Functional assessment of neuronal cannabinoid receptors in the muscular layers of human ileum and colon.

BACKGROUND & AIMS: The notion that specific receptors account for the ability of natural and synthetic cannabinoids to alter physiological functions, prompted this study aimed at assessing their functional presence in the human gut. METHODS: The effects have been studied of cannabinoids and selective antagonists of their receptors on chemically or electrically evoked contractions in preparations of human intestinal smooth muscle in vitro. RESULTS: Atropine prevented the contractions of longitudinal and circular muscle strips of ileum and colon induced by carbachol or electrical field stimulation; tetrodotoxin abolished only the latter which suggests they do involve activation of cholinergic neurons. The synthetic cannabinoid (+)WIN 55,212-2 had no effect on carbachol contractions, but in a concentration-dependent fashion prevented those elicited by electrical field stimulation - which were insensitive to the putative endogenous cannabinoid anandamide - more potently in longitudinal than in circular strips. The selective CB1 receptor antagonist SR141716, which had no effect in the absence of (+)WIN 55,212-2, competitively antagonised its inhibition of electrical field stimulation contractions, unlike the selective CB2 antagonist SR144528. CONCLUSIONS: Cannabinoid CB1 receptors are functionally present in the human ileum and colon; their pharmacological activation apparently results in inhibition of excitatory cholinergic pathways subserving smooth muscle contraction.

Mutational analysis and molecular modelling of the antagonist SR 144528 binding site on the human cannabinoid CB(2) receptor.

We have investigated the binding site of the subtype specific antagonist SR 144528, (N-[(1S)-endo-1,3,3-trimethyl bicyclo [2.2. 1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methoxybenzyl)- pyrazo le-3-carboxamide) on the human cannabinoid CB(2) receptor based on functional studies with mutated receptors. Two serine residues in the fourth transmembrane region, Ser(161) and Ser(165), were singly mutated to the cognate cannabinoid CB(1) receptor residue, alanine, and each gave receptors with wild-type properties for the cannabinoid agonists CP 55,940 (1R,3R,4R)-3-[2-hydroxy-4-(1, 1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol) and WIN 55212-2 (R)-(+)[2, 3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1, 4-benzoxazin-6-yl](1-naphthalenyl) methanone, which SR 144528 completely failed to antagonise. Molecular modelling studies show that SR 144528 interacts with residues in transmembrane domains 3, 4, and 5 of the cannabinoid CB(2) receptor through a combination of hydrogen bonds and aromatic and hydrophobic interactions. In addition, the replacement by serine of a nearby cannabinoid CB(2) receptor-specific residue, Cys(175) resulted in wild-type receptor properties with CP 55,940, loss of SR 144528 binding and eight-fold reduced binding and activity of WIN 55212-2, a result compatible with a recently-proposed binding site model for WIN 55212-2.

Biodistribution of [18F] SR144385 and [18F] SR147963: selective radioligands for positron emission tomographic studies of brain cannabinoid receptors.

ABSTRACT. [(18)F] SR144385 and [(18)F] SR147963 were synthesized in a multistep reaction in which fluorine-18 was introduced by nucleophilic halogen displacement on a bromo precursor. The fluorine-18-labeled intermediate was deprotected and coupled with the appropriate alkyl amine to give the final products. Both radioligands had appropriate regional brain distribution for cannabinoid receptors with a target to nontarget ratio of 1.7 for [(18)F] SR147963 and 2.5 for [(18)F] SR144385 at 60 and 90 min postinjection, respectively. The uptake of both tracers was blocked with a 1 mg/kg dose of SR141716A.

The development of cannabinoid antagonists.

The discovery of two distinct cannabinoid receptors (CB1 and CB2) in the early 1990's has revived the research on cannabinoid antagonists. While the search for antagonists based on the structure of agonists (classical cannabinoids or aminoalkylindoles) appeared rather disappointing, the first potent cannabinoid antagonists were developed in a new chemical series: the diarylpyrazoles. Since its discovery in 1994, the selective CB1 antagonist SR 141716 has became a major pharmacological tool to elucidate the physiological role of the CB1 cannabinoid receptor and its endogenous ligand. The selective CB2 antagonist SR 144528 is expected to play the same role for the CB2 receptors, while the recent development of cannabinoid antagonists belonging to other chemical series illustrates the interest of these compounds which are now considered as interesting therapeutic targets by many pharmaceutical companies.

