The effects of anandamide transport inhibitor AM404 on voltage-dependent calcium channels

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 3μM 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 10μM AM404, B_max values were 51±6 and 27±5pM/mg, and KD values were 236±43 and 220±37pM, 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 (1μM), 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 3.2μM. In conclusion, results indicate that AM404 inhibits directly the function of L-type voltage-dependent Ca²⁺ channels in mammalian skeletal muscles.