Figure 3
From: The Trans Golgi Region is a Labile Intracellular Ca2+ Store Sensitive to Emetine
The acidic intracellular Ca2+ stores sensitive to emetine are not lysosomes. All experiments were carried out in the absence of external Ca2+ and in the presence of 0.1 mM EGTA (-Ca2+) in HeLa cells (2 × 105 cells/ml). (A) Representative simultaneous recordings of changes in both [Ca2+]c (top) and [Ca2+]L (bottom) in cells exposed to 10 μM ATP and 1 μM Tg followed by 50 μM emetine, at the time indicated. Cells were untreated or incubated with either 18 mM NH4Cl or 100 nM bafilomycin before initiating recording of Ca2+ changes. (B) Bar graphs represent the average peak [Ca2+]c response (top) elicited by ATP and Tg or the reduction in the [Ca2+]L (bottom) elicited by ATP and Tg (left side bars) followed by emetine (right side bars) in control (black bars, n = 10), NH4Cl-treated cells (red bars, n = 3) or baf-treated cells (blue bars, n = 4). (C) Representative traces of changes in the [Ca2+]c (top) and the [Ca2+]L (bottom) from cells treated with 200 μM GPN (red trace) versus control cells (black trace). (D) Bar graphs represent the average peak [Ca2+]c (top) or the maximal decrease of the [Ca2+]L (bottom) elicited both by ATP plus Tg and the emetine-induced [Ca2+]L response (right side bars) in control (black bars, n = 4) or GPN-treated cells (red bars, n = 5). (E) Representative simultaneous recordings of changes in both [Ca2+]i (top) and [Ca2+]L (bottom) in response to 10 nM NAADP-AM and followed by 50 μM emetine, added where indicated (black trace). Another set of cells were treated with NAADPR blockers BZ194 (100 μM, red trace) or Ned 19 (50 μM, blue trace) before the addition of NAADP-AM and emetine. (F) Bar graphs on the left side represent the average reduction in the [Ca2+]L elicited by NAADP-AM (black, n = 8), and the inhibition of this effect in cells treated with BZ194 (red, n = 4) or Ned 19 (blue, n = 4). Bars graphs on the right side indicate the control emetine-induced reduction in the [Ca2+]L (black bar) and the effect of treating cells with NAADP-AM (gray bar, n = 4); with NAADP-AM plus BZ149 (red bar, n = 4) and NAADP-AM plus Ned 19 (blue bar, n = 4) previous to the addition of emetine. Data are presented as mean ± SEM of n number of independent experiments.
