Ceramide is involved in r(+)-methanandamide-induced cyclooxygenase-2 expression in human neuroglioma cells

Cannabinoids have recently been shown to induce the expression of the cyclooxygenase-2 (COX-2) isoenzyme in H4 human neuroglioma cells. Using this cell line, the present study investigates the contribution of the second messenger ceramide to this signaling pathway. Incubation of cells with the endocannabinoid analog R(+)-methanandamide (R(+)-MA) was associated with an increase of intracellular ceramide levels. Enhancement of ceramide formation by R(+)-MA was abolished by fumonisin B1, a ceramide synthase inhibitor, whereas inhibitors of neutral sphingomyelinase (spiroepoxide, glutathione) and serine palmitoyltransferase (l-cycloserine, ISP-1) were inactive in this respect. R(+)-MA caused a biphasic activation of the p38 and p42/44 mitogen-activated protein kinases (MAPKs), with phosphorylation peaks occurring after 15-min and 4- to 8-h treatments, respectively. Inhibition of ceramide synthesis with fumonisin B1 was associated with a suppression of R(+)-MA-induced delayed phosphorylations of p38 and p42/44 MAPKs and subsequent COX-2 expression. The involvement of ceramide in COX-2 expression was corroborated by findings showing that C2-ceramide and neutral sphingomyelinase from Bacillus cereus caused concentration-dependent increases of COX-2 expression that were suppressed in the presence of 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)imidazol (SB203580, a p38 MAPK inhibitor) or 2'-amino-3'-methoxyflavone (PD98059, a p42/44 MAPK activation inhibitor). In contrast, dihydro-C2-ceramide being used as a negative control did not induce MAPK phosphorylation and COX-2 expression. Collectively, our results demonstrate that R(+)-MA induces COX-2 expression in human neuroglioma cells via synthesis of ceramide and subsequent activation of p38 and p42/44 MAPK pathways. Induction of COX-2 expression via ceramide represents a hitherto unknown mechanism by which cannabinoids mediate biological effects within the central nervous system.     

Cannabinoids inhibit GABAergic synaptic transmission in mice nucleus accumbens

In mice nucleus accumbens slices, whole-cell patch clamp recording of medium-spiny neurons revealed that cannabimimetics ((R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphtalenylmethanone) (WIN-2) and ((-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)-phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol) inhibit stimulus-evoked gamma-aminobutyric acid mediated inhibitory post-synaptic currents (IPSC). The actions of WIN-2 were reversed by the selective cannabinoid CB(1) receptor antagonist [N-piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride] (SR141716A). WIN-2 modified paired-pulse ratio of evoked IPSCs and decreased miniature IPSC frequency indicating a presynaptic localization of cannabinoid CB(1) receptors.     

Up-regulation of cyclooxygenase-2 expression is involved in R(+)-methanandamide-induced apoptotic death of human neuroglioma cells

Cannabinoids have been implicated in the reduction of glioma growth. The present study investigated a possible relationship between the recently shown induction of cyclooxygenase (COX)-2 expression by the endocannabinoid analog R(+)methanandamide [R(+)-MA] and its effect on the viability of H4 human neuroglioma cells. Incubation with R(+)-MA for up to 72 h decreased the cellular viability and enhanced accumulation of cytoplasmic DNA fragments in a time-dependent manner. Suppression of R(+)-MA-induced prostaglandin (PG) E2 synthesis with the selective COX-2 inhibitor celecoxib (0.01-1 microM) or inhibition of COX-2 expression by COX-2-silencing small-interfering RNA was accompanied by inhibition of R(+)-MA-mediated DNA fragmentation and cell death. In contrast, the selective COX-1 inhibitor SC-560 was inactive in this respect. Cells were also protected from apoptotic cell death by other COX-2 inhibitors (NS-398 [[N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide]] and diclofenac) and by the ceramide synthase inhibitor fumonisin B1, which interferes with COX-2 expression by R(+)-MA. Moreover, the proapoptotic action of R(+)-MA was mimicked by the major COX-2 product PGE2. Apoptosis and cell death by R(+)-MA were not affected by antagonists of cannabinoid receptors (CB1, CB2) and vanilloid receptor 1. In further experiments, celecoxib was demonstrated to suppress apoptotic cell death elicited by anandamide, which is structurally similar to R(+)-MA. As a whole, this study defines COX-2 as a hitherto unknown target by which a cannabinoid induces apoptotic death of glioma cells. Furthermore, our data show that pharmacological concentrations of celecoxib may interfere with the proapoptotic action of R(+)-MA and anandamide, suggesting that cotreatment with COX-2 inhibitors could diminish glioma regression induced by these compounds.     

