title: Release, neuronal effects and removal of extracellular β-nicotinamide adenine dinucleotide (β-NAD⁺) in the rat brain.
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.
authors.affiliation: Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA.
authors.forename: Leonie
authors.initials: L
authors.lastname: Durnin
authors.forename: Yanping
authors.initials: Y
authors.lastname: Dai
authors.forename: Isamu
authors.initials: I
authors.lastname: Aiba
authors.forename: C William
authors.initials: CW
authors.lastname: Shuttleworth
authors.forename: Ilia A
authors.initials: IA
authors.lastname: Yamboliev
authors.forename: Violeta N
authors.initials: VN
authors.lastname: Mutafova-Yambolieva
mesh_terms: D000818:Animals; D001921:Brain / Q000166:cytology / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D005260:Female; D008297:Male; D009243:NAD / Q000031:analogs & derivatives* / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000378:metabolism; D011247:Pregnancy; D011687:Purines / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D013574:Synaptosomes / Q000378:metabolism
chemical_list: D018377:Neurotransmitter Agents; D011687:Purines; D009243:NAD; D002118:Calcium