Influence of chronic bromocriptine and levodopa administration on cerebral type 1 cannabinoid receptor binding.

OBJECTIVES: The endocannabinoid system is an important modulatory system in the brain. Complex interactions with brain dopaminergic circuits have been demonstrated. The aim of this study was to investigate the in vivo effect of the commonly used antiparkinsonian drugs, levodopa (L-DOPA) and bromocriptine, on type 1 cannabinoid (CB1) receptors, using the PET radioligand [(18)F]MK-9470. EXPERIMENTAL APPROACH: Seventeen female Wistar rats were studied at baseline and after chronic exposure to either L-DOPA (6 mg/kg/day with 1.5 mg/kg/day carbidopa; n = 6), bromocriptine (4 mg/kg/day; n = 5), or saline (n = 6). [(18)F]MK-9470 binding was assessed in vivo using small animal PET imaging. [(18)F]MK-9470 parametric images were generated, anatomically standardized to Paxinos space and analyzed by voxel-based statistical parametric mapping (SPM2) and a predefined volume-of-interest (VOI) approach. RESULTS: In a 2 x 2 analysis design (condition vs. treatment), no significant changes in absolute or relative [(18)F]MK-9470 binding were present upon chronic exposure to L-DOPA or bromocriptine as compared to saline treatment. The post hoc comparison of chronic scans to baseline within each treatment modality showed regional increases in relative [(18)F]MK-9470 binding in the thalamus (peak average value +6.3%) and in the sensorimotor cortex and hippocampus (peak average value +10.2%) after bromocriptine exposure, while no changes were found for L-DOPA. CONCLUSION: Chronic administration of L-DOPA and bromocriptine at the applied doses does not produce major cerebral changes in in vivo cannabinoid CB1 receptor binding of [(18)F]MK-9470 in the rat brain. These results also suggest that similar chronic L-DOPA and bromocriptine usage is unlikely to interfere with human PET imaging in healthy conditions using this radioligand.

Versatility of GPCR recognition by drugs: from biological implications to therapeutic relevance.

Most drugs acting on G-protein-coupled receptors (GPCRs) are classically defined as agonists, partial agonists or antagonists. This simplified classification seems sufficient to explain most of their therapeutic properties. The more recent description of inverse agonism has helped to revise theoretical models of GPCR function, but the therapeutic implications of the new concepts remain clearly restricted. Further complexity has arisen with demonstrations that a given receptor can adopt various conformations that support coupling with distinct G proteins. Because the related signaling pathways seem to be differentially affected by some ligands, the concept of 'functional selectivity' has been proposed, calling for a revision of the definitions of agonism and intrinsic efficacy. Evidence of complexity in G-protein coupling and examples of functional selectivity are accumulating, opening perspectives for drug development. Although such complexity should be regarded as an opportunity to gain pharmacological specificity, unraveling the physiological implications of these concepts is essential before their therapeutic relevance can be defined.

Reciprocal influences of CB1 cannabinoid receptor agonists on ERK and JNK signalling in N1E-115 cells.

Agonists acting at the CB1 cannabinoid receptor in N1E-115 neuroblastoma cells were found to activate MAPK family members with reciprocal efficacies. Thus, HU 210 robustly increased phosphorylation of ERK1/2 whereas CP 55,940 was more effective in activating JNK. The use of selected kinase inhibitors confirmed that distinct signalling cascades were involved in these responses. This reciprocal control of MAPK activity was correlated with the observation that HU 210- and CP 55,940-mediated regulations of tyrosine hydroxylase gene expression were respectively impaired by MEK and JNK inhibitors. These data indicate that complex interactions of the CB1 receptor with intracellular signalling partners controlling MAPK activities may explain the apparent disparities in cellular responses to functional selective agonists.

An in vivo [18F]MK-9470 microPET study of type 1 cannabinoid receptor binding in Wistar rats after chronic administration of valproate and levetiracetam.

