Figure 7.
Ca2+-Independent Effects of the NBCs
(A) To induce volume-regulated Cl− currents (VRAC), LPS-primed (1 μg mL−1, 2 hr) iBMDMs were superfused with hypotonic solution. Representative current traces are shown, which have been measured in the absence (VRAC) or presence of 30 μM NBC6 (Ai), 30 μM BC7 (Aii), or 30 μM BC23 (Aiii).
(B–E) LPS-primed (1 μg mL−1, 2 hr) iBMDMs were kept untreated or were pre-treated for 2 min with 75 μM 2APB or 30 μM NBC6. Subsequently, 100 μM ATP was added to the bath solution. (B–D) Representative Ca2+ traces of ATP-stimulated cells in the absence (B, n = 12) or presence of 75 μM 2APB (C, n = 14), or 30 μM NBC6 (D, n = 12). (E) Mean peak Ca2+ concentrations determined in cells treated with ATP alone (ATP) or with ATP in the presence of inhibitors. ns, no significant difference; ***p < 0.001, significant difference from Ca2+ signals of ATP-stimulated cells determined in the absence of inhibitors (Holm-Sidak corrected one-sample t test).
(F and G) LPS-primed iBMDMs were also stimulated with 5 mM ATP. Following development of sustained Ca2+ increases, 75 μM 2APB or 30 μM NBC6 was added to the ATP-containing solution. Images show representative examples of Ca2+ responses following treatment with ATP and the addition of 2APB (n = 12, F) or the addition of NBC6 (n = 12, G).
(H and I) LPS-primed iBMDMs were treated with concentrations of the inhibitors maximal for blocking IL-1β release (i.e., 2APB = 75 μM, NBC6 = 30 μM). Inhibitors were added to the iBMDMs 5 min before or 5 min after the addition of ATP (5 mM, 1 hr) (H) with IL-1β release measured by ELISA (I). ***p < 0.001, significant difference from corresponding vehicle control; ns, no significant effect of ATP administration time.
Data are presented as representative traces from calcium imaging experiments (B–D, F, and G), mean + SEM peak Ca2+ concentrations versus treatment with ATP alone (E), or mean + SEM IL-1β release as detected by ELISA (I).