title: Role of cannabinoid CB1 receptors and Gi/o protein activation in the modulation of synaptosomal Na+,K+-ATPase activity by WIN55,212-2 and delta(9)-THC.
abstract: In the present study, we evaluated the effects of the synthetic cannabinoid receptor agonist (R)-(+)-[2,3-Dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN55,212-2) and the active component of Cannabis delta-9-tetrahydrocannabinol (triangle up(9)-THC) on Na(+),K(+)-ATPase activity in synaptosomal mice brain preparation. Additionally, the potential exogenous cannabinoids and endogenous opioid peptides interaction as well as the role of G(i/o) proteins in mediating Na(+),K(+)-ATPase activation were also explored. The ouabain-sensitive Na(+),K(+)-ATPase activity was measured in whole-brain pure intact synaptosomes (obtained by Percoll gradient method) of female CF-1 mice and was calculated as the difference between the total and the ouabain (1 mM)-insensitive Na(+),K(+)-ATPase activities. Incubation in vitro of the synaptosomes with WIN55,212-2 (0.1 pM-10 microM) or triangle up(9)-THC (0.1 pM-0.1 microM), in a concentration-dependent manner, stimulated ouabain-sensitive Na(+),K(+)-ATPase activity. WIN55,212-2 was less potent but more efficacious than triangle up(9)-THC. N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM-251) (10 nM), a CB(1) cannabinoid receptor selective antagonist, had not effect per se but antagonized the enhancement of Na(+),K(+)-ATPase activity induced by both, WIN55,212-2 and triangle up(9)-THC. AM-251 produced a significant reduction in the E(max) of cannabinoid-induced increase in Na(+),K(+)-ATPase activity, but did not significantly modify their EC(50). On the other hand, co-incubation with naloxone (1 microM), an opioid receptor antagonist, did not significantly modify the effect of WIN55,212-2 and completely failed to modify the effect of triangle up(9)-THC on synaptosomal Na(+),K(+)-ATPase. Finally, pre-incubation with 0.5 microg of pertussis toxin (G(i/o) protein blocker) completely abolished the enhancement of ouabain-sensitive Na(+),K(+)-ATPase activity induced by WIN55,212-2. A lower dose, 0.25 microg, decreased the E(max) of WIN55,212-2 by 70% but did not significantly affect its EC(50). These results suggest that WIN55212-2 and triangle up(9)-THC indirectly enhance Na(+),K(+)-ATPase activity in the brain by activating cannabinoid CB(1) receptors in a naloxone-insensitive manner. In addition, the effect of WIN55,212-2 on neuronal Na(+),K(+)-ATPase is apparently due to activation of G(i/o) proteins.
authors.affiliation: Department of Pharmacy (Division of Molecular Pharmacology), Faculty of Chemistry, Pontificia Universidad Católica de Chile, Casilla 306, Santiago, 6094411, Chile.
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Araya
authors.forename: C
authors.initials: C
authors.lastname: David Pessoa Mahana
authors.forename: Luis G
authors.initials: LG
authors.lastname: González
mesh_terms: D000818:Animals; D048588:Benzoxazines / Q000494:pharmacology*; D001921:Brain / Q000187:drug effects* / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D013759:Dronabinol / Q000494:pharmacology*; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000819:agonists*; D066298:In Vitro Techniques; D051379:Mice; D009025:Morpholines / Q000494:pharmacology*; D009270:Naloxone / Q000494:pharmacology; D009281:Naphthalenes / Q000494:pharmacology*; D009292:Narcotic Antagonists / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D037342:Pertussis Toxin / Q000008:administration & dosage / Q000494:pharmacology; D010880:Piperidines / Q000494:pharmacology; D011720:Pyrazoles / Q000494:pharmacology; D043884:Receptor, Cannabinoid, CB1 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000502:physiology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013574:Synaptosomes / Q000187:drug effects / Q000201:enzymology
chemical_list: D048588:Benzoxazines; D004791:Enzyme Inhibitors; D009025:Morpholines; D009281:Naphthalenes; D009292:Narcotic Antagonists; D010880:Piperidines; D011720:Pyrazoles; D043884:Receptor, Cannabinoid, CB1; D009270:Naloxone; C103505:AM 251; D010042:Ouabain; C070417:(3R)-((2,3-dihydro-5-methyl-3-((4-morpholinyl)methyl)pyrrolo-(1,2,3-de)-1,4-benzoxazin-6-yl)(1-naphthalenyl))methanone; D013759:Dronabinol; D037342:Pertussis Toxin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000254:Sodium-Potassium-Exchanging ATPase