A role for cannabinoid receptors, but not endogenous opioids, in the antinociceptive activity of the CB2-selective agonist, GW405833

Several recent reports have demonstrated a role for selective cannabinoid CB2 receptor agonists in pain modulation, showing both analgesic and antihyperalgesic activities. While the mechanism of action is poorly understood, it has been postulated that these effects may be indirect, involving release of endogenous opioids. We have previously reported that administration of the selective cannabinoid CB2 receptor agonist GW405833 (2,3-dichloro-phenyl)-[5-methoxy-2-methyl-3-(2-morpholin-4-yl-ethyl)-indol-1-yl]-methanone) to rats elicits potent and efficacious antihyperalgesic effects against neuropathic and inflammatory pain and, at high dose (100 mg/kg), is analgesic and ataxic [Valenzano, K.J., Tafesse, L., Lee, G., Harrison, J.E., Boulet, J., Gottshall, S.L., Mark, L., Pearson, M.S., Miller, W., Shan, S., Rabadi, L., Rotstheyn, Y., Chaffer, S.M., Turchin, P.I., Elsemore, D.A., Toth, M., Koetzner, L., Whiteside, G.T., 2005. Pharmacological and pharmacokinetic characterization of the cannabinoid receptor 2 agonist, GW405833, utilizing rodent models of acute and chronic pain, anxiety, ataxia and catalepsy. Neuropharmacology 48, 658-672]. In the current study, we confirm these properties using mouse models and investigate the role of cannabinoid CB2 receptors using knockout animals. Furthermore, we provide evidence that the antinociceptive properties of GW405833 are opioid independent. GW405833 elicited robust antihyperalgesic effects in mouse models of inflammatory (Freund's complete adjuvant) and neuropathic (Seltzer) pain. In contrast, GW405833 showed no antihyperalgesic activity against Freund's complete adjuvant-mediated inflammatory pain in cannabinoid CB2 receptor knockout mice. As in rats, high-dose GW405833 (100 mg/kg) showed both analgesic and sedative activities in wild-type mice, activities that were also apparent in cannabinoid CB2 receptor knockout mice. In rats, neither the antihyperalgesic effect in the Freund's complete adjuvant model nor the analgesic effects in tail flick and hot plate assays were inhibited by pre-treatment with the non-selective opioid receptor antagonist, naltrexone. These data demonstrate that the antihyperalgesic effects of GW405833 are mediated via the cannabinoid CB2 receptor, whereas the analgesic and sedative effects are not. Furthermore, these data suggest that the mechanism of action for GW405833 does not depend on the release of endogenous opioids.     

Analgesic and antiinflammatory effects of cannabinoid receptor agonists in a rat model of neuropathic pain

Cannabinoid receptor (CB) agonists are known to attenuate allodynia in a range of pain models, but their long-term effects and their mechanisms of action are controversial. The present study compares the antiallodynic effects of long-term treatment with a mixed CB1/CB2 (WIN55,212-2) and a selective CB2 (GW405833) cannabinoid receptor agonist and correlates these effects with their influences on spinal cord (SC) glial activation. The substances were applied daily in a rat neuropathic pain model. Tactile allodynia was assessed, and the development of gliosis was illustrated with immunohistochemical methods. Both substances reduced mechanical allodynia. Their analgesic effect was accompanied by a significant reduction in reactive gliosis and cathepsins (CAT) X and S expression. A daily injection of either substance for 8 days was sufficient to induce a sustained antiallodynic effect, which persisted up to 6 days after the last injection. The re-appearance of mechanical allodynia after this period was associated with a breakout of a strong gliotic response in the lumbar SC. Our results emphasize the therapeutic efficacy of cannabinoid receptor agonists and their inhibitory effects on the formation of gliosis.     

