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CELLULAR AND MOLECULAR

The Endogenous Cannabinoid Anandamide Inhibits Cromakalim-Activated K⁺ Currents in Follicle-Enclosed Xenopus Oocytes

Intramural Research Program, Integrative Neuroscience Section, Department of Health and Human Services, National Institute on Drug Abuse, National Institutes of Health, Baltimore, Maryland (M.O., T.S.S.); Laboratory of Neural Control, Section on Developmental Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland (K.-H.Y.); and Department of Pulmonary Diseases, Oncology Training and Research Hospital, Ankara, Turkey (M.D.)

The effect of the endogenous cannabinoid anandamide on K⁺ currents activated by the ATP-sensitive potassium (K_ATP) channel opener cromakalim was investigated in follicle-enclosed Xenopus oocytes using the two-electrode voltage-clamp technique. Anandamide (1–90 µM) reversibly inhibited cromakalim-induced K⁺ currents, with an IC₅₀ value of 8.1 ± 2 µM. Inhibition was noncompetitive and independent of membrane potential. Coapplication of anandamide with the cannabinoid type 1 (CB₁) receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR 141716A) (1 µM), the CB₂ receptor antagonist N-[(1S)endo-1,3,3-trimethyl bicyclo heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528) (1 µM), or pertussis toxin (5 µg/ml) did not alter the inhibitory effect of anandamide, suggesting that known cannabinoid receptors are not involved in anandamide inhibition of K⁺ currents. Similarly, neither the amidohydrolase inhibitor phenylmethylsulfonyl fluoride (0.2 mM) nor the cyclooxygenase inhibitor indomethacin (5 µM) affected anandamide inhibition of K⁺ currents, suggesting that the effects of anandamide are not mediated by its metabolic products. In radioligand binding studies, anandamide inhibited the specific binding of the K_ATP ligand [³H]glibenclamide in the oocyte microsomal fractions, with an IC₅₀ value of 6.3 ± 0.4 µM. Gonadotropin-induced oocyte maturation and the cromakalim-acceleration of progesterone-induced oocyte maturation were significantly inhibited in the presence of 10 µM anandamide. Collectively, these results indicate that cromakalim-activated K⁺ currents in follicular cells of Xenopus oocytes are modulated by anandamide via a cannabinoid receptor-independent mechanism and that the inhibition of these channels by anandamide alters the responsiveness of oocytes to gonadotropin and progesterone.

Address correspondence to: Dr. Murat Oz, National Institute on Drug Abuse/Intramural Research Program Integrative Neuroscience Section, 333 Cassell Dr., Baltimore, MD 21224. E-mail: moz{at}intra.nida.nih.gov