The effects of anandamide transport inhibitor AM404 were investigated on depolarization-induced 45Ca2+ fluxes in transverse tubule membrane vesicles from rabbit skeletal muscle and on Ba2+ currents through L-type voltage-dependent Ca2+ channels in rat myotubes. AM404, at the concentration of 3 microM and higher, caused a significant inhibition of 45Ca2+ fluxes. Radioligand binding studies indicated that the specific binding of [3H]Isradipine to transverse tubule membranes was also inhibited significantly by AM404. In controls and in presence of 10 microM AM404, B(max) values were 51+/-6 and 27+/-5 pM/mg, and KD values were 236+/-43 and 220+/-37 pM, respectively. Inhibitory effects of AEA and arachidonic acid on 45Ca2+ flux and [3H]Isradipine binding reported in earlier studies, were also enhanced significantly in the presence of AM404. In the presence of VDM11 (1 microM), another anandamide transport inhibitor, AM404 continued to inhibit 45Ca2+ fluxes and [3H]Isradipine binding. In rat myotubes, Ca2+ currents through L-type Ca2+ channels recorded in whole-cell configuration of patch clamp technique were inhibited by AM404 in a concentration-dependent manner with an IC50 value of 3.2 microM. In conclusion, results indicate that AM404 inhibits directly the function of L-type voltage-dependent Ca2+ channels in mammalian skeletal muscles.
Published by Elsevier B.V.