Contrasting effects of different cannabinoid receptor ligands on mouse ingestive behaviour

This study characterized the effects of seven diverse cannabinoid receptor agonists (and one antagonist) on ingestive behaviour in nondeprived adult, male CD1 mice. Microstructural analysis of licking for a range of concentrations of condensed milk (10, 15 and 20%) was carried out following administration of vehicle or: Δ-tetrahydrocannabinol (Δ-THC) at 1, 3 or 6 mg/kg; CP55,940 at 10, 30 or 50 μg/kg; Win 55,212-2 at 0.5, 1 or 3 mg/kg; HU-210 at 0.01, 0.03 or 0.1 mg/kg; methanandamide at 1, 3 or 6 mg/kg; arachidonyl-2'-chloroethylamide at 1, 3 or 6 mg/kg and JWH133 at 1, 3 or 6 mg/kg. The cannabinoid receptor antagonist/inverse agonist rimonabant was also tested at 0.3, 1 or 3 mg/kg. Test sessions comprised three 30 s presentations of the milk concentrations separated by 10 s interpresentation intervals. The nonselective CB1 receptor agonists Δ-THC, CP55,940 and Win 55,212-2 increased the number of licks for condensed milk, primarily by a significant increase in bout number. The potent and nonselective CB1 receptor agonist HU-210 and the selective CB1 receptor agonists methanandamide and arachidonyl-2'-chloroethylamide did not significantly affect licking behaviour but did significantly increase the latency to start licking. The CB1 receptor antagonist rimonabant produced effects that were opposite in direction to those produced by Δ-THC, CP55,940 and Win 55,212-2. Finally, the selective CB2 receptor agonist JWH133 had no significant effects on behaviour. These data add to reports that cannabinoid agonists can enhance the appetitive aspects of feeding, but they also demonstrate that not all CB1 receptor agonists do this, and therefore the relationship between action at CB1 receptors and appetitive feeding effects is not straightforward.     

Discriminative stimulus effects of BAY 38-7271, a novel cannabinoid receptor agonist

BAY 38-7271 [(-)-(R)-3-(2-hydroxymethylindanyl-4-oxy)phenyl-4,4,4-trifluoro-1-sulfonate] is a novel, highly potent and selective cannabinoid CB(1)/CB(2) receptor agonist with neuroprotective properties. It was the aim of the present study to further confirm its cannabinoid CB(1) receptor agonist properties in a highly sensitive in vivo assay. Male Wistar rats (n=24) were trained to discriminate BAY 38-7271 (0.05 mg/kg, i.p., t-30 min) from vehicle in a fixed-ratio:10, food-reinforced two-lever standard procedure. The animals acquired the discrimination after a median number of 52 training sessions. BAY 38-7271 generalized dose-dependently when tested after different routes of administration (ED(50): 0.018 mg/kg, i.p.; 0.001 microg/kg, i.v.; 0.18 mg/kg, p.o.). A time-dependency study indicated that the cue (0.05 mg/kg, i.p.) was detectable between 15 min and 4 h, with a maximum of generalization obtained at 30 min after administration. Pretreatment with the selective cannabinoid CB(1) receptor antagonist SR 141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride] completely antagonized the effects of BAY 38-7271 (ID(50): 1.1 mg/kg, i.p.). Dose-dependent and complete generalization was also obtained after i.p. administration of the reference cannabinoid CB(1) receptor agonists HU-210 [(-)-11-OH-Delta(8)-tetrahydrocannabinol-dimethylheptyl, ED(50): 0.003 mg/kg], CP 55,940 [(-)-cis-3-[2-hydroxy-4(1,1-dimethyl-heptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol, 0.007 mg/kg], WIN 55,212-2 [(R)-4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphtalenylcarbonyl)-6H-pyrrolo [3,2,1-ij] quinolin-6-one, 0.28 mg/kg] and (-)-Delta(9)-tetrahydrocannabinol (0.34 mg/kg). The present study confirms that BAY 38-7271 is a highly potent cannabinoid CB(1) receptor agonist in vivo.     

CB1 and CB2 receptor agonists promote analgesia through synergy in a murine model of tumor pain

In light of the adverse side-effects of opioids, cannabinoid receptor agonists may provide an effective alternative for the treatment of cancer pain. This study examined the potency and efficacy of synthetic CB1 and CB2 receptor agonists in a murine model of tumor pain. Intraplantar injection of the CB1 receptor agonist arachidonylcyclopropylamide (ED(50) of 18.4 μg) reduced tumor-related mechanical hyperalgesia by activation of peripheral CB1 but not CB2 receptors. Similar injection of the CB2 receptor agonist AM1241 (ED50 of 19.5 μg) reduced mechanical hyperalgesia by activation of peripheral CB2 but not CB1 receptors. Both agonists had an efficacy comparable with that of morphine (intraplantar), but their analgesic effects were independent of opioid receptors. Isobolographic analysis of the coinjection of arachidonylcyclopropylamide and AM1241 determined that the CB1 and CB2 receptor agonists interacted synergistically to reduce mechanical hyperalgesia in the tumor-bearing paw. These data extend our previous findings that the peripheral cannabinoid receptors are a promising target for the management of cancer pain and mixed cannabinoid receptor agonists may have a therapeutic advantage over selective agonists.     

