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.

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.

SR141716A, a potent and selective antagonist of the brain cannabinoid receptor.

SR141716A is the first selective and orally active antagonist of the brain cannabinoid receptor. This compound displays nanomolar affinity for the central cannabinoid receptor but is not active on the peripheral cannabinoid receptor. In vitro, SR141716A antagonises the inhibitory effects of cannabinoid receptor agonists on both mouse vas deferens contractions and adenylyl cyclase activity in rat brain membranes. After intraperitoneal or oral administration SR141716A antagonises classical pharmacological and behavioural effects of cannabinoid receptor agonists. This compound should prove to be a powerful tool for investigating the in vivo functions of the anandamide/cannabinoid system.

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.

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.

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.

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.

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.

Up-regulation of 5-HT2 receptors in the rat brain by repeated administration of SR 46349B, a selective 5-HT2 receptor antagonist.

Chronic administration (twice a day for three days and on the morning of the fourth day) of SR 46349B (trans-4-[(3Z)3-(2-dimethylaminoethyl)oxyimino-3-(2-fluoroph enyl)propen-1- yl]phenol hemifumarate) (10 mg/kg, orally), a selective 5-HT2 receptor antagonist, caused 24 h later a marked increase (+42%) of the maximum binding capacity of [3H]ketanserin in rat brain cortical membranes without change in its affinity constant. Further, administration of the 5-HT2 receptor agonist, (+/-)-DOI((+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane) (1 mg/kg, i.p.), produced in chronic SR 46349B treated rats a significant increase in the amount of [3H]-inositol phosphate compared to corresponding controls. In addition, subacute administration of SR 46349B caused a 2-fold increase in the head-twitch response to (+/-)-DOI (0.5 mg/kg, i.p.). This enhanced response was blocked by an acute administration of ritanserin (6-(2-[4-[bis(4-fluorophenyl)methylene]-1-piperidinyl]ethyl]-7- methyl-5H-thiazolo[3,2-a]pyrimidin-5-one) (10 mg/kg). Finally, a significant enhancement (+29%) of 5-HT2 receptor mRNA levels was observed in the cortex. Taken together, these data showed that an up-regulation of 5-HT2 receptors occurred in rats following repeated treatment with a selective 5-HT2 receptor antagonist. The effects of SR 46349B on 5-HT2 receptors might implicate pre-translational regulation.

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.

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.

Identification of binding sites for SR 46349B, a 5-hydroxytryptamine2 receptor antagonist, in rodent brain.

SR 46349B belongs to a new class of compounds (propenone oxime ether derivative) that inhibit 5-hydroxytryptamine (HT)2 receptors in vitro and in vivo. (3H) SR 46349B has been shown to bind with high affinity (Kd = 1.20 nM) to a single class of sites in rat prefrontal cortical membranes. The maximum binding capacity (Bmax = 0.262 pmol/mg of protein) is similar to that found for other classes of 5-HT2 receptor antagonists. Although the highest density of specific (3H) SR 46349B binding was found in cortex tissue, specific binding was also detectable in other brain areas. Among various receptor or channel ligands [including alpha or beta adrenergic, dopamine (D1 or D2), histamine (H1 or H2), 5-HT subclasses (5-HT1, 5-HT3), muscarinic and Na+ and Ca2+ channel blockers] only 5-HT2 receptor effectors were able to displace (3H) SR 46349B. In addition, the type of inhibition exerted by known 5-HT2 receptor antagonists such as ketanserin and ritanserin was investigated by saturation studies. In vivo, (3H) SR 46349B bound predominantly in mouse brain regions containing 5-HT2 receptors. This binding was displaced by SR 46349B, ketanserin and ritanserin following oral administration. From these results we suggest that SR 46349B in its tritiated form is a useful tool to label the 5-HT2 receptor in vitro and in vivo.

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.

Biochemical and pharmacological characterisation of SR141716A, the first potent and selective brain cannabinoid receptor antagonist.

