Second messenger function of nicotinic acid adenine dinucleotide phosphate revealed by an improved enzymatic cycling assay.
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.
Gasser
Andreas
A
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.
Bruhn
Sören
S
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.
Guse
Andreas H
AH
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. Electronic address: guse@uke.uni-hamburg.de.
D001671:Biochemistry / Q000379:methods*; D017252:CD3 Complex / Q000096:biosynthesis; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D004305:Dose-Response Relationship, Drug; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D008954:Models, Biological; D009244:NAD+ Nucleosidase / Q000378:metabolism; D009249:NADP / Q000031:analogs & derivatives* / Q000737:chemistry / Q000502:physiology; D015290:Second Messenger Systems; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000201:enzymology*
D017252:CD3 Complex; D009249:NADP; C024376:NAADP; D009244:NAD+ Nucleosidase; D002118:Calcium