Role of TRPV1 and cannabinoid CB1 receptors in AM 404-evoked hypothermia in rats

AM 404 inhibits endocannabinoid uptake and enhances the cannabinoid CB(1)-mediated effects of endogenous cannabinoids. Accumulating evidence also suggests that AM 404 acts at sites other than the endocannabinoid system. One site is the transient receptor potential vanilloid 1 cation channel (TRPV1). A useful endpoint for discriminating between TRPV1- or CB(1)-mediated effects of AM 404 is hypothermia. This is because TRPV1 or CB(1) receptor activation produces a significant hypothermia in rats. The present study investigated the effects of AM 404 (1, 5, 10 and 20 mg/kg, i.p.) on body temperature in rats and the involvement of TRPV1 and CB(1) receptors in the effects of AM 404. Doses of 10 and 20 mg/kg of AM 404 produced significant hypothermia. Pre-treatment with capsazepine (30 mg/kg, i.p.) blocked the hypothermia caused by 10 and 20 mg/kg of AM 404. Pre-treatment with SB 366791 (2 mg/kg, i.p.), a new TRPV1 antagonist, also abolished the hypothermia evoked by AM 404 (20 mg/kg, i.p.). In contrast, pre-treatment with SR 141716A (Rimonabant), a CB(1) antagonist, or AA-5-HT, a fatty acid amide hydrolase (FAAH) blocker, did not affect AM 404-evoked hypothermia. The present data demonstrate that AM 404 evokes a significant hypothermia in rats that is dependent on TRPV1 receptor activation.     

Constitutive activity of cannabinoid-2 (CB2) receptors plays an essential role in the protean agonism of (+)AM1241 and L768242

Background and purpose:

Cannabinoid-2 (CB₂) receptor-selective agonists have shown anti-nociceptive activity in models of neuropathic and inflammatory pain, and the two agonists most widely used, (+/−)AM1241 [(2-iodo-5-nitrophenyl)-[1-(1-methylpiperidin-2-ylmethyl)-1H-indol-3-yl-methanone] and L768242 [(2,3-dichloro-phenyl)-[5-methoxy-2-methyl-3-(2-morpholin-4-yl-ethyl)-indol-1-yl]-methanone] ({"type":"entrez-nucleotide","attrs":{"text":"GW405833","term_id":"288331434","term_text":"GW405833"}}GW405833), have been suggested to be protean agonists. Here we investigated the role of the constitutive activity of CB₂ receptors in (+)AM1241 and L768242 protean agonism.

Experimental approach:

Pharmacological profiles of CB₂ receptor ligands were evaluated in Chinese hamster ovary cells expressing recombinant human (hCB₂) or rat (rCB₂) receptors, by measuring modulation of cAMP. To assess the influence of constitutive activity on pharmacological profile, constitutive activity was abolished by pretreatment with AM630 [(6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl) methanone)], followed by extensive washing.

Key results:

In cell lines expressing either hCB₂ or rCB₂ receptors, (+)AM1241 did not reverse forskolin stimulation of cAMP levels. Conversely, L768242 was an inverse agonist at both hCB₂ and rCB₂ receptors. Abolition of constitutive activity disclosed (+)AM1241 and L768242 agonist activity, while activity of CP55940 [5-(1,1-dimethylheptyl)-2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxy-propyl)-cyclohexyl]-phenol] was unaffected and AM630 became a neutral antagonist. In presence of constitutively active CB₂ receptors, (+)AM1241 antagonized CP55940, but when constitutive activity was abolished, it acted as a partial agonist with additive or antagonistic behaviour, depending on concentration.

Conclusions and implications:

These results show that (+)AM1241 and L768242 are protean agonists at both hCB₂ and rCB₂ receptors. Abolition of constitutive activity reveals the agonist activity of these compounds. Thus, differences between in vivo and in vitro profiles of CB₂ receptor agonists could be due to different levels of constitutive activity in recombinant versus native CB₂ receptors.     

