Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA.
Eur J Neurosci. 2012 Feb;35(3):423-35. doi: 10.1111/j.1460-9568.2011.07957.x. Epub 2012 Jan 25.
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.
最近的证据支持β-烟酰胺腺嘌呤二核苷酸（β-NAD(+)）作为一种新型神经递质和神经调质在周围神经系统中的新兴作用 -β-NAD(+)在神经平滑肌制剂和肾上腺嗜铬细胞中以一种神经递质的特征方式释放。目前尚不清楚这是否适用于中枢神经系统。使用小室超滤液测定和高灵敏度高压液相色谱技术，我们证明高钾刺激大鼠前脑突触体可引起β-NAD(+)、三磷酸腺苷及其代谢物二磷酸腺苷（ADP）、一磷酸腺苷、腺苷、ADP-核糖（ADPR）和环 ADPR 的溢出。SNAP-25 用肉毒杆菌神经毒素 A 切割、N 型电压依赖性 Ca(2+)通道用 ω-芋螺毒素 GVIA 抑制以及突触小泡质子梯度用巴弗洛霉素 A1 抑制均可减轻高钾诱导的β-NAD(+)溢出，表明β-NAD(+)可能通过囊泡胞吐释放。Western 分析表明，CD38 是一种多功能蛋白，可代谢β-NAD(+)，存在于突触体膜和细胞质中。完整的突触体降解β-NAD(+)。1,N (6) -etheno-NAD，β-NAD(+)的荧光类似物，被突触体摄取，这种摄取被真实的β-NAD(+)抑制，但不是间隙连接蛋白 43 抑制剂 Gap 27 抑制。在皮质神经元中，β-NAD(+)的局部应用会导致快速的 Ca(2+)瞬变，可能是由于细胞外 Ca(2+)的流入。因此，大鼠脑突触体可以主动释放、降解和摄取β-NAD(+)，并且β-NAD(+)可以刺激突触后神经元，所有这些标准都是一种物质被认为是大脑中候选神经递质的必要条件。
