Cannabinoid receptor and WIN-55,212-2-stimulated [S]GTPγS binding and cannabinoid receptor mRNA levels in several brain structures of adult male rats chronically exposed to R-methanandamide

We and others have recently demonstrated that the pharmacological tolerance observed after prolonged exposure to plant and synthetic cannabinoids in adult individuals seems to have a pharmacodynamic basis, based on the observed down-regulation of cannabinoid receptors in the brain of cannabinoid-tolerant rats. However, we were unable to elicit a similar receptor down-regulation after a chronic exposure to anandamide, the first discovered endogenous cannabinoid, possibly because of its rapid metabolic breakdown in arachidonic acid and ethanolamine. The present study was designed to progress in these previous studies, by using R-methanandamide, a more stable analog, instead anandamide. In addition, we examined not only cannabinoid receptor binding, but also WIN-55,212-2-stimulated [³⁵S]-GTPγS binding, by autoradiography, and cannabinoid receptor mRNA levels, by in situ hybridization. Results were as follows. The daily administration of R-methanandamide for a period of five days produced decreases in cannabinoid receptor binding in the lateral caudate-putamen, cerebellum, entopeduncular nucleus and substantia nigra. The remaining areas, the medial caudate-putamen, globus pallidus, cerebral cortex (layers I and VI), hippocampus (dentate gyrus and Ammon’s horn) and several limbic structures (nucleus accumbens, septum nuclei and basolateral amygdaloid nucleus), exhibited no changes in cannabinoid receptor binding. Similarly, the levels of cannabinoid receptor mRNA expression decreased in the lateral and medial caudate-putamen and in the CA1 and CA2 subfields of the Ammon’s horn in the hippocampus after the chronic exposure to R-methanandamide, whereas the remaining areas showed no changes. WIN-55,212-2-stimulated [³⁵S]-GTPγS binding did not change in the lateral caudate-putamen, cerebral cortex (layer I), septum nuclei and hippocampal structures (dentate gyrus and Ammon’s horn) of animals chronically exposed to R-methanandamide, whereas a certain trend to decrease could be observed in the substantia nigra and deep layer (VI) of the cerebral cortex in these animals. In summary, as reported for other cannabinoid receptor agonists, the prolonged exposure of rats to R-methanandamide, a more stable analog of anandamide, was able to produce cannabinoid receptor-related changes in contrast with the absence of changes observed early with the metabolically labile anandamide. The observed changes exhibited an evident regional pattern with areas, such as basal ganglia, cerebellum and hippocampus, responding to chronic R-methanandamide treatment while regions, such as the cerebral cortex and limbic nuclei, not responding.     

Enhancement of anandamide formation in the limbic forebrain and reduction of endocannabinoid contents in the striatum of delta9-tetrahydrocannabinol-tolerant rats

Recent studies have shown that the pharmacological tolerance observed after prolonged exposure to synthetic or plant-derived cannabinoids in adult rats is accompanied by down-regulation/desensitization of brain cannabinoid receptors. However, no evidence exists on possible changes in the contents of the endogenous ligands of cannabinoid receptors in the brain of cannabinoid-tolerant rats. The present study was designed to elucidate this possibility by measuring, by means of isotope dilution gas chromatography/mass spectrometry, the contents of both anandamide (arachidonoylethanolamide; AEA) and its biosynthetic precursor, N-arachidonoylphosphatidylethanolamine (NArPE), and 2-arachidonoylglycerol (2-AG) in several brain regions of adult male rats treated daily with delta9-tetrahydrocannabinol (delta9-THC) for a period of 8 days. The areas analyzed included cerebellum, striatum, limbic forebrain, hippocampus, cerebral cortex, and brainstem. The same regions were also analyzed for cannabinoid receptor binding and WIN-55,212-2-stimulated guanylyl-5'-O-(gamma-[35S]thio)-triphosphate ([35S]GTPgammaS) binding to test the development of the well known down-regulation/desensitization phenomenon. Results were as follows: As expected, cannabinoid receptor binding and WIN-55,212-2-stimulated [35S]GTPgammaS binding decreased in most of the brain areas of delta9-THC-tolerant rats. The only region exhibiting no changes in both parameters was the limbic forebrain. This same region exhibited a marked (almost fourfold) increase in the content of AEA after 8 days of delta9-THC treatment. By contrast, the striatum exhibited a decrease in AEA contents, whereas no changes were found in the brainstem, hippocampus, cerebellum, or cerebral cortex. The increase in AEA contents observed in the limbic forebrain was accompanied by a tendency of NArPE levels to decrease, whereas in the striatum, no significant change in NArPE contents was found. The contents of 2-AG were unchanged in brain regions from delta9-THC-tolerant rats, except for the striatum where they dropped significantly. In summary, the present results show that prolonged activation of cannabinoid receptors leads to decreased endocannabinoid contents and signaling in the striatum and to increased AEA formation in the limbic forebrain. The pathophysiological implications of these findings are discussed in view of the proposed roles of endocannabinoids in the control of motor behavior and emotional states.     