Actions of the endocannabinoid transport inhibitor AM404 in neuropathic and inflammatory pain models

1. Although cannabinoid receptor agonists have analgesic activity in chronic pain states, they produce a spectrum of central cannabinoid CB(1) receptor-mediated motor and psychotropic side-effects. The actions of endocannabinoids, such as anandamide, are terminated by uptake and subsequent intracellular enzymatic degradation. In the present study, we examined the effect of acute administration of the anandamide transport inhibitor AM404 in rat models of chronic neuropathic and inflammatory pain. 2. Systemic administration of AM404 (10 mg/kg) reduced mechanical allodynia in the partial sciatic nerve ligation (PNL) model of neuropathic pain, but not in the complete Freund's adjuvant (CFA) model of inflammatory pain. 3. The effect of AM404 in the PNL model was abolished by coapplication with the selective cannabinoid CB(1) receptor antagonist AM251 (1 mg/kg). AM404 did not produce a reduction in motor performance in either the PNL or CFA models. 4. These findings suggest that acute administration of AM404 reduces allodynia in a neuropathic pain model via cannabinoid CB(1) receptor activation, without causing the undesirable motor disruption associated with cannabinoid receptor agonists.     

Differential effects of two cannabinoid receptor agonists on progressive ratio responding for food and free-feeding in rats

The cannabinoid receptor agonists delta9-tetrahydrocannabinol (delta9-THC) and HU-210 were compared in terms of their effects on: (1) progressive ratio (PR) responding for food, and (2) free food intake. In the first experiment, food-deprived Wistar rats were trained on a time-constrained (60 min) PR-5 schedule for food reinforcement, in which the response requirement incremented by five lever presses for each successive reinforcer. One group of rats received vehicle, 0.5, 1 or 3 mg/kg delta9-THC (i.p.), and three other groups received HU-210 (i.p.) at three different dose ranges, spanning 0.001-0.1 mg/kg. In the second experiment, the effects of the two drugs on free food intake were tested in a separate group of non-deprived rats. For PR responding, delta9-THC significantly increased the break point (final ratio completed) and the total number of lever presses emitted. The same drug also significantly increased free food intake. However, the effects of HU-210 were quite different: it did not alter PR responding at any dose; instead, its only significant effect was to reduce free food intake at 0.06 mg/kg. These data suggest that increased motivation to obtain food might underlie the hyperphagic effects of delta9-THC. However, the synthetic agonist HU-210 has different effects: it only acts to reduce feeding behaviour, an outcome that probably reflects non-specific behavioural disruption. These findings suggest important differences between the two CB1 receptor agonists in terms of their pharmacological effects.     

Cannabinoid agonists but not inhibitors of endogenous cannabinoid transport or metabolism enhance the reinforcing efficacy of heroin in rats.

Accumulating evidence suggests that the endogenous cannabinoid system is involved in the reinforcing effects of heroin. In rats intravenously self-administering heroin, we investigated effects of cannabinoid CB1 receptor agonists and compounds that block transport or metabolism of the endogenous cannabinoid anandamide. The natural cannnabinoid CB1 receptor agonist delta-9-tetrahydrocannabinol (THC, 0.3-3 mg/kg i.p.) did not alter self-administration of heroin under a fixed-ratio one (FR1) schedule, except at a high 3 mg/kg dose which decreased heroin self-administration. Under a progressive-ratio schedule, however, THC dose-dependently increased the number of 50 mug/kg heroin injections self-administered per session and the maximal ratio completed (break-point), with peak increases at 1 mg/kg THC. In addition, 1 mg/kg THC increased break-points and injections self-administered over a wide range of heroin injection doses (25-100 microg/kg), indicating an increase in heroin's reinforcing efficacy and not its potency. The synthetic cannabinoid CB1 receptor agonist WIN55,212-2 (0.3-3 mg/kg i.p.) had effects similar to THC under the progressive-ratio schedule. In contrast, AM-404 (1-10 mg/kg i.p.), an inhibitor of transport of anandamide, and URB-597 (0.01-0.3 mg/kg i.p.), an inhibitor of the enzyme fatty acid amide hydrolase (FAAH) that degrades anandamide, or their combination, did not increase reinforcing efficacy of heroin at any dose tested. Thus, activation of cannabinoid CB1 receptors facilitates the reinforcing efficacy of heroin and this appears to be mediated by interactions between cannabinoid CB1 receptors and mu-opioid receptors and their signaling pathways, rather than by an opioid-induced release of endogenous cannabinoids.     