Cannabinoid receptor interactions with the antagonists SR 141716A and SR 144528.

The G protein-coupled cannabinoid receptor subtypes CB1 and CB2 have been cloned from several species. The CB1 receptor is highly conserved across species, whereas the CB2 receptor shows considerable cross-species variations. The two human receptors share only 44% overall identity, ranging from 35% to 82% in the transmembrane regions. Despite this structural disparity, the most potent cannabinoid agonists currently available are largely undiscriminating and are therefore unsatisfactory tools for investigating the architecture of ligand binding sites. However, the availability of two highly specific antagonists, SR 141716A for the CB1 receptor and SR 144528 for the CB2 receptor, has allowed us to adopt a systematic approach to defining their respective binding sites through the use of chimeric CB1 receptor/CB2 receptor constructs, coupled with site-directed mutagenesis. We identified the region encompassed by the fourth and fifth transmembrane helices as being critical for antagonist specificity. Both the wild type human receptors overexpressed in heterologous systems are autoactivated; SR 141716A and SR 144528 exhibit classical inverse agonist properties with their respective target receptors. In addition, through its interaction with the CB1 receptor SR 141716A blocks the Gi protein-mediated activation of mitogen-activated protein kinase stimulated by insulin or insulin-like growth factor I. An in-depth analysis of this discovery has led to a modified three-state model for the CB1 receptor, one of which implicates the SR 141716A-mediated sequestration of Gi proteins, with the result that the growth factor-stimulated intracellular pathways are effectively impeded.

SR 144528, an antagonist for the peripheral cannabinoid receptor that behaves as an inverse agonist.

In the present report, we investigated in detail the effects of SR 144528, a selective antagonist of the peripheral cannabinoid receptor (CB2), on two well-characterized functions mediated by CB2: the induction of the early response gene krox24 and the inhibition of adenylyl cyclase. We generated Chinese hamster ovary cells doubly transfected with human CB2 and a luciferase reporter gene linked to either the murine krox24 regulatory sequence or multiple cAMP responsive elements. Our results show that (1) SR 144528 antagonizes the effect of receptor agonists-it inhibits the krox24 reporter activity and prevents the inhibition of forskolin-induced cAMP reporter activity mediated by CP 55,940; (2) CB2 is autoactivated-CB2 mediates signaling in the absence of ligand, and this basal activity is reduced by pretreating the cells with pertussis toxin; (3) SR 144528 is an inverse agonist-it reproduces the effects of pertussis toxin; and (4) inhibition of precoupled CB2 by a long-term pretreatment of cells with SR 144528 potentiates krox24 response to cannabinoid receptor agonists and restores activation of adenylyl cyclase. Taken together, these data provide evidences for the inverse agonist property of SR 144528 and the constitutive activation of CB2 in Chinese hamster ovary-expressing cells.

Modulation of CB1 cannabinoid receptor functions after a long-term exposure to agonist or inverse agonist in the Chinese hamster ovary cell expression system.

We have investigated the adaptive changes of the human central cannabinoid receptor (CB1) stably expressed in Chinese hamster ovary cells (CHO-CB1), after agonist (CP 55,940) or selective CB1 inverse agonist (SR 141716) treatment. CB1 receptor density and affinity constant as measured by binding assays with both tritiated ligands remained essentially unchanged after varying period exposure of CHO-CB1 cells (from 30 min to 72 hr) to saturating concentrations of CP 55,940 or SR 141716. However, using a C-myc-tagged version of the CB1 receptor, FACS analysis and confocal microscopy studies on CB1 expression indicated that the agonist promoted a disappearance of cell surface receptor although inverse agonist increased its cell surface density. Taken together these results suggest that 1) agonist induces internalization of the receptor into a cellular compartment that would be still accessible to both the hydrophobic ligands CP 55,940 or SR 141716; 2) inverse-agonist promotes externalization of the receptor from an intracellular preexisting pool to the cell surface. In parallel, we also investigated the associated effects of CP 55,940 and SR 141716 on CB1 receptor-coupled second messengers. We showed that preexposure of cells to CP 55,940 induced a rapid desensitization of the CB1 to the agonist response. The ability of CP 55,940 to inhibit the forskolin-stimulated adenylyl cyclase and to activate the mitogen-activated protein kinase activity was dramatically reduced. By striking contrast, SR 141716 pretreatment of CHO-CB1 cells not only had no significant effect on the potency of CP 55,940 to inhibit the forskolin-stimulated adenylyl cyclase but also induced a significant enhancement of the CP 55,940 ability to stimulate the mitogen-activated protein kinase activity. These results suggest that the modulation of the number of cell surface receptor could lead to functional desensitization or sensitization of the CB1 receptors.