WIN-55,212-2 and SR-141716A alter nicotine-induced changes in locomotor activity, but do not alter nicotine-evoked [3H]dopamine release

Nicotine, the main psychoactive ingredient in tobacco, plays a key role in the development of cigarette smoking addiction. The endocannabinoid system has been demonstrated to have an important role in the motivational and reinforcing effects of drugs. The present study used behavioral and neurochemical techniques to study the interaction of cannabinoid receptors and nicotine pharmacology. In a locomotor activity experiment in rats, the CB(1)/CB(2) cannabinoid receptor agonist WIN-55,212-2 (0.28-2.8 mg/kg) attenuated nicotine (0.4 mg/kg)-induced hyperactivity, but did not alter nicotine (1.0 mg/kg)-induced hypoactivity. In contrast, the selective CB(1) cannabinoid receptor antagonist SR-141716A (1.0 mg/kg) diminished nicotine-induced hypoactivity, but did not alter nicotine-induced hyperactivity. In a neurochemical experiment, rat striatal slices preloaded with [(3)H]dopamine were superfused with WIN-55,212-2 or SR-141716A. A high concentration (100 microM) of WIN-55,212-2 evoked [(3)H]overflow, but this effect was not blocked by the cannabinoid receptor antagonist AM-251. SR-141716A did not evoke [(3)H]overflow, and neither WIN-55,212-2 nor SR-141716A altered nicotine-evoked [(3)H]overflow. Overall, these results indicate a behavioral interaction between cannabinoid receptors and nicotine pharmacology. Likely, WIN-55,212-2 and SR-141716A block nicotine-induced changes in behavior through an indirect mechanism, such as alteration in endocannabinoid regulation of motor circuits, rather than directly through blockade of nicotinic acetylcholine receptors.     

Role of receptor internalization in the agonist-induced desensitization of cannabinoid type 1 receptors

Agonist-induced internalization of G protein-coupled receptors (GPCRs) is an important mechanism for regulating signaling transduction of functional receptors at the plasma membrane. We demonstrate here that both caveolae/lipid-rafts- and clathrin-coated-pits-mediated pathways were involved in agonist-induced endocytosis of the cannabinoid type 1 receptor (CB1R) in stably transfected human embryonic kidney (HEK) 293 cells and that the internalized receptors were predominantly sorted into recycling pathway for reactivation. The treatment of CB1 receptors with the low endocytotic agonist Δ⁹-THC induced a faster receptor desensitization and slower resensitization than the high endocytotic agonist WIN 55,212-2. In addition, the blockade of receptor endocytosis or recycling pathway markedly enhanced agonist-induced CB1 receptor desensitization. Furthermore, co-expression of phospholipase D2, an enhancer of receptor endocytosis, reduced CB1 receptor desensitization, whereas co-expression of a phospholipase D2 negative mutant significantly increased the desensitization after WIN 55,212-2 treatment. These findings provide evidences for the importance of receptor endocytosis in counteracting CB1 receptor desensitization by facilitating receptor reactivation. Moreover, in primary cultured neurons, the low endocytotic agonist Δ⁹-THC or anandamide exhibited a greater desensitization of endogenous CB1 receptors than the high endocytotic agonist WIN 55,212-2, CP 55940 or 2-arachidonoyl glycerol, indicating that cannabinoids with high endocytotic efficacy might cause reduced development of cannabinoid tolerance to some kind cannabinoid-mediated effects.     