Inhibition of small intestinal secretion by cannabinoids is CB1 receptor-mediated in rats

We tested the hypothesis that cannabinoids, acting via a neuronal mechanism of action decrease small intestinal secretion. In vitro electrical stimulation induced ileal secretion in rats, that was attenuated by a cannabinoid receptor agonist, WIN 55212-2, (mesylate(R)-(+)-[2, 3-dihydro-5-methyl-3-[4-morpholino)methyl]pyrrolo-[1,2,3-de]-1, 4-benzoxazin-6-yl](1-naphthyl)methanone) but not its optical isomer WIN 55212-3. The inhibition of secretion induced by WIN 55212-2 was reversed by SR141716A (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride), a cannabinoid CB1 receptor antagonist. An ileal secretory response stimulated by acetylcholine was unaffected by WIN 55212-2. These findings show that cannabinoids inhibit neurally mediated secretion via cannabinoid CB1 receptors. Thus, cannabinoids may have therapeutic potential for diarrhea unresponsive to available therapies.     

Effects of cannabinoids on non-adrenergic non-cholinergic-mediated relaxation in guinea-pig trachea

The effects of cannabinoid receptor agonists on the non-adrenergic non-cholinergic (NANC) inhibitory responses to electrical field stimulation in guinea-pig trachea were assessed. R-(+)-[2,3-dihydro-5-methyl-3-[(morpholilinyl) methyl]pyrrolo [1,2,3-de]-1,4-benzoxazin-6-yl]-(1-naphthalenyl)methanone mesylate (WIN 55,212-2; 10(-5) M) significantly enhanced the frequency-dependent response to electrical stimulation. The same concentration of R-(N)-(2-hydroxy-1-methylethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (R(+)methanandamide) and 1-propyl-2-methyl-3-(1-naphthoyl)indole (JWH-015) did not affect significantly the electrically induced inhibitory NANC responses. The effect of WIN 55,212-2 was not modified by the cannabinoid CB1 and CB2 receptor-selective antagonists, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A; 10(-5) M) and 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; 10(-5) M), respectively. Moreover, the nitric oxide synthase inhibitor, L-NG-nitro-arginine methyl ester (L-NAME; 10(-4) M), but not the peptidase, alpha-chymotrypsin (2 U/ml), blocked the effect of WIN 55,212-2. Postsynaptically, WIN 55,212-2 did not produce any change of tracheal smooth muscle tone, either basal or histamine-induced, and did not interfere with the relaxant activity of the nitric oxide donor, sodium nitroprusside (10(-8)-10(-4) M). In conclusion, our results suggest that (a) cannabinoid CB1 and CB2 receptor stimulation does not alter the inhibitory NANC transmission in guinea-pig trachea, and (b) WIN 55,212-2 potentiates the NO-mediated component of the NANC relaxant response to electrical stimulation through a cannabinoid receptor-independent mechanism.     

