Figure 1.

NAADP-Induced Ca²⁺ Release from Acidic Intracellular Stores in Cytotoxic T Lymphocytes

(A) Single-cell Ca²⁺ traces normalized to initial fluorescence (F/F₀). Bottom panels show fluorescence confocal images of fluo-3-loaded cytotoxic T lymphocytes (CTLs) taken at the time indicated in seconds at basal, during NAADP/AM application and upon addition of 1 μM ionomycin. NAADP/AM (2.5–10 μM) elicited Ca²⁺ oscillations, which were blocked by the NAADP antagonist trans-Ned-19 (10 μM). No Ca²⁺ responses were observed in response to 2.5–10 μM nonesterified (free) NAADP/DMSO.

(B) Fluorescence changes (ΔF/F₀) were calculated as shown in the inset. MER (mean elevated ratio) was defined as the mean fluorescence divided by F₀ for the period after the addition of NAADP/AM.

(C and D) NAADP/AM-induced Ca²⁺ release displayed a “bell-shaped” concentration-response curve, characteristic of this second messenger.

(E and F) Ca²⁺ signals to 2.5–10 μM NAADP/AM were inhibited by preincubation with 1 μM bafilomycin A1, 10 μM nigericin, 1 μM monensin, or 50 μM GPN. n = 383−1,030 cells.

(G and H) CTLs were treated with 10 μM CPA in Ca²⁺-free buffer (G) or 2 μM 2-APB (H) prior to application of 10 μM NAADP/AM. n = 81–178 cells.

(I) Ca²⁺ signals with 2.5–10 μM NAADP/AM in 0.5 mM extracellular Ca²⁺ (+Ca²⁺_o) or Ca²⁺-free medium containing 100 μM EGTA (–Ca²⁺_o). n = 730–1,030 cells.

(J–O) Traces from single cells (J, L, and N) and Ca²⁺ changes (K, M, and O) (n = 66–99 cells) upon addition of 1 μM NAADP/AM followed by 5 μM IP₃/BM (J and K) or vice versa (L and M). Cells responded to ionomycin (1 μM) at the end of the recording. The dotted line represents the sum of NAADP/AM alone and IP₃/BM alone responses. In (N) and (O), 5 μM IP₃/BM and 1 μM NAADP/AM were added simultaneously. ^∗∗∗p < 0.001 versus Ctrl. All phases of the Ca²⁺ signal for free NAADP versus NAADP/AM + inhibitor are not significantly different.

Error bars are mean ± SEM. See also Figure S1.