International Union of Basic and Clinical Pharmacology. LXXIX. Cannabinoid receptors and their ligands: beyond CB1 and CB2.

There are at least two types of cannabinoid receptors (CB(1) and CB(2)). Ligands activating these G protein-coupled receptors (GPCRs) include the phytocannabinoid Δ(9)-tetrahydrocannabinol, numerous synthetic compounds, and endogenous compounds known as endocannabinoids. Cannabinoid receptor antagonists have also been developed. Some of these ligands activate or block one type of cannabinoid receptor more potently than the other type. This review summarizes current data indicating the extent to which cannabinoid receptor ligands undergo orthosteric or allosteric interactions with non-CB(1), non-CB(2) established GPCRs, deorphanized receptors such as GPR55, ligand-gated ion channels, transient receptor potential (TRP) channels, and other ion channels or peroxisome proliferator-activated nuclear receptors. From these data, it is clear that some ligands that interact similarly with CB(1) and/or CB(2) receptors are likely to display significantly different pharmacological profiles. The review also lists some criteria that any novel "CB(3)" cannabinoid receptor or channel should fulfil and concludes that these criteria are not currently met by any non-CB(1), non-CB(2) pharmacological receptor or channel. However, it does identify certain pharmacological targets that should be investigated further as potential CB(3) receptors or channels. These include TRP vanilloid 1, which possibly functions as an ionotropic cannabinoid receptor under physiological and/or pathological conditions, and some deorphanized GPCRs. Also discussed are 1) the ability of CB(1) receptors to form heteromeric complexes with certain other GPCRs, 2) phylogenetic relationships that exist between CB(1)/CB(2) receptors and other GPCRs, 3) evidence for the existence of several as-yet-uncharacterized non-CB(1), non-CB(2) cannabinoid receptors; and 4) current cannabinoid receptor nomenclature.

The cannabinoid receptor: biochemical, anatomical and behavioral characterization.

The actions of the active principle of marihuana, delta 9-tetrahydrocannabinol, are mimicked by synthetic cannabinoid agonists showing high potency and enantio-selectivity in behavioral assays. These drugs have been used to characterize cannabinoid receptor binding, biochemistry and pharmacology, leading to a better understanding of the effects of cannabinoids in the CNS of humans and experimental animals.

Differential receptor-G-protein coupling evoked by dissimilar cannabinoid receptor agonists.

Multiple affinity states are revealed by agonist competition for radioantagonist [3H]SR141716A binding to rat brain CB1 cannabinoid receptors. Desacetyllevonantradol (DALN), a tricyclic cannabinoid, and WIN55212-2, an aminoalkylindole, both bound in two discrete affinity states (30% high affinity), but the ratios of the IC50 revealed distinct differences. Other affinity-state differences between the agonists were: Na+ reduced the affinity for the membrane-bound receptor by 10-fold for DALN but minimally for WIN55212-2; a nonhydrolysable GTP analogue decreased the fraction of high-affinity WIN55212-2 binding but not that of DALN unless Na+ was also present. Detergent solubilisation increased the fraction of high-affinity binding for both agonists but eliminated any effect of Na+ on the agonist affinities. In detergent solution, the GTP analogue reduced the WIN55212-2 high-affinity fraction but not that of DALN, even though the IC50 values increased for both DALN and WIN55212-2. The differential modulation of CB1 receptor-G-protein coupling by Na+ and guanine nucleotides is dependent upon the cannabimimetic agonist bound.

Involvement of a pertussis toxin-sensitive G protein in the regulation of angiotensinogen production by an angiotensin II analog in HepG2 cells.

The cellular mechanism by which the angiotensin II (AII) agonist, Sar1-AII, inhibits production and release of angiotensinogen in human hepatoma HepG2 cells was examined. Pretreatment of HepG2 cells with pertussis toxin attenuated the ability of Sar1-AII to block angiotensinogen production. This effect could be correlated with the in situ ADP-ribosylation of a protein(s) of apparent molecular weight 39,000-41,000 on SDS-PAGE, and attenuation of the ability of Sar1-AII to inhibit cAMP accumulation. The role of cAMP in angiotensinogen production was examined. A transient increase in cAMP accumulation above basal could be evoked by forskolin (8-fold) or by glucagon (5-fold) using insulin-deficient media. Although neither forskolin nor glucagon had a significant effect on angiotensinogen production agents producing a sustained increase in intracellular cAMP (8-bromo-cAMP, dibutyryl-cAMP, cholera toxin) were able to increase angiotensinogen production. Although these data indicate that intracellular cAMP is a regulatory factor in angiotensinogen production other evidence suggests that modulation of intracellular cAMP is not entirely responsible for the effects of Sar1-AII.