Inhibition of nitric oxide production in RAW264.7 macrophages by cannabinoids and palmitoylethanolamide

We have investigated the inhibition of lipopolysaccharide stimulated nitric oxide production in RAW264.7 macrophages by the cannabinoids and the putative cannabinoid CB(2)-like receptor ligand, palmitoylethanolamide. (R)-(+)-[2, 3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo-[1,2,3-de]-1, 4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate ((+)-WIN55212) and, to a lesser extent (-)-cis-3-[2-hydroxy-4-(1, 1-dimethylheptyl)phenyl]-trans-4-(3-hydroxy-propyl)cyclohexan++ +-1-ol (CP55940), significantly inhibited lipopolysaccharide stimulated nitric oxide production. The level of inhibition was found to be dependent on the concentration of lipopolysaccharide used to induce nitric oxide production. Palmitoylethanolamide significantly inhibited nitric oxide production induced by lipopolysaccharide. The inhibition of nitric oxide production by (+)-WIN55212 but not palmitoylethanolamide was significantly attenuated in the presence of 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). (+)-WIN55212 produced a pertussis toxin-sensitive parallel rightward shift in the log concentration-response curve for lipopolysaccharide, causing a fivefold increase in the EC(50) value for lipopolysaccharide with no change in the E(max) value. (-)-WIN55212 had no effect on the log concentration-response curve for lipopolysaccharide. Palmitoylethanolamide did not produce a rightward shift in the lipopolysaccharide concentration-response curve. However, it did produce a pertussis toxin-insensitive reduction in the E(max) value. The results suggest that the inhibition of lipopolysaccharide mediated nitric oxide release by (+)-WIN55212 in murine macrophages is mediated by cannabinoid CB(2) receptors. In contrast, the inhibition by palmitoylethanolamide does not appear to be mediated by cannabinoid receptors.     

Inhibition of interleukin-8 release in the human colonic epithelial cell line HT-29 by cannabinoids

We have investigated the effects of cannabinoid agonists and antagonists on tumour necrosis factor-alpha (TNF-alpha)-induced secretion of interleukin-8 from the colonic epithelial cell line, HT-29. The cannabinoid receptor agonists [(-)-3-[2-hydroxy-4-(1,1-dimethyl-heptyl)-phenyl]4-[3-hydroxypropyl]cyclo-hexan-1-ol] (CP55,940); Delta-9-tetrahydrocannabinol; [R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl) methyl] pyrrolo[1,2,3-de]1,4-benzoxazin-6-yl](1-naphthyl) methanone mesylate] (WIN55,212-2) and 1-propyl-2-methyl-3-naphthoyl-indole (JWH 015) inhibited TNF-alpha induced release of interleukin-8 in a concentration-dependent manner. The less active enantiomer of WIN55212-2, [S(-)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]1,4-benzoxazin-6-yl](1-naphthyl) methanone mesylate (WIN55212-3), and the cannabinoid CB(1) receptor agonist arachidonoyl-2-chloroethylamide (ACEA) had no significant effect on TNF-alpha-induced release of interleukin-8. The cannabinoid CB(1) receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1,4-pyrazole-3-carboxamide hydrochloride (SR141716A; 10(-6) M) antagonised the inhibitory effect of CP55,940 (pA(2)=8.3+/-0.2, n=6) but did not antagonise the inhibitory effects of WIN55212-2 and JWH 015. The cannabinoid CB(2) receptor antagonist N-(1,S)-endo1,3,3-trimethylbicyclo(2,2,1)heptan-2-yl)-5(4-chloro-3-methyl-phenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528; 10(-6) M) antagonised the inhibitory effects of CP55,940 (pA(2)=8.2+/-0.8, n=6), WIN55212-2 (pA(2)=7.1+/-0.3, n=6) and JWH 015 (pA(2)=7.6+/-0.3, n=6), respectively. Western immunoblotting of HT-29 cell lysates revealed a protein with a size that is consistent with the presence of cannabinoid CB(2) receptors. We conclude that in HT-29 cells, TNF-alpha-induced interleukin-8 release is inhibited by cannabinoids through activation of cannabinoid CB(2) receptors.     