Antinociceptive effects induced through the stimulation of spinal cannabinoid type 2 receptors in chronically inflamed mice

The stimulation of spinal cannabinoid type 2 (CB(2)) receptors is a suitable strategy for the alleviation of experimental pain symptoms. Several reports have described the up-regulation of spinal cannabinoid CB(2) receptors in neuropathic settings together with the analgesic effects derived from their activation. Besides, we have recently reported in two murine bone cancer models that the intrathecal administration of cannabinoid CB(2) receptor agonists completely abolishes hyperalgesia and allodynia, whereas spinal cannabinoid CB(2) receptor expression remains unaltered. The present experiments were designed to measure the expression of spinal cannabinoid CB(2) receptors as well as the analgesic efficacy derived from their stimulation in mice chronically inflamed by the intraplantar injection of complete Freund's adjuvant 1 week before. Both spinal cannabinoid CB(2) receptors mRNA measured by real-time PCR and cannabinoid CB(2) receptor protein levels measured by western blot remained unaltered in inflamed mice. Besides, the intrathecal (i.t.) administration of the cannabinoid CB(2) receptor agonists AM1241, (R,S)-3-(2-Iodo-5-nitrobenzoyl)-1-(1-methyl-2-piperidinylmethyl)-1H-indole, (0.03-1 μg) and JWH 133, (6aR,10aR)-3-(1,1-Dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran, (3-30 μg) dose-dependently blocked inflammatory thermal hyperalgesia and mechanical allodynia. The analgesic effects induced by both agonists were counteracted by the coadministration of the selective cannabinoid CB(2) receptor antagonist SR144528, 5-(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-N-[(1S,2S,4R)-1,3,3-trimethylbicyclo[2.2.1]hept-2-yl]-1H-pyrazole-3-carboxamide, (5 μg) but not by the cannabinoid CB(1) receptor antagonist AM251, N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide, (10 μg). The effects induced by AM1241 were also inhibited by the coadministration of the opioid receptor antagonist, naloxone (1 μg). These results demonstrate that effective analgesia can be achieved in chronic inflammatory settings through the stimulation of spinal cannabinoid CB(2) receptors even if this receptor population is not up-regulated.     

Effects of cannabinoids on lithium-induced conditioned rejection reactions in a rat model of nausea

Rationale. Marijuana has been reported to suppress nausea produced by chemotherapy treatment in human cancer patients. Although there is abundant evidence that cannabinoid agonists attenuate vomiting in emetic species, there has been little experimental evidence of their anti-nausea potential. Considerable evidence suggests that conditioned rejection reactions in rats reflect nausea. The present experiments evaluated the potential of low doses of the cannabinoid agonists, Δ-9-tetrahydrocannabinol (THC; 0.5 mg/kg, i.p.), and HU-210 (0.001 mg/kg and 0.01 mg/kg, i.p.), and the CB₁ antagonist SR-141716A in modulating the establishment and the expression of lithium-induced conditioned rejection reactions in rats. Objectives. To evaluate the effect of cannabinoids on conditioned rejection reactions, a rat model of nausea. Methods. In experiments 1 and 2, respectively, rats were injected with cannabinoid agonists, THC (0.5 mg/kg, i.p.) and HU-210 (0.001, 0.005 or 0.01 mg/kg), 30 min prior to exposure to 0.1% saccharin solution by intraoral infusion. Immediately following saccharin exposure, they were injected with 20 ml/kg 0.15 M lithium chloride or saline. On each of two test trials, the rats were injected with the cannabinoid or vehicle 30 min prior to exposure to saccharin. In experiment 3, rats were injected with the CB₁ antagonist, SR-141716A (2.5 mg/kg) or a combination of SR-141716A and HU-210 (0.01 mg/kg) 30 min prior to an infusion of saccharin followed by injection of lithium or saline. They were given a single drug-free test trial. Experiment 4 replicated and extended the findings of experiment 3. Results. Δ-9-THC and HU-210 interfered with the establishment and the expression of lithium-induced conditioned rejection reactions. The suppressive effect of HU-210 on rejection reactions was reversed by pretreatment with SR-141716A. Administration of SR-141716A prior to conditioning potentiated lithium-induced conditioned rejection reactions. Conclusions. These results indicate that the establishment and the expression of lithium-induced conditioned rejection reactions are suppressed by pretreatment with cannabinoid agents. These effects appear to be mediated by their action on the CB₁ receptor, because they are reversed by pretreatment with SR-141716A. Finally, our results suggest that endogenous cannabinoids play a role in modulation of nausea, because the antagonist potentiated lithium-induced nausea. Electronic Publication     

