Activation of peripheral CB1 cannabinoid receptors in haemorrhagic shock

Anandamide, an endogenous cannabinoid ligand, binds to CB1 cannabinoid receptors in the brain and mimics the neurobehavioural actions of marijuana. Cannabinoids and anandamide also elicit hypotension mediated by peripheral CB1 receptors. Here we report that a selective CB1 receptor antagonist, SR141716A, elicits an increase in blood pressure in rats subjected to haemorrhagic shock, whereas similar treatment of normotensive rats or intracerebroventricular administration of the antagonist during shock do not affect blood pressure. Blood from haemorrhaged rats causes hypotension in normal rats, which can be prevented by SR141716A but not by inhibition of nitric oxide synthase in the recipient. Macrophages and platelets from haemorrhaged rats elicit CB1 receptor-mediated hypotension in normotensive recipients, and incorporate arachidonic acid or ethanolamine into a product that co-elutes with anandamide on reverse-phase high-performance liquid chromatography. Also, macrophages from control rats stimulated with ionomycin or bacterial phospholipase D produce anandamide, as identified by gas chromatography and mass spectrometry. These findings indicate that activation of peripheral CB1 cannabinoid receptors contributes to haemorrhagic hypotension, and anandamide produced by macrophages may be a mediator of this effect.     

Platelet- and macrophage-derived endogenous cannabinoids are involved in endotoxin-induced hypotension

Macrophages are the primary cellular targets of bacterial lipopolysaccharide (LPS), but the role of macrophage-derived cytokines in LPS-induced septic shock is uncertain. Recent evidence indicates that activation of peripheral CB1 cannabinoid receptors contributes to hemorrhagic hypotension and that macrophage-derived anandamide as well as unidentified platelet-derived substances may be contributing factors. Here we demonstrate that rat platelets contain the endogenous cannabinoid 2-arachidonyl glyceride (2-AG), as identified by reverse phase high-performance liquid chromatography, gas chromatography, and mass spectrometry, and that in vitro exposure of platelets to LPS (200 microg/ml) markedly increases 2-AG levels. LPS-stimulated, but not control, macrophages contain anandamide, which is undetectable in either control or LPS-stimulated platelets. Prolonged hypotension and tachycardia are elicited in urethane-anesthetized rats treated 1) with LPS (15 mg/kg i.v.); 2) with macrophages plus platelets isolated from 3 ml of blood from an LPS-treated donor rat; or 3) with rat macrophages or 4) platelets preincubated in vitro with LPS (200 microg/ml). In all four cases, the hypotension but not the tachycardia is prevented by pretreatment of the recipient rat with the CB1 receptor antagonist SR141716A (3 mg/kg i.v.), which also inhibits the hypotensive response to anandamide or 2-AG. The hypotension elicited by LPS-treated macrophages or platelets remains unchanged in the absence of sympathetic tone or after blockade of nitric oxide synthase. These findings indicate that platelets and macrophages generate different endogenous cannabinoids, and that both 2-AG and anandamide may be paracrine mediators of endotoxin-induced hypotension via activation of vascular CB1 receptors.     

Control of pain initiation by endogenous cannabinoids

The potent analgesic effects of cannabis-like drugs and the presence of CB1-type cannabinoid receptors in pain-processing areas of the brain and spinal cord indicate that endogenous cannabinoids such as anandamide may contribute to the control of pain transmission within the central nervous system (CNS). Here we show that anandamide attenuates the pain behaviour produced by chemical damage to cutaneous tissue by interacting with CB1-like cannabinoid receptors located outside the CNS. Palmitylethanolamide (PEA), which is released together with anandamide from a common phospholipid precursor, exerts a similar effect by activating peripheral CB2-like receptors. When administered together, the two compounds act synergistically, reducing pain responses 100-fold more potently than does each compound alone. Gas-chromatography/mass-spectrometry measurements indicate that the levels of anandamide and PEA in the skin are enough to cause a tonic activation of local cannabinoid receptors. In agreement with this possibility, the CB1 antagonist SR141716A and the CB2 antagonist SR144528 prolong and enhance the pain behaviour produced by tissue damage. These results indicate that peripheral CB1-like and CB2-like receptors participate in the intrinsic control of pain initiation and that locally generated anandamide and PEA may mediate this effect.     

