The European Journal Of Neuroscience
Recent evidence supports an emerging role of -nicotinamide adenine dinucleotide (-NAD(+) ) as a novel neurotransmitter and neuromodulator in the peripheral nervous system --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 -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 -NAD(+) is attenuated by cleavage of SNAP-25 with botulinum neurotoxin A, by inhibition of N-type voltage-dependent Ca(2+) channels with -conotoxin GVIA, and by inhibition of the proton gradient of synaptic vesicles with bafilomycin A1, suggesting that -NAD(+) is likely released via vesicle exocytosis. Western analysis demonstrates that CD38, a multifunctional protein that metabolizes -NAD(+) , is present on synaptosomal membranes and in the cytosol. Intact synaptosomes degrade -NAD(+) . 1,N(6) -etheno-NAD, a fluorescent analog of -NAD(+) , is taken by synaptosomes and this uptake is attenuated by authentic -NAD(+) , but not by the connexin 43 inhibitor Gap 27. In cortical neurons local applications of -NAD(+) cause rapid Ca(2+) transients, likely due to influx of extracellular Ca(2+) . Therefore, rat brain synaptosomes can actively release, degrade and uptake -NAD(+) , and -NAD(+) can stimulate postsynaptic neurons, all criteria needed for a substance to be considered a candidate neurotransmitter in the brain.