5-HT1A receptors are involved in the anxiolytic effect of Delta9-tetrahydrocannabinol and AM 404, the anandamide transport inhibitor, in Sprague-Dawley rats

The mechanism mediating the effects of cannabinoids on anxiety-related responses appear to involve cannabinoid CB1 and non-CB1 receptors. However, other neurotransmitters may play a role in such effect. This study shows evidence of an interaction between endocannabinoid system and serotonin (5-HT), 1A receptor subtype on anxiety-like behavior in Sprague-Dawley rats. The exogenous cannabinoid agonist, Delta9-tetrahydrocannabinol (THC), and N-(4-hydroxyphenyl)-arachidonylamide, the anandamide transporter inhibitor (AM 404) were evaluated in the elevated plus maze test. THC (0.075-0.75 mg/kg i.p.), given 30 min and AM 404 (0.75-1.25 mg/kg i. p.), given 60 min before the test, exhibited a dose-response anxiolytic effect evaluated in terms of increase in the percentage of total entries and time spent in the open and decrease of total entries and time spent in the closed arms. The anxiolytic effect obtained with the maximal active dose of both THC (0.75 mg/kg) and AM 404 (1.25 mg/kg) was blocked by the 5-HT1A receptor antagonist, N-[2-[4-(2-methoxyphenyl) piperazin-1-yl]ethyl]-N-pyridin-2-yl-cyclohexanecarboxamide dihydro chloride (WAY-100635 (300 microg/kg, s.c.), given 30 min before THC or 15 min before AM 404. The combination of an ineffective dose of THC (0.015 mg/kg) or AM 404 (0.015 mg/kg) on anxiety-related responses with an ineffective dose of the 5HT(1A) receptor agonist, 8-Hydroxy-2-(di-n-propylamino) tetralin hydrobromide (8-OH-DPAT) (7.5 microg/kg, i.p.), led to a synergistic effect. No interference with spontaneous motor activity, evaluated in an activity cage for 5 min, in rats given the drugs alone or in combination, was found. These data suggest that the anxiolytic effect produced by endo- and eso-cannabinoids is modulated by 5-HT1A receptors.     

Antiinflammatory action of endocannabinoid palmitoylethanolamide and the synthetic cannabinoid nabilone in a model of acute inflammation in the rat.

1. The antiinflammatory activity of synthetic cannabinoid nabilone in the rat model of carrageenan-induced acute hindpaw inflammation was compared with that of the endocannabinoid palmitoylethanolamide and the nonsteroidal antiinflammatory drug indomethacin. 2. Preliminary experiments in rats used a tetrad of behavioural tests, specific for tetrahydrocannabinol-type activity in the CNS. These showed that the oral dose of nabilone 2.5 mg kg(-1) had no cannabinoid psychoactivity. 3. Intraplantar injection of carrageenan (1% w v(-1)) elicited a time-dependent increase in paw volume and thermal hyperalgesia. 4. Nabilone (0.75, 1.5, 2.5 mg kg(-1), p.o.), given 1 h before carrageenan, reduced the development of oedema and the associated hyperalgesia in a dose-related manner. Nabilone 2.5 mg kg(-1), palmitoylethanolamide 10 mg kg(-1) and indomethacin 5 mg kg(-1), given p.o. 1 h before carrageenan, also reduced the inflammatory parameters in a time-dependent manner. 5. The selective CB(2) cannabinoid receptor antagonist [N-[(1S)-endo-1,3,3-trimethyl bicyclo [2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)pyrazole-3 carboxamide] (SR 144528), 3 mg kg(-1) p.o. 1 h before nabilone and palmitoylethanolamide, prevented the anti-oedema and antihyperalgesic effects of the two cannabinoid agonists 3 h after carrageenan. 6. Our findings show the antiinflammatory effect of nabilone and confirm that of palmitoylethanolamide indicating that these actions are mediated by an uncharacterized CB(2)-like cannabinoid receptor.     