Cannabinoid-induced hypotension and bradycardia in rats mediated by CB1-like cannabinoid receptors

Previous studies indicate that the CB1 cannabinoid receptor antagonist, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-met hyl-1H-pyrazole-3-carboxamide HCl (SR141716A), inhibits the anandamide- and delta9-tetrahydrocannabinol- (THC) induced hypotension and bradycardia in anesthetized rats with a potency similar to that observed for SR141716A antagonism of THC-induced neurobehavioral effects. To further test the role of CB1 receptors in the cardiovascular effects of cannabinoids, we examined two additional criteria for receptor-specific interactions: the rank order of potency of agonists and stereoselectivity. A series of cannabinoid analogs including the enantiomeric pair (-)-11-OH-delta9-THC dimethylheptyl (+)-11-OH-delta9-THC dimethylheptyl were evaluated for their effects on arterial blood pressure and heart rate in urethane anesthetized rats. Six analogs elicited pronounced and long lasting hypotension and bradycardia that were blocked by 3 mg/kg of SR141716A. The rank order of potency was (-)-11-OH-delta9-THC dimethylheptyl > or = (-)-3-[2-hydroxy-4-(1,1-dimethyl-heptyl)phenyl]-4-[3-hydroxy-propyl]c yclohexan-1-ol > (-)-3-[2-hydroxy-4-(1,1-dimethyl-heptyl)phenyl]-4-[3-hydroxy-propyl]c yclohexan-1-ol > THC > anandamide > or = (-)-3-[2-hydroxy-4-(1,1-dimethyl-heptyl)phenyl]-4-[3-hydroxy-propyl]c yclohexan-1-ol, which correlated well with CB1 receptor affinity or analgesic potency (r = 0.96-0.99). There was no hypotension or bradycardia after palmitoylethanolamine or (+)-11-OH-delta9-THC dimethylheptyl. An initial pressor response was also observed with THC and anandamide, which was not antagonized by SR141716A. We conclude that the similar rank orders of potency, stereoselectivity and sensitivity to blockade by SR141716A indicate the involvement of CB1-like receptors in the hypotensive and bradycardic actions of cannabinoids, whereas the mechanism of the pressor effect of THC and anandamide remains unclear.     

Classifying extraintestinal non-typhoid Salmonella infections

The cannabinoid receptors expressed in the mouse neuroblastoma X rat glioma NG108-15 cell and the rat pituitary tumor GH4C1 cell were determined by polymerase chain reaction, dideoxysequencing and pharmacologically. The CB1 but not the CB2 or CB1A cannabinoid receptor was found in both cell lines. The cDNA identified in GH4C1 cells corresponds to the rat CB1 receptor. Interestingly, NG108-15 cells express two distinct cDNAs, one corresponds to the rat and the other to the mouse CB1 receptor. The newly developed CB1 receptor selective antagonist SR141716A was found to reverse cannabinoid agonist (WIN55212-2 or CP55940)-induced adenylyl cyclase inhibition. These results provide more direct evidence that the CB1 receptor is mediating the pharmacological actions of cannabinoids in NG108-15 and GH4C1 cells.     

Evidence that the cannabinoid CB1 receptor is a 2-arachidonoylglycerol receptor. Structure-activity relationship of 2-arachidonoylglycerol, ether-linked analogues, and related compounds