There is substantial evidence that the endocannabinoid system and in particular the type 1 cannabinoid receptor (CB1R) is involved in epilepsy. We evaluated the in vivo effect of chronic administration of the anti-epileptic drugs valproate (VPA) and levetiracetam (LEV) on rat brain CB1 receptors using the positron emission tomography (PET) tracer [(18)F]MK-9470. Six Wistar rats were treated with VPA (200mg/kg) or LEV (50mg/kg) IP daily for 2 weeks. Dynamic imaging after intravenous injection of 18 MBq [(18)F]MK-9470 was performed on a FOCUS 220 microPET at baseline and after chronic treatment. Six animals were used as controls and were injected with saline, using the same protocol. Parametric images based on standardized uptake values (SUV) were generated and were spatially normalized to Paxinos space. These CB1R images were analyzed using a predefined volume of interest (VOI)-based analysis. Differences in SUV values between chronic and baseline scans in each condition (saline, VPA and LEV treatment) were calculated in each VOI. Direct binding affinity of the drugs at CB1R was assessed by competitive binding assay in Chinese hamster ovarian cells expressing human CB1R. Chronic injections of saline did not produce significant changes in global [(18)F]MK-9470 binding (p=0.43), nor in tracer binding in individual VOIs. We found a significant increase in global cerebral [(18)F]MK-9470 binding after chronic VPA administration compared to sham treated animals (+32.5%, p<0.001), as well as in tracer binding in all individual VOIs. After chronic administration of LEV, there was no significant change in global cerebral CB1R binding (+6.9%, p=0.81), nor in tracer binding in individual VOIs. As VPA does not exhibit high affinity for CB1R (displacement of [(3)H]-SR141716A 1.3+/-14.0%), such upregulation is most likely caused by an indirect effect on the endocannabinoid system. This increase in CB1R tracer binding and possibly signaling may represent a supplementary and new mechanism of VPA, but not LEV, since activation of CB1Rs has been shown to decrease excitability and excitotoxicity on-demand.

Labelling and biological evaluation of [(11)C]methoxy-Sch225336: a radioligand for the cannabinoid-type 2 receptor.

INTRODUCTION: The cannabinoid type 2 receptor (CB(2) receptor) is part of the endocannabinoid system and has been suggested as mediator of a number of central and peripheral inflammatory processes. In the present study, we have synthesized N-[(1s)-1-[4-[[4-methoxy-2-[(4-[(11)C]methoxyphenyl)sulfonyl)-phenyl]sulfonyl] phenyl]ethyl]methanesulfonamide ([(11)C]methoxy-Sch225336) and evaluated this new tracer agent as a potential positron emission tomography radioligand for the in vivo visualization of CB(2) receptors. METHODS: Sch225336 was demethylated and the resulting phenol precursor was radiolabelled with a carbon-11 methyl group by methylation using [(11)C]methyl iodide, followed by purification by high-performance liquid chromatography. The log P of [(11)C]methoxy-Sch225336 and its biodistribution in normal mice were determined. Enhancement of brain uptake by inhibition of blood-brain barrier (BBB) efflux transporters was studied. Mouse plasma was analysed to quantify the formation of radiometabolites. The affinity of Sch225336 for the human cannabinoid type 1 and type 2 receptor was determined. RESULTS: [(11)C]methoxy-Sch225336 was obtained with a decay corrected radiochemical yield of about 30% and a specific activity of 88.8 GBq/mumol (end of synthesis). After intravenous injection in mice, the compound is rapidly cleared from the blood through the hepatobiliary pathway and does not show particular retention in any of the major organs. Polar metabolites were found in mouse plasma. Brain uptake was low despite the favourable log P value of 2.15, which is partly due to efflux by BBB pumps. CONCLUSION: [(11)C]methoxy-Sch225336 is a good candidate for in vivo imaging of the CB(2) receptor, although the low blood-brain barrier penetration limits its potential for central nervous system imaging.

Astroglial CB1 Receptors Determine Synaptic D-Serine Availability to Enable Recognition Memory.

Bidirectional communication between neurons and astrocytes shapes synaptic plasticity and behavior. D-serine is a necessary co-agonist of synaptic N-methyl-D-aspartate receptors (NMDARs), but the physiological factors regulating its impact on memory processes are scantly known. We show that astroglial CB1 receptors are key determinants of object recognition memory by determining the availability of D-serine at hippocampal synapses. Mutant mice lacking CB1 receptors from astroglial cells (GFAP-CB1-KO) displayed impaired object recognition memory and decreased in vivo and in vitro long-term potentiation (LTP) at CA3-CA1 hippocampal synapses. Activation of CB1 receptors increased intracellular astroglial Ca2+ levels and extracellular levels of D-serine in hippocampal slices. Accordingly, GFAP-CB1-KO displayed lower occupancy of the co-agonist binding site of synaptic hippocampal NMDARs. Finally, elevation of D-serine levels fully rescued LTP and memory impairments of GFAP-CB1-KO mice. These data reveal a novel mechanism of in vivo astroglial control of memory and synaptic plasticity via the D-serine-dependent control of NMDARs.

