Role of cannabinoid CB1 receptors and Gi/o protein activation in the modulation of synaptosomal Na+,K+-ATPase activity by WIN55,212-2 and ▵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 (▵ 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 μM) or ▵ 9 -THC (0.1 pM–0.1 μM), in a concentration-dependent manner, stimulated ouabain-sensitive Na + ,K + -ATPase activity. WIN55,212-2 was less potent but more efficacious than ▵ 9 -THC. N -(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H -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 ▵ 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 μM), an opioid receptor antagonist, did not significantly modify the effect of WIN55,212-2 and completely failed to modify the effect of ▵ 9 -THC on synaptosomal Na + ,K + -ATPase. Finally, pre-incubation with 0.5 μg 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 μg, 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 ▵ 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.