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

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 Gi/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-1H-pyrazole-3-carboxamide (AM-251) (10 nM), a CB1 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 Emax of cannabinoid-induced increase in Na+,K+-ATPase activity, but did not significantly modify their EC50. 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 (Gi/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 Emax of WIN55,212-2 by 70% but did not significantly affect its EC50. These results suggest that WIN55212-2 and ▵9-THC indirectly enhance Na+,K+-ATPase activity in the brain by activating cannabinoid CB1 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 Gi/o proteins.