The anti-inflammatory activities of cannabinoid receptor ligands in mouse peritonitis models.

In this report, we describe experiments in which cannabinoid receptor ligands were evaluated for effects on the development of a peritoneal inflammation when elicited in mice with thioglycollate broth or staphylococcus enterotoxin A. The cannabinoid receptor agonists [(-)-11-hydoxy-Delta(8) tetrahydrocannabinol-dimethylheptyl] (HU-210) and [(R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl[pyrrolo[1,2,3-de]1,4-benzoxazin-6-yl](1-naphthalenyl) methanone] (WIN 55212-2) blocked the migration of neutrophils into the peritoneal cavity in response to these inflammatory stimuli. This effect was caused by a delay in the production of the neutrophil chemoattractants, KC and macrophage inflammatory protein-2. HU-210 and WIN 55212-2 blocked neutrophil chemokines and neutrophil migration whether administered subcutaneously (s.c.) or intracerebroventricularly (i.c.v.). Their modulatory effects on the inflammation were antagonized by centrally administered [N-(piperdin-1-yl)-5-(4-chloropheny)-1-(2,4-dichloropheny)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride] (SR141716A), a selective cannabinoid CB(1) receptor antagonist. This latter observation, and the ability of the cannabinoid receptor agonists to suppress the peritoneal inflammation at relatively low doses when administered i.c.v., indicated a role for central cannabinoid CB(1) receptors in the anti-inflammatory activities of HU-210 and WIN 55212-2. The cannabinoid receptor agonists had no effect on monocyte migration elicited by thioglycollate, despite their ability to suppress monocyte chemotactic protein-1 levels in lavage fluids. The cannabinoid CB(2) receptor antagonist, [N-[(1S)-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]5-(4-choro-3 methylphenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide] (SR144528) inhibited the peritoneal inflammation in a manner analogous to that of HU-210 and WIN 55212-2 when administered i.c.v., but it did not appear to act through central cannabinoid CB(1) receptors. The present results add to the body of literature indicating that cannabinoid receptor ligands have diverse anti-inflammatory properties.     

(+)-Cannabidiol analogues which bind cannabinoid receptors but exert peripheral activity only

Delta9-Tetrahydrocannabinol (Delta9-THC) and (-)-cannabidiol are major constituents of the Cannabis sativa plant with different pharmacological profiles: (-)-Delta9-tetrahydrocannabinol, but not (-)-cannabidiol, activates cannabinoid CB1 and CB2 receptors and induces psychoactive and peripheral effects. We have tested a series of (+)-cannabidiol derivatives, namely, (+)-cannabidiol-DMH (DMH-1,1-dimethylheptyl-), (+)-7-OH-cannabidiol-DMH, (+)-7-OH- cannabidiol, (+)-7-COOH- cannabidiol and (+)-7-COOH-cannabidiol-DMH, for central and peripheral (intestinal, antiinflammatory and peripheral pain) effects in mice. Although all (+)-cannabidiols bind to cannabinoid CB1 and CB2 receptors, only (+)-7-OH-cannabidiol-DMH was centrally active, while all (+)-cannabidiol analogues completely arrested defecation. The effects of (+)-cannabidiol-DMH and (+)-7-OH-cannabidiol-DMH were partially antagonized by the cannabinoid CB1 receptor antagonist N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716), but not by the cannabinoid CB2 receptor antagonist N-[-(1S)-endo-1,3,3-trimethil bicyclo [2.2.1] heptan-2-yl-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528), and had no effect on CB1(-/-) receptor knockout mice. (+)-Cannabidiol-DMH inhibited the peripheral pain response and arachidonic-acid-induced inflammation of the ear. We conclude that centrally inactive (+)-cannabidiol analogues should be further developed as antidiarrheal, antiinflammatory and analgesic drugs for gastrointestinal and other peripheral conditions.     

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.     