The endogenous cannabinoid anandamide is a lipid messenger activating cell growth via a cannabinoid receptor-independent pathway in hematopoietic cell lines.

The effect of anandamide, an endogenous ligand for central (CB1) and peripheral (CB2) cannabinoid receptors, was investigated on the growth of the murine IL-6-dependent lymphoid cell line B9 and the murine IL-3-dependent myeloblastic cell line FDC-P1. In conditions of low serum level, anandamide potentiated the growth of both cytokine-dependent cell lines. Comparison with other fatty acid cannabinoid ligands such as (R)-methanandamide, a ligand with improved selectivity for the CB1 receptor, or palmitylethanolamide, an endogenous ligand for the CB2 receptor, showed a very similar effect, suggesting that cell growth enhancement by anandamide or its analogs could be mediated through either receptor subtype. However, several lines of evidence indicated that this growth-promoting effect was cannabinoid receptor-independent. First, the potent synthetic cannabinoid agonist CP 55940, which displays high affinity for both receptors, was inactive in this model. Second, SR 141716A and SR 144528, which are potent and specific antagonists of CB1 and CB2 receptors respectively, were unable, alone or in combination, to block the anandamide-induced effect. Third, inactivation of both receptors by pretreatment of cells with pertussis toxin did not affect the potentiation of cell growth by anandamide. These data demonstrated that neither CB1 nor CB2 receptors were involved in the anandamide-induced effect. Moreover, using CB2-transfected Chinese hamster ovary cells, we demonstrated that after complete blockade of the receptors by the specific antagonist SR 144528, anandamide was still able to strongly stimulate a mitogen-activated protein (MAP) kinase activity, clearly indicating that the endogenous cannabinoid can transduce a mitogenic signal in the absence of available receptors. Finally, arachidonic acid, a structurally related compound and an important lipid messenger without known affinity for cannabinoid receptors, was shown to trigger MAP kinase activity and cell growth enhancement similar to those observed with anandamide. These findings provide clear evidence for a functional role of anandamide in activating a signal transduction pathway leading to cell activation and proliferation via a non-cannabinoid receptor-mediated process.

SR 144528, the first potent and selective antagonist of the CB2 cannabinoid receptor.

Based on both binding and functional data, this study introduces SR 144528 as the first, highly potent, selective and orally active antagonist for the CB2 receptor. This compound which displays subnanomolar affinity (Ki = 0.6 nM) for both the rat spleen and cloned human CB2 receptors has a 700-fold lower affinity (Ki = 400 nM) for both the rat brain and cloned human CB1 receptors. Furthermore it shows no affinity for any of the more than 70 receptors, ion channels or enzymes investigated (IC50 > 10 microM). In vitro, SR 144528 antagonizes the inhibitory effects of the cannabinoid receptor agonist CP 55,940 on forskolin-stimulated adenylyl cyclase activity in cell lines permanently expressing the h CB2 receptor (EC50 = 10 nM) but not in cells expressing the h CB1 (no effect at 10 microM). Furthermore, SR 144528 is able to selectively block the mitogen-activated protein kinase activity induced by CP 55,940 in cell lines expressing h CB2 (IC50 = 39 nM) whereas in cells expressing h CB1 an IC50 value of more than 1 microM is found. In addition, SR 144528 is shown to antagonize the stimulating effects of CP 55,940 on human tonsillar B-cell activation evoked by cross-linking of surface Igs (IC50 = 20 nM). In vivo, after oral administration SR 144528 totally displaced the ex vivo [3H]-CP 55,940 binding to mouse spleen membranes (ED50 = 0.35 mg/kg) with a long duration of action. In contrast, after the oral route it does not interact with the cannabinoid receptor expressed in the mouse brain (CB1). It is expected that SR 144528 will provide a powerful tool to investigate the in vivo functions of the cannabinoid system in the immune response.