An endogenous cannabimimetic molecule, 2-arachidonoylglycerol, induces a rapid, transient increase in intracellular free Ca2+ concentrations in NG108-15 cells through a cannabinoid CB1 receptor-dependent mechanism. We examined the activities of 24 relevant compounds (2-arachidonoylglycerol, its structural analogues, and several synthetic cannabinoids). We found that 2-arachidonoylglycerol is the most potent compound examined so far: its activity was detectable from as low as 0.3 nM, and the maximal response induced by 2-arachidonoylglycerol exceeded the responses induced by others. Activities of HU-210 and CP55940, potent cannabinoid receptor agonists, were also detectable from as low as 0.3 nM, whereas the maximal responses induced by these compounds were low compared with 2-arachidonoylglycerol. Anandamide was also found to act as a partial agonist in this assay system. We confirmed that free arachidonic acid failed to elicit a response. Furthermore, we found that a metabolically stable ether-linked analogue of 2-arachidonoylglycerol possesses appreciable agonistic activity, although its activity was apparently lower than that of 2-arachidonoylglycerol. We also confirmed that pretreating cells with various cannabinoid receptor agonists nullified the response induced by 2-arachidonoylglycerol, whereas pretreating cells with other neurotransmitters or neuromodulators did not affect the response. These results strongly suggested that the cannabinoid CB1 receptor is originally a 2-arachidonoylglycerol receptor, and 2-arachidonoylglycerol is the intrinsic physiological ligand for the cannabinoid CB1 receptor.     

Assessment of anandamide interaction with the cannabinoid brain receptor: SR 141716A antagonism studies in mice and autoradiographic analysis of receptor binding in rat brain

Anandamide is the newly discovered endogenous cannabinoid ligand that binds to brain cannabinoid receptors and shares most, but not all, of the pharmacological properties of delta 9-THC. Therefore, this study was undertaken to determine whether its interaction with the CB1 receptor in brain was identical to that of delta 9-THC. Anandamide depressed spontaneous activity and produced hypothermia, antinociception and immobility in mice after i.v. administration. However, none of these effects was blocked by pretreatment with the selective CB1 antagonist, SR 141716A. However, the metabolically stable analog 2-methyl-2'-fluoroethylanandamide produced reductions in motor activity and antinociception in mice, effects that were blocked by the antagonist. To determine whether anandamide's receptor binding mimicked that of other cannabinoids, an autoradiographic comparison of anandamide, SR 141716A and CP 55,940 competition for [3H]CP55,940 binding was conducted throughout rat brain. The receptor affinities for all three compounds did not change according to brain area. As expected, Bmax values differed dramatically among differ brain areas. However, the Bmax values for each brain area were similar regardless of the compound used for displacement. These data suggest that anandamide, SR 141716A and CP 55,940 compete for the same cannabinoid receptor throughout brain despite SR 141716A's failure to block anandamide's pharmacological effects. Although there is no question that anandamide binds to the cannabinoid receptor, failure of SR 141716A to block its pharmacological effects in mice poses a dilemma. The results presented herein raise the possibility that anandamide may not be producing all of its effects by a direct interaction with the CB1 receptor.     

Unresponsiveness to cannabinoids and reduced addictive effects of opiates in CB1 receptor knockout mice

The function of the central cannabinoid receptor (CB1) was investigated by invalidating its gene. Mutant mice did not respond to cannabinoid drugs, demonstrating the exclusive role of the CB1 receptor in mediating analgesia, reinforcement, hypothermia, hypolocomotion, and hypotension. The acute effects of opiates were unaffected, but the reinforcing properties of morphine and the severity of the withdrawal syndrome were strongly reduced. These observations suggest that the CB1 receptor is involved in the motivational properties of opiates and in the development of physical dependence and extend the concept of an interconnected role of CB1 and opiate receptors in the brain areas mediating addictive behavior.     

Repression of Drosophila photoreceptor cell fate through cooperative action of two transcriptional repressors Yan and Tramtrack

Using the endogenous cannabinoid receptor agonist anandamide, the synthetic agonist CP 55940 [[1alpha,2beta(R)5alpha]-(-)-5-(1,1-dimethylheptyl+ ++)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]phenol], and the specific antagonist SR 141716 [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide hydrochloride], second messenger activation of the central cannabinoid receptor (CB1) was examined in rat striatal and cortical slices. The effects of these cannabinoid ligands on electrically evoked dopamine (DA) release from [3H] dopamine-prelabelled striatal slices were also investigated. CP 55940 (1 microM) and anandamide (10 microM) caused significant reductions in forskolin-stimulated cyclic AMP accumulation in rat striatal slices, which were reversed in the presence of SR 141716 (1 microM). CP 55940 (1 microM) had no effect on either KCl- or neurotransmitter-stimulated 3H-inositol phosphate accumulation in rat cortical slices. CP 55940 and anandamide caused significant reductions in the release of dopamine after electrical stimulation of [3H]dopamine-prelabelied striatal slices, which were antagonised by SR 141716. SR 141716 alone had no effect on electrically evoked dopamine release from rat striatal slices. These data indicate that the CB1 receptors in rat striatum are negatively linked to adenylyl cyclase and dopamine release. That the CB1 receptor may influence dopamine release in the striatum suggests that cannabinoids play a modulatory role in dopaminergic neuronal pathways.     