Pharmacological characterisation of cannabinoid receptors inhibiting interleukin 2 release from human peripheral blood mononuclear cells

The effects of a range of cannabinoid receptor agonists and antagonists on phytohaemagglutinin-induced secretion of interleukin-2 from human peripheral blood mononuclear cells were investigated. The nonselective cannabinoid receptor agonist WIN55212-2 ((R)-(+)-[2,3-dihydro-5-methyl-3-[4-morpholinylmethyl]pyrrolo[1,2,3-de]1,4-benzoxazin-6-yl](1-naphthyl) methanone mesylate) and the selective cannabinoid CB(2) receptor agonist JWH 015 ((2-methyl-1-propyl-1H-indol-3-yl)-1-napthalenylmethanone) inhibited phytohaemagglutinin (10 microg/ml)-induced release of interleukin-2 in a concentration-dependent manner (IC(1/2max), WIN55212-2=8.8 x 10(-7) M, 95% confidence limits (C.L.)=2.2 x 10(-7)-3.5 x 10(-6) M; JWH 015=1.8 x 10(-6) M, 95% C.L.=1.2 x 10(-6)-2.9 x 10(-6) M, n=5). The nonselective cannabinoid receptor agonists CP55,940 ((-)-3-[2-hydroxy-4-(1,1-dimethyl-hepthyl)-phenyl]4-[3-hydroxypropyl]cyclo-hexan-1-ol), Delta(9)-tetrahydrocannabinol and the selective cannabinoid CB(1) receptor agonist ACEA (arachidonoyl-2-chloroethylamide) had no significant (P>0.05) inhibitory effect on phytohaemagglutinin-induced release of interleukin-2. Dexamethasone significantly (P<0.05) inhibited phytohaemagglutinin-induced release of interleukin-2 in a concentration-dependent manner (IC(1/2max)=1.3 x 10(-8) M, 95% C.L.=1.4 x 10(-9)-3.2 x 10(-8) M). The cannabinoid CB(1) receptor antagonist SR141716A (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride) (10(-6) M) did not antagonise the inhibitory effect of WIN55212-2 whereas the cannabinoid CB(2) receptor antagonist SR144528 (N-(1,S)-endo-1,3,3-trimethyl bicyclo(2,2,1)heptan-2-yl)-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide) antagonised the inhibitory effect of WIN55212-2 (pA(2)=6.3+/-0.1, n=5). In addition, CP55,940 (10(-6) M) and Delta(9)-tetrahydrocannabinol (10(-6) M) also antagonised the inhibitory effects of WIN55212-2 (pA(2)=6.1+/-0.1, n=5 and pA(2)=6.9+/-0.2, n=5). In summary, WIN55,212-2 and JWH 015 inhibited interleukin-2 release from human peripheral blood mononuclear cells via the cannabinoid CB(2) receptor. In contrast, CP55,940 and Delta(9)-tetrahydrocannabinol behaved as partial agonists/antagonists in these cells.     

p38 and JNK MAPK, but not ERK1/2 MAPK, play important role in colchicine-induced cortical neurons apoptosis

Colchicine is a microtubule interfering agent and is able to induce neural apoptosis. However, the intracellular pathway involved in its neurotoxicity is still unclear. In the present study, three of mitogen-activated protein kinases (MAPKs): p38, c-Jun N-terminal kinase (JNK) and extracellular-regulated kinase 1/2 (ERK1/2) were investigated in colchicine-induced apoptosis on cortical neurons for the first time. Our results showed that 1 microM colchicine administration in primarily cultured cortical neurons led to typical neuronal apoptosis, and the apoptosis was attenuated by taxol, a microtubule stabilizer. Moreover, activation of p38 MAPK was found for the first time, as well as that of JNK MAPK, but not of ERK1/2 MAPK, after colchicine exposure. Apoptosis was inhibited by p38 MAPK inhibitors, SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole), SB239063 (trans-1-(4-hydroxycyclohexyl)-4-(fluorophenyl)-5-(2-methoxypyrimidin-4-yl) imidazole), and JNK MAPK pathway inhibitors, CEP11004 (9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid, 2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-5,16-bis[[(1-methylethyl)thio]methyl]-1-oxo-, methyl ester, (9S,10R,12R)-), SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one). However, PD98059 (2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one) and U0126 (1,4-diamino-2,3-dicyano-1, 4-bis[2-aminophenylthio]butadiene), ERK1/2 MAPK inhibitors, did not work. Furthermore, better neuronal protective effects were achieved by using JNK and the p38 MAPK inhibitors together as compared to that by using either alone. The results suggested that p38 MAPK, JNK MAPK, but not ERK1/2 MAPK may play pivotal role in colchicine's neurotoxicity in primarily cultured cortical neurons, and the protective effects of the inhibition of p38 or JNK MPAK on cortical neurons were synergistically.     

