Second messenger function of nicotinic acid adenine dinucleotide phosphate revealed by an improved enzymatic cycling assay
Second messenger function of nicotinic acid adenine dinucleotide phosphate revealed by an improved enzymatic cycling assay
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DOI：
10.1074/jbc.m601347200
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发表时间：
2006-06-23
影响因子：
4.8
通讯作者：
Guse, Andreas H.
中科院分区：
文献类型：
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作者：
Gasser, Andreas;Bruhn, Soren;Guse, Andreas H.
烟酸腺嘌呤二核苷酸磷酸（NAADP）是目前已知的最有效的细胞内钙释放激活剂。虽然最近的报道表明NAADP在人T淋巴细胞中的重要功能，但在这些细胞中还缺少受体诱导的NAADP形成的直接证据。因此，我们开发了一种高灵敏度和特异性的酶测定，能够定量低fmol量的NAADP。在未刺激的T细胞中，NAADP浓度为4.4 +/- 1.6 nM（0.055 +/- 0.028 pmol/mg蛋白质）。通过T细胞受体/CD 3复合物刺激细胞导致细胞NAADP水平的双相升高动力学，其特征在于NAADP的钟形浓度-反应曲线。与此相反，NAADP浓度升高时，既不激活ADP-核糖/TRPM 2通道Ca 2+信号系统，也不增加细胞内Ca 2+浓度thapsigargin刺激。T细胞受体/CD 3复合物介导的NAADP形成依赖于酪氨酸激酶的活性，因为染料木素完全阻断NAADP升高。因此，我们提出了一种调节形成NAADP后，T细胞受体/CD 3复合物的特异性刺激，这表明在T细胞活化过程中，NAADP作为Ca 2+动员第二信使的功能。
Nicotinic acid adenine dinucleotide phosphate ( NAADP) is the most potent activator of Ca2+ release from intracellular stores known today. Although recent reports have suggested an important function of NAADP in human T lymphocytes, direct evidence for receptor-induced formation of NAADP is yet missing in these cells. Thus, we developed a highly sensitive and specific enzyme assay capable of quantifying low fmol amounts of NAADP. In unstimulated T cells, the NAADP concentration amounted to 4.4 +/- 1.6 nM (0.055 +/- 0.028 pmol/mg of protein). Stimulation of the cells via the T cell receptor/CD3 complex resulted in biphasic elevation kinetics of cellular NAADP levels and was characterized by a bell-shaped concentration-response curve for NAADP. In contrast, the NAADP concentration was elevated neither upon activation of the ADP-ribose/TRPM2 channel Ca2+ signaling system nor by an increase of the intracellular Ca2+ concentration upon thapsigargin stimulation. T cell receptor/CD3 complex-mediated NAADP formation was dependent on the activity of tyrosine kinases because genistein completely blocked NAADP elevation. Thus, we propose a regulated formation of NAADP upon specific stimulation of the T cell receptor/CD3 complex, suggesting a function of NAADP as a Ca2+ mobilizing second messenger during T cell activation.