Increase in meso-prefrontal dopaminergic activity after stimulation of CB1 receptors by cannabinoids

The intravenous administration of the psychoactive constituent of marijuana, delta9-tetrahydrocannabinol (delta9-THC) (62.5-1000 microg/kg), and the synthetic cannabinoid agonist WIN 55212,2 (WIN) (62.5-500 microg/kg), produced a dose-related increase in the firing rate and burst firing in the majority of antidromically identified meso-prefrontal dopaminergic neurons. In a restricted number of neurons (n=4), WIN administration did not increase firing rate but produced an increment of bursting activity. These effects of the cannabinoids were reversed by the intravenous administration of SR 141716 A, a selective cannabinoid antagonist (1 mg/kg), per se ineffective to modify the electrical activity of dopaminergic neurons. The results indicate that stimulation of cannabinoid CB1 receptors produces an activation of meso-prefrontal dopaminergic transmission. Considering that supranormal stimulation of D1 dopamine receptors in the prefrontal cortex has been shown to impair working memory, the present results suggest that the negative effects of cannabinoids on cognitive processes might be related to the activation of dopaminergic transmission in the prefrontal cortex.     

Protein kinase C disrupts cannabinoid actions by phosphorylation of the CB1 cannabinoid receptor

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

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.     

CB1 cannabinoid receptor antagonist-induced opiate withdrawal in morphine-dependent rats

Recent reports have provided evidence of a link between the endogenous brain cannabinoid system and the endogenous central opioid systems. Here we report that the selective CB1 receptor antagonist SR 141716A induced behavioral and endocrine alterations associated with opiate withdrawal in morphine-dependent animals in a dose-dependent manner and that naloxone induced an opiate withdrawal syndrome in animals made cannabinoid-dependent by repeated administration of the potent cannabinoid agonist HU-210. Additionally CB1 and mu-opioid receptor mRNAs were co-localized in brain areas relevant for opiate withdrawal such as the nucleus accumbens, septum, dorsal striatum, the central amygdaloid nucleus and the habenular complex. These results suggest that CB1 cannabinoid receptors may play a role in the neuroadaptive processes associated with opiate dependence, and they lend further support for the hypothesis of a potential role of cannabinoid receptors in the neurobiological changes that culminate in drug addiction.     

A new perspective on cannabinoid signalling: complementary localization of fatty acid amide hydrolase and the CB1 receptor in rat brain

CB1-type cannabinoid receptors in the brain mediate effects of the drug cannabis. Anandamide and sn-2 arachidonylglycerol (2-AG) are putative endogenous ligands for CB1 receptors, but it is not known which cells in the brain produce these molecules. Recently, an enzyme which catalyses hydrolysis of anandamide and 2-AG, known as fatty acid amide hydrolase (FAAH), was identified in mammals. Here we have analysed the distribution of FAAH in rat brain and compared its cellular localization with CB1-type cannabinoid receptors using immunocytochemistry. High concentrations of FAAH activity were detected in the cerebellum, hippocampus and neocortex, regions of the rat brain which are enriched with cannabinoid receptors. Immunocytochemical analysis of these brain regions revealed a complementary pattern of FAAH and CB1 expression with CB1 immunoreactivity occurring in fibres surrounding FAAH-immunoreactive cell bodies and/or dendrites. In the cerebellum, FAAH was expressed in the cell bodies of Purkinje cells and CB1 was expressed in the axons of granule cells and basket cells, neurons which are presynaptic to Purkinje cells. The close correspondence in the distribution of FAAH and CB1 in rat brain and the complementary pattern of FAAH and CB1 expression at the cellular level provides important new evidence that FAAH may participate in cannabinoid signalling mechanisms of the brain.     

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.     