Gastric antisecretory role and immunohistochemical localization of cannabinoid receptors in the rat stomach

1. The role of cannabinoid (CB) receptors in the regulation of gastric acid secretion was investigated in the rat by means of functional experiments and by immunohistochemistry. 2. In anaesthetized rats with lumen-perfused stomach, the non selective CB-receptor agonist WIN 55,212-2 (0.30 - 4.00 micromol kg(-1), i.v.) and the selective CB(1)-receptor agonist HU-210 (0.03 - 1.50 micromol kg(-1), i.v.), dose-dependently decreased the acid secretion induced by both pentagastrin (30 nmol kg(-1) h(-1)) and 2-deoxy-D-glucose (1.25 mmol kg(-1), i.v.). By contrast, neither WIN 55,212-2 (1 - 4 micromol kg(-1), i.v.) nor HU-210 (0.03 - 1.50 micromol kg(-1), i.v.) did modify histamine-induced acid secretion (20 micromol kg(-1) h(-1)). The selective CB(2)-receptor agonist JWH-015 (3 - 10 micromol kg(-1), i.v.) was ineffective. 3. The gastric antisecretory effects of WIN 55,212-2 and HU-210 on pentagastrin-induced acid secretion were prevented by the selective CB(1)-receptor antagonist SR141716A (0.65 micromol kg(-1), i.v.) and unaffected by the selective CB(2)-receptor antagonist SR144528 (0.65 - 2 micromol kg(-1), i.v.). 4. Bilateral cervical vagotomy and ganglionic blockade with hexamethonium (10 mg kg(-1), i.v., followed by continuous infusion of 10 mg kg(-1) h(-1)) significantly reduced, but not abolished, the maximal inhibitory effect of HU-210 (0.3 micromol kg(-1), i.v.) on pentagastrin-induced acid secretion; by contrast, pretreatment with atropine (1 mg kg(-1), i.v.) did not modify the antisecretory effect of HU-210. 5. Immunoreactivity to the CB(1) receptor was co-localized with that of the cholinergic marker choline acetyltransferase in neural elements innervating smooth muscle, mucosa and submucosal blood vessels of rat stomach fundus, corpus and antrum. In contrast, CB(2) receptor-like immunoreactivity was not observed. 6. These results indicate that gastric antisecretory effects of cannabinoids in the rat are mediated by suppression of vagal drive to the stomach through activation of CB(1) receptors, located on pre- and postganglionic cholinergic pathways. However, the ineffectiveness of atropine in reducing the effect of HU-210 suggests that the release of non cholinergic excitatory neurotransmitters may be regulated by CB(1) receptors.     

Antipsychotics improve Delta9-tetrahydrocannabinol-induced impairment of the prepulse inhibition of the startle reflex in mice

Recently, cannabinoid receptor agonists have been reported to impair prepulse inhibition (PPI) of the startle reflex. In the current study, we examined the effect of Delta9-tetrahydrocannabinol (THC), the principal psychoactive component of cannabis, on the PPI, and found that THC (10 mg/kg, i.p.) impaired the PPI concomitant with a decrease in the startle response. Antipsychotics such as haloperidol (0.3 mg/kg, i.p.) and risperidone (0.1 mg/kg, i.p.), which are potent dopamine D2 receptor antagonists, and SR141716 (10 mg/kg, i.p.), a CB1 cannabinoid receptor antagonist, reversed these THC-induced PPI deficits. Moreover, THC (10 mg/kg) increased dopamine (DA) release in the nucleus accumbens but not medial prefrontal cortex over a 50-100-min period (time of PPI test) after treatment, and SR141716 (10 mg/kg) reversed this increase in DA release induced by THC. These results suggest that dopaminergic hyperfunction in the nucleus accumbens may be involved in THC-induced PPI deficits.     

The role of cannabinoid receptors in intestinal motility, defaecation and diarrhoea in rats

