Smyth Lisa M, Bobalova Janette, Mendoza Michael G, Lew Christy, Mutafova-Yambolieva Violeta N
Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557-0271, USA.
J Biol Chem. 2004 Nov 19;279(47):48893-903. doi: 10.1074/jbc.M407266200. Epub 2004 Sep 13.
Chemical signaling in autonomic neuromuscular transmission involves agents that function as neurotransmitters and/or neuromodulators. Using high performance liquid chromatography techniques with fluorescence and electrochemical detection we observed that, in addition to ATP and norepinephrine (NE), electrical field stimulation (EFS, 4-16 Hz, 0.1-0.3 ms, 15 V, 60-120 s) of isolated vascular and non-vascular preparations co-releases a previously unidentified compound with apparent nucleotide or nucleoside structure. Extensive screening of more than 25 nucleotides and nucleosides followed by detailed peak identification revealed that beta-nicotinamide adenine dinucleotide (beta-NAD) is released in tissue superfusates upon EFS of canine mesenteric artery (CMA), canine urinary bladder, and murine urinary bladder in the amounts of 7.1 +/- 0.7, 26.5 +/- 4.5, and 15.1 +/- 3.2 fmol/mg of tissue, respectively. Smaller amounts of the beta-NAD metabolites cyclic adenosine 5'-diphosphoribose (cADPR) and ADPR were also present in the superfusates collected during EFS of CMA (2.5 +/- 0.9 and 5.8 +/- 0.8 fmol/mg of tissue, respectively), canine urinary bladder (1.8 +/- 0.5 and 9.0 +/- 6.0 fmol/mg of tissue, respectively), and murine urinary bladder (1.4 +/- 0.1 and 6.2 +/- 2.4 fmol/mg of tissue, respectively). The three nucleotides were also detected in the samples collected before EFS (0.2-1.6 fmol/mg of tissue). Exogenous beta-NAD, cADPR, and ADPR (all 100 nm) reduced the release of NE in CMA at 16 Hz from 27.8 +/- 6.0 fmol/mg of tissue to 15.5 +/- 5.0, 12 +/- 3.0, and 10.0 +/- 4.0 fmol/mg of tissue, respectively. In conclusion, we detected constitutive and nerve-evoked overflow of beta-NAD, cADPR, and ADPR in vascular and non-vascular smooth muscles, beta-NAD being the prevailing compound. These substances modulate the release of NE, implicating novel nucleotide mechanisms of autonomic nervous system control of smooth muscle.
自主神经肌肉传递中的化学信号传导涉及充当神经递质和/或神经调节剂的物质。使用具有荧光和电化学检测功能的高效液相色谱技术，我们观察到，除了三磷酸腺苷（ATP）和去甲肾上腺素（NE）之外，对分离的血管和非血管制剂进行电场刺激（EFS，4 - 16Hz，0.1 - 0.3ms，15V，60 - 120s）会共同释放一种先前未鉴定的具有明显核苷酸或核苷结构的化合物。对25种以上核苷酸和核苷进行广泛筛选，随后进行详细的峰鉴定，结果显示，在对犬肠系膜动脉（CMA）、犬膀胱和小鼠膀胱进行EFS后，组织灌流液中会释放β - 烟酰胺腺嘌呤二核苷酸（β - NAD），释放量分别为7.1±0.7、26.5±4.5和15.1±3.2 fmol/mg组织。在对CMA进行EFS期间收集的灌流液中，还存在少量的β - NAD代谢物环腺苷5'-二磷酸核糖（cADPR）和腺苷二磷酸核糖（ADPR）（分别为2.5±0.9和5.8±0.8 fmol/mg组织），犬膀胱（分别为1.8±0.5和9.0±6.0 fmol/mg组织）和小鼠膀胱（分别为1.4±0.1和6.2±2.4 fmol/mg组织）。在EFS之前收集的样品中也检测到了这三种核苷酸（0.2 - 1.6 fmol/mg组织）。外源性β - NAD、cADPR和ADPR（均为100 nM）使CMA中16Hz时NE的释放量从27.8±6.0 fmol/mg组织分别降至15.5±5.0、12±3.0和10.0±4.0 fmol/mg组织。总之，我们检测到血管和非血管平滑肌中β - NAD、cADPR和ADPR的组成性和神经诱发的溢出，β - NAD是主要的化合物。这些物质调节NE的释放，暗示了自主神经系统控制平滑肌的新核苷酸机制。
