Activation of the CB1 cannabinoid receptor protects cultured mouse spinal neurons against excitotoxicity.

Significant advances are being made towards understanding the genetic basis for spinal neurodegenerative diseases, however, effective pharmacotherapy remains elusive. One of the primary theories underlying neuron vulnerability is susceptibility to excitotoxicity. We present for the first time evidence that the activation of the CB(1) cannabinoid receptor effectively modulates kainate toxicity in primary neuronal cultures prepared from mouse spinal cord. Addition of Delta(9)-tetrahydrocannabinol to the culture medium attenuated the toxicity produced by kainate. The CB(1) receptors were localized to spinal neurons and astrocytes. The neuroprotective effect was blocked with the CB(1) receptor antagonist, SR141716A, indicating a receptor-mediated effect.

Cannabinoid receptors can activate and inhibit G protein-coupled inwardly rectifying potassium channels in a xenopus oocyte expression system.

In this study, we focused on the pharmacological characterization of cannabinoid receptor coupling to G protein-gated inwardly rectifying potassium (GIRK) channels. Cannabinoids were tested on Xenopus laevis oocytes coexpressing the CB(1) receptor and GIRK1 and GIRK4 channels (CB(1)/GIRK1/4) or the CB(2) receptor and GIRK1/4 channels (CB(2)/GIRK1/4). WIN 55,212-2 enhanced currents carried by GIRK channels in the CB(1)/GIRK1/4 and CB(2)/GIRK1/4 system; however, the CB(2) receptor did not couple efficiently to GIRK1/4 channels. In the CB(1)/GIRK1/4 system, WIN 55,212-2 was the most efficacious compound tested. CP 55,940 and anandamide acted as partial agonists. The rank order of potency was CP 55,940 > WIN 55,212-2 = anandamide. The CB(1)-selective antagonist SR141716A alone acted as a inverse agonist by inhibiting GIRK currents in oocytes expressing CB(1)/GIRK1/4, suggesting the CB(1) receptor is constitutively activated. A conserved aspartate residue, which was previously shown to be critical for G protein coupling in cannabinoid receptors, was mutated (to asparagine, D163N) and analyzed. Oocytes coexpressing CB(1)/GIRK1/4 or D163N/GIRK1/4 were compared. The potency of WIN 55, 212-2 at the mutant receptor was similar to wild type, but its efficacy was substantially reduced. CP 55,940 did not elicit currents in oocytes expressing D163N/GIRK1/4. In summary, it appears the CB(1) and CB(2) receptors couple differently to GIRK1/4 channels. In the CB(1)/GIRK1/4 system, cannabinoids evaluated demonstrated the ability to enhance or inhibit GIRK currents. Furthermore, a conserved aspartate residue in the CB(1) receptor is required for normal communication with GIRK channels in oocytes demonstrating the interaction between receptor and channels is G protein dependent.

Evaluation of the cannabinoid CB2 receptor-selective antagonist, SR144528: further evidence for cannabinoid CB2 receptor absence in the rat central nervous system.

The aim of this study was to characterize the activity of the cannabinoid CB2 receptor selective 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) in a number of biochemical assays and to look for evidence of cannabinoid CB2 receptors in the rat central nervous system. SR144528 displaced [3H]CP 55,940 ((-)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)-phenyl]-4-[3-hydroxyprop yl]cyclohexan-1-ol) from binding sites in CB2- and CB1-transfected cells (Ki = 0.67+/-0.30 and 33.0+/-5.09 nM) and from rat cerebellum and whole brain membrane homogenates (Ki = 54.7+/-9.70 and 54.8+/-7.86 nM). In the GTPgammaS binding assay, SR144528 antagonized a number of cannabinoid receptor agonists (K(B) values ranging from 26.3 to 76.6 nM) in rat cerebellar membranes and in rat whole brain membranes (K(B) = 50.8 nM). SR144528 also antagonized CP 55,940-stimulated GTPgammaS binding in a CB2-expressing cell line (K(B) = 6.34 nM). In Xenopus oocytes co-expressing the CB1 receptor and G-protein coupled inwardly rectifying K+ channels (GIRK 1/4), SR144528 antagonized WIN 55212-2((R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrolo [1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone) -stimulated K+ currents (K(B) = 558 nM). In summary, this report characterizes the cannabinoid CB2 receptor-selective cannabinoid antagonist, SR144528, and additionally suggests an absence of cannabinoid CB2 receptors in the rat central nervous system, an observation confirmed by Northern blot.

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.