Endogenous ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase that is not regulated by nitric oxide in Dictyostelium discoideum.

A 41,000 M(r) cytosolic protein (p41) in Dictyostelium discoideum was shown to be modified by ADP-ribosylation that was not regulated by nitric oxide (NO). This endogenous ADP-ribosylation was optimal at conditions distinct from those optimal for the NO-stimulated ADP-ribosylation of p41. These two activities were also differentially sensitive to reducing agents and modified different amino acids. The addition of haemoglobin, which sequesters NO, and of NO synthase inhibitors failed to block the endogenous ADP-ribosylation. P41 was purified to homogeneity. The N-terminal sequence of the purified protein was shown to be highly homologous to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both endogenous and NO-stimulated activities ADP-ribosylated three isoforms of the protein, with pI values of 6.6, 6.8 and 7.0. In each case, the isoform with pI 6.8 was preferentially modified. Experiments using purified GAPDH indicate that both the endogenous and NO-stimulated ADP-ribosylation are self-catalysed modifications.

Cannabinoid receptor signaling.

The cannabinoid receptor family currently includes two types: CB1, characterized in neuronal cells and brain, and CB2, characterized in immune cells and tissues. CB1 and CB2 receptors are members of the superfamily of seven-transmembrane-spanning (7-TM) receptors, having a protein structure defined by an array of seven membrane-spanning helices with intervening intracellular loops and a C-terminal domain that can associate with G proteins. Cannabinoid receptors are associated with G proteins of the Gi/o family (Gi1, 2 and 3, and Go1 and 2). Signal transduction via Gi inhibits adenylyl cyclase in most tissues and cells, although signaling via Gs stimulates adenylyl cyclase in some experimental models. Evidence exists for cannabinoid receptor-mediated Ca2+ fluxes and stimulation of phospholipases A and C. Stimulation of CB1 and CB2 cannabinoid receptors leads to phosphorylation and activation of p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK and Jun N-terminal kinase (JNK) as signaling pathways to regulate nuclear transcription factors. The CB1 receptor regulates K+ and Ca2+ ion channels, probably via Go. Ion channel regulation serves as an important component of neurotransmission modulation by endogenous cannabinoid compounds released in response to neuronal depolarization. Cannabinoid receptor signaling via G proteins results from interactions with the second, third and fourth intracellular loops of the receptor. Desensitization of signal transduction pathways that couple through the G proteins probably entails phosphorylation of critical amino acid residues on these intracellular surfaces.

Thyroid effects on adenosine 3',5'-monophosphate levels and adenylate cyclase in cultured neuroblastoma cells.

Using neuroblastoma cells as a model of developing neurons, we have tested the hypothesis that thyroid hormones alter cAMP metabolism. Neuroblastoma cells were grown in serum-free defined medium for 48 h with or without thyroid hormones. Treatment with 20-200 nM 3,5,3'-triiodo-L-thyronine (T3) increased the accumulation of cAMP by intact cells without altering growth, gross morphology, or DNA or protein content. The increase in cAMP accumulation could be detected 5 h after the addition of T3 and was abolished by the addition of cycloheximide. The maximum stimulation produced by prostaglandin E1 was increased in T3 cells without a significant alteration of the half-maximal concentration. T4 and D-T3 in concentrations up to 20 microM did not increase cAMP accumulation. Adenylate cyclase activity in response to forskolin, guanine nucleotides, and stimulatory hormones was increased in purified membranes from cells grown in T3, suggesting that increased adenylate cyclase is probably the major mechanism of the observed response to thyroid hormone.