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.     

Modulation of gastric emptying and gastrointestinal transit in rats through intestinal cannabinoid CB(1) receptors

We studied the delay in gastric emptying and gastrointestinal transit induced by the cannabinoid receptor agonists (+)-WIN 55,212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate) and CP 55,940 ((-)-cis-3[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol), as prevented by the selective cannabinoid CB(1)-receptor antagonist SR141716 ((N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide)) in rats after systemic or central drug administration. Oral SR141716 showed comparable potency (ID(50) range 1.0-3.9 mg/kg) in antagonizing gastric emptying and gastrointestinal transit delay by (+)-WIN 55,212-2 or CP 55,940. Gastric emptying and gastrointestinal transit delay after intracerebroventricular (i.c.v.) (+)-WIN 55,212-2 was prevented by oral or i.c.v. SR141716, but i.c.v. SR141716 did not significantly reduce the effect of i.p. (+)-WIN 55,212-2. Pertussis toxin prevented the delaying action of i.c.v. (+)-WIN 55,212-2 on both gastric emptying and gastrointestinal transit, but had no effect on (+)-WIN 55,212-2 i.p. These findings are consistent with a primary role of peripheral cannabinoid CB(1) receptor mechanisms in gastrointestinal transit delay by specific agonists.     

Vasorelaxant effect of Win 55,212-2 in rat aorta: new mechanisms involved

R(+)-[2,3-dihydro-5-methyl-3-[(moroholinyl)methyl] pyrrolo [1,2,3-de]-1,4benzoxazinyl]-1(1-naphthalenyl) methanone mesylate (Win 55,212-2) is a synthetic cannabinoid classically classified as a potent CB(1) and CB(2) agonist with high stereoselectivity and a slight preference for CB(2) cannabinoid receptors. Its vascular actions are not always explained by its binding to these cannabinoid receptors and new targets are being proposed. The aim of this study was to further assess the vascular actions of Win 55,212-2. Isometric tension changes in response to a cumulative concentration-response curve of Win 55,212-2 (10(-9) M-10(-4) M) were recorded in aortic rings from male Wistar rats. The involvement of the endothelium, cannabinoid receptors, vanilloid receptors, and the release of calcitonin gene related peptide (CGRP) was tested. Win 55,212-2 caused a concentration-dependent vasorelaxation in rat aorta. This vascular effect was significantly inhibited by endothelial denudation, inhibition of nitric oxide synthesis, a CB(1) receptor antagonist, a transient receptor potential vanilloid-1 antagonist, capsaicin desensibilization, and a CGRP receptor antagonist (P<0.001). CB(2) and non-CB(1)/non-CB(2) receptor antagonists only caused a slight inhibitory effect in vasorelaxation to Win 55,212-2. The present findings indicate that endothelium and nitric oxide-dependent vasorelaxation induced by Win 55,212-2 mainly involves vanilloid receptors while CB(1), CB(2) and nonCB(1)/nonCB(2) cannabinoid receptors have a minor participation in its vascular effect.     

Conformationally restrained analogues of pravadoline: nanomolar potent, enantioselective, (aminoalkyl)indole agonists of the cannabinoid receptor.

