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(1/1038) Cannabinoid suppression of noxious heat-evoked activity in wide dynamic range neurons in the lumbar dorsal horn of the rat.

The effects of cannabinoid agonists on noxious heat-evoked firing of 62 spinal wide dynamic range (WDR) neurons were examined in urethan-anesthetized rats (1 cell/animal). Noxious thermal stimulation was applied with a Peltier device to the receptive fields in the ipsilateral hindpaw of isolated WDR neurons. To assess the site of action, cannabinoids were administered systemically in intact and spinally transected rats and intraventricularly. Both the aminoalkylindole cannabinoid WIN55,212-2 (125 microg/kg iv) and the bicyclic cannabinoid CP55,940 (125 microg/kg iv) suppressed noxious heat-evoked activity. Responses evoked by mild pressure in nonnociceptive neurons were not altered by CP55,940 (125 microg/kg iv), consistent with previous observations with another cannabinoid agonist, WIN55,212-2. The cannabinoid induced-suppression of noxious heat-evoked activity was blocked by pretreatment with SR141716A (1 mg/kg iv), a competitive antagonist for central cannabinoid CB1 receptors. By contrast, intravenous administration of either vehicle or the receptor-inactive enantiomer WIN55,212-3 (125 microg/kg) failed to alter noxious heat-evoked activity. The suppression of noxious heat-evoked activity induced by WIN55,212-2 in the lumbar dorsal horn of intact animals was markedly attenuated in spinal rats. Moreover, intraventricular administration of WIN55,212-2 suppressed noxious heat-evoked activity in spinal WDR neurons. By contrast, both vehicle and enantiomer were inactive. These findings suggest that cannabinoids selectively modulate the activity of nociceptive neurons in the spinal dorsal horn by actions at CB1 receptors. This modulation represents a suppression of pain neurotransmission because the inhibitory effects are selective for pain-sensitive neurons and are observed with different modalities of noxious stimulation. The data also provide converging lines of evidence for a role for descending antinociceptive mechanisms in cannabinoid modulation of spinal nociceptive processing.  (+info)

(2/1038) Role of a conserved lysine residue in the peripheral cannabinoid receptor (CB2): evidence for subtype specificity.

The human cannabinoid receptors, central cannabinoid receptor (CB1) and peripheral cannabinoid receptor (CB2), share only 44% amino acid identity overall, yet most ligands do not discriminate between receptor subtypes. Site-directed mutagenesis was employed as a means of mapping the ligand recognition site for the human CB2 cannabinoid receptor. A lysine residue in the third transmembrane domain of the CB2 receptor (K109), which is conserved between the CB1 and CB2 receptors, was mutated to alanine or arginine to determine the role of this charged amino acid in receptor function. The analogous mutation in the CB1 receptor (K192A) was found to be crucial for recognition of several cannabinoid compounds excluding (R)-(+)-[2, 3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1, 4-benzoxazin-6-yl](1-naphthalenyl)methanone (WIN 55,212-2). In contrast, in human embryonic kidney (HEK)-293 cells expressing the mutant or wild-type CB2 receptors, we found no significant differences in either the binding profile of several cannabinoid ligands nor in inhibition of cAMP accumulation. We identified a high-affinity site for (-)-3-[2-hydroxyl-4-(1, 1-dimethylheptyl)phenyl]-4-[3-hydroxyl propyl] cyclohexan-1-ol (CP-55,940) in the region of helices 3, 6, and 7, with S3.31(112), T3.35(116), and N7.49(295) in the K109A mutant using molecular modeling. The serine residue, unique to the CB2 receptor, was then mutated to glycine in the K109A mutant. This double mutant, K109AS112G, retains the ability to bind aminoalkylindoles but loses affinity for classical cannabinoids, as predicted by the molecular model. Distinct cellular localization of the mutant receptors observed with immunofluorescence also suggests differences in receptor function. In summary, we identified amino acid residues in the CB2 receptor that could lead to subtype specificity.  (+info)

(3/1038) Effect of the cannabinoid receptor agonist WIN55212-2 on sympathetic cardiovascular regulation.