Pharmacomodulations around the 4-oxo-1,4-dihydroquinoline-3-carboxamides, a class of potent CB2-selective cannabinoid receptor ligands: consequences in receptor affinity and functionality.

CB2 receptor selective ligands are becoming increasingly attractive drugs due to the potential role of this receptor in several physiopathological processes. Thus, the development of our previously described series of 4-oxo-1,4-dihydroquinoline-3-carboxamides was pursued with the aim to further characterize the structure-affinity and structure-functionality relationships of these derivatives. The influence of the side chain was investigated by synthesizing compounds bearing various carboxamido and keto substituents. On the other hand, the role of the quinoline central scaffold was studied by synthesizing several 6-, 7-, or 8-chloro-4-oxo-1,4-dihydroquinolines, as well as 4-oxo-1,4-dihydronaphthyridine and 4-oxo-1,4-dihydrocinnoline derivatives. The effect of these modifications on the affinity and functionality at the CB2 receptor was studied and allowed for the characterization of new selective CB2 receptor ligands.

A new model of nerve injury in the rat reveals a role of Regulator of G protein Signaling 4 in tactile hypersensitivity.

Tactile hypersensitivity is one of the most debilitating symptoms of neuropathic pain syndromes. Clinical studies have suggested that its presence at early postoperative stages may predict chronic (neuropathic) pain after surgery. Currently available animal models are typically associated with consistent tactile hypersensitivity and are therefore limited to distinguish between mechanisms that underlie tactile hypersensitivity as opposed to mechanisms that protect against it. In this study we have modified the rat model of spared nerve injury, restricting the surgical lesion to a single peripheral branch of the sciatic nerve. This modification reduced the prevalence of tactile hypersensitivity from nearly 100% to approximately 50%. With this model, we here also demonstrated that the Regulator of G protein Signaling 4 (RGS4) was specifically up-regulated in the lumbar dorsal root ganglia and dorsal horn of rats developing tactile hypersensitivity. Intrathecal delivery of the RGS4 inhibitor CCG63802 was found to reverse tactile hypersensitivity for a 1h period. Moreover, tactile hypersensitivity after modified spared nerve injury was most frequently persistent for at least four weeks and associated with higher reactivity of glial cells in the lumbar dorsal horn. Based on these data we suggest that this new animal model of nerve injury represents an asset in understanding divergent neuropathic pain outcomes, so far unravelling a role of RGS4 in tactile hypersensitivity. Whether this model also holds promise in the study of the transition from acute to chronic pain will have to be seen in future investigations.

Inhibition of the regulator of G protein signalling RGS4 in the spinal cord decreases neuropathic hyperalgesia and restores cannabinoid CB1 receptor signalling.

BACKGROUND AND PURPOSE: Regulators of G protein signalling (RGS) are major determinants of metabotropic receptor activity, reducing the lifespan of the GTP-bound state of G proteins. Because the reduced potency of analgesic agents in neuropathic pain may reflect alterations in RGS, we assessed the effects of CCG 63802, a specific RGS4 inhibitor, on pain hypersensitivity and signalling through cannabinoid receptors, in a model of neuropathic pain. EXPERIMENTAL APPROACH: The partial sciatic nerve ligation (PSNL) model in male Sprague Dawley rats was used to measure paw withdrawal thresholds to mechanical (von Frey hairs) or thermal (Hargreaves method) stimuli, during and after intrathecal injection of CCG 63802. HEK293 cells expressing CB1 receptors and conditional expression of RGS4 were used to correlate cAMP production and ERK phosphorylation with receptor activation and RGS4 action. KEY RESULTS: Treatment of PSNL rats with CCG 63802, twice daily for 7 days after nerve injury, attenuated thermal hyperalgesia during treatment. Spinal levels of anandamide were higher in PSNL animals, irrespective of the treatment. Although expression of CB1 receptors was unaffected, HU210-induced CB1 receptor signalling was inhibited in PSNL rats and restored after intrathecal CCG 63802. In transfected HEK cells expressing CB1 receptors and RGS4, inhibition of cAMP production, a downstream effect of CB1 receptor signalling, was blunted after RGS4 overexpression. RGS4 expression also attenuated the CB1 receptor-controlled activation of ERK1/2. CONCLUSIONS AND IMPLICATIONS: Inhibition of spinal RGS4 restored endogenous analgesic signalling pathways and mitigated neuropathic pain. Signalling through CB1 receptors may be involved in this beneficial effect.