We have studied the effects of the cannabinoid receptor agonists (R)-(+)[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2, 3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN 55,212-2, 0. 3-5 mg/kg, i.p.) and (-)-cis-3-[2-hydroxy-4-(1, 1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol) (CP 55,940, 0.03-1 mg/kg, i.p.), the cannabinoid CB(1) receptor antagonist (N-piperidin-1-yl)-5-(4-chlorophenyl)-1-2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A, 0. 3-5 mg/kg, i.p.) and the cannabinoid CB(2) receptor antagonist N-[-(1S)-endo-1,3,3-trimethyl bicyclo [2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazo le- 3-carboxamide (SR144528, 1 mg/kg, i.p.) on intestinal motility, defaecation and castor-oil (1 ml/100 g rat, orally)-induced diarrhoea in the rat. SR141716A, but not SR144528, increased defaecation and upper gastrointestinal transit, while WIN 55,212-2 and CP 55,940 decreased upper gastrointestinal transit but not defaecation. WIN 55,212-3 (5 mg/kg), the less active enantiomer of WIN 55,212-2, was without effect. A per se non-effective dose of SR141716A (0.3 mg/kg), but not of SR144528 (1 mg/kg) or the opioid receptor antagonist, naloxone (2 mg/kg i.p.), counteracted the inhibitory effect of both WIN 55,212-2 (1 mg/kg) and CP 55,940 (0.1 mg/kg) on gastrointestinal motility. WIN 55,212-2 did not modify castor-oil-induced diarrhoea, while CP 55,940 produced a transient delay in castor-oil-induced diarrhoea at the highest dose tested (1 mg/kg), an effect counteracted by SR141715A (5 mg/kg). These results suggest that (i) intestinal motility and defaecation could be tonically inhibited by the endogenous cannabinoid system, (ii) exogenous activation of cannabinoid CB(1) receptors produces a reduction in intestinal motility in the upper gastrointestinal tract but not in defaecation, (iii) endogenous or exogenous activation of cannabinoid CB(2) receptors does not affect defaecation or intestinal motility and (iv) the cannabinoid receptor agonist, CP 55, 940, possesses a weak and transient antidiarrhoeal effect while the cannabinoid receptor agonist, WIN 55,212-2, does not possess antidiarrhoeal activity.     

Cannabinoids modulate neuronal firing in the rat basolateral amygdala: evidence for CB1- and non-CB1-mediated actions

Recent evidence indicates that the basolateral amygdala (BLA) may be involved in behavioural effects induced by cannabinoids. High levels of CB1 cannabinoid receptors have been shown in this region, where they modulate excitatory and inhibitory synaptic transmission. However, the neurophysiological effects of these opposing synaptic actions have not been investigated in vivo. To this purpose, single-unit extracellular recordings were performed in urethane anaesthetized rats in order to determine whether exogenously applied cannabinoids influenced the spontaneous or evoked electrical activity of neurons in the BLA. The effects of cannabinoids were found to be dependent on the characteristics of the neurons examined and on the properties of the agents used. We tested and compared two structurally different synthetic cannabinoid receptor agonists, the highly potent HU-210 (0.125-1.0 mg/kg, i.v.) and WIN55212-2 (WIN, 0.125-1.0 mg/kg, i.v.). With a CB1 cannabinoid receptor-dependent mechanism, HU-210 potently inhibited the firing rate of BLA interneurons whereas WIN modulated the discharge rate in a biphasic manner. By contrast, BLA projection neurons, antidromically identified from the shell of the nucleus accumbens, were significantly inhibited by WIN at all doses tested, while HU-210 administration led to less consistent effects, since only 1.0 mg/kg inhibited firing rate in the majority of recorded neurons. Additionally, WIN, but not HU-210, significantly attenuated short-latency spiking activity in BLA projection neurons evoked by electrical stimulation of the medial prefrontal cortex. In these neurons, WIN-induced effects were antagonised by the non-selective cannabinoid receptor antagonist SR141716A and by the vanilloid receptor antagonist capsazepine, but not by the selective CB1 antagonist AM-251. Taken together, our findings indicate that the overall excitability of efferent neurons in the BLA is strongly reduced by WIN in a non-CB1-dependent manner. In this effect, the contribution of a novel cannabinoid-vanilloid-sensitive putative non-CB1 receptors, the existence of which was postulated in recent reports, might play a role.     

Antiarrhythmic properties of a cannabinoid (CB) receptor agonist

The cannabinoid (CB1) receptor agonist HU-210 (0.5 mg/kg, i.v.) exhibited a pronounced antiarrhythmic effect in rats with the adrenaline (epinephrine) and aconitine induced arrhythmia models. At the same time, the intracerebrovascular introduction of HU-210 (500 or 5000 ng) did not affect the adrenaline-induced arrhythmia. The CB1 receptor pretreatment (blocking) with the SR 141716 antagonist (3 mg/kg) and the introduction of a ganglion blocker (hexamethonium, 10 mg/kg) did not inhibit the antiarrhythmic effect of HU-210. It is concluded that the antiarrhythmic effect of intravenously injected HU-210 is neither related to the CB 1 receptor activation nor mediated by the autonomous (vegetative) nervous system.