In vitro functional evidence of neuronal cannabinoid CB1 receptors in human ileum.

We investigated the effect of the cannabinoid agonist (+)WIN-55212-2 on human ileum longitudinal smooth muscle preparations, either electrically stimulated or contracted by carbachol. Electrical field stimulation mostly activated cholinergic neurons, since atropine and tetrodotoxin (TTX), alone or coincubated, reduced twitch responses to a similar degree (85%). (+)WIN-55212-2 concentration-dependently inhibited twitch responses (IC50 73 nM), but had no additive effect with atropine or TTX. The cannabinoid CB1 receptor antagonist SR 141716 (pA2 8.2), but not the CB2 receptor antagonist, SR 144528, competitively antagonized twitch inhibition by (+)WIN-55212-2. Atropine but not (+)WIN-55212-2 or TTX prevented carbachol-induced tonic contraction. These results provide functional evidence of the existence of prejunctional cannabinoid CB1-receptors in the human ileum longitudinal smooth muscle. Agonist activation of these receptors prevents responses to electrical field stimulation, presumably by inhibiting acetylcholine release. SR 141716 is a potent and competitive antagonist of cannabinoid CB1 receptors naturally expressed in the human gut.

A selective inverse agonist for central cannabinoid receptor inhibits mitogen-activated protein kinase activation stimulated by insulin or insulin-like growth factor 1. Evidence for a new model of receptor/ligand interactions.

In the present study, we showed that Chinese hamster ovary (CHO) cells transfected with human central cannabinoid receptor (CB1) exhibit high constitutive activity at both levels of mitogen-activated protein kinase (MAPK) and adenylyl cyclase. These activities could be blocked by the CB1-selective ligand, SR 141716A, that functions as an inverse agonist. Moreover, binding studies showed that guanine nucleotides decreased the binding of the agonist CP-55,940, an effect usually observed with agonists, whereas it enhanced the binding of SR 141716A, a property of inverse agonists. Unexpectedly, we found that CB1-mediated effects of SR 141716A included inhibition of MAPK activation by pertussis toxin-sensitive receptor-tyrosine kinase such as insulin or insulin-like growth factor 1 receptors but not by pertussis toxin-insensitive receptor-tyrosine kinase such as the fibroblast growth factor receptor. We also observed similar results when cells were stimulated with Mas-7, a mastoparan analog, that directly activates the Gi protein. Furthermore, SR 141716A inhibited guanosine 5'-0-(thiotriphosphate) uptake induced by CP-55,940 or Mas-7 in CHO-CB1 cell membranes. This indicates that, in addition to the inhibition of autoactivated CB1, SR 141716A can deliver a biological signal that blocks the Gi protein and consequently abrogates most of the Gi-mediated responses. By contrast, SR 141716A had no effect on MAPK activation by insulin or IGF1 in CHO cells lacking CB1 receptors, ruling out the possibility of a direct interaction of SR 141716A with the Gi protein. This supports the notion that the Gi protein may act as a negative intracellular signaling cross-talk molecule. From these original results, which considerably enlarge the biological properties of the inverse agonist, we propose a novel model for receptor/ligand interactions.

Characterization of CB1 receptors on rat neuronal cell cultures: binding and functional studies using the selective receptor antagonist SR 141716A.

