Nicotinic acid adenine dinucleotide phosphate (NAADP(+)) is an essential regulator of T-lymphocyte Ca(2+)-signaling

Abstract

Microinjection of human Jurkat T-lymphocytes with nicotinic acid adenine dinucleotide phosphate (NAADP(+)) dose-dependently stimulated intracellular Ca(2+)-signaling. At a concentration of 10 nM NAADP(+) evoked repetitive and long-lasting Ca(2+)-oscillations of low amplitude, whereas at 50 and 100 nM, a rapid and high initial Ca(2+)-peak followed by trains of smaller Ca(2+)-oscillations was observed. Higher concentrations of NAADP(+) (1 and 10 microM) gradually reduced the initial Ca(2+)-peak, and a complete self-inactivation of Ca(2+)-signals was seen at 100 microM. The effect of NAADP(+) was specific as it was not observed with nicotinamide adenine dinucleotide phosphate. Both inositol 1,4, 5-trisphosphate- and cyclic adenosine diphosphoribose-mediated Ca(2+)-signaling were efficiently inhibited by coinjection of a self-inactivating concentration of NAADP(+). Most importantly, microinjection of a self-inactivating concentration of NAADP(+) completely abolished subsequent stimulation of Ca(2+)-signaling via the T cell receptor/CD3 complex, indicating that a functional NAADP(+) Ca(2+)-release system is essential for T-lymphocyte Ca(2+)-signaling.

Figure 1

Concentration-response curves of Ca²+ signals in single intact T-lymphocytes microinjected with NAADP⁺. Jurkat T-lymphocytes were loaded with Fura2/AM and Ca²⁺ was measured as detailed in Materials and Methods. Cells were injected as described in Materials and Methods in the presence of 1 mM extracellular Ca²⁺. Data are presented as overlays of single tracings of individual cells (left). The right shows the corresponding averages from these measurements (number of cells displayed, n = 5–17). As a control, vehicle buffer without NAADP⁺ was injected (A and B). The time points of microinjection are indicated by arrows.

Figure 3

NADP⁺ does not mediate Ca²⁺-signaling. Jurkat T-lymphocytes were loaded with Fura2/AM and ratiometric Ca²⁺ imaging, and parallel microinjection in the presence of 1 mM extracellular Ca²⁺ was carried out as detailed under Materials and Methods. Cells were injected with 50 nM NAADP⁺ (A) or NADP⁺ (B). Shown are the averages from 13 (A) and 5 (B) cells. The time points of microinjection are indicated by arrows.

Figure 2

Dose-response curve for NAADP⁺ in Jurkat T cells. Data from Fig. 1 are shown as mean values (n = 5–17) from time point 80 s (Ca²⁺-peak; A) or 400 s (Ca²⁺-plateau; B).

Figure 5

Influence of Ins(1,4,5)P₃ and its antagonist Ins(1,4,6)PS₃ on NAADP⁺-mediated Ca²⁺-signaling. Jurkat T-lymphocytes were loaded with Fura2/AM, and ratiometric Ca²⁺ imaging and parallel microinjection in the presence of 1 mM extracellular Ca²⁺ was carried out as detailed under Materials and Methods. Left, Overlays of single tracings of individual cells after injection (A–E); right, demonstrates the corresponding averages from these overlays (F–J). Shown are: A/F, coinjection of NAADP⁺ (50 nM) and Ins(1,4,6)PS₃ (40 μM; n = 7); B/G, injection of NAADP⁺ (50 nM; n = 10); C/H, coinjection of NAADP⁺ (10 μM) and Ins(1,4,5)P₃ (4 μM; n = 9); D/I, coinjection of NAADP⁺ (50 nM) and Ins(1,4,5)P₃ (4 μM; n = 3); and E/J injection of Ins(1,4,5)P₃ (4 μM; n = 8).

Figure 4

Influence of cADPR and its antagonist 8-OCH₃-cADPR on NAADP⁺-mediated Ca²⁺-signaling. Jurkat T-lymphocytes were loaded with Fura2/AM, and ratiometric Ca²⁺ imaging and parallel microinjection in the presence of 1 mM extracellular Ca²⁺ was carried out as detailed under Materials and Methods. Left, Overlays of single tracings of individual cells after injection (A–E); right, demonstrates the corresponding averages from these overlays (F–J). Shown are (n = number of experiments): A/F, coinjection of NAADP (50 nM) and 8-OCH₃-cADPR (100 μM; n = 7); B/G, injection of NAADP⁺ (50 nM, n = 10); C/H, coinjection of NAADP⁺ (10 μM) and cADPR (10μM; n = 7); D/I, coinjection of NAADP⁺ (50 nM) and cADPR (10 μM; n = 5); and E/J, injection of cADPR (10 μM, n = 5).

Figure 6

Effect of NAADP⁺ on OKT3-induced Ca²⁺-signaling in single Jurkat T-lymphocytes. Jurkat T-lymphocytes were loaded with Fura2/AM, and ratiometric Ca²⁺ imaging and parallel microinjection in the presence of 1 mM extracellular Ca²⁺ was carried out as detailed under Materials and Methods. The cells were injected with different concentrations of NAADP⁺ and then OKT3 (10 μg/ml) was added. Injection of intracellular buffer (A), NAADP⁺ (50 nM [B] and 10 μM [C]), and addition of OKT3 is indicated by arrows. Data are presented as a typical tracing from one individual cell; for each condition at least three experiments were carried out.