Rat Cannabinoid Research Articles

The C-terminal helix 9 motif in rat cannabinoid receptor type 1 regulates axonal trafficking and surface expression.

Cannabinoid type one receptor (CB1R) is only stably surface expressed in axons, where it downregulates neurotransmitter release. How this tightly regulated axonal surface polarity is established and maintained is unclear. To address this question, we used time-resolved imaging to determine the trafficking of CB1R from biosynthesis to mature polarised localisation in cultured rat hippocampal neurons. We show that the secretory pathway delivery of CB1R is axonally biased and that surface expressed CB1R is more stable in axons than in dendrites. This dual mechanism is mediated by the CB1R C-terminus and involves the Helix 9 (H9) domain. Removal of the H9 domain increases secretory pathway delivery to dendrites and decreases surface stability. Furthermore, CB1RΔH9 is more sensitive to agonist-induced internalisation and less efficient at downstream signalling than CB1RWT. Together, these results shed new light on how polarity of CB1R is mediated and indicate that the C-terminal H9 domain plays key roles in this process.

Dietary conditions and highly palatable food access alter rat cannabinoid receptor expression and binding density

Endogenous cannabinoid signaling, mediated predominately by CB1 receptor activation, is involved in food intake control and body weight regulation. Despite advances in determining the role of the CB1 receptor in obesity, its involvement in the driven nature of eating pathologies has received little attention. The present study examined CB1 receptor alterations as a consequence of dietary-induced binge eating in female Sprague Dawley rats. Four control groups were used to control for calorie restriction and highly palatable food variables characterizing this behavioral model. All groups were kept on their respective feeding schedules for 6-weeks and were given a uniform 33% calorie restriction (~22h food deprivation) prior to sacrifice. Our findings indicate that regional CB1 mRNA and density were influenced by dietary conditions, but were not specific to the dietary-induced binge eating paradigm used. An increase of approximately 50% (compared with naive controls) in CB1 receptor mRNA levels in the nucleus of the solitary tract as measured by in situ hybridization was found in animals receiving continuous access to a highly palatable food (i.e., vegetable shortening with 10% sucrose). This group also had a significant increase in body weight and adiposity. An approximate 20% reduction in CB1 mRNA was observed in the cingulate cortex (areas 1 and 2) in animals exposed to an intermittent schedule of feeding, compared with groups that had ad libitum feeding schedules (i.e., continuous access and naive controls). Receptor density as measured by [3H]CP55,940 autoradiography, was reduced by approximately 30% in the nucleus accumbens shell region in groups receiving repeated access to the highly palatable food. Taken together, these findings indicate that dietary conditions can differentially influence CB1 receptors in forebrain and hindbrain regions.

Peripheral and central sites of action for the non‐selective cannabinoid agonist WIN 55,212‐2 in a rat model of post‐operative pain

Activation of cannabinoid (CB) receptors decreases nociceptive transmission in inflammatory or neuropathic pain states. However, the effects of CB receptor agonists in post-operative pain remain to be investigated. Here, we characterized the anti-allodynic effects of WIN 55,212-2 (WIN) in a rat model of post-operative pain. WIN 55,212-2 was characterized in radioligand binding and in vitro functional assays at rat and human CB(1) and CB(2) receptors. Analgesic activity and site(s) of action of WIN were assessed in the skin incision-induced post-operative pain model in rats; receptor specificity was investigated using selective CB(1) and CB(2) receptor antagonists. WIN 55,212-2 exhibited non-selective affinity and agonist efficacy at human and rat CB(1) versus CB(2) receptors. Systemic administration of WIN decreased injury-induced mechanical allodynia and these effects were reversed by pretreatment with a CB(1) receptor antagonist, but not with a CB(2) receptor antagonist, given by systemic, intrathecal and supraspinal routes. In addition, peripheral administration of both CB(1) and CB(2) antagonists blocked systemic WIN-induced analgesic activity. Both CB(1) and CB(2) receptors were involved in the peripheral anti-allodynic effect of systemic WIN in a pre-clinical model of post-operative pain. In contrast, the centrally mediated anti-allodynic activity of systemic WIN is mostly due to the activation of CB(1) but not CB(2) receptors at both the spinal cord and brain levels. However, the increased potency of WIN following i.c.v. administration suggests that its main site of action is at CB(1) receptors in the brain.