This study was undertaken to characterize further the central cannabinoid receptors in rat primary neuronal cell cultures from selected brain structures. By using [3H]SR 141716A, the specific CB1 receptor antagonist, we demonstrate in cortical neurons the presence of a high density of specific binding sites (Bmax = 139 +/- 9 fmol/mg of protein) displaying a high affinity (KD = 0.76 +/- 0.09 nM). The two cannabinoid receptor agonists, CP 55940 and WIN 55212-2, inhibited in a concentration-dependent manner cyclic AMP production induced by either 1 microM forskolin or isoproterenol with EC50 values in the nanomolar range (4.6 and 65 nM with forskolin and 1.0 and 5.1 nM with isoproterenol for CP 55940 and WIN 55212-2, respectively). Moreover, in striatal neurons and cerebellar granule cells, CP 55940 was also able to reduce the cyclic AMP accumulation induced by 1 microM forskolin with a potency similar to that observed in cortical neurons (EC50 values of 3.5 and 1.9 nM in striatum and cerebellum, respectively). SR 141716A antagonized the CP 55940- and WIN 55212-2-induced inhibition of cyclic AMP accumulation, suggesting CB1 receptor-specific mediation of these effects on all primary cultures tested. Furthermore, CP 55940 was unable to induce mitogen-activated protein kinase activation in either cortical or striatal neurons. In conclusion, our results show nanomolar efficiencies for CP 55940 and WIN 55212-2 on adenylyl cyclase activity and no effect on any other signal transduction pathway investigated in primary neuronal cultures.

Characterization of two cloned human CB1 cannabinoid receptor isoforms.

We have investigated the pharmacology of two central human cannabinoid receptor isoforms, designated CB1 and CB1A, stably expressed in Chinese hamster ovary cell lines, designated as CHO-CB1 and CHO-CB1A, respectively. In direct binding assays on isolated membranes the agonist [3H]CP 55,940 bound in a saturable and highly specific manner to both cannabinoid receptor isoforms. Competition binding experiments performed with other commonly used receptor agonists showed the following rank order of potency: CP 55,940 > tetrahydrocannabinol > WIN 55212-2 > anandamide. Except for the endogenous ligand anandamide (CB1, Ki = 359.6 nM vs. CB1A, Ki = 298 nM), these agonists bound to CB1A (CP 55,940, WIN 55212-2 and delta 9-THC, Ki = 7.24,345 and 26.7 nM, respectively) with about 3-fold less affinity than to CB1 (CP 55,940, WIN 55212-2 and delta 9-THC, Ki = 2.26, 93 and 7.1 nM, respectively). The cannabinoid receptor antagonist SR 141716A also bound to CB1A (Ki = 43.3 nM) with slightly less affinity than to CB1 (Ki = 4.9 nM). Cannabinoid receptor-linked second messenger system studies performed in the CHO-CB1 and CHO-CB1A cells showed that both receptors mediated their action through the agonist-induced inhibition of forskolin-stimulated cAMP accumulation. This activity was totally blocked by pretreatment with PTX. Additionally, both isoforms activated mitogen-activated protein kinase. The selective antagonist SR 141716A was able to selectively block these responses in both cell lines, to an extent that reflected its binding characteristics. Our results show that the amino-truncated and -modified CB1 isoform CB1A exhibits all the properties of CB1 to a slightly attenuated extent.

Improvement of memory in rodents by the selective CB1 cannabinoid receptor antagonist, SR 141716.

Social short-term memory in rodents is based on the recognition of a juvenile by an adult conspecific when the juvenile is presented on two successive occasions. Cannabimimetics are claimed to induce memory deficits in both humans and animals. In the brain, they mainly bind to CB1 receptors for which anandamide is a purported endogenous ligand. SR 141716, a specific antagonist of CB1 receptors, dose-dependently reverses biochemical and pharmacological effects of cannabimimetics. More particularly, it antagonizes the inhibition of hippocampal long-term potentiation induced by WIN 55,212-2 and anandamide, and it increases arousal when given alone. The present experiments study the ability of SR 141716 (from 0.03 to 3 mg/kg SC) to facilitate short-term olfactory memory in the social recognition test in rodents. SR 141716 improved social recognition in a long intertrial paradigm with a threshold dose of 0.1 mg/kg SC. At 1 mg/kg, it antagonized the memory disturbance elicited by retroactive inhibition. Scopolamine (0.06 mg/kg IP) partially reversed its memory-enhancing effect. Moreover, SR 141716 reduced memory deficit in aged rats (0.03-0.1 mg/kg) and mice (0.3-1 mg/kg). As SR 141716 is not known to exhibit any pharmacological activity which is not mediated by CB1 receptors, the results strongly support the concept that blockade of CB1 receptors plays an important role in consolidation of short-term memory in rodents and suggest there may be a role for an endogenous cannabinoid agonist tone (anandaminergic) in forgetting.