1. The aim of the present study was to analyse the cardiovascular actions of the synthetic CB1/CB2 cannabinoid receptor agonist WIN55212-2, and specifically to determine its sites of action on sympathetic cardiovascular regulation. 2. Pithed rabbits in which the sympathetic outflow was continuously stimulated electrically or which received a pressor infusion of noradrenaline were used to study peripheral prejunctional and direct vascular effects, respectively. For studying effects on brain stem cardiovascular regulatory centres, drugs were administered into the cisterna cerebellomedullaris in conscious rabbits. Overall cardiovascular effects of the cannabinoid were studied in conscious rabbits with intravenous drug administration. 3. In pithed rabbits in which the sympathetic outflow was continuously electrically stimulated, intravenous injection of WIN55212-2 (5, 50 and 500 microg kg(-1)) markedly reduced blood pressure, the spillover of noradrenaline into plasma and the plasma noradrenaline concentration, and these effects were antagonized by the CB1 cannabinoid receptor-selective antagonist SR141716A. The hypotensive and the sympathoinhibitory effect of WIN55212-2 was shared by CP55940, another mixed CB1/CB2 cannabinoid receptor agonist, but not by WIN55212-3, the enantiomer of WIN55212-2, which lacks affinity for cannabinoid binding sites. WIN55212-2 had no effect on vascular tone established by infusion of noradrenaline in pithed rabbits. 4. Intracisternal application of WIN55212-2 (0.1, 1 and 10 microg kg(-1)) in conscious rabbits increased blood pressure and the plasma noradrenaline concentration and elicited bradycardia; this latter effect was antagonized by atropine. 5. In conscious animals, intravenous injection of WIN55212-2 (5 and 50 microg kg(-1)) caused bradycardia, slight hypotension, no change in the plasma noradrenaline concentration, and an increase in renal sympathetic nerve firing. The highest dose of WIN55212-2 (500 microg kg(-1)) elicited hypotension and tachycardia, and sympathetic nerve activity and the plasma noradrenaline concentration declined. 6. The results obtained in pithed rabbits indicate that activation of CB1 cannabinoid receptors leads to marked peripheral prejunctional inhibition of noradrenaline release from postganglionic sympathetic axons. Intracisternal application of WIN55212-2 uncovered two effects on brain stem cardiovascular centres: sympathoexcitation and activation of cardiac vagal fibres. The highest dose of systemically administered WIN55212-2 produced central sympathoinhibition; the primary site of this action is not known.  (+info)

(4/1038) Agonist-inverse agonist characterization at CB1 and CB2 cannabinoid receptors of L759633, L759656, and AM630.

We have tested our prediction that AM630 is a CB2 cannabinoid receptor ligand and also investigated whether L759633 and L759656, are CB2 receptor agonists. Binding assays with membranes from CHO cells stably transfected with human CB1 or CB2 receptors using [3H]-CP55940, confirmed the CB2-selectivity of L759633 and L759656 (CB2/CB1 affinity ratios = 163 and 414 respectively) and showed AM630 to have a Ki at CB2 receptors of 31.2 nM and a CB2/CB1 affinity ratio of 165. In CB2-transfected cells, L759633 and L759656 were potent inhibitors of forskolin-stimulated cyclic AMP production, with EC50 values of 8.1 and 3.1 nM respectively and CB1/CB2 EC50 ratios of > 1000 and > 3000 respectively. AM630 inhibited [35S]-GTPgammaS binding to CB2 receptor membranes (EC50 = 76.6 nM), enhanced forskolin-stimulated cyclic AMP production in CB2-transfected cells (5.2 fold by 1 microM), and antagonized the inhibition of forskolin-stimulated cyclic AMP production in this cell line induced by CP55940. In CB1-transfected cells, forskolin-stimulated cyclic AMP production was significantly inhibited by AM630 (22.6% at 1 microM and 45.9% at 10 microM) and by L759633 at 10 microM (48%) but not 1 microM. L759656 (10 microM) was not inhibitory. AM630 also produced a slight decrease in the mean inhibitory effect of CP55940 on cyclic AMP production which was not statistically significant. We conclude that AM630 is a CB2-selective ligand that behaves as an inverse agonist at CB2 receptors and as a weak partial agonist at CB1 receptors. L759633 and L759656 are both potent CB2-selective agonists.  (+info)

(5/1038) Cannabinoids and neuroprotection in global and focal cerebral ischemia and in neuronal cultures.

Marijuana and related drugs (cannabinoids) have been proposed as treatments for a widening spectrum of medical disorders. R(+)-[2, 3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1, 4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate (R(+)-WIN 55212-2), a synthetic cannabinoid agonist, decreased hippocampal neuronal loss after transient global cerebral ischemia and reduced infarct volume after permanent focal cerebral ischemia induced by middle cerebral artery occlusion in rats. The less active enantiomer S(-)-WIN 55212-3 was ineffective, and the protective effect of R(+)-WIN 55212-2 was blocked by the specific central cannabinoid (CB1) cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide-hydrochloride. R(+)-WIN 55212-2 also protected cultured cerebral cortical neurons from in vitro hypoxia and glucose deprivation, but in contrast to the receptor-mediated neuroprotection observed in vivo, this in vitro effect was not stereoselective and was insensitive to CB1 and CB2 receptor antagonists. Cannabinoids may have therapeutic potential in disorders resulting from cerebral ischemia, including stroke, and may protect neurons from injury through a variety of mechanisms.  (+info)

(6/1038) Involvement of CB1 cannabinoid receptors in the EDHF-dependent vasorelaxation in rabbits.

