European Journal of Neuroscience 2012-02-01

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

Leonie Durnin, Yanping Dai, Isamu Aiba, C William Shuttleworth, Ilia A Yamboliev, Violeta N Mutafova-Yambolieva

Index: Eur. J. Neurosci. 35 , 423-35, (2012)

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Abstract

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.© 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.