Molecular cloning, expression and function of the murine CB2 peripheral cannabinoid receptor.

We have cloned the peripheral cannabinoid receptor, mCB2, from a mouse splenocyte cDNA library. The 3.7 kb sequence contains an open reading frame encoding a protein of 347 residues sharing 82% overall identity with the only other known peripheral receptor, human CB2 (hCB2) and shorter than hCB2 by 13 amino acids at the carboxyl terminus. Binding experiments with membranes from COS-3 cells transiently expressing mCB2 showed that the synthetic cannabinoid WIN 55212-2 had a 6-fold lower affinity for mCB2 than for hCB2, whereas both receptors showed similar affinities for the agonists CP 55,940, delta(9)-THC and anandamide and almost no affinity for the central receptor- (CB1) specific antagonist SR 141716A. Both hCB2 and mCB2 mediate agonist-stimulated inhibition of forskolin-induced cAMP production in CHO cell lines permanently expressing the receptors. SR 141716A failed to antagonize this activity in either cell line, confirming its specificity for CB1.

Signaling pathway associated with stimulation of CB2 peripheral cannabinoid receptor. Involvement of both mitogen-activated protein kinase and induction of Krox-24 expression.

Cannabinoids, known for their psychoactive effects, also possess immunomodulatory properties. The recent isolation and cloning of the G-protein-coupled peripheral cannabinoid receptor (CB2), mainly expressed in immune tissues, have provided molecular tools to determine how cannabinoid compounds may mediate immunomodulation. We here investigated the CB2 signaling properties using stably transfected Chinese hamster ovary cells expressing human CB2. First, we showed that stimulation by a cannabinoid agonist activated mitogen-activated protein (MAP) kinase in time- and dose-dependent manners. The rank order of potency for MAP kinase activation of cannabinoid agonists correlated well with their binding capacities. Second, we demonstrated that, following MAP kinase activation, cannabinoids induced the expression of the growth-related gene Krox-24, also known as NGFI-A, zif/268, and egr-1. Pertussis toxin completely prevented both MAP kinase activation and Krox-24 induction, even more these responses appeared to be dependent of specific protein kinase C isoforms and independent of inhibition of adenylyl cyclase. A similar coupling of CB2 to a mitogenic pathway and to the regulation of Krox-24 expression was also observed in human promyelocytic cells HL60. Taken together, these findings provide evidence for a functional role of the CB2 receptor in gene induction mediated by the MAP kinase network.

Central mediation of the cannabinoid cue: activity of a selective CB1 antagonist, SR 141716A.

Active cannabimimetic drugs are known to bind to two receptor subtypes: one, called CB1, is mainly localised in the central nervous system while the other (CB2) is expressed preferentially in the immune system. SR 141716A has been demonstrated to have a nanomolar affinity for CB1 receptor subtypes and a micromolar affinity for CB2 receptors. Moreover, it is an effective antagonist at these receptors both in vitro (antagonism of cannabinoid activity in vas deferens) and in vivo (suppression of the hypothermia elicited by WIN 55,212-2). The present experiments were thus undertaken to investigate the role of CB1 receptors in cannabinoid discrimination. Rats were trained to discriminate WIN 55,212-2 (0.3mg/kg s.c.) from saline in a standard operant (FR10) food rewarded discrimination procedure. Acquisition of the discrimination required 16 days on average and the ED(50) of WIN 55,212-2 was 0.032mg/kg s.c. CP55,940 and delta-9-tetrahydrocannabinol (Delta(9)-THC) generalised to the WIN 55,212-2 stimulus with the respective ED(50)s of 0.007mg/kg (s.c.) and 0.64mg/kg (p.o.). Pretreatment with SR 141716A antagonised the cue elicited by WIN 55,212-2 (ED(50) = 1.6mg/kg) as well as the generalisation to CP 55,940 (ED(50) = 0.08mg/kg) and to Delta(9)-THC (ED(50) = 0.15mg/kg). SR 140098 is a CB1 antagonist as potent as SR 141716A in vitro. This compound is unlikely to pass into the brain since it failed to displace [(3)H]-CP55, 940 from rat brain membranes ex vivo, and to reverse WIN 55,212-2-induced hypothermia. SR 140098, in contrast to SR 141716A, did not antagonise the WIN 55,212-2 stimulus. Taken together, the present results demonstrate that the brain CB1 receptor subtype mediates the cannabinoid cue.

