Yamboliev Ilia A, Smyth Lisa M, Durnin Leonie, Dai Yanping, Mutafova-Yambolieva Violeta N
Department of Pharmacology, University of Nevada School of Medicine, Reno, NV 89557, USA.
Eur J Neurosci. 2009 Sep;30(5):756-68. doi: 10.1111/j.1460-9568.2009.06869.x. Epub 2009 Aug 27.
In nerve-smooth muscle preparations beta-nicotinamide adenine dinucleotide (beta-NAD) has emerged as a novel extracellular substance with putative neurotransmitter and neuromodulator functions. beta-NAD is released, along with noradrenaline and adenosine 5'-triphosphate (ATP), upon firing of action potentials in blood vessels, urinary bladder and large intestine. At present it is unclear whether noradrenaline, ATP and beta-NAD are stored in and released from common populations of synaptic vesicles. The answer is unattainable in complex systems such as nerve-smooth muscle preparations. Adrenal chromaffin cells are thus used here as a single-cell model to examine mechanisms of concomitant neurosecretion. Using high-performance liquid chromatography techniques with electrochemical and fluorescence detection we simultaneously evaluated secretion of dopamine (DA), ATP, adenosine 5'-diphosphate, adenosine 5'-monophosphate, adenosine, beta-NAD and its immediate metabolites ADP-ribose and cyclic ADP-ribose in superfused nerve growth factor-differentiated rat pheochromocytoma PC12 cells. beta-NAD, DA and ATP were released constitutively and upon stimulation with high-K(+) solution or nicotine. Botulinum neurotoxin A tended to increase the spontaneous secretion of all substances and abolished the high-K(+)-evoked release of beta-NAD and DA but not of ATP. Subcellular fractionation by continuous glycerol and sucrose gradients along with immunoblot analysis of the vesicular marker proteins synaptophysin and secretogranin II revealed that beta-NAD, ATP and DA are stored in both small synaptic-like vesicles and large dense-core-like vesicles. However, the three substances appear to have different preferential sites of release upon membrane depolarization including sites associated with SNAP-25 and sites not associated with SNAP-25.
在神经-平滑肌制剂中，β-烟酰胺腺嘌呤二核苷酸（β-NAD）已成为一种具有假定神经递质和神经调节剂功能的新型细胞外物质。在血管、膀胱和大肠中，动作电位发放时，β-NAD与去甲肾上腺素和5'-三磷酸腺苷（ATP）一起释放。目前尚不清楚去甲肾上腺素、ATP和β-NAD是否储存在共同的突触小泡群体中并从中释放。在神经-平滑肌制剂等复杂系统中无法得到答案。因此，这里使用肾上腺嗜铬细胞作为单细胞模型来研究伴随神经分泌的机制。我们使用带有电化学和荧光检测的高效液相色谱技术，同时评估了在经神经生长因子分化的大鼠嗜铬细胞瘤PC12细胞中，多巴胺（DA）、ATP、5'-二磷酸腺苷、5'-单磷酸腺苷、腺苷、β-NAD及其直接代谢产物ADP-核糖和环ADP-核糖的分泌情况。β-NAD、DA和ATP在基础状态下以及用高钾溶液或尼古丁刺激时都会释放。肉毒杆菌神经毒素A倾向于增加所有物质的自发分泌，并消除高钾诱发的β-NAD和DA的释放，但不影响ATP的释放。通过连续甘油和蔗糖梯度进行亚细胞分级分离，以及对囊泡标记蛋白突触素和分泌粒蛋白II进行免疫印迹分析，结果显示β-NAD、ATP和DA既储存在小的突触样囊泡中，也储存在大的致密核心样囊泡中。然而，这三种物质在膜去极化时似乎有不同的优先释放位点，包括与SNAP-25相关的位点和与SNAP-25不相关的位点。