Partial agonism and fast dissociation of LASSBio-579 at dopamine D2 receptor.

In an attempt to better understand the molecular mechanism of action of the antipsychotic lead LASSBio-579 and of its main metabolite LQFM 037, the aim of this work was to evaluate their intrinsic activity and binding kinetics at the dopamine D2 receptor. In transfected HEK cells expressing the D2L receptor under an inducible promoter, LASSBio-579 and LQFM 037, but not clozapine, behaved as weak partial agonists in [(35)S]-GTPγS binding assays performed in optimized conditions previously shown to evidence the partial agonist profile of aripiprazole. Besides, data obtained in radioligand competition assays on rat striatal membranes suggested a rapid association to and dissociation from the D2-like receptors. Using the kinetic rate index based on the strategy of the dual-point competition association assay, we showed that our compounds share a similar kinetic profile with clozapine, distinct from the typical antipsychotic haloperidol. These two characteristics could contribute to the atypical-like profile observed after administration of LASSBio-579 to rodents, in models of positive and negative symptoms of schizophrenia.

The FAAH inhibitor URB597 efficiently reduces tyrosine hydroxylase expression through CB1- and FAAH-independent mechanisms.

BACKGROUND: Anandamide and 2-arachidonoylglycerol are neuromodulatory lipids interacting with cannabinoid receptors, whose availability is regulated by the balance between 'on demand' generation and enzymatic degradation [by fatty acid amide hydrolase (FAAH)/monoacylglycerol lipase]. Given the reported effects of anandamide on dopamine transmission, we investigated the influence of endocannabinoids and URB597, a well-known FAAH inhibitor, on the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis. EXPERIMENTAL APPROACH: We investigated TH expression in N1E115 neuroblastoma using a reporter gene assay, as well as mRNA and protein quantifications. FAAH inhibition was confirmed by measuring radiolabelled substrate hydrolysis and endogenous endocannabinoids. KEY RESULTS: Anandamide decreased TH promoter activity in N1E115 cells through CB1 receptor activation. Unexpectedly, URB597 reduced TH expression (pEC50 = 8.7 ± 0.2) through FAAH-independent mechanisms. Indeed, four structurally unrelated inhibitors of FAAH had no influence on TH expression, although all the inhibitors increased endocannabinoid levels. At variance with the endocannabinoid responses, the use of selective antagonists indicated that the URB597-mediated decrease in TH expression was not directed by the CB1 receptor, but rather by abnormal-cannabidiol-sensitive receptors and PPARs. Further supporting the physiological relevance of these in vitro data, URB597 administration resulted in reduced TH mRNA levels in mice brain. CONCLUSIONS: While confirming the implication of endocannabinoids on the modulation of TH, we provide strong evidence for additional physiologically relevant off-target effects of URB597. In light of the numerous preclinical studies involving URB597, particularly in anxiety and depression, the existence of non-CB1 and non-FAAH mediated influences of URB597 on key enzymes of the catecholaminergic transmission system should be taken into account when interpreting the data.

Astroglial CB1 cannabinoid receptors regulate leptin signaling in mouse brain astrocytes.

Type-1 cannabinoid (CB1) and leptin (ObR) receptors regulate metabolic and astroglial functions, but the potential links between the two systems in astrocytes were not investigated so far. Genetic and pharmacological manipulations of CB1 receptor expression and activity in cultured cortical and hypothalamic astrocytes demonstrated that cannabinoid signaling controls the levels of ObR expression. Lack of CB1 receptors also markedly impaired leptin-mediated activation of signal transducers and activators of transcription 3 and 5 (STAT3 and STAT5) in astrocytes. In particular, CB1 deletion determined a basal overactivation of STAT5, thereby leading to the downregulation of ObR expression, and leptin failed to regulate STAT5-dependent glycogen storage in the absence of CB1 receptors. These results show that CB1 receptors directly interfere with leptin signaling and its ability to regulate glycogen storage, thereby representing a novel mechanism linking endocannabinoid and leptin signaling in the regulation of brain energy storage and neuronal functions.

