Synaptic action of anandamide and related substances in mammalian brain

Anandamide and the synthetic cannabimimetic drugs AM 404 and WIN 55,212-2 were found to inhibit the binding of [3H]batrachotoxinin A 20--benzoate (BTX) to voltage-gated sodium channels (VGSCs) and also to depress VGSC-dependent release of GABA and L-glutamic acid. These effects occur independently of CB-1 receptor activation since they were not attenuated by AM251 at concentrations known to antagonize CB-1 receptors, although at higher concentrations AM251 inhibited VGSCs also. These results suggest that anandamide and endocannabimimetics have the ability to depress synaptic transmission by reducing the capacity of VGSCs to support action potentials. In other experiments, I observed that anandamide is synthesized by both resting and depolarized synaptoneurosomes, however, data from filtration and superfusion experiments failed to support the hypothesis that depolarization activates the release of anandamide from these synaptic preparations, even though substantial quantities of anandamide were clearly present in the intracellular compartments. In these investigations, I unexpectedly found that [3H]ethanolamine (a potential precursor of anandamide) is accumulated by synaptosomes and synaptoneurosomes during extended incubation and released (as [3H]ethanolamine) in a calcium-dependent fashion during a depolarizing challenge with 30 mM KCl. Ethanolamine also stimulates the release of the pH fluoroprobe acridine orange (AO) from synaptosomes and decreases AO release when synaptosomes are depolarized with KCl. However, ethanolamine and other amino alcohols (methylethanolamine and dimethylethanolamine) stimulate resting and KCl-evoked release of [3H]-D-aspartate from synaptosomes. All amino alcohols rapidly access synaptic vesicles where they sequester protons, thus accounting for AO efflux. Proton sequestration by amino alcohols increases the ATP-dependent transvesicular membrane potential which in turn enhances D-aspartate uptake into synaptic vesicles. My data suggest a potential role for ethanolamine and related amino alcohols in the regulation of synaptic vesicle filling and release of amino acid neurotransmitters. Depolarization evoked release of ethanolamine from synaptosomes supports the idea that this amino alcohol may also have an important postsynaptic role.