Cannabinoid inhibition of adenylate cyclase: relative activity of constituents and metabolites of marihuana.

delta 9Tetrahydrocannabinol (THC) has been shown to inhibit the activity of adenylate cyclase in the N18TG2 clone of murine neuroblastoma cells. The concentration of delta 9THC exhibiting half-maximal inhibition was 500 nM. delta 8Tetrahydrocannabinol was less active, and cannabinol was only partially active. Cannabidiol, cannabigerol, cannabichromene, olivetol and compounds having a reduced length of the C3 alkyl side chain were inactive. The metabolites of delta 8THC and delta 9THC hydroxylated at the C11 position were more potent than the parent drugs. However, hydroxylation at the C8 position of the terpenoid ring resulted in loss of activity. Compounds hydroxylated along the C3 alkyl side chain were equally efficacious but less potent than delta 9THC. These findings are compared to the pharmacology of cannabinoids reported for psychological effects in humans and behavioral effects in a variety of animal models.

Signal transduction interactions between CB1 cannabinoid and dopamine receptors in the rat and monkey striatum.

Signal transduction interactions between the CB1 cannabinoid and 1 and D2 dopamine receptor systems were studied in rat (Sprague Dawley) and monkey (Macaca fascilaris) striatal membranes. The D2 agonist quinelorane inhibited forskolin (10 microM)-stimulated adenylyl cyclase in a dose-dependent manner (26% and 20% maximal inhibition; EC50 = 2 and 0.5 microM, in rats and monkeys, respectively) and maximal inhibition was completely blocked by the D2 antagonist sulpiride (10 microM). The CB1 agonist desacetyllevonantradol inhibited forskolin-stimulated adenylyl cyclase (18% and 36% maximal inhibition; EC50 = 160 and 73 nM, in rats and monkeys, respectively) and the CB1 antagonist SR141716A (10 microM) completely blocked the maximal inhibition. Combined addition of > EC(90) concentrations of quinelorane (10, 30 microM) and desacetyllevonantradol (1 microM) resulted in no greater inhibition than that produced by either drug alone, indicative of signal transduction convergence between the D2 and CB1 receptor systems. The D1 agonist 6-Br-APB (3-allyl-6-bromo-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepin) produced a dose-dependent stimulation of adenylyl cyclase (45% and 26% stimulation; EC50 = 24 and 32 nM, in rat and monkey, respectively), and maximal stimulation was completely blocked by the D1 antagonist SCH23390 (1 microM). D1 agonist-stimulated activity could be inhibited to basal levels with desacetyllevonantradol (1 microM), indicative of D1 and CB1 signal transduction convergence. The data suggest that CB1 receptors are co-localized with D1 or D2 receptors on the same population of striatal membranes and can interact at the level of G-protein/adenylyl cyclase signal transduction. Similar results obtained with both rat and monkey membranes indicate that striatal dopamine and cannabinoid interactions are conserved for these two species.

Stereochemical effects of 11-OH-delta 8-tetrahydrocannabinol-dimethylheptyl to inhibit adenylate cyclase and bind to the cannabinoid receptor.

The recent preparation of the enantiomers of 11-OH-delta 8-tetrahydrocannabinol-dimethylheptyl (THC-DMH), recrystallized to absolute enantiomeric purity, has made it possible to examine the requirement for stereospecificity for the interaction of this component with the cannabinoid receptor, defined by the binding of [3H]CP-55,940 and the adenylate cyclase enzyme. The enantiomer (-)11-OH-delta 8-THC-DMH exhibited a fully efficacious and potent (IC50 = 1.8 nM) inhibition of the accumulation of cAMP in intact N18TG2 cells. The (-)enantiomer was as efficacious and potent (Kinh = 7.2 nM) as desacetyllevonantradol in inhibiting adenylate cyclase activity in membrane preparations. The (-)enantiomer was able to compete fully for the specific binding of [3H]CP-55,940 to membranes from the brain of the rat in homologous displacement studies (Ki = 234 pM). The potency ratios exhibited by the (-) to (+)enantiomers of 11-OH-delta 8-THC-DMH exceeded 1000 for each of these activities.

Derivation of a pharmacophore model for anandamide using constrained conformational searching and comparative molecular field analysis.

Constrained molecular dynamics simulations on anandamide, together with a systematic distance comparison search, have revealed a specific low-energy conformer whose spatial disposition of the pharmacophoric elements closely matches that of HHC. This conformer enables near superposition of the following: (1) the oxygen of the carboxyamide and the phenolic hydroxyl group of HHC, (2) the hydroxyl group of the ethanol and the cyclohexyl hydroxyl group of HHC, (3) the alkyl tail and the lipophilic side chain of HHC, and (4) the polyolefin loop and the tricyclic ring structure of HHC. The close matching of common pharmacophoric elements of anandamide with HHC offers persuasive evidence of the biological relevance of this conformer. The proposed pharmacophore model was capable of discriminating between structurally related compounds exhibiting different pharmacological potency for the CB1 cannabinoid receptor, i.e., anandamide and N-(2-hydroxyethyl)prostaglandinamide. Furthermore, a 3D-QSAR model was derived using CoMFA for a training set of 29 classical and nonclassical analogues which rationalized the binding affinity in terms of steric and electrostatic properties and, more importantly, which predicted the potency of anandamide in excellent agreement with experimental data. The ABC tricyclic HU-210/HU-211 and ACD tricyclic CP55,243/CP55,244 enantiomeric pairs were employed as test compounds to validate the present CoMFA model. For each enantiomeric pair, the CoMFA-predicted log Ki values correctly identified that enantiomer exhibiting the higher affinity for the receptor.

(Aminoalkyl)indole isothiocyanates as potential electrophilic affinity ligands for the brain cannabinoid receptor.

A series of (aminoalkyl)indole compounds, naphthalene analogs of pravadoline (1), has been shown to exhibit cannabinoid agonist activities such as antinociception in animals, inhibition of adenylate cyclase in brain membranes, and binding to the cannabinoid receptor. These pravadoline analogs were selected for the preparation of potential electrophilic affinity ligands based on the synthesis of isothiocyanate derivatives. One isothiocyanatonaphthalene derivative (8) displaced [3H]CP-55940 binding to a rat brain P2 membrane preparation with an IC50 of 690 nM, which was 10-fold less potent than the parent molecule (IC50 = 73 nM). Isothiocyanate substitution at various positions on the naphthalene moiety of the desmethyl analog 10 gave compounds that displaced [3H]CP-55940 with IC50 values between 400 and 1000 nM, compared with 46 nM for the parent compound 10. However, 6-isothiocyanato substitution on the indole ring of the desmethyl analog provided isothiocyanate 12 that displaced [3H]CP-55940 binding with an IC50 and 160 nM. After pretreatment of brain membranes with this high-affinity isothiocyanato ligand followed by washing out the ligand, the membranes were depleted of 90% of the cannabinoid receptor binding capacity. Loss of receptor binding capacity was half-maximal at 300 nM of the derivative under standard assay conditions. As a control, pretreatment with the parent compound at concentrations that were 20 times the Kd failed to alter subsequent binding activity. This study demonstrates that an isothiocyanato (aminoalkyl)-indole (12) can behave as an affinity ligand which binds irreversibly to the cannabinoid receptor in brain and which precludes subsequent binding of the cannabinoid ligand [3H]CP-55940.

Three-dimensional quantitative structure-activity relationship study of the cannabimimetic (aminoalkyl)indoles using comparative molecular field analysis.

The present study describes the implementation of comparative molecular field analysis (CoMFA) to develop two 3D-QSAR (quantitative structure-activity relationship) models (CoMFA models 1 and 2) of the cannabimimetic (aminoalkyl)indoles (AAIs) for CB1 cannabinoid receptor binding affinity, based on pKi values measured using radioligand binding assays that displace two different agonist ligands, [3H]CP-55940 and [3H]WIN-55212-2. Both models exhibited a strong correlation between the calculated steric-electrostatic fields and the observed biological activity for the respective training set compounds. In light of the basicity of the morpholine nitrogen in the AAIs, separate CoMFA models were built for the AAIs as unprotonated and protonated species. Comparison of the statistical parameters resulting from these CoMFA models failed to provide unequivocal evidence as to whether the AAIs are protonated or neutral as receptor-bound species. Although the training sets of CoMFA model 1 and CoMFA model 2 differed with respect to composition and to the choice of displacement radioligand in each biological assay, their CoMFA StDevCoeff contour plots reveal similarities in terms of identifying those regions around the AAIs that are important for CB1 cannabinoid receptor binding such as the sterically favored region around the C3 aroyl group and the sterically forbidden region around the indole ring. When the experimental pKi values for the training set compounds to displace the AAI radioligand [3H]WIN-55212-2 were plotted against the pKi values as predicted for the same compounds to displace the cannabinoid radioligand [3H]CP-55940, the correlation was moderately strong (r = 0.73). However, the degree of correlation may have been lowered by the structural differences in the compounds comprising the training sets for CoMFA model 1 and CoMFA model 2. Taken together, the results of this study suggest that the binding site region within the CB1 cannabinoid receptor can accommodate a wide range of structurally diverse cannabimimetic analogues including the AAIs.

Cannabinoid receptor binding and messenger RNA expression in human brain: an in vitro receptor autoradiography and in situ hybridization histochemistry study of normal aged and Alzheimer's brains.

The distribution and density of cannabinoid receptor binding and messenger RNA expression in aged human brain were examined in several forebrain and basal ganglia structures. In vitro binding of [3H]CP-55,940, a synthetic cannabinoid, was examined by autoradiography in fresh frozen brain sections from normal aged humans (n = 3), patients who died with Alzheimer's disease (n = 5) and patients who died with other forms of cortical pathology (n = 5). In the structures examined--hippocampal formation, neocortex, basal ganglia and parts of the brainstem--receptor binding showed a characteristic pattern of high densities in the dentate gyrus molecular layer, globus pallidus and substantia nigra pars reticulata, moderate densities in the hippocampus, neocortex, amygdala and striatum, and low densities in the white matter and brainstem. In situ hybridization histochemistry of human cannabinoid receptor, a ribonucleotide probe for the human cannabinoid receptor messenger RNA, showed a pattern of extremely dense transcript levels in subpopulations of cells in the hippocampus and cortex, moderate levels in hippocampal pyramidal neurons and neurons of the striatum, amygdala and hypothalamus, and no signal over dentate gyrus granule cells and most of the cells of the thalamus and upper brainstem, including the substantia nigra. In Alzheimer's brains, compared to normal brains, [3H]CP-55,940 binding was reduced by 37-45% in all of the subfields of the hippocampal formation and by 49% in the caudate. Lesser reductions (20-24%) occurred in the substantia nigra and globus pallidus, internal segment. Other neocortical and basal ganglia structures were not different from control levels. Levels of messenger RNA expression did not differ between Alzheimer's and control brains, but there were regionally discrete statistically significant losses of the intensely expressing cells in the hippocampus. The reductions in binding did not correlate with or localize to areas showing histopathology, estimated either on the basis of overall tissue quality or silver staining of neuritic plaques and neurofibrillary tangles. Reduced [3H]55,940 binding was associated with increasing age and with other forms of cortical pathology, suggesting that receptor losses are related to the generalized aging and/or disease process and are not selectively associated with the pathology characteristic of Alzheimer's disease, nor with overall decrements in levels of cannabinoid receptor gene expression.

Inhibition of adenylate cyclase by p-bromophenacyl bromide.

p-Bromophenacyl bromide (BPB) is an alkylating agent which has been used in biochemical studies as an inhibitor of phospholipase A2 activity. We report here that BPB irreversibly inhibited adenylate cyclase activity stimulated by hormones, forskolin, GppNHp, NaF, and cholera toxin. The action of BPB in S49 lymphoma cell membranes (wild type and cyc-) indicates that it can inhibit adenylate cyclase function in the absence of Gs. In the presence of Gs, however, inhibition of adenylate cyclase by BPB was enhanced, suggesting that BPB may covalently modify the catalytic protein on a site involved in activated catalytic functioning or critical to its interaction with Gs and/or additionally on the alpha s protein.

Regulation of adenylate cyclase by cannabinoid drugs. Insights based on thermodynamic studies.

The abilities of lipophilic cannabinoid drugs to regulate adenylate cyclase activity in neuroblastoma cell membranes were analyzed by thermodynamic studies. Arrhenius plots of hormone-stimulated adenylate cyclase activity exhibited a break point at 20 degrees. The break point was reduced to 14 degrees by benzyl alcohol, consistent with results from other laboratories that have correlated this response with the increase in membrane fluidity induced by benzyl alcohol. Because cannabinoid drugs partition into membrane lipids and alter membrane fluidity parameters in a number of model systems, it was of interest to examine the influence of delta 9-tetrahydrocannabinol and cannabidiol on enzyme activity analyzed by the Arrhenius plot. delta 9-Tetrahydrocannabinol, known to inhibit adenylate cyclase, failed to decrease the transition temperature either at 1 microM or at concentrations exceeding its aqueous solubility (30 microM), suggesting that delta 9-tetrahydrocannabinol could not mimic the effects observed with benzyl alcohol. In contrast, 30 microM cannabidiol, which stimulated enzyme activity slightly, decreased the Arrhenius plot break point to 17.5 degrees. The decrease in the transition temperature in response to benzyl alcohol or cannabidiol was not accompanied by a change in activation energies above or below the transition temperature. delta 9-Tetrahydrocannabinol inhibits adenylate cyclase activity via Gi as does the muscarinic agonist carbachol (Howlett et al., Mol Pharmacol 29: 307-313, 1986). Both carbachol and delta 9-tetrahydrocannabinol decreased the enthalpy and entropy of activation. The net free energy of activation at 37 degrees was increased in the presence of both of these inhibitory agonists. These data suggest that, for the entropy-driven hormone-stimulated adenylate cyclase enzyme, less disorder of the system occurs in the presence of regulators that inhibit the enzyme via Gi. In summary, thermodynamic data suggest that cannabidiol can influence adenylate cyclase by increasing membrane fluidity, but that the inhibition of adenylate cyclase by delta 9-tetrahydrocannabinol is not related to membrane fluidization.

Failure of cannabinoid compounds to stimulate estrogen receptors.

delta 9-Tetrahydrocannabinol (THC), the primary active compound in Cannabis sativa (marihuana), and other cannabinoid receptor agonists exert potent effects on luteinizing hormone and prolactin release in animal models and humans. Compounds possessing the tricyclic cannabinoid structure, including delta 9-THC and cannabidiol, have been reported to interact with rodent uterine estrogen receptors in ligand binding assays. The present study tested the hypothesis that cannabinoid compounds produce a direct activation of estrogen receptors. We investigated whether cannabinoid compounds exhibit estrogen-induced mitogenesis in MCF-7 breast cancer cells. Under conditions in which 10 pM estradiol promoted MCF-7 cell proliferation, no response was observed with biologically relevant concentrations (< = 10 microM) of delta 9-THC or its tricyclic analog desacetyllevonantradol. No response was observed with cannabidiol, a bicyclic cannabinoid compound that exhibits no cannabimimetic behavioral effects but has been reported to bind to the estrogen receptor in vitro. delta 9-THC also failed to antagonize the response to estradiol under conditions in which the antiestrogen LY156758 (keoxifene; raloxifene) was effective. The phytoestrogen formononetin behaved as an estrogen at high concentrations, and this response was antagonized by LY156758. We also investigated the ability of cannabinoid compounds to stimulate transcription of an EREtkCAT reporter gene transiently transfected into MCF-7 cells. Neither delta 9-THC, desacetyllevonantradol, nor cannabidiol stimulated transcriptional activity. We conclude that psychoactive or inactive compounds of the cannabinoid structural class fail to behave as agonists in appropriate assays of estrogen receptor responses in vitro.

Inverse agonist properties of N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide HCl (SR141716A) and 1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carboxyl ic acid phenylamide (CP-272871) for the CB(1) cannabinoid receptor.

Two subtypes of cannabinoid receptors are currently recognized, CB(1), found in brain and neuronal cells, and CB(2), found in spleen and immune cells. We have characterized 1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carboxyl ic acid phenylamide (CP-272871) as a novel aryl pyrazole antagonist for the CB(1) receptor. CP-272871 competed for binding of the cannabinoid agonist (3)H-labeled (-)-3-[2-hydroxy-4-(1, 1-dimethylheptyl)-phenyl]-4-[3-hydroxypropyl]cyclohexan-1-ol ([(3)H]CP-55940) at the CB(1) receptor in rat brain membranes with a K(d) value 20-fold greater than that of N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide HCl (SR141716A). CP-272871 also competed for binding with the aminoalkylindole agonist (3)H-labeled (R)-(+)-[2, 3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]1, 4-benzoxazin-6-yl](1-naphthyl)methanone ([(3)H]WIN-55212-2), as well as the aryl pyrazole antagonist [(3)H]SR141716A. Inverse agonist as well as antagonist properties were observed for both SR141716A and CP-272871 in signal transduction assays in biological preparations in which the CB(1) receptor is endogenously expressed. SR141716A augmented secretin-stimulated cyclic AMP (cAMP) accumulation in intact N18TG2 neuroblastoma cells, and this response was reversed by the agonist desacetyllevonantradol. CP-272871 antagonized desacetyllevonantradol-mediated inhibition of adenylyl cyclase in N18TG2 membranes, and increased adenylyl cyclase activity in the absence of agonist. SR141716A and CP-272871 antagonized desacetyllevonantradol-stimulated (35)S-labeled guanosine-5'-O-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) binding to brain membrane G-proteins, and decreased basal [(35)S]GTPgammaS binding to G-proteins. K(+) enhanced CP-272871 and SR141716A inverse agonist activity compared with Na(+) or NMDG(+) in the assay. These results demonstrated that the aryl pyrazoles SR141716A and CP-272871 behave as antagonists and as inverse agonists in G-protein-mediated signal transduction in preparations of endogenously expressed CB(1) receptors.

CB1 cannabinoid receptor: cellular regulation and distribution in N18TG2 neuroblastoma cells.

In order to characterize cellular regulation of CB1 cannabinoid receptors, synthesis and turnover studies were performed. Metabolic labeling of N18TG2 cells with 35S-labeled amino acids was followed by immunoprecipitation from cell lysates using an affinity-purified antibody generated to the N-terminal 14-amino-acid segment of the CB1 receptor. During a 2 h labeling period, CB1 receptors were rapidly and constitutively synthesized (rate: 0.86%/min). The majority of newly synthesized CB1 cannabinoid receptors (70%) was degraded rapidly by a first-order process (t1/2=4.8 h). The remaining nascent receptors, which were degraded slowly (t1/2>24 h), may represent the pool of potentially functional receptors. Trypsin treatment of intact confluent cells, designed to cleave the extracellular antibody recognition site, did not alter the recovery of metabolically labeled immunoprecipitated CB1 receptors. This suggests that a large percentage of newly synthesized receptors was inaccessible to the protease and is probably intracellular. Immunocytochemistry revealed CB1 cannabinoid immunoreactivity both intracellularly and on the cell surface. Subcellular membrane fractions exhibited receptor binding activity on plasma membranes and nuclear-associated membranes. Only low-affinity binding was seen in the chromatin fraction. An hypothesis has been developed to explain these results and form the basis for future studies.

Cannabinoid receptor agonist and antagonist effects on motor function in normal and 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP)-treated non-human primates.

RATIONALE: Although cannabinoid effects on motor function have been extensively studied in rodents, the role of cannabinoids in regulating behavior in primates is relatively unknown. OBJECTIVES: We compared the effects of cannabinoid agonists and dopamine antagonists on unconditioned behaviors in cynomolgus monkeys (Macaca fascicularis). We further investigated the therapeutic potential of cannabinoid antagonists in a primate model of Parkinson's disease. METHODS: Drugs were administered i.m., and sessions were videotaped and rated by a "blind" observer using a rating scale. RESULTS: The dopamine antagonist haloperidol decreased locomotor activity and increased bradykinesia in three subjects. Haloperidol also produced a dose-dependent increase in freezing and catalepsy in two out of the three subjects. The cannabinoid agonist levonantradol dose-dependently decreased general and locomotor activity and increased bradykinesia. In contrast to haloperidol, levonantradol failed to produce freezing or catalepsy. At the dose range studied, tetrahydrocannabinol did not affect general or locomotor activity, but increased bradykinesia. In view of the psychomotor slowing induced by cannabinoid agonists, we investigated the therapeutic potential of the cannabinoid receptor antagonist SR141716A in an early and advanced stage of 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine-induced parkinsonism. In both models of Parkinson's disease, SR141716A failed to alleviate the motor deficits of parkinsonism. CONCLUSIONS: Cannabinoid agonists do not induce catalepsy in primates, a finding that differs from their effects in rodents. The primate may be more suitable than rodents for predicting the effects of cannabinoids and their therapeutic potential on select primate behaviors.