Increasing the density of the D2L receptor and manipulating the receptor environment are required to evidence the partial agonist properties of aripiprazole.

The clinical efficacy of aripiprazole in the treatment of psychosis relies on a partial agonism at D2 receptors. As the expression of this receptor differs physiologically between pre- and post-synaptic sites and is affected by pathological conditions or pharmacological treatments, it appears difficult to predict the clinical response to partial agonists. In addition, the response to this novel antipsychotic was shown to depend on the cell-line and the pathway analyzed, suggesting a functional selective profile at the D2 receptor. This study aims at examining the influence of receptor density and ionic environment on the pharmacological properties of aripiprazole. A cell line was developed in which the expression of the recombinant D2 receptor can be tightly manipulated using doxycycline and sodium butyrate. The potency and efficacy of aripiprazole and other reference D2 receptor ligands were examined in [35S]GTPγS binding assays, in buffers containing either NaCl or N-methyl-D-glucamine (NMDG) which is proposed to enhance G protein coupling. Increasing the density of D2 receptors considerably enhanced the [35S]GTPγS binding induced by dopamine and the full agonist NPA. In maximally induced cells, the agonist properties of the partial agonist (-)-3-PPP was revealed in a buffer containing NaCl, whereas the response to aripiprazole was not evidenced. Substituting NMDG for NaCl promoted the response to dopamine and (-)3-PPP and was proven efficient to reveal the partial agonist profile of aripiprazole. While NMDG substitution for NaCl strongly enhanced receptor-G protein coupling, these ionic manipulations are likely to influence receptor conformations, thereby modulating the activation of signaling pathways. Our data obtained with partial agonists acting at the D2 receptor suggest that these changes in the experimental conditions could contribute to reveal the functional selective profile of GPCR ligands. They also emphasize that the properties of functional selective ligands do not only depend on receptor density but also on the surrounding environment which likely differs between brain structures.

Differential modulations of striatal tyrosine hydroxylase and dopamine metabolism by cannabinoid agonists as evidence for functional selectivity in vivo.

It is generally assumed that cannabinoids induce transient modulations of dopamine transmission through indirect regulation of its release. However, we previously described a direct cannabinoid-mediated control of tyrosine hydroxylase (TH) expression, in vitro. We herein report on the influence of cannabinoid agonists on the expression of this key enzyme in catecholamine synthesis as well as on the modification of dopamine content in adult rats. As expected for cannabinoid agonists, the exposure to either Δ(9)-THC, HU 210 or CP 55,940 induced both catalepsy and hypolocomotion. Supporting a possible long-lasting control on dopaminergic activity, we noticed a significant HU 210-mediated increase in TH expression in the striatum that was concomitant with an increase in striatal dopamine content. Surprisingly, while a similar trend was reported with Δ(9)-THC, CP 55,940 completely failed to modulate TH expression or dopamine content. Nevertheless, the access of CP 55,940 to brain structures was validated by determinations of drug concentrations in the tissue and by ex vivo binding experiments. Furthermore, confirming the central activity of CP 55,940, the analysis of dopamine metabolites revealed a reduction in striatal DOPAC concentrations. Consistent with the involvement of the CB(1) cannabinoid receptor in these different responses, both HU 210- and CP 55,940-mediated effects were prevented by SR 141716A. Therefore, the present data suggest that both HU 210 and CP 55,940 cause a delayed/persistent regulation of the dopamine neurotransmission system. Nevertheless, these commonly used cannabinoid agonists endowed with similar pharmacodynamic properties clearly triggered distinct biochemical responses highlighting the existence of functional selectivity in vivo.

A PET brain reporter gene system based on type 2 cannabinoid receptors.

