethanol on loss of righting reflex and hypothermia but show normal responses to flunitrazepam or pentobarbital. Likewise, GABAA receptor function of isolated brain membranes showed that the mutation abolished the action of ethanol but did not alter actions of flunitrazepam or pentobarbital. These studies show the unique interactions of ethanol with GABAA receptors and suggest protein kinase isoenzymes as possible determinants of genetic differences in response to ethanol.

https://www.pnas.org/content/pnas/92/9/3658.full.pdf

Mutant mice lacking the gamma isoform of protein kinase C show decreased behavioral actions of ethanol and altered function of gamma-aminobutyrate type A receptors.

The effect of ethanol exposure in vitro on the GABA receptor-operated chloride channel was evaluated by monitoring 36Cl- influx in a membrane vesicle suspension (microsacs) prepared from mouse cerebellum. These experiments directly demonstrate ethanol augmentation of muscimol-stimulated chloride flux. DBA/2J mice were made tolerant to and dependent on ethanol by administration of an ethanol containing liquid diet for 7 days. Exposure to physiologically relevant concentrations of ethanol (10-45 mM) in vitro potentiated muscimol stimulation of 36Cl- uptake in control (pair-fed) membranes, but had no effect on cerebellar microsacs from tolerant/dependent mice. Muscimol stimulation of 36Cl- uptake was not different for pair-fed and ethanol-treated mice. Augmentation of muscimol-induced 36Cl- flux by in vitro ethanol was abolished by a single 4 g/kg injection of ethanol. This "acute tolerance" occurred within 5 min and disappeared within 24 hr after ethanol treatment. The reduced sensitivity of ethanol treated (chronic and acute) mice to ethanol potentiation of muscimol stimulated 36Cl- uptake offers a biochemical correlate to the phenomenon of ethanol tolerance. Moreover, the findings suggest that this biochemical tolerance develops rapidly following a single hypnotic dose of ethanol.

Acute and chronic ethanol treatments alter GABA receptor-operated chloride channels.

In vitro addition of ethanol or pentobarbital to synaptosomes isolated from rat or mouse brain inhibited the depolarization-dependent uptake of calcium without affecting uptake under nondepolarizing conditions. Ethanol inhibited the uptake in a concentration-dependent manner over the range of 45 to 720 mM. Analysis of the effects of ethanol and pentobarbital on calcium uptake at different temperatures indicated that the drugs did not change the activation energy of the process but shifted the Arrhenius curves toward higher temperatures. Synaptosomes isolated from mice chronically ingesting ethanol were found to be tolerant to the inhibitory effects of ethanol and pentobarbital but not tolerant to the inhibitory effects of acetaldehyde. Synaptosomes from ethanol tolerant-dependent mice accumulated less calcium in the absence of ethanol than did synaptosomes from control mice. This depression of uptake was reversed by in vitro exposure of the synaptosomes to a low concentration of ethanol, suggesting that it represents a biochemical response to withdrawal of alcohol. A single acute injection of ethanol 1 hr before sacrifice did not alter the calcium uptake activity or the drug sensitivity of the synaptosomes. These results suggest that the known inhibitory effects of ethanol and pentobarbital on the stimulated release of neurotransmitters and the development of tolerance of these effects may be mediated by the inhibition of the depolarization-dependent influx of calcium by these drugs.

Inhibition of synaptosomal calcium uptake by ethanol.

Molecular cloning of cDNAs coding for ligand-gated ion channel subunits makes it possible to study the pharmacology of recombinant receptors with defined subunit compositions. Many laboratories have used these techniques recently to study actions of agents that produce general anesthesia. We review the effects of volatile and intravenous anesthetics on recombinant GABAA, glycine, AMPA, kainate, NMDA, and 5HT3 receptors. Evidence for and against specific ligand-gated ion channel subunits as targets responsible for anesthesia or the side effects of anesthetic agents is discussed for each type of receptor. Subunit specific actions of some of the agents suggest that construction and testing of certain chimeric receptor subunits may be useful for defining the amino acid sequences responsible for anesthetic actions.

R A Harris

Papers

Functional coupling of γ-aminobutyric acid receptors to chloride channels in brain membranes

Mutant mice lacking the gamma isoform of protein kinase C show decreased behavioral actions of ethanol and altered function of gamma-aminobutyrate type A receptors.

Acute and chronic ethanol treatments alter GABA receptor-operated chloride channels.

Inhibition of synaptosomal calcium uptake by ethanol.

Actions of anesthetics on ligand-gated ion channels: role of receptor subunit composition