Anandamide transport inhibitor AM404 and structurally related compounds inhibit synaptic transmission between rat hippocampal neurons in culture independent of cannabinoid CB₁ receptors

N-(hydroxyphenyl)-arachidonamide (AM404) is an inhibitor of endocannabinoid transport. We examined the effects of AM404 on glutamatergic synaptic transmission using network-driven increases in intracellular Ca²⁺ concentration ([Ca²⁺] spikes) as an assay. At a concentration of 1 μM AM404 inhibited [Ca²⁺]_i spiking by 73±8%. The cannabinoid CB₁ receptor antagonist N-(piperidin-1-yl)-5-(4- chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A), the vanilloid VR₁ receptor antagonist capsazepine (CPZ), and treatment with pertussis toxin failed to block AM404-mediated inhibition. AM404 (3 μM) inhibited action-potential-evoked Ca²⁺ influx by 58±3% but failed to affect calcium influx evoked by depolarization with 30 mM K⁺, suggesting that the inhibition of electrically evoked [Ca²⁺]_i increases and that [Ca²⁺]_i spiking was due to inhibition of Na ⁺ channels. Palmitoylethanolamide (PMEA), capsaicin (CAP) and (5Z,8Z,11Z,14Z)-N-(4-hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide (VDM11), compounds structurally similar to AM404, inhibited [Ca ²⁺]_i spiking by 34±10%, 42±18% and 67±12%, respectively. Thus, AM404 and related compounds inhibit depolarization-induced Ca²⁺ influx independent of cannabinoid receptors, suggesting caution when using these agents as pharmacological probes to study synaptic transmission.