Pravadoline (1) is an (aminoalkyl)indole analgesic agent which is an inhibitor of cyclooxygenase and, in contrast to other NSAIDs, inhibits neuronally stimulated contractions in mouse vas deferens (MVD) preparations (IC50 = 0.45 microM). A number of conformationally restrained heterocyclic analogues of pravadoline were synthesized in which the morpholinoethyl side chain was tethered to the indole nucleus. Restraining the morpholine diminished the ability of these pravadoline analogues to inhibit prostaglandin synthesis in vitro. In contrast, mouse vas deferens inhibitory activity was enhanced in [2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl] pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-(4-methoxyphenyl)methano ne (20). Only the R enantiomer of 20 was active (IC50 = 0.044 microM). An optimal orientation of the morpholine nitrogen for MVD inhibitory activity within the analogues studied was in the lower right quadrant, below the plane defined by the indole ring. A subseries of analogues of 20 and a radioligand of the most potent analogue, (R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo [1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone (21) were prepared. Inhibition of radioligand binding in rat cerebellar membranes was observed to correlate with functional activity in mouse vas deferens preparations. Binding studies with this ligand (Win 55212-2) have helped demonstrate that the (aminoalkyl)indole binding site is functionally equivalent with the CP-55,940 cannabinoid binding site. These compounds represent a new class of cannabinoid receptor agonists.     

Effects of NRA0045, NRA0160, and NRA0215 on regional Fos-like immunoreactivity in the rat brain.

Pharmacological characteristics of NRA compounds, novel atypical antipsychotics, were compared with those of clozapine and haloperidol, in regard to modification of Fos-like immunoreactivity (FLI) in rats. (R)-(+)-2-Amino-4-(4-fluorophenyl)-5-[1-[4-(4-fluorophenyl)-4-oxobutyl] pyrrolidin-3-yl] thiazole (NRA0045) and 2-carbamoyl-4-phenyl-5-[2-[4-(4-fluorobenzylidene) piperidin-1-yl] ethyl] thiazole (NRA0215) have a high affinity for dopamine D4 receptors, serotonin2A receptors, and the alpha1 adrenoceptor. 2-Carbamoyl-4-(4-fluorophenyl)-5-[2-[4-(3-fluorobenzylidene) piperidin-1-yl] ethyl] thiazole (NRA0160) has a selective and high affinity for dopamine D4 receptors. NRA0045 and clozapine (10 and 30 mg/kg, IP) produced significant increases in FLI in both the nucleus accumbens (N. Acc.) and the medial prefrontal cortex (mPFC) but not in the dorsolateral striatum (DLS). In contrast, NRA0160 and NRA0215 (10 and 30 mg/kg, IP) significantly increased FLI in the mPFC but not in the N. Acc. and the DLS. Haloperidol (0.1 and 1 mg/kg, IP) significantly produced FLI in the N. Acc., the DLS, and the mPFC. These data indicate that the antagonistic effects of dopamine D4 receptors may contribute, at least in part, to the actions of NRA0045, NRA0160, and NRA0215 in the mPFC.     

Functional selectivity in CB(2) cannabinoid receptor signaling and regulation: implications for the therapeutic potential of CB(2) ligands