Agonist efficacy and receptor efficiency in heterozygous CB1 knockout mice: relationship of reduced CB1 receptor density to G-protein activation

Heterozygous CB1 receptor knockout mice were used to examine the effect of reduced CB1 receptor density on G-protein activation in membranes prepared from four brain regions: cerebellum, hippocampus, striatum/globus pallidus (striatum/GP) and cingulate cortex. Results showed that CB1 receptor levels were approximately 50% lower in heterozygous mice in all regions examined. However, maximal stimulation of [(35)S]guanosine-5'-(gamma-O-thio) triphosphate ([(35)S]GTPgammaS) binding by the high efficacy agonist WIN 55,212-2 was reduced by only 20-25% in most brain regions, with the exception of striatum/GP where the decrease in stimulation was as predicted (approximately 50%). Furthermore, although the efficacies of the cannabinoid partial agonists, methanandamide and (9)-tetrahydrocannabinol, were similarly lower in heterozygous mice, their relative efficacies compared with WIN 55,212-2 were generally unchanged. Saturation analysis of net WIN 55,212-2-stimulated [(35)S]GTPgammaS binding showed that decreased stimulation by WIN 55,212-2 in striatum/GP of heterozygous mice was caused by a decrease in the apparent affinity of net-stimulated [(35)S]GTPgammaS binding. The apparent maximal number of binding sites (B(max)) values of net WIN 55,212-2-stimulated [(35)S]GTPgammaS binding were unchanged in cerebellum and striatum/GP of heterozygous mice, but decreased in cingulate cortex, with a similar trend in hippocampus. Moreover, in every region except cingulate cortex, the maximal number of net-stimulated [(35)S]GTPgammaS binding sites per receptor was significantly increased in heterozygous mice. These results indicate region-dependent increases in the apparent efficiency of CB1 receptor-mediated G-protein activation in heterozygous CB1 knockout mice.     

Loss of cannabinoid-stimulated guanosine 5'-O-(3-[(35)S]Thiotriphosphate) binding without receptor down-regulation in brain regions of anandamide-tolerant rats

The endogenous cannabinoid anandamide has been reported to produce well-defined behavioral tolerance, but studies on the possible mechanisms underlying this process are few and often contradictory. The present study was designed to survey the cellular events involved in anandamide tolerance, in terms of the effects on receptor number, coupling with G proteins, and activation of the cyclic AMP (cAMP) cascade. Chronic treatment of rats with anandamide (20 mg/kg i.p. for 15 days) resulted in behavioral tolerance without any change in cannabinoid receptor binding in the brain regions studied (striatum, cortex, hippocampus, and cerebellum), suggesting that receptor down-regulation was not involved in the development of anandamide behavioral tolerance. In contrast, prolonged exposure to anandamide significantly reduced agonist-stimulated guanosine 5'-O:-(3-[(35)S]thiotriphosphate) binding in the same areas, with losses of >50%, suggesting that receptor desensitization may be part of the molecular mechanism underlying this tolerance. Finally, concerning the cAMP cascade-the most well-known intracellular signaling pathways activated by CB(1) receptors-in the brain regions from rats tolerant to anandamide, we found no alteration in cAMP levels or in protein kinase A activity. We propose that anandamide, unlike Delta(9)-tetrahydrocannabinol and other cannabinoids, does not alter the receptor system at multiple levels but that desensitization of the CB(1) receptor might account for behavioral tolerance to the drug.     

Identification and characterization of a novel synthetic cannabinoid CP 55,940 binder in rat brain cytosol

