FIGURE 7.

Caspase-insensitive ATP release stimulated by chemotherapeutic drugs is resistant to carbenoxolone blockade but suppressed by intracellular Ca²⁺ buffering. A, schematic of alternative ATP release pathways. When caspases are inhibited, ATP may be secreted via activation of noncleaved Panx1 channels or exocytosis of secretory lysosomes or autophagolysosomes. B and C, Jurkat T cells were incubated with no stimulus or with 3 μm STS for 8 h in the absence or presence of 100 μm Z-VAD, 500 μm CBX, or both inhibitors. Samples of the extracellular medium were processed for analysis of ATP only (B) or summed ATP + ADP + AMP (C). Data indicate mean ± S.E. for n = 6 wells from two experiments; ***, p < 0.001. D, Jurkat T cells were incubated for 1 h at 37 °C in either standard BSS (1.5 mm CaCl₂) or calcium-free BSS (0 mm CaCl₂) in the absence or presence 25 μm BAPTA-AM and then resuspended in fresh 1.5 calcium-BSS or 0 calcium BSS. The BAPTA-loaded or mock-loaded cells were then incubated with no stimulus or with 3 μm STS for 8 h in the absence or presence of 100 μm Z-VAD. Samples of the extracellular medium were processed for analysis of ATP. Data indicate mean ± S.E. for one experiment performed in triplicate; ns, p > 0.05; ***, p < 0.001. E and F, Jurkat T cells were incubated for 1 h at 37 °C in standard BSS (1.5 mm CaCl₂) in the absence or presence 25 μm BAPTA-AM and then resuspended in fresh BSS. The BAPTA-loaded or mock-loaded cells were then incubated with no stimulus or with 3 μm STS for 8 h in the absence or presence of 100 μm Z-VAD. Samples of the extracellular medium were processed for analysis of ATP only (E) or summed ATP + ADP + AMP (F). Data indicate mean ± S.E. for n = 3 experiments; *, p < 0.05; **, p < 0.01; ***, p < 0.001. G, Jurkat cells were preincubated for 1 h in the absence or presence of 250 nm bafilomycin A (BafA), 100 μm Z-VAD, or both inhibitors. The cells were incubated for an additional 4 h with or without 3 μm STS and then assayed for LysoTracker Red accumulation. Data indicate average ± range from one experiment performed in duplicate. H, Jurkat T cells were incubated with no stimulus or with 3 μm STS for 8 h in the absence or presence of 100 μm Z-VAD, 250 nm bafilomycin A, or both inhibitors. Samples of the extracellular medium were processed for analysis of ATP. Data indicates mean ± S.E. for n = 3 experiments each performed in duplicate; ns, p > 0.05; *, p < 0.05. I, Jurkat T cells were incubated with no stimulus or with 3 μm STS for 8 h in the absence or presence of 100 μm Z-VAD, 5 mm 3-methyladenine (3MA), or both inhibitors. Samples of the extracellular medium were processed for analysis of ATP. Data indicate mean ± S.E. for n = 6 wells from two experiments; ns, p > 0.05; ***, p < 0.001.