Figure 2.

NAADP induces Ca²⁺ release from acidic Ca²⁺ stores

• A, B Representative single-cell Ca²⁺ traces showing 350/380 ratios of fura-2 fluorescence (A) and maximum Ca²⁺ changes (B) upon addition of 10 μM extracellular NAADP/AM in WT MEFs, which were blocked by pre-incubation with 10 μM trans-Ned-19 (Ned-19; 45 min), 1 μM bafilomycin A1 (Baf; 45 min), 5 μM nigericin (Nig; 30 min), or 200 μM GPN (5 min); control (Ctrl) was pre-incubated with DMSO (vehicle); n = 121–272; ***P < 0.001 relative to control using the ANOVA-Tukey test.
• C, D Ca²⁺ signals with 10 μM NAADP/AM in 1.8 mM extracellular Ca²⁺ (+ Ca²⁺_o) or Ca²⁺-free medium containing 100 μM EGTA (− Ca²⁺_o) in WT MEFs. (C) Representative single-cell fura-2 Ca²⁺ traces upon addition of 10 μM NAADP/AM and 100 μM ATP. (D) Maximum Ca²⁺ changes (max) and mean Ca²⁺ release over a period of 300 s post-addition of 10 μM NAADP/AM;n = 233–385 cells; ***P < 0.001 relative to + Ca²⁺_o using an unpaired t-test.
• E–H Cells treated with 200 μM CPA or 0.1% DMSO in medium + Ca²⁺_o for 50 min. Cells were then briefly washed and maintained in Ca²⁺-free medium (+100 µM EGTA) in which they were stimulated with 2 μM ionomycin (E, F) or 10 μM NAADP/AM (G, H). Maximum Ca²⁺ changes (E, G) and representative single-cell fura-2 Ca²⁺ traces (F, H); n = 49–148 cells; ***P < 0.001 relative to DMSO control, using the unpaired t-test.
• I, J MEFs express all three IP₃ receptor subtypes (IP₃R 1–3) and ryanodine receptor (RyR) types 1 and 2, detected by RT–PCR analysis. Blank refers to no RNA. Positive control for expression for RyR type 3 is shown in Supplementary Fig S1.
• K, L Cells treated with 2 μM 2-APB, 20 μM ryanodine, or 0.1% DMSO prior to application of 10 μM NAADP/AM. Representative single-cell fura-2 Ca²⁺ traces (K) and maximum Ca²⁺ changes (L); n = 142–374; ***P < 0.001 relative to control using the unpaired t-test.

Data information: Error bars represent SEM. See also Supplementary Fig S1. Source data are available online for this figure.