Release, neuronal effects and removal of extracellular β‐nicotinamide adenine dinucleotide (β‐NAD + ) in the rat brain

Recent evidence supports an emerging role of β‐nicotinamide adenine dinucleotide (β‐NAD + ) as a novel neurotransmitter and neuromodulator in the peripheral nervous system –β‐NAD + is released in nerve‐smooth muscle preparations and adrenal chromaffin cells in a manner characteristic of a neurotransmitter. It is currently unclear whether this holds true for the CNS. Using a small‐chamber superfusion assay and high‐sensitivity high‐pressure liquid chromatography techniques, we demonstrate that high‐K + stimulation of rat forebrain synaptosomes evokes overflow of β‐NAD + , adenosine 5′‐triphosphate, and their metabolites adenosine 5′‐diphosphate (ADP), adenosine 5′‐monophosphate, adenosine, ADP‐ribose (ADPR) and cyclic ADPR. The high‐K + ‐evoked overflow of β‐NAD + is attenuated by cleavage of SNAP‐25 with botulinum neurotoxin A, by inhibition of N‐type voltage‐dependent Ca 2+ channels with ω‐conotoxin GVIA, and by inhibition of the proton gradient of synaptic vesicles with bafilomycin A1, suggesting that β‐NAD + is likely released via vesicle exocytosis. Western analysis demonstrates that CD38, a multifunctional protein that metabolizes β‐NAD + , is present on synaptosomal membranes and in the cytosol. Intact synaptosomes degrade β‐NAD + . 1, N   6 ‐etheno‐NAD, a fluorescent analog of β‐NAD + , is taken by synaptosomes and this uptake is attenuated by authentic β‐NAD + , but not by the connexin 43 inhibitor Gap 27. In cortical neurons local applications of β‐NAD + cause rapid Ca 2+ transients, likely due to influx of extracellular Ca 2+ . Therefore, rat brain synaptosomes can actively release, degrade and uptake β‐NAD + , and β‐NAD + can stimulate postsynaptic neurons, all criteria needed for a substance to be considered a candidate neurotransmitter in the brain.