Protein Kinase C Disrupts Cannabinoid Actions by Phosphorylation of the CB1 Cannabinoid Receptor

We have found that phosphorylation of a G-protein-coupled receptor by protein kinase C (PKC) disrupts modulation of ion channels by the receptor. In AtT-20 cells transfected with rat cannabinoid receptor (CB1), the activation of an inwardly rectifying potassium current (Kir current) and depression of P/Q-type calcium channels by cannabinoids were prevented by stimulation of protein kinase C by 100 nM phorbol 12-myristate 13-acetate (PMA). In contrast, activation of Kir current by somatostatin was unaffected, and inhibition of calcium channels was only modestly attenuated. The possibility that PKC acted by phosphorylating CB1 receptors was confirmed by demonstrating that PKC phosphorylated a single serine (S317) of a fusion protein incorporating the third intracellular loop of CB1. Mutating this serine to alanine did not affect the ability of CB1 to modulate currents, but it eliminated disruption by PMA, demonstrating that PKC can disrupt ion channel modulation by receptor phosphorylation.

Interaction of brain cannabinoid receptors with guanine nucleotide binding protein: A radioligand binding study

The binding of a classical cannabinoid agonist, [ 3H]R-(+)-(2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrol[1,2,3-de]-l,4-benzoxazin-6-yl)(1-napthalenyl)methanone monomethanesulfonate ([ 3H] WIN55212-2), and a selective cannabinoid receptor (CB 1) antagonist, N-(piperidin-1-yl)-5-(4-chlorophenyl)- l-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride ([ 3H]SR141716A), to rat cannabinoid receptors was evaluated using rat cerebellar membranes. Guanine nucleotides inhibited [ 3H]WIN55212-2 binding by approximately 50% at 10 (JtM and enhanced [5H]SR141716A binding very slightly. In the same tissue, the binding of guanosine 5′-O-[γ-[ 35S]thio]triphosphate ([ 35S]GTP-γ-S) was characterized and the influence of cannabinomimetics evaluated on this binding. Cannabinoid receptor agonists enhanced [ 35 S]GTP-γ-S binding, whereas SR141716A was devoid of action by itself but antagonized the action of cannabinoid receptor agonists. The good correlation obtained between the half maximum efficient concentration ( ec 50 ) values in [ 35S]GTP-γ-S binding and the ic 50 values [ 3H]WIN55212-2 binding shows that [ 35S]GTP-γ-S binding could be a good functional assay for brain cannabinoid receptors.

Identification and characterization of the leech CNS cannabinoid receptor: coupling to nitric oxide release

The present study demonstrates that stereoselective binding sites for anandamide, a naturally occurring cannabinoid substance, can be found in leech ( Theromyzon tessulatum and Hirudo medicinalis) central nervous system. The anandamide binding site is monophasic and of high affinity exhibiting a K d of approximately 32 nM with a B max of 550 fmol/mg protein in both animals. These sites are highly select as demonstrated by the inability of other types of signaling molecules to displace [ 3H]anandamide. Furthermore, this binding site is coupled to nitric oxide release. A deduced amino acid sequence (153 residues) analysis from a 480 pb amplified RT-PCR fragment cDNA exhibits a 49.3% and 47.2% sequence identity with human and rat cannabinoid receptors (CB1R), respectively. Thus, the leech cannabinoid receptor may be a G-protein coupled receptor with seven transmembrane domains as in CB1R. Moreover, this sequence exhibits highly conserved regions, particularly in the putative transmembrane domains 1 and 2. The presence of a cannabinoid receptor in these organisms indicates that this signaling system has been conserved during evolution. © 1977 Elsevier Science B.V. All rights reserved.

