The Endogenous Cannabinoid Anandamide Inhibits alpha_7 Nicotinic Acetylcholine Receptor-Mediated Responses in Xenopus Oocytes

摘要

The effect of the endogenous cannabinoid ligand anandamide on the function of the cloned alpha_7 subunit of the nicotinic acetylcholine (Ach) receptor expressed in Xenopus oocytes was investigated by using the two-electrode voltage-clamp technique. Anandamide reversibly inhibited nicotine (10 muM) induced-currents in a concentration-dependent manner (10 nM to 30 muM), with an IC_50 value of 229.7+-20.4 nM. The effect of anandamide was neither dependent on the membrane potential nor meditated by endogenous Ca~2+ dependent Cl~_ channels since it was unaffected by intracellularly injected BAPTA and perfusion with Ca~2+- free bathing solution containing 2 mM Ba~2+. Anandamide decreased the maximal nicotine-induced responses without significantly affecting its potency, indicating that it acts as a noncompetitive antagonist on nicotinic acetylcholine (nACh) alpha_7 receptors. This effect was not mediated by CB_1 or CB_2 receptors, as neither the selective CB_1 receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorphenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR 141716A) nor CB_2 receptor antagonist N-((IS)-endo-1,3,3-trimethyl-bicyclo-heptan-2-yl)-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR 144528) reduced the inhibition by anandamide. In addition, inhibition of nicotinic responses by anandamide was not sensitive to either pertussis toxin treatment or to the membrane permeable camp analog 8-Br-Camp(0.2 mM). Inhibitors of enzymes involved in anandamide metabolism including phenylmethylsulfonyl fluoride, superoxide dismutase, and indomethacin, or the anandamide transport inhibitor AM404 did not prevent anandamide inhibition of nicotinic responses, suggesting that anandamide itself acted on nicotinic receptors In conclusion, these results demonstrate that the endogenous cannabinoid anandamide inhibits the function of nACH alpha_7 receptors expressed in Xenopus oocytes in a cannabinoid receptor-independent and noncompetitive manner.