UNLABELLED: PET of gene expression in the brain may greatly facilitate neuroscience research and potential clinical implementation of gene or cell therapy of central nervous system diseases. To date, no adequate PET reporter system is available for the central nervous system because available tracers either do not cross the intact blood-brain barrier or have high background signals. Here we report the first, to our knowledge, PET reporter system for imaging gene expression in the intact brain. METHODS: We selected the human type 2 cannabinoid receptor (hCB(2)) as a reporter because of its low basal expression in the brain. An inactive mutant (D80N) was chosen so as not to interfere with signal transduction. As a reporter probe we used the (11)C-labeled CB(2) ligand, (11)C-GW405833, which readily crosses the blood-brain barrier. Dual-modality imaging lentiviral and adeno-associated viral vectors encoding both hCB(2)(D80N) and firefly luciferase or enhanced green fluorescent protein were engineered and validated in cell culture. Next, hCB(2)(D80N) was locoregionally overexpressed in rat striatum by stereotactic injection of lentiviral and adeno-associated viral vectors. RESULTS: Kinetic PET revealed specific and reversible CB(2) binding of (11)C-GW405833 in the transduced rat striatum. hCB(2) and firefly luciferase expression was followed until 9 mo and showed similar kinetics. Both hCB(2) expression and enhanced green fluorescent protein expression were confirmed by immunohistochemistry. CONCLUSION: Dual-modality imaging viral vectors encoding hCB(2)(D80N) were engineered, and the reporter system was validated in different animal species. The results support the potential future clinical use of CB(2) as a PET reporter in the intact brain.

Functionally selective cannabinoid receptor signalling: therapeutic implications and opportunities.

The CB(1) and CB(2) cannabinoid receptors are G protein-coupled receptors (GPCRs) recognized by a variety of endogenous ligands and activating multiple signalling pathways. This multiplicity of ligands and intracellular transduction mechanisms supports a complex control of physiological functions by the endocannabinoid system, but requires a finely tuned regulation of the signalling events triggered on receptor activation. Here we review the diverse signalling pathways activated by the cannabinoid receptors and discuss the mechanisms allowing for specificity in the associated functional responses triggered by endogenous or exogenous ligands. At variance with the classical concept that all agonists at a given GPCR induce a similar repertoire of downstream events in all tissues, we also summarize the experimental evidence supporting the existence of functional selectivity and protean agonism at cannabinoid receptors. By placing emphasis on the ligand- or constitutive activity-dependent specifications of receptor-G protein coupling, these concepts explain how distinct cannabinoid ligands may activate specific downstream mediators. Finally, although both the diversity and specificity in cannabinoid signalling are now established in vitro, few data are available from in vivo studies. Therefore, we conclude this review by examining the experimental evidence supporting the physiological relevance of this complexity in the cannabinoid system. The ability to selectively manipulate physiological functions, through activation of defined signalling cascades, will in all likelihood help in the development of efficacious and safe cannabinoid-based therapeutics for a variety of indications.

Revisiting the complex influences of cannabinoids on motor functions unravels pharmacodynamic differences between cannabinoid agonists.

While numerous cannabinoid ligands were historically characterized using the tetrad test (hypomobility, catalepsy, hypothermia, analgesia), only few studies have extensively compared HU 210 and CP 55,940 which are nowadays classically used as reference agonists. Therefore, we herein re-examined the acute and the sustained changes in motor activities mediated by these two agonists in adult rats. As expected for cannabinoid agonists, exposure to either HU 210 or CP 55,940 induced a marked reduction in spontaneous locomotion. This reduction observed as early as 15 min after injection was correlated with the typical rearing and cataleptic responses, and was reversed by co-administration of the CB(1) cannabinoid receptor antagonist SR 141716A. Nevertheless, HU 210, but not CP 55,940, was found to induce persistent responses, lasting for at least 24h. Also suggesting the involvement of additional targets for HU 210, 10mg/kg SR 141716A failed to reverse the persistent HU 210-mediated decline in locomotion and rearing, while 1mg/kg was sufficient to completely abolish the behavioural responses measured 6h after the injection. Beside pharmacokinetic differences, these data therefore denote distinct pharmacodynamic profiles for HU 210 and CP 55,940. Together, these results suggest that HU 210 displays multicomponent responses that should be taken into account when interpreting data from in vivo/ex vivo studies.

Influence of chronic nicotine administration on cerebral type 1 cannabinoid receptor binding: an in vivo micro-PET study in the rat using [18F]MK-9470.

