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 Ca2+ and membrane potentials were generated by establishing K+ gradients across the vesicle using the ionophore, valinomycin. Anandamide, in the range of 1-100 mu M, inhibited depolarization-induced efflux responses. Anandamide also functionally modulated the effects of nifedipine (1-10 mu M) and Bay K 8644 (1 mu M) on Ca2+ flux responses. Pretreatment with the specific cannabinoid receptor antagonist, SR141716A (1 mu M), pertussis toxin (5 mu g/ml), the amidohydrolase inhibitor, phenylmethylsulfonyl fluoride (0.2 mM) or the cyclooxygenase inhibitor, indomethacin (5 mu M) did not alter the inhibition of efflux responses by anandamide. Arachidonic acid (10-100 mu M) also effectively inhibited Ca-45(2+) efflux from membrane vesicles. in radioligand binding studies, it was found that both anandamide and arachidonic acid inhibited the specific binding of [H-3]PN 200-110 to transverse tubule membranes with IC50 values of 4.4 +/- 0.7 and 13.4 +/- 3,5 mu M, 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. All rights reserved.