Fig. 1. ATP_e-induced inward currents and Ca²⁺ signaling in HEK-P2X₇ cells and macrophages. (A-C) Whole-cell voltage clamp recordings of macrophages (A), HEK-293 cells (B), and HEK-P2X₇ cells (C). Cells were kept at room temperature and ATP (1 mM) was applied (arrows) pneumatically. (D-F) Changes in free cytoplasmic Ca²⁺ concentration of macrophages (D), HEK-293 cells (E), and HEK-P2X₇ cells (F) loaded with Fura-2-AM. Cells were kept at 37°C and 5 mM ATP (final concentration) was applied as indicated by the horizontal bars. Ca²⁺ concentration was measured for 20-30 cells in the same microscope field using arbitrary fluorescence ratio units (a.u.). (G) Dose-effect curve of ATP_e in HEK-P2X₇ cells. Cells were exposed to different ATP_e concentrations in the same conditions as in F and the fluorescence signal was normalized taking the maximum fluorescence obtained after the addition of 0.1% saponin. (H) Anti-P2X₇ western blotting of macrophages (lane 1) HEK-293 cells (lane 2), and HEK-P2X₇ cells (lane 3). Each result is representative of at least three independent experiments. In G, the bars represent the mean ± s.d. of at least three measurements per data point.