Characterization and distribution of binding sites for [3H]-SR 141716A, a selective brain (CB1) cannabinoid receptor antagonist, in rodent brain.

SR 141716A belongs to a new class of compounds (diarylpyrazole) that inhibits brain cannabinoid receptors (CB1) in vitro and in vivo. The present study showed that [3H]-SR 141716A binds with high affinity (Kd=0.61 +/- 0.06 nM) to a homogenous population of binding sites (Bmax=0.72 +/- 0.05 pmol/mg of protein) in rate whole brain (minus cerebellum) synaptosomes. This specific binding was displaced by known cannabinoid receptor ligands with the following rank order of potency SR 141716A > CP 55,940 > WIN 55212-2 = delta9-THC > anandamide. Apart from anandamide, all these compounds were found to interact competitively with the binding sites labeled by [3H]-SR 141716A. On the other hand, agents lacking affinity for cannabinoid receptors were unable to displace [3H]-SR 141716A from its binding sites (IC50 > 10 microM). In addition, the binding of [3H]-SR 141716A was insensitive to guanyl nucleotides. Regional rat brain distribution of CB1 cannabinoid receptors detected by [3H]-SR 141716A saturation binding and autoradiographic studies, showed that this distribution was very similar to that found for [3H]-CP 55,940. In vivo, the [3H]-SR 141716A binding was displaced by SR 141716A with ED50 values of 0.39 +/- 0.07 and 1.43 +/- 0.29 mg/kg following intraperitoneal and oral administration, respectively. Finally, the [3H]-SR 141716A binding sites remained significantly occupied for at least 12 hr following oral administration of 3 mg/kg SR 141716A. Taken together, these results suggest that SR 141716A in its tritiated form is a useful research tool for labeling brain cannabinoid receptors (CB1) in vitro and in vivo.

Activation of mitogen-activated protein kinases by stimulation of the central cannabinoid receptor CB1.

The G-protein-coupled central cannabinoid receptor (CB1) has been shown to be functionally associated with several biological responses including inhibition of adenylate cyclase, modulation of ion channels and induction of the immediate-early gene Krox-24. Using stably transfected Chinese Hamster Ovary cells expressing human CB1 we show here that cannabinoid treatment induces both phosphorylation and activation of mitogen-activated protein (MAP) kinases, and that these effects are inhibited by SR 141716A, a selective CB1 antagonist. The two p42 and p44 kDa MAP kinases are activated in a time- and dose-dependent manner. The rank order of potency for the activation of MAP kinases with various cannabinoid agonists is CP-55940 > delta 9-tetrahydrocannabinol > WIN 55212.2, in agreement with the pharmacological profile of CB1. The activation of MAP kinases is blocked by pertussis toxin but not by treatment with hydrolysis-resistant cyclic AMP analogues. This suggests that the signal transduction pathway between CB1 and MAP kinases involves a pertussis-toxin-sensitive GTP-binding protein and is independent of cyclic AMP metabolism. This coupling of CB1 subtype and mitogenic signal pathway, also observed in the human astrocytoma cell line U373 MG, may explain the mechanism of action underlying cannabinoid-induced Krox-24 induction.

The CB1 cannabinoid receptor antagonist SR 141716A affects A9 dopamine neuronal activity in the rat.

CB1 receptors and their putative natural ligand anandamide, have been tentatively involved in the control of midbrain extrapyramidal function. Electrophysiological activity of dopamine neurones was measured after acute and repeated administration of the CB1 receptor antagonist SR 141716A (0.3-3 mg kg-1) in rats. Acute SR 141716A increased A9, but not A10 cell population response without affecting either their spontaneous firing rate or apomorphine-induced rate inhibition and prevented amphetamine-induced inhibition of A9, but not of A10 cell firing. After repeated administration SR 141716A (1 or 5 mg kg-1) decreased population response of A9 cells, which was reversed by apomorphine. These results suggest that CB1 receptor blockade by SR 141716A interrupts a cannabinoid-like endogenous tone controlling extrapyramidal function.