Evaluation of binding in a transfected cell line expressing a peripheral cannabinoid receptor (CB2): identification of cannabinoid receptor subtype selective ligands

Two cannabinoid receptors have been identified to date; one is located predominantly in the central nervous system (CB1), whereas the other is located exclusively in the periphery (CB2). The purposes of this study were to explore further the binding requirements of the CB2 receptor and to search for compounds displaying distinct affinities for either cannabinoid receptor. The binding affinities of a series of cannabinoids tested previously at the CB1 receptor were determined at cloned human CB1 and CB2 receptors using a filtration assay. In addition, possible allosteric regulation of the CB2 receptor was examined. Sodium and a GTP analog elicited a concentration-dependent decrease in specific binding to the CB2 receptor. The affinity of cannabinol for CB2 receptors (Ki = 96.3 +/- 14 nM) was confirmed to be in approximately the same range as that of delta 9-THC (Ki = 36.4 +/- 10 nM). Affinities at cloned CB1 and CB2 receptors were compared with affinities determined in the brain. Although most of the chosen compounds did not discriminate between CB1 and CB2, several ligands were identified that showed selectivity. Affinity ratios demonstrated that two 2'-fluoro analogs of anandamide were over 23-fold selective for the CB1 receptor and confirmed the CB1 selectivity of SR141716A {N- (piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4- methyl-1H-pyrazole-3-carboxamidehydrochloride}. In addition, WIN-55, 212-2 {(R)-(+)-[2, 3-dihydro-5-methyl-3-[(4-morpholinyl) methyl] pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl) methanone} and a closely related propyl indole analog were shown to be 6.75- and 27.5- fold selective, respectively, for the CB2 receptor. These ligands can now serve as a basis for the design of compounds with even greater selectivity.     

Effects of cannabinoids on prolactin and gonadotrophin secretion: involvement of changes in hypothalamic gamma-aminobutyric acid (GABA) inputs

CB1 cannabinoid receptors are located in hypothalamic nuclei and their activation alters several hypothalamic neurotransmitters resulting in, among other things, decreased prolactin (PRL) and luteinizing hormone (LH) secretion from the anterior pituitary gland. In the present study, we addressed two related objectives to further explore this complex regulation. First, we examined whether changes in gamma-aminobutyric acid (GABA) and/or dopamine (DA) inputs in the medial basal hypothalamus might occur in parallel to the effects resulting from the activation of CB1 receptors on PRL and gonadotrophin secretion in male rats. Thus, the acute administration of (-)-delta9-tetrahydrocannnabinol (delta9-THC) produced, as expected, a marked decrease in plasma PRL and LH levels, with no changes in follicle-stimulating hormone (FSH) levels. This was paralleled by an increase in the contents of GABA, but not of DA, in the medial basal hypothalamus and, to a lesser extent, in the anterior pituitary gland. The co-administration of delta9-THC and SR141716, a specific antagonist for CB1 receptors, attenuated both PRL and LH decrease and GABA increase, thus asserting the involvement of the activation of CB1 receptors in these effects. As a second objective, we tested whether the prolonged activation of these receptors might induce tolerance with regard to the decrease in PRL and LH release, and whether this potential tolerance might be related to changes in CB1-receptor binding and/or mRNA expression. The chronic administration of R-methanandamide (AM356), a more stable analog of anandamide, the putative endogenous cannabinoid ligand, produced a marked decrease in plasma PRL and LH levels, with no changes in FSH. The decreases were of similar magnitude to those caused by a single injection of this cannabimimetic ligand, thus suggesting the absence of tolerance. In parallel, the analysis of CB1-receptor binding and mRNA expression in several hypothalamic structures proved that the acute or chronic administration of AM356 did not affect either the binding or the synthesis of these receptors. In summary, the activation of CB1 receptors in hypothalamic nuclei produced the expected decrease in PRL and LH secretion, an effect which might be related to an increase in GABAergic activity in the hypothalamus-anterior pituitary axis. The prolonged activation of these receptors for five days did not elicit tolerance in terms of an attenuation in the magnitude of the decrease in PRL and LH, and, accordingly, did not alter CB1-receptor binding and mRNA levels in the hypothalamic nuclei examined.     

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.