Gasser Andreas, Bruhn Sören, Guse Andreas H
Calcium Signalling Group, Institute of Biochemistry and Molecular Biology I: Cellular Signal Transduction, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, D-20246 Hamburg, Germany.
Calcium Signalling Group, Institute of Biochemistry and Molecular Biology I: Cellular Signal Transduction, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, D-20246 Hamburg, Germany.
J Biol Chem. 2006 Jun 23;281(25):16906-16913. doi: 10.1074/jbc.M601347200. Epub 2006 Apr 20.
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.
烟酰胺腺嘌呤二核苷酸磷酸（NAADP）是目前已知的从细胞内储存库释放钙离子的最有效激活剂。尽管最近的报告表明NAADP在人T淋巴细胞中具有重要功能，但在这些细胞中，受体诱导形成NAADP的直接证据仍然缺失。因此，我们开发了一种高度灵敏且特异的酶测定法，能够定量低至飞摩尔量的NAADP。在未受刺激的T细胞中，NAADP浓度为4.4±1.6纳摩尔（0.055±0.028皮摩尔/毫克蛋白质）。通过T细胞受体/CD3复合物刺激细胞，导致细胞内NAADP水平呈现双相升高动力学，其特征为NAADP的钟形浓度-反应曲线。相比之下，激活ADP-核糖/TRPM2通道钙离子信号系统或在毒胡萝卜素刺激后细胞内钙离子浓度升高时，NAADP浓度均未升高。T细胞受体/CD3复合物介导的NAADP形成依赖于酪氨酸激酶的活性，因为染料木黄酮完全阻断了NAADP的升高。因此，我们提出在特异性刺激T细胞受体/CD3复合物后，NAADP的形成受到调控，这表明NAADP在T细胞激活过程中作为一种钙离子动员的第二信使发挥作用。
