title: Functional ryanodine receptor expression is required for NAADP-mediated local Ca2+ signaling in T-lymphocytes.
abstract: Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+-mobilizing nucleotide involved in T cell Ca2+ signaling (Berg, I., Potter, B. V. L., Mayr, G. W., and Guse, A. H. (2000) J. Cell Biol. 150, 581-588). The objective of this study was to analyze whether the first subcellular Ca2+ signals obtained upon NAADP stimulation of T-lymphocytes depend on the functional expression of ryanodine receptors. Using combined microinjection and high resolution confocal calcium imaging, we demonstrate here that subcellular Ca2+ signals, characterized by amplitudes between approximately 30 and 100 nM and diameters of approximately 0.5 microM, preceded global Ca2+ signals. Co-injection of the ryanodine receptor antagonists ruthenium red and ryanodine together with NAADP abolished the effects of NAADP, whereas the D-myo-inositol 1,4,5-trisphosphate antagonist heparin and the Ca2+ entry blocker SKF&96365 were without effect. This pharmacological approach was confirmed by a molecular knock-down approach. Jurkat T cell clones with largely reduced expression of ryanodine receptors did not respond to microinjections of NAADP. Taken together, our data suggest that the Ca2+ release channel sensitive to NAADP in T-lymphocytes is the ryanodine receptor.
authors.affiliation: University Hospital Hamburg-Eppendorf, Center of Experimental Medicine, Institute of Biochemistry and Molecular Biology I: Cellular Signal Transduction, Martinistrasse 52, 20246 Hamburg, Germany.
authors.forename: Werner
authors.initials: W
authors.lastname: Dammermann
authors.forename: Andreas H
authors.initials: AH
authors.lastname: Guse
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D036563:Cyclic ADP-Ribose / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D019169:Jurkat Cells; D008213:Lymphocyte Activation; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009249:NADP / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000502:physiology*; D012430:Ruthenium Red / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000096:biosynthesis* / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000378:metabolism*; D013997:Time Factors
chemical_list: D002121:Calcium Channel Blockers; D007093:Imidazoles; D019837:Ryanodine Receptor Calcium Release Channel; D012430:Ruthenium Red; D036563:Cyclic ADP-Ribose; D009249:NADP; C024376:NAADP; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D002118:Calcium