Expression of a cannabinoid receptor in baculo virus-infected insect cells

A cannabinoid receptor recombinant baculovirus (AcNPV-THCR) has been constructed and employed to express rat neural cannabinoid receptors. Northern analysis of total RNA from Spodoptera frugiperda (Sf9) insect cells infected with AcNPV-THCR revealed novel hyper-production of a 3.3 kb transcript when probed with nick-translated rat cannabinoid receptor cDNA. Optimal viral protein expression was observed in 35S-metabolically labeled AcNPV-THCR-infected Sf9 cells at a multiplicity of infection of 2.5. Transmission electron microscopy of AcNPV-THCR-infected Sf9 cells showed extensive membrane perturbation and electron-dense cytoplasmic perinuclear accumulation, indicative of receptor glycoprotein expression. Immunofluorescence staining using antiserum produced to a fusion protein consisting of the external domain of the cannabinoid receptor and hepatitis B core antigen revealed cannabinoid receptor expression in AcNPV-THCR-infected Sf9 cells. Scatchard-Rosenthal analysis of [ 3H]CP55,940 receptor binding indicated a K d of 3.4 nM and a B max equal to 3.17 pmol/mg protein. Western immunoblotting performed on AcNPV-THCR-infected Sf9 cell lysates revealed immunoreactive bands with relative molecular weights ranging from 45 to 79 kDa. The predominant species (55 kDa) exhibited a relative molecular weight consistent with that predicted for the translational product obtained from the cannabinoid receptor cDNA coding sequence. In vitro translation using AcNPV-THCR mRNA also yielded a 55 kDa immunoreactive species. These data indicate that the baculovirus expression system is a viable means of expressing relatively large quantities of cannabinoid receptor recombinant protein.

Developmental expression of cannabinoid receptor mRNA

Cloning of the cannabinoid receptor affords the opportunity to examine its developmental expression. Other G-protein-coupled receptor systems, those for the opioids for example, exhibit distinct ontogenies. For the initial study, therefore, cannabinoid receptor mRNA expression was assessed in rat pups postnatal days 3, 5, 8, 10, 12, 15, 18 and 21. The brains were grossly dissected into cerebellum/brainstem and forebrain, and total RNA was extracted by a modified acid-extraction method. Expression of the cannabinoid receptor was analyzed by two methods: polymerase chain reaction (PCR) and Northern blot analysis. Oligonucleotide primers based on bp 1–21 and bp 824–843 on the opposite strand were chosen for use in the PCR. The probe used in the Northern blot analysis was a full length cDNA corresponding to the rat cannabinoid receptor and was cloned in our lab based on published sequence information. Our results indicate that by postnatal day 3, cannabinoid receptor mRNA can be detected in the brain. Our results further indicate that cannabinoid mRNA expression steadily increases in the cerebellum/brainstem until postnatal days 18–21, while expression in the forebrain does not change. The findings from the present study indicate that cannabinoid receptor mRNA is present in very young rats. Our data also suggest, however, regional differences in the relative expression of message which may parallel cerebellar proliferation and organization.

Anandamide, an endogenous cannabimimetic eicosanoid, binds to the cloned human cannabinoid receptor and stimulates receptor-mediated signal transduction.

Arachidonylethanolamide (anandamide), a candidate endogenous cannabinoid ligand, has recently been isolated from porcine brain and displayed cannabinoid-like binding activity to synaptosomal membrane preparations and mimicked cannabinoid-induced inhibition of the twitch response in isolated murine vas deferens. In this study, anandamide and several congeners were evaluated as cannabinoid agonists by examining their ability to bind to the cloned cannabinoid receptor, inhibit forskolin-stimulated cAMP accumulation, inhibit N-type calcium channels, and stimulate one or more functional second messenger responses. Synthetic anandamide, and all but one congener, competed for [3H]CP55,940 binding to plasma membranes prepared from L cells expressing the rat cannabinoid receptor. The ability of anandamide to activate receptor-mediated signal transduction was evaluated in Chinese hamster ovary (CHO) cells expressing the human cannabinoid receptor (HCR, termed CHO-HCR cells) and compared to control CHO cells expressing the muscarinic m5 receptor (CHOm5 cells). Anandamide inhibited forskolin-stimulated cAMP accumulation in CHO-HCR cells, but not in CHOm5 cells, and this response was blocked with pertussis toxin. N-type calcium channels were inhibited by anandamide and several active congeners in N18 neuroblastoma cells. Anandamide stimulated arachidonic acid and intracellular calcium release in both CHOm5 and CHO-HCR cells and had no effect on the release of inositol phosphates or phosphatidylethanol, generated after activation of phospholipase C and D, respectively. Anandamide appears to exhibit the essential criteria required to be classified as a cannabinoid/anandamide receptor agonist and shares similar nonreceptor effects on arachidonic acid and intracellular calcium release as other cannabinoid agonists.