We have detected the presence of a specific [³H] CP 55,940 binder in the cytosol of rat cerebral cortex. Competition studies showed that only cold CP 55,940 and to a lesser extent D⁹THC was able to compete with [³H] CP 55,940; little competition was observed with either D⁸;THC or anandamide. Scatchard analysis of the data indicate the presence of two distinct binding components having affinity constants (Kd) of 0.97 ± 0.03 nM, 5.83 ± 0.08 nM, and B_max of 3.31 ± 0.06 pmol/mg protein, 22.2 ± 1.2 pmol/mg protein respectively. The cytosolic CP 55,940 binder was heat stable up to 30øC. Besides the brain cytosol, lesser amounts of binding were also detected in the spleen, and testis. Liver, kidney and muscle cytosol preparations were found to be devoid of this binder. Unlike the previously characterized brain membrane cannabinoid receptor, this binder was found to be salt, sulfhydryl blocking reagents and nucleotide resistant. Interestingly, dithiothreitol (DTT), a protein-disulfide group reducing agent, inhibited the binding of [³H] CP-55,940 to the receptor and approximately 80% binding inhibition was obtained at a 5 mM concentration. Western blot analysis using anti-receptor antibody reveal the presence of a 95-110, 50 and 38 kDa band in the brain, spleen and testis cytosolic preparations. In conclusion, we have identified the presence of a novel CP 55,940 binder in rat cerebral cortex cytosol possessing biochemical properties distinct from those previously observed using rat cerebral cortex membrane cannabinoid receptor.     

Changes in the cannabinoid receptor binding, G protein coupling, and cyclic AMP cascade in the CNS of rats tolerant to and dependent on the synthetic cannabinoid compound CP55,940

Chronic exposure to CP55,940 produced a significant down-regulation of cannabinoid receptors in the striatum, cortex, hippocampus, and cerebellum of rat brain. At 24 h after SR141716-precipitated withdrawal, we observed a tendency to return to basal levels in the striatum and cortex, whereas the specific binding remained lower in the hippocampus and cerebellum. When we surveyed cannabinoid receptor-activated G proteins, in chronic CP55,940-treated rats the guanosine 5'-O:-(3-[(35)S]thiotriphosphate) ([(35)S]GTPgammaS) binding assay revealed a decrease of activated G proteins in the striatum, cortex, and hippocampus, whereas no significant changes were seen in the cerebellum. At 24 h after the SR141716-precipitated withdrawal, [(35)S]GTPgammaS binding increased compared with that of rats chronically exposed to CP55,940, attaining the control level except for cerebellum, where we observed a trend to overcome the control amounts. Concerning the cyclic AMP (cAMP) cascade, which represents the major intracellular signaling pathway activated by cannabinoid receptors, in the cerebral areas from rats chronically exposed to CP55,940 we found alteration in neither cAMP levels nor protein kinase A activity. In the brain regions taken from CP55, 940-withdrawn rats, we only observed a significant up-regulation in the cerebellum. Our findings suggest that receptor desensitization and down-regulation are strictly involved in the development of cannabinoid tolerance, whereas alterations in the cAMP cascade in the cerebellum could be relevant in the mediation of the motor component of cannabinoid abstinence.     

Activation of capsaicin-sensitive primary sensory neurones induces anandamide production and release

The inhibitory cannabinoid 1 receptor and the excitatory vanilloid receptor 1, both of which are responsive to the endogenous ligand anandamide, are co-expressed on a subpopulation of primary sensory neurones. We report that activation of the cannabinoid 1 receptor/vanilloid receptor 1-co-expressing primary sensory neurones induces the production and release of anandamide. Application of capsaicin (3 nm-1 micro m) to cultured primary sensory neurones evoked calcitonin gene-related peptide release, which was significantly increased by the selective cannabinoid 1 receptor antagonist, SR141716A (200 nm). Mass spectrometric analyses of the extracellular solution revealed that exposure to capsaicin (10 nm or 100 nm) enhanced the anandamide concentration of the medium from less then 0.05 pmol/ micro L to more then 2 pmol/ micro L. Depolarization of the neurones with 50 mm KCl also enhanced the anandamide content of the buffer. Both the capsaicin- and KCl-induced anandamide release depended on extracellular Ca2+. Prolonged treatment of the cultures with capsaicin (10 micro m) reduced both the capsaicin- and KCl-induced anandamide release. These findings indicate that activation of capsaicin-sensitive primary sensory neurones evokes anandamide production and release, and that anandamide might be a key endogenous regulator of the excitability of these neurones.     