Several lines of evidence suggest a functional interaction between central nicotinic and endocannabinoid systems. Furthermore, type 1 cannabinoid receptor (CB1R) antagonism is evaluated as antismoking therapy, and nicotine usage can be an important confound in positron emission tomography (PET) imaging studies of the CB1R. We evaluated CB1R binding in the rat brain using the PET radioligand [(18)F]MK-9470 after chronic administration of nicotine. Twelve female Wistar rats were scanned at baseline and after chronic administration of either nicotine (1 mg/kg; 2 weeks daily intraperitoneal (IP)) or saline as control. In vivo micro-PET images of CB1R binding were anatomically standardized and analyzed by voxel-based statistical parametric mapping and a predefined volume-of-interest approach. We did not observe changes in [(18)F]MK-9470 binding (p (height) < 0.001 level; uncorrected) on a group basis in either condition. Only at a less stringent threshold of p (height) < 0.005 (uncorrected) was a modest increase observed in tracer binding in the cerebellum for nicotine (peak voxel value + 6.8%, p (cluster) = 0.002 corrected). In conclusion, chronic IP administration of nicotine does not produce major cerebral changes in CB1R binding of [(18)F]MK-9470 in the rat. These results also suggest that chronic nicotine usage is unlikely to interfere with human PET imaging using this radioligand.

Concomitant activation of adenylyl cyclase suppresses the opposite influences of CB(1) cannabinoid receptor agonists on tyrosine hydroxylase expression.

The CB(1) cannabinoid receptor shows complex interactions with intracellular signalling partners, and responses to cannabinoid ligands are likely to be influenced by concomitant inputs modifying the overall tone of signalling cascades. This appears even more relevant as we previously evidenced opposite regulations of tyrosine hydroxylase (TH) expression by the two common cannabinoid agonists HU 210 and CP 55,940. Therefore, we studied the consequences of manipulating adenylyl cyclase activity with forskolin on the regulation of TH gene transcription in neuroblastoma cells (N1E-115). Reporter gene experiments performed with the luciferase sequence cloned under the control of modified fragments of the TH gene promoter revealed that the AP-1 consensus sequence is essential for cannabinoid-mediated regulation of TH expression. Consistently, inhibition of PKC totally blocked the responses mediated by both HU 210 and CP 55,940. In addition, forskolin which boosts adenylyl cyclase activity remarkably modified the responses to the cannabinoid agonists. Thus, in these conditions, both agonists efficiently reduced TH gene promoter activity, a response requiring functional PKA/CRE-dependent signallings. Finally, the modulations of the promoter were inhibited in pertussis toxin treated cells, suggesting that responses to both agonists are mediated through G(i/o)-dependent mechanisms. Emphasising on the importance of functional selectivity at GPCRs, these data demonstrate that the concomitant activation of adenylyl cyclase by forskolin strongly influences the biochemical responses triggered by distinct cannabinoid agonists. Together our results suggest that the physiological modulation of TH expression by cannabinoid agonists in dopaminergic neurons would be influenced by additional endogenous inputs.

Agonist selective modulation of tyrosine hydroxylase expression by cannabinoid ligands in a murine neuroblastoma cell line.

Functional interactions between catecholamines and cannabinoid transmission systems could explain the influence of Delta(9)-tetrahydrocannabinol on several central activities. Hence, the presence of cannabinoid CB(1) receptors in tyrosine hydroxylase (TH) containing cells has been suggested, providing clue for a direct control of catecholamines synthesis. In the present study, we evidenced the constitutive expression of functional cannabinoid CB(1) receptors in N1E-115 neuroblastoma and reported on the use of this model to examine the influence of diverse cannabinoid ligands on TH expression. Exposure of the cells to the high-affinity agonist HU 210 (5 h) resulted in a significant decrease in TH content (pEC(50): 6.40). In contrast, no change was observed after a similar treatment with the structurally unrelated agonist CP 55,940. Besides, the use of a luciferase reporter assay revealed that these two agonists showed opposite influences on TH gene promoter activity. Thus, in cells expressing pTH-luc constructs, inhibition and induction of luciferase activity were respectively observed with HU 210 (pEC(50): 8.95) and CP 55,940 (pEC(50): 9.09). Pharmacological characterisation revealed that these reciprocal responses were both related to the specific activation of cannabinoid CB(1) receptor, suggesting an agonist-dependent modulation of distinct signalling pathways. While these data points out the possible pharmacological manipulation of TH expression by cannabinoid ligands, such approach should take into account the existence of agonist selective trafficking of cannabinoid CB(1) receptor signalling.