1. It was recently suggested that an endogenous cannabinoid could represent an endothelium-derived hyperpolarizing factor (EDHF). The aim of the present study was to clarify whether CB1 cannabinoid receptors are involved in the nitric oxide (NO)- and prostanoid-independent vasodilation produced by acetylcholine in rabbits. 2. Pithed rabbits received indomethacin. Noradrenaline was infused to raise blood pressure, and vasodilation was elicited by bolus injections of acetylcholine. The NO-synthase inhibitor Nomega-nitro-L-arginine methylester inhibited the acetylcholine-evoked vasodilation by about 40%. The remaining vasodilation was unaffected by the CB1 cannabinoid receptor antagonist SR141716A, but was inhibited by the potassium channel blocker tetraethylammonium. In addition, the mixed CB1/CB2 cannabinoid receptor agonist WIN55212-2 did not elicit vasodilation. 3. No CB1 cannabinoid receptors were involved in the prostanoid- and NO-independent vasodilation produced by acetylcholine. An exogenous cannabinoid also did not cause vasodilation. Therefore, it is unlikely that an endogenous cannabinoid serves as an EDHF acting at smooth muscle CB1 cannabinoid receptors in the rabbit.  (+info)

(7/1038) Distinct domains of the CB1 cannabinoid receptor mediate desensitization and internalization.

Desensitization of cannabinoid receptor signaling by a G-protein coupled receptor kinase (GRK) was examined using the Xenopus oocyte expression system. Application of a CB1 agonist, WIN 55,212-2, evoked a concentration-dependent increase in K+ conductance (Kir3) in oocytes coexpressing rat CB1 with the G-protein-gated, inwardly rectifying K+ channels Kir3.1 and Kir3.4. Desensitization was slight during continuous agonist application in the absence of GRK and arrestin. However, coexpression of GRK3 and beta-arrestin 2 (beta-arr2) caused profound homologous CB1 receptor desensitization, supporting the hypothesis that GRK3 and beta-arr2 effectively produce CB1 receptor desensitization. To identify the regions of the CB1 receptor responsible for GRK3- and beta-arr2-mediated desensitization, we constructed several CB1 receptor mutants. Truncation of the C-terminal tail of CB1 receptor at residue 418 (Delta418) almost completely abolished desensitization but did not affect agonist activation of Kir3. In contrast, truncation at residues 439 and 460 did not significantly affect GRK3- and beta-arr2-dependent desensitization. A deletion mutant (Delta418-439) did not desensitize, indicating that residues within this region are important for GRK3- and beta-arr2-mediated desensitization. Phosphorylation in this region was likely involved in desensitization, because mutation of either of two putative phosphorylation sites (S426A or S430A) significantly attenuated desensitization. CB1 receptors rapidly internalize after activation by agonist. Phosphorylation of S426 or S430 was not necessary for internalization, because the S426A/S430A CB1 mutant internalized when stably expressed in AtT20 cells. These studies establish that CB1 desensitization can be regulated by a GRK and that different receptor domains are involved in GRK- and beta-arrestin-dependent desensitization and CB1 internalization.  (+info)

(8/1038) Cannabinoid CB1 receptor of cat cerebral arterial muscle functions to inhibit L-type Ca2+ channel current.

The CB1 subtype of the cannabinoid receptor is present on neurons in the brain and mediates the perceptual effects of Delta9-tetrahydrocannabinol and other cannabinoids. We found that cat cerebral arterial smooth muscle cells (VSMC) contain the protein for the CB1 receptor and express a cDNA that has >98% amino acid homology to the CB1 cDNA expressed in rat and human neurons. Activation of the CB1 cannabinoid receptor has been shown to decrease the opening of N-type voltage-gated Ca2+ channels in neurons through a pertussis toxin-sensitive GTP-binding protein. In the present study we tested the hypothesis that activation of the cannabinoid CB1 receptor in cerebral VSMC inhibits voltage-gated Ca2+ channels and results in cerebral vasodilation. The predominant Ca2+ current identified in cat cerebral VSMC is a voltage-gated, dihydropyridine-sensitive, L-type Ca2+ current. The cannabimimetic drug WIN-55,212-2 (10-100 nM) induced concentration-dependent inhibition of peak L-type Ca2+ current, which reached a maximum of 82 +/- 4% at 100 nM (n = 14). This effect was mimicked by the putative endogenous CB1-receptor agonist anandamide, which produced a concentration-related reduction of peak L-type Ca2+ current with a maximum inhibition (at 300 nM) of 39 +/- 4% (n = 12). The inhibitory effects of both ligands on peak L-type Ca2+ currents were abolished by pertussis toxin pretreatment and application of the CB1-receptor antagonist SR-141716A (100 nM, n = 5). Both WIN-55,212-2 and anandamide produced concentration-dependent relaxation of preconstricted cerebral arterial segments that was abolished by SR-141716A. These results indicate that the CB1 receptor is expressed in cat cerebral VSMC and that the cerebral vasculature is one of the targets for endogenous cannabinoids. These findings suggest that the CB1 receptor and its endogenous ligand may play a fundamental role in the regulation of cerebral arterial tone and reactivity by modulating the influx of Ca2+ through L-type Ca2+ channels.  (+info)