The lack of CB1 receptors prevents neuroadapatations of both NMDA and GABA(A) receptors after chronic ethanol exposure

As the contribution of cannabinoid (CB1) receptors in the neuroadaptations following chronic alcohol exposure is unknown, we investigated the neuroadaptations induced by chronic alcohol exposure on both NMDA and GABA(A) receptors in CB1-/- mice. Our results show that basal levels of hippocampal [(3)H]MK-801 ((1)-5-methyl-10,11-dihydro-5Hdibenzo[a,d]cyclohepten-5,10-imine) binding sites were decreased in CB1-/- mice and that these mice were also less sensitive to the locomotor effects of MK-801. Basal level of both hippocampal and cerebellar [(3)H]muscimol binding was lower and sensitivity to the hypothermic effects of diazepam and pentobarbital was increased in CB1-/- mice. GABA(A)alpha1, beta2, and gamma2 and NMDA receptor (NR) 1 and 2B subunit mRNA levels were altered in striatum of CB1-/- mice. Our results also showed that [(3)H]MK-801 binding sites were increased in cerebral cortex and hippocampus after chronic ethanol ingestion only in wild-type mice. Chronic ethanol ingestion did not modify the sensitivity to the locomotor effects of MK-801 in both genotypes. Similarly, chronic ethanol ingestion reduced the number of [(3)H]muscimol binding sites in cerebral cortex, but not in cerebellum, only in CB1+/+ mice. We conclude that lifelong deletion of CB1 receptors impairs neuroadaptations of both NMDA and GABA(A) receptors after chronic ethanol exposure and that the endocannabinoid/CB1 receptor system is involved in alcohol dependence.     

Lack of CB1 receptor activity impairs serotonergic negative feedback

Serotonergic and endocannabinoid systems are important substrates for the control of emotional behaviour and growing evidence show an involvement in the pathophysiology of mood disorders. In the present study, the absence of the activity of the CB₁ cannabinoid receptor impaired serotonergic negative feedback in mice. Thus, in vivo microdialysis experiments revealed increased basal 5-HT extracellular levels and attenuated fluoxetine-induced increase of 5-HT extracellular levels in the prefrontal cortex of CB₁ knockout compared with wild-type mice. These observations could be related to the significant reduction in the 5-HT transporter binding site density detected in frontal cortex and hippocampus of CB₁ knockout mice. The lack of CB₁ receptor also altered some 5-HT receptors related to the 5-HT feedback. Extracellular recordings in the dorsal raphe nucleus (DRN) revealed that the genetic and pharmacological blockade of CB₁ receptor induced a 5-HT_1A autoreceptor functional desensitization. In situ hybridization studies showed a reduction in the expression of the 5-HT_2C receptor within several brain areas related to the control of the emotional responses, such as the DRN, the nucleus accumbens and the paraventricular nucleus of the hypothalamus, whereas an over-expression was observed in the CA3 area of the ventral hippocampus. These results reveal that the lack of CB₁ receptor induces a facilitation of the activity of serotonergic neurons in the DRN by altering different components of the 5-HT feedback as well as an increase in 5-HT extracellular levels in the prefrontal cortex in mice.     

Cannabinoid receptor ligands mediate growth inhibition and cell death in mantle cell lymphoma

We have earlier reported overexpression of the central and peripheral cannabinoid receptors CB1 and CB2 in mantle cell lymphoma (MCL), a B cell non-Hodgkin lymphoma. In this study, treatment with cannabinoid receptor ligands caused a decrease in viability of MCL cells, while control cells lacking CB1 were not affected. Interestingly, equipotent doses of the CB1 antagonist SR141716A and the CB1/CB2 agonist anandamide inflicted additive negative effects on viability. Moreover, treatment with the CB1/CB2 agonist Win-55,212-2 caused a decrease in long-term growth of MCL cells in culture. Induction of apoptosis, as measured by FACS/Annexin V-FITC, contributed to the growth suppressive effect of Win-55,212-2. Our data suggest that cannabinoid receptors may be considered as potential therapeutic targets in MCL.     

