FIGURE 8.

CytC and CT9 inhibit BK-induced Ca²⁺ oscillations. A, cells were loaded with CytC or with vehicle only (DTR) by in situ electroporation. Loaded cells are in red (Load), and non-loaded cells are gray (Ctrl). The scale bar is 25 μm. B, representative traces of CytC loaded cells (Load) and control cells (Ctrl). Parallel experiments were done with the cells loaded with vehicle only (DTR). C, average data of experiments as illustrated in the previous panel. The loading procedure resulted by itself in some suppressive effect on the oscillations (percentage of oscillating cells). CytC strongly reduced the oscillations and co-loading together with IP₃RCYT peptide removed the inhibitory effect of CytC. The stars indicate a significant difference between the bars indicated. D, CytC did not affect the initial [Ca²⁺]_i transient triggered by 0.5 μm BK (2 min in zero Ca²⁺ medium). Subsequent addition of thapsigargin (2 μm, 3 min, in zero Ca²⁺ medium) liberated less Ca²