Introduction Subcellular Ca 2+ signals are the fundamental pacemaking events preceding global Ca 2+ waves and/or Ca 2+ oscillations. These global Ca 2+ signals are then converted into cellular responses such as cell proliferation, differentiation, fertilization, exocytosis and muscle contraction (reviewed by Guse 2000; da Silva and Guse, 2000; Galione and Churchill, 2002; Guse, 2002; Berridge et al., 2003). The different phases of the development of the cellular Ca 2+ signal were described as (1) the basal phase, (2) the pacemaker phase, in which subcellular Ca 2+ signals were observed, and (3) the global signal (Bootman et al., 1997a). Subcellular Ca 2+ signals during the pacemaker phase were analyzed in several non-excitable and excitable cell types (Thorn et al., 1993; Tsugorka et al., 1995; Yao et al., 1995; Lipp and Niggli, 1996; Klein et al., 1996; Thomas et al., 2000; Tovey et al., 2001). The molecular mechanisms underlying subcellular Ca 2+ signals were analyzed by stimulation of cells with agonists that are thought exclusively, or at least mainly, to stimulate one of the intracellular systems for Ca 2+ mobilization; for example, histamine to stimulate the D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ]/Ca 2+ -signaling pathway (Bootman et al., 1997b) or caffeine to stimulate exclusively the ryanodine receptor (RyR) (Conklin et al., 1999; Rossi et al., 2002). However, the assumption that exclusive stimulation of one of the Ca 2+ - mobilizing systems is experimentally possible is problematic for two reasons. First, in a growing number of cell types, more than one system is functionally expressed (reviewed by da Silva and Guse, 2000; Yusufi et al., 2002). Second, the D-myo- Ins(1,4,5)P3 receptor (D-myo-Ins(1,4,5)P 3 R) and the RyR might respond to [Ca 2+ ] elevations by Ca 2+ -induced Ca 2+ release (CICR) and thus could be recruited even if the primary effect was targeted to one of the other systems for Ca 2+ mobilization (reviewed by Galione and Churchill, 2002). Recently, we generated and thoroughly characterized a type 3 RyR-knockdown Jurkat T-cell clone (Schwarzmann et al., 2002). In this clone, RyR expression was substantially decreased whereas Ins(1,4,5)P3 R expression remained unchanged. At both the cell-population level and the single- cell level, reduced Ca 2+ signaling was observed during the 2141 Ligation of the T-cell receptor/CD3 complex results in global Ca 2+ signals that are essential for T-cell activation. We have recently reported that these global Ca 2+ signals are preceded by localized pacemaker Ca 2+ signals. Here, we demonstrate for the first time for human T cells that an increase in signal frequency of subcellular pacemaker Ca 2+ signals at sites close to the plasma membrane, in the cytosol and in the nucleus depends on the type 3 ryanodine receptor (RyR) and its modulation by cyclic ADP-ribose. The spatial distribution of D-myo-inositol 1,4,5- trisphosphate receptors and RyRs indicates a concerted action of both of these receptors/Ca 2+ channels in the generation of initial pacemaker signals localized close to the plasma membrane. Inhibition or knockdown of RyRs resulted in significant decreases in (1) the frequency of initial pacemaker signals localized close to the plasma membrane, and (2) the frequency of localized pacemaker Ca 2+ signals in the inner cytosol. Moreover, upon microinjection of cyclic ADP-ribose or upon extracellular addition of its novel membrane-permeant mimic N-1- ethoxymethyl-substituted cyclic inosine diphosphoribose, similarly decreased Ca 2+ signals were observed in both type 3 RyR-knockdown cells and in control cells microinjected with the RyR antagonist Ruthenium Red. Taken together, our results show that, under physiological conditions in human T cells, RyRs play crucial roles in the local amplification and the spatiotemporal development of subcellular Ca 2+ pacemaker signals. Key words: Ca 2+ signaling, Ryanodine receptor, Cyclic ADP-ribose, T cell, D-myo-inositol 1,4,5-trisphosphate receptor Summary Amplification and propagation of pacemaker Ca 2+ signals by cyclic ADP-ribose and the type 3 ryanodine receptor in T cells Svenja Kunerth 1 , Matthias F. Langhorst 1 , Nadine Schwarzmann 1, *, Xianfeng Gu 2 , Lijun Huang 2 , Zhenjun Yang 2 , Liangren Zhang 2 , Steven J. Mills 3 , Li-he Zhang 2 , Barry V.L. Potter 3 and Andreas H. Guse 1,‡ 1 University Hospital Hamburg-Eppendorf, Center for Experimental Medicine, Institute of Biochemistry and Molecular Biology I: Cellular Signal Transduction, Martinistr. 52, 20246 Hamburg, Germany 2 National Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xue Yuan Road, Beijing 100083, China 3 Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath, BA2 7AY, UK *Present address: AstraZeneca, Tinsdaler Weg 183, 22876 Wedel, Germany ‡ Author for correspondence (e-mail: guse@uke.uni-hamburg.de) Accepted 17 December 2003 Journal of Cell Science 117, 2141-2149 Published by The Company of Biologists 2004 doi:10.1242/jcs.01063 Research Article