SR141716A is a selective, potent and orally active antagonist of the brain cannabinoid receptor with a long duration of action. This compound shows high affinity for the central cannabinoid receptor (Ki = 2 nM), displays low affinity for the peripheral cannabinoid receptor (Ki > 1000 nM). In vitro, SR141716A antagonizes the inhibitory effects of cannabinoid receptor agonists on both mouse vas deferens contractions and dopamine-stimulated adenylyl cyclase activities in rat brain membranes. After oral administration SR141716A totally inhibited the ex vivo [3H]-CP55,940 binding to cerebral membranes with a ED50 value of 3.5 mg/kg. Furthermore SR141716A antagonizes the classical pharmacological responses elicited by cannabinoid receptor agonists. In addition, SR141716A reverses the inhibitory effect of WIN55212-2 on isoniazid-induced elevation of cGMP in rat cerebellum. This compound will provide a powerful tool for studying the in vivo functions of the anandamide/cannabinoid system.

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.

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.

Intrastriatal injection of cannabinoid receptor agonists induced turning behavior in mice.

When injected unilaterally into the mouse striatum, cannabinoid agonists such as Win 55212-2 (1-100 ng/mouse), CP 55940 (0.1-50 ng/mouse), and anandamide (0.5-50 ng/mouse), the putative endogenous ligand of CB1 receptor, dose-dependently induced turning behavior. SR 141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H- pyrazole-3-carboxamide hydrochloride], the selective antagonist of CB1 receptor, antagonized the three cannabinoid receptor agonists-induced turning with similar ED50s (0.13-0.15 mg/kg, IP). Spiroperidol (a D2 receptor blocker), (+)-SCH 23390 (a D1 receptor blocker), or prior 6-OHDA lesions of the striatum blocked Win 55212-2- and CP 55940-induced turning, thus suggesting the involvement of DA transmission in cannabinoid-induced turning. Taken together, these findings reinforce the notion of a cannabinoid receptor-mediated control of nigrostriatal function.

Interaction of SR 33557 with skeletal muscle calcium channel blocker receptors in the baboon: characterization of its binding sites.

A procedure for the isolation of primate skeletal microsomal membranes was initiated. Membranes exhibited specific enzymatic markers such as 5'-nucleotidase, Ca++,Mg(++)-adenosine triphosphatase and an ATP-dependent calcium uptake. Baboon skeletal microsomes bound specifically with high-affinity potent Ca++ channel blockers such as dihydropyridine, phenylalkylamine and benzothiazepine derivatives. Scatchard analysis of equilibrium binding assays with [3H](+)-PN 200-110, [3H](-)-desmethoxyverapamil [( 3H](-)-D888) and [3H]-d-cis-dilitiazem were consistent with a single class of binding sites for the three radioligands. The pharmacological profile of SR 33557, an original compound with calcium antagonist properties, was investigated using radioligand binding studies. SR 33557 totally inhibited the specific binding of the three main classes of Ca++ channel effectors and interacted allosterically with them. In addition, SR 33557 bound with high affinity to a homogeneous population of binding sites in baboon skeletal muscle.

Stimulation of cannabinoid receptor CB1 induces krox-24 expression in human astrocytoma cells.

The recent isolation and cloning of the G protein-coupled central cannabinoid receptor (CB1) from brain tissue has provided a molecular basis to elucidate how cannabinoid compounds may mediate their psychoactive effects. Here we report the high expression of cannabinoid receptors in human astrocytoma tumors of different grades, in the astrocytoma cell lines U373 MG and GL-15, as well as in normal astrocytes. From an analysis of the coupling mechanisms of functional CB1 receptors in U373 MG, we show that, in addition to the inhibition of adenylyl cyclase, activation by the cannabinoid agonist CP-55940 induces the expression of the immediate-early gene krox-24, also known as NGFI-A, zif/268, egr-1, and TIS8. The amount of Krox-24 protein and the level of Krox-24 DNA binding activity, as measured by Western blot and electrophoretic mobility shift assay, respectively, were also increased by the addition of CP-55940. These effects were blocked by incubation with pertussis toxin but not by treatment with hydrolysis-resistant cAMP analogues, suggesting that the transduction pathway between the cannabinoid receptor and krox-24 involves a pertussis toxin-sensitive GTP-binding protein and is independent of cAMP metabolism. The specific involvement of CB1 in Krox-24 induction was demonstrated in Chinese hamster ovary cells transfected with the human CB1 receptor and also in experiments using the CB1-selective cannabinoid antagonist SR 141716A.

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.