Age-related loss of cannabinoid receptor binding sites and mRNA in the rat striatum

Differences in the distribution of cannabinoid receptor (studied by receptor binding radioautography using the synthetic psychoactive cannabinoid ligand [3H]CP55,940) and its mRNA (studied by in situ hybridization histochemistry using oligonucleotide probes complementary to rat cannabinoid receptor cDNA) have been investigated in the rat striatum during ageing. The striata of old rats (24 months) show a 50% reduction in expression of cannabinoid receptors and mRNA levels when compared with young (3 months) equivalents. Thus, changes in cannabinoid receptor gene expression appear to be partially responsible for age-related loss of these receptors.

Distribution of neuronal cannabinoid receptor in the adult rat brain: A comparative receptor binding radioautography and in situ hybridization histochemistry

The neuronal distribution of cannabinoid receptor in the adult rat brain is reported, combining receptor binding radioautography using the synthetic psychoactive cannabinoid ligand CP55,940 with in situ hybridization histochemistry using oligonucleotide probes complementary to rat cannabinoid receptor cDNA. In the cerebral cortex, especially in the frontal and cingulate cortex, dense binding was found in layers I and VI together with slight mRNA levels in a majority of both pyramidal and non-pyramidal-shaped neurons and of high mRNA levels in a moderate number of non-pyramidal-shaped neurons especially in layers II–III and V–VI. In the hippocampal dentate gyrus, very dense staining was found in the molecular layer together with high mRNA levels in a moderate number of hilar neurons close to the granular layer. In Ammon's horn, especially in the CA3 sector, very dense binding was found in the dendritic layers together with slight mRNA levels in the majority of the pyramidal cells and high mRNA levels in a moderate number of interneurons. In the basal ganglia, binding was very dense in the lateral putamen, substantia nigra pars reticulata, globus pallidus and entopeduncular nucleus, moderate in the medial putamen and caudate; and slight in the accumbens, together with slight to moderate mRNA levels in the striatal medium-sized neurons. Together with slight binding, slight to moderate mRNA levels were found in the majority of the neurons in the subthalamic nucleus. No binding and mRNA were found in the substantia nigra pars compacta and ventral tegmental area. Slight to moderate binding was found together with slight to moderate mRNA levels in the majority of neurons in the anterior olfactory nucleus; septum, especially medial septum and diagonal band of Broca; amygdala, especially basolateral amygdala; lateral habenula; ventromedial hypothalamic nucleus; lateral interpeduncular nucleus; central gray, dorsal cochlear nucleus; parabrachial nucleus; dorsal pontine tegmentum; pontine nuclei; commissural part of the nucleus tractus solitarius; inferior olive and dorsal horn of the spinal cord. In the cerebellum, very dense binding was found in the molecular layer together with slight mRNA levels in the majority of the granule cells and moderate mRNA levels in the basket and stellate cells. In conclusion, this study provides, for the first time, indirect assessment of the neurons containing cannabinoid receptor in the entire adult rat brain and will serve as a basis for future direct morphological confirmation using receptor immunohistochemistry and for functional studies.

Molecular cloning of a human cannabinoid receptor which is also expressed in testis

A cDNA clone encoding a receptor protein which presents all the characteristics of a guanine-nucleotide-binding protein (G-protein)-coupled receptor was isolated from a human brain stem cDNA library. The probe used (HGMP08) was a 600 bp DNA fragment amplified by a low-stringency PCR, using human genomic DNA as template and degenerate oligonucleotide primers corresponding to conserved sequences amongst the known G-protein-coupled receptors. The deduced amino acid sequence encodes a protein of 472 residues which shares 97.3% identity with the rat cannabinoid receptor cloned recently [Matsuda, Lolait, Brownstein, Young & Bronner (1990) Nature (London) 346, 561-564]. Abundant transcripts were detected in the brain, as expected, but lower amounts were also found in the testis. The same probe was used to screen a human testis cDNA library. The cDNA clones obtained were partially sequenced, demonstrating the identity of the cannabinoid receptors expressed in both tissues. Specific binding of the synthetic cannabinoid ligand [3H]CP55940 was observed on membranes from Cos-7 cells transfected with the recombinant receptor clone. In stably transfected CHO-K1 cell lines, cannabinoid agonists mediated a dose-dependent and stereoselective inhibition of forskolin-induced cyclic AMP accumulation. The ability to express the human cannabinoid receptor in mammalian cells should help in developing more selective drugs, and should facilitate the search for the endogenous cannabinoid ligand(s).