Receptor internalization increases the flexibility and scope of G protein-coupled receptor (GPCR) signaling. CB(1) and CB(2) cannabinoid receptors undergo internalization after sustained exposure to agonists. However, it is not known whether different agonists internalize CB(2) to different extents. Because CB(2) is a promising therapeutic target, understanding its trafficking in response to different agonists is necessary for a complete understanding of its biology. Here we profile a number of cannabinoid receptor ligands and provide evidence for marked functional selectivity of cannabinoid receptor internalization. Classic, aminoalkylindole, bicyclic, cannabilactone, iminothiazole cannabinoid, and endocannabinoid ligands varied greatly in their effects on CB(1) and CB(2) trafficking. Our most striking finding was that (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo-[1,2,3-d,e]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone (WIN55,212-2) (and other aminoalkylindoles) failed to promote CB(2) receptor internalization, whereas 5-(1,1-dimethylheptyl)-2-(5-hydroxy-2-(3-hydroxypropyl)cyclohexyl)phenol (CP55,940) robustly internalized CB(2) receptors. Furthermore, WIN55,212-2 competitively antagonized CP55,940-induced CB(2) internalization. Despite these differences in internalization, both compounds activated CB(2) receptors as measured by extracellular signal-regulated kinase 1/2 phosphorylation and recruitment of β-arrestin(2) to the membrane. In contrast, whereas CP55,940 inhibited voltage-gated calcium channels via CB(2) receptor activation, WIN55,212-2 was ineffective on its own and antagonized the effects of CP55,940. On the basis of the differences we found between these two ligands, we also tested the effects of other cannabinoids on these signaling pathways and found additional evidence for functional selectivity of CB(2) ligands. These novel data highlight that WIN55,212-2 and other cannabinoids show strong functional selectivity at CB(2) receptors and suggest that different classes of CB(2) ligands may produce diverse physiological effects, emphasizing that each class needs to be separately evaluated for therapeutic efficacy.     

The cannabinoid receptor agonist WIN 55212-2 inhibits neurogenic inflammations in airway tissues

We examined the effects of a cannabinoid receptor agonist, (R)-(+)-[2,3-dihydro-5-methyl-3-[(4-merpholino)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthyl)methanone (WIN 55212-2), on various respiratory reactions induced by the activation of capsaicin-sensitive afferent sensory nerves (C-fibers). WIN 55212-2 significantly inhibited capsaicin-induced guinea pig bronchoconstriction, but not the neurokinin A-induced reaction. Intravenous injection of WIN 55212-2 also blocked cigarette smoke-induced rat tracheal plasma extravasation. However, substance P-induced rat tracheal plasma extravasation was not affected by the administration of WIN 55212-2. A cannabinoid CB(2) receptor antagonist, {N-[(1S)-endo-1,3,3-trimethylbicyclo[2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide} (SR 144528) reduced the inhibitory effects of WIN 55212-2, but not a cannabinoid CB(1) antagonist, [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride] (SR 141716A). A Maxi-K(+) channel opener, 1-(2'-hydroxy-5'-trifluoromethylphenyl)-5-trifluoromethyl-2(3H)benzimidazolone (NS 1619), specifically inhibited capsaicin-induced guinea pig bronchoconstriction and cigarette smoke-induced rat tracheal plasma extravasation. These findings suggest that WIN 55212-2 inhibits the activation of C-fibers via cannabinoid CB(2) receptors and Maxi-K(+) channels and reduces airway neurogenic inflammatory reactions in vivo.     

Cannabinoid penetration into mouse brain as determined by ex vivo binding.

We have used an ex vivo binding assay in the mouse to evaluate the brain penetration of cannabinoid receptor ligands. After intraperitoneal or oral administration, the pharmacological activity linked to the compound was assessed by using by [3H]WIN 55212-2 binding on cerebellar membranes. The brain penetration was high for compounds like methanandamide or delta9-tetrahydrocannabinol but poor for synthetic agonists such as (cis)-3-(2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-(trans)-4-(3-hydr oxypropyl)cyclohexanol (CP 55940) or, R-(+)-(2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrol[1,2,3-d e]-1,4-benzoxazin-6-yl)(1-napthalenyl)methanone monomethane-sulfonate (WIN 55212-2). After oral administration the duration of action of delta9-tetrahydrocannabinol, methanandamide and WIN 55212-2 is limited and decreased 4 h after administration. The cannabinoid CB1 receptor antagonist: N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-met hyl-1 H-pyrazole-3-carboxamide hydrochloride (SR141716A) exhibited a good brain penetration and a long duration of action.