Analogs of SR-141716A (Rimonabant) alter d-amphetamine-evoked [3H] dopamine overflow from preloaded striatal slices and amphetamine-induced hyperactivity

The CB(1) cannabinoid receptor antagonist SR-141716A (Rimonabant) markedly diminishes the behavioral effects of opiates and nicotine and has been an important tool to ascertain the role of cannabinoid receptors in drug addiction. The present goal was to determine the less-explored interaction of SR-141716A and d-amphetamine in neurochemical and behavioral assays. Additionally, the effect of the substituents and substitution patterns on the phenyl ring located at the 5 position of SR-141716A (4-chlorophenyl), and of the CB(1)/CB(2) cannabinoid receptor agonist WIN-55,212-2, was determined. SR-141716A, AM-251 (4-iodophenyl) and NIDA-41020 (4-methoxyphenyl) did not alter amphetamine-evoked [(3)H]overflow from rat striatal slices preloaded with [(3)H]dopamine. MRI-8273-30-1 (4-fluorophenyl; 0.1-10 microM) attenuated amphetamine (3 microM)-evoked [(3)H]overflow, and MRI-8273-59 (3,4-dichlorphenyl; 0.01-10 microM) augmented amphetamine (0.3-3 microM)-evoked [(3)H]overflow. WIN-55,212-2 was without effect. In a locomotor activity experiment, SR-141716A and MRI-8273-30-1 did not alter amphetamine-induced hyperactivity. However, MRI-8273-59 (1-3 mg/kg) dose-dependently attenuated amphetamine (1 mg/kg)-induced hyperactivity. The present results suggest that SR-141716A is less efficacious to alter amphetamine effects than its reported efficacy to diminish the effects of opiates and nicotine. Modification of the 5-phenyl position of SR-141716A affords compounds that do interact with amphetamine in vitro and in vivo.     

Autoradiographic Visualization of the Receptor Subclasses for Vasoactive Intestinal Polypeptide (VIP) in Rat Brain

Vertongen, P., S. N. Schiffmann, P. Gourlet and P. Robberecht. Autoradiographic visualization of the receptor subclasses for Vasoactive Intestinal Polypeptide (VIP) in rat brain. Peptides 18(10) 1547–1554, 1997.—Vasoactive Intestinal Polypeptide (VIP) exerts its biological effects through interaction with two high affinity receptors named the VIP₁- and the VIP₂ receptors. Their messenger RNAs have been mapped in rat brain by in situ hybridization. A cyclic peptide (RO 25-1553) and a secretin analogue ([R¹⁶]chicken secretin) were identified as selective agonist peptides for the VIP₂- and VIP₁ receptors, respectively. The iodinated peptides retained the high affinity and selectivity of the unlabelled peptides and were used for the mapping of each receptor subclass in rat brain. VIP₁ receptors were present in the cerebral cortex, the piriform cortex, the claustrum, the caudate-putamen, the dentate gyrus, the lateral amygdaloïd nucleus, the anteroventral thalamic nucleus, the rhomboïd nucleus, the supraoptic nucleus and the choroïd plexus. VIP₂ receptors were present in the cerebral cortex, the claustrum, the caudate-putamen, the nucleus accumbens, the lateral septal nuclei, the bed nucleus of the stria terminalis, the basolateral amygdaloïd nucleus, the Ammon’s horn, the thalamic nuclei except some centromedial nuclei, the medial habenula, the suprachiasmatic nucleus, the periventricular nucleus, the mammilary nucleus, the superior colliculus and the choroïd plexus.     

Cellular localization of cannabinoid receptors and activated G-proteins in rat anterior cingulate cortex

Cannabinoid receptors are found in moderate density throughout the cerebral cortex. The anterior cingulate cortex (ACC) is of particular interest due its high level of cannabinoid receptors and role in behaviors known to be modulated by cannabinoids. These studies were conducted to determine the cellular localization of cannabinoid receptors and to compare the level of cannabinoid receptor binding with receptor-mediated G-protein activity in the rat ACC. Either ibotenic acid or undercut lesions were made in ACC, and brains were processed for [3H]WIN 55,212-2 and WIN 55,212-2-stimulated [35S]GTPgammaS autoradiography. Both cannabinoid receptors and receptor-activated G-proteins were highest in laminae I and VI of ACC in control tissue. Although similar levels of receptor binding were found in these laminae, significantly higher levels of receptor-activated G-proteins were found in lamina VI. Ibotenic acid lesions that destroyed ACC neurons decreased [3H]WIN 55,212-2 binding by 60-70% and eliminated WIN 55,212-2-stimulated [35S]GTPgammaS binding. In contrast, deafferentation of the ACC with undercut lesions had no significant effect on cannabinoid receptor binding or G-protein activation. These results indicate that cannabinoid receptors in laminae I and VI of the ACC are located on somatodendritic elements or axons intrinsic to the ACC. In addition, differences in the relative levels of cannabinoid binding sites and activated G-proteins between cortical laminae indicate that the efficiency of cannabinoid receptors for G-protein activation may vary within a specific brain region.     

Occurrence and possible biological role of the endocannabinoid system in the sea squirt Ciona intestinalis

A cannabinoid receptor orthologue (CiCBR) has been described in the sea squirt Ciona intestinalis. Here we report that CiCBR mRNA expression is highest in cerebral ganglion, branchial pharynx, heart and testis of C. intestinalis, and that this organism also contains cannabinoid receptor ligands and some of the enzymes for ligand biosynthesis and inactivation. Using liquid chromatography-mass spectrometry, the endocannabinoid anandamide was found in all tissues analysed (0.063-5.423 pmol/mg of lipid extract), with the highest concentrations being found in brain and heart. The endocannabinoid 2-arachidonoylglycerol (2-AG) was fivefold more abundant than anandamide, and was most abundant in stomach and intestine and least abundant in heart and ovaries (2.677-50.607 pmol/mg of lipid extract). Using phylogenomic analysis, we identified orthologues of several endocannabinoid synthesizing and degrading enzymes. In particular, we identified and partly sequenced a fatty acid amide hydrolase (FAAH) orthologue, showing 44% identity with human FAAH and containing nearly all the amino acids necessary for a functional FAAH enzyme. Ciona intestinalis also contained specific binding sites for cannabinoid receptor ligands, and an amidase enzyme with pH-dependency and subcellular/tissue distribution similar to mammalian FAAHs. Finally, a typical C. intestinalis behavioural response, siphon reopening after closure induced by mechanical stimulation, was inhibited by the cannabinoid receptor agonist HU-210, and this effect was significantly attenuated by mammalian cannabinoid receptor antagonists.     

Internalization and Recycling of the CB1 Cannabinoid Receptor

Tolerance develops rapidly to cannabis, cannabinoids, and related drugs acting at the CB1 cannabinoid receptor. However, little is known about what happens to the receptor as tolerance is developing. In this study, we have found that CB1 receptors are rapidly internalized following agonist binding and receptor activation. Efficacious cannabinoid agonists (WIN 55,212-2, CP 55,940, and HU 210) caused rapid internalization. Methanandamide (an analogue of an endogenous cannabinoid, anandamide) was less effective, causing internalization only at high concentration, whereas delta9-tetrahydrocannabinol caused little internalization, even at 3 microM. CB1 internalized via clathrin-coated pits as sequestration was inhibited by hypertonic sucrose. Internalization did not require activated G protein alpha(i), alpha(o), or alpha(s) subunits. A region of the extreme carboxy terminus of the receptor was necessary for internalization, as a mutant CB1 receptor lacking the last 14 residues did not internalize, whereas a mutant lacking the last 10 residues did. Steps involved in the recycling of sequestered receptor were also investigated. Recovery of CB1 to the cell surface after short (20 min) but not long (90 min) agonist treatment was independent of new protein synthesis. Recycling also required endosomal acidification and dephosphorylation. These results show that CB1 receptor trafficking is dynamically regulated by cannabimimetic drugs.     

Ligand Binding and Modulation of Cyclic AMP Levels Depend on the Chemical Nature of Residue 192 of the Human Cannabinoid Receptor 1

Abstract: The human cannabinoid receptor associated with the CNS (CB1) binds Δ⁹-tetrahydrocannabinol, the psychoactive component of marijuana, and other cannabimimetic compounds. This receptor is a member of the seven transmembrane domain G protein-coupled receptor family and mediates its effects through inhibition of adenylyl cyclase. An understanding of the molecular mechanisms involved in ligand binding and receptor activation requires identification of the active site residues and their role. Lys¹⁹² of the third transmembrane domain of the receptor is noteworthy because it is the only nonconserved, charged residue in the transmembrane region. To investigate the properties of this residue, which are important for both ligand binding and receptor activation, we generated mutant receptors in which this amino acid was changed to either Arg (K192R), Gln (K192Q), or Glu (K192E). Wild-type and mutant receptors were stably expressed in Chinese hamster ovary cells and were evaluated in binding assays with the bicyclic cannabinoid CP-55,940 and the aminoalkylindole WIN 55,212-2. We found that only the most conservative change of Lys to Arg allowed retention of binding affinity to CP-55,940, whereas WIN 55,212-2 bound to all of the mutant receptors in the same range as it bound the wild type. Analysis of the ligand-induced inhibition of cyclic AMP production in cells expressing each of the receptors gave an EC₅₀ value for each agonist that was comparable to its binding affinity, with one exception. Although the mutant K192E receptor displayed similar binding affinity as the wild type with WIN 55,212-2, an order of magnitude difference was observed for the EC₅₀ for cyclic AMP inhibition with this compound. The results of this study indicate that binding of CP-55,940 is highly sensitive to the chemical nature of residue 192. In contrast, although this residue is not critical for WIN 55,212-2 binding, the data suggest a role for Lys¹⁹² in WIN 55,212-2-induced receptor activation.     

In-Vitro and In-Vivo Action of Cannabinoids

The discovery of endocannabinoids such as anandamide and the wide spread localization of cannabinoid receptors in the brain and peripheral tissues, suggests that the cannabinoid system represents a previously unrecognized ubiquitous net work in the nervous system, whose physiology and function is unfolding. In this study, we tested the hypothesis that some of the actions of anandamide are independent of a cannabinoid receptor mechanism. This was accomplished by the use of cannabinoid agonist and antagonist interaction in an in-vitro and in-vivo test systems. In-vitro, we used Xenopus laevis oocytes expression system and two-voltage clamp technique in combination with differential display polymerase chain reaction to determine whether the differential display of genes following treatment with anandamide may be linked to AMPA glutamate receptor. The differential expression of genes in vivo after the sub-acute administration of anandamide could not be directly linked with the AMPA glutamate receptor. In the voltage clamp studies we investigated the effects of anandamide on recombinant AMPA GluR3 sub-unit currents generated by kainic acid in oocytes expressing the AMPA glutamate receptor. In the in-vitro studies, we present evidence that anandamide inhibited the kainate activated currents in oocytes expressing AMPA glutamate receptor involves cAMP transduction via a cannabinoid receptor independent mechanism. In the in-vivo studies, SR141716A, the CB1 antagonist, induced anxiolysis, that was dependent on the mouse strain used in the anxiety model and blocked the anxiogenic effects of anandamide or methanandamide whereas SR141716A had no effect on the anandamide inhibition of kainate activated currents in-vitro.     

Cannabinoid-serotonin interactions in alcohol-preferring vs. alcohol-avoiding mice

Because cannabinoid and serotonin (5-HT) systems have been proposed to play an important role in drug craving, we investigated whether cannabinoid 1 (CB1) and 5-HT(1A) receptor ligands could affect voluntary alcohol intake in two mouse strains, C57BL/6 J and DBA/2 J, with marked differences in native alcohol preference. When offered progressively (3-10% ethanol) in drinking water, in a free-choice procedure, alcohol intake was markedly lower (approximately 70%) in DBA/2 J than in C57BL/6 J mice. In DBA/2 J mice, chronic treatment with the cannabinoid receptor agonist WIN 55,212-2 increased alcohol intake. WIN 55,212-2 effect was prevented by concomitant, chronic CB1 receptor blockade by rimonabant or chronic 5-HT(1A) receptor stimulation by 8-hydroxy-2-(di-n-propylamino)-tetralin, which, on their own, did not affect alcohol intake. In C57BL/6 J mice, chronic treatment with WIN 55,212-2 had no effect but chronic CB1 receptor blockade or chronic 5-HT(1A) receptor stimulation significantly decreased alcohol intake. Parallel autoradiographic investigations showed that chronic treatment with WIN 55,212-2 significantly decreased 5-HT(1A)-mediated [35S]guanosine triphosphate-gamma-S binding in the hippocampus of both mouse strains. Conversely, chronic rimonabant increased this binding in C57BL/6 J mice. These results show that cannabinoid neurotransmission can exert a permissive control on alcohol intake, possibly through CB1-5-HT(1A) interactions. However, the differences between C57BL/6 J and DBA/2 J mice indicate that such modulations of alcohol intake are under genetic control.