Figure 4

There is a perinuclear intracellular Ca²⁺ store that is labile and sensitive to emetine. All these experiments were carried out in the presence of 1.8 mM external [Ca²⁺] ( + Ca²⁺) and in single HeLa cells. (A) Confocal image of cells loaded with Mag-Fluo-4. The dash line delineates a cell where cytosolic (ROI 1) and perinuclear (ROI 2) regions of interest have been marked for analysis. (B) Representative recording showing the effect of 50 µM emetine on the Mag-Fluo-4 fluorescence in the ROIs identified in panel A). (C) Average maximal reduction in the Mag-Fluo-4 fluorescence in response to emetine in cytosolic (black bar) and perinuclear ROIs (red bar, n = 24 cells). (D) Representative recording from Fluo-4/AM-loaded HeLa cells showing the changes in [Ca²⁺]_c in response to the perfusion with 30 μM ATP followed by perfusion with 500 μM emetine. (E) In this case, emetine was perfused to naïve cells followed by ATP, and then there was a second addition of emetine as indicated. (F) The bar graph shows that the average peak [Ca²⁺]_c response to ATP was decreased after the addition of emetine (left red bar, n = 42 cells) when compared with the control (left black bar, n = 52 cells). The [Ca²⁺]_c response was absent when emetine was applied as the first stimulus (right red bar), but there was a significant increase in [Ca²⁺]_c when emetine was applied after ATP (right black bar). Similar results were obtained with 50 μM emetine but the amplitude was smaller due to the perfusion system being slow (F, blue bar). Data are presented as mean ± SEM where n indicates the number of cells studied.