Endogenous cannabinoid anandamide directly inhibits voltage-dependent Ca²⁺ fluxes in rabbit T-tubule membranes

The effect of the endogenous cannabinoid, anandamide on Ca²⁺ flux responses mediated by voltage-dependent Ca²⁺ channels was studied in transverse tubule membrane vesicles from rabbit skeletal muscle. Vesicles were loaded with ⁴⁵Ca²⁺ and membrane potentials were generated by establishing K⁺ gradients across the vesicle using the ionophore, valinomycin. Anandamide, in the range of 1-100 μM, inhibited depolarization- induced efflux responses. Anandamide also functionally modulated the effects of nifedipine (1-10 μM) and Bay K 8644 (1 μM) on Ca²⁺ flux responses. Pretreatment with the specific cannabinoid receptor antagonist, SR141716A (1 μM), pertussis toxin (5 μg/ml), the amidohydrolase inhibitor, phenylmethylsulfonyl fluoride (0.2 mM) or the cyclooxygenase inhibitor, indomethacin (5 μM) did not alter the inhibition of efflux responses by anandamide. Arachidonic acid (10-100 μM) also effectively inhibited ⁴⁵Ca²⁺ efflux from membrane vesicles. In radioligand binding studies, it was found that both anandamide and arachidonic acid inhibited the specific binding of [³H]PN 200-110 to transverse tubule membranes with IC₅₀ values of 4.4 ± 0.7 and 13.4 ± 3.5 μM, respectively. These results indicate that anandamide, independent of cannabinoid receptor activation, directly inhibits the function of voltage-dependent calcium channels and modulates the specific binding of calcium channel ligands of the dihydropyridine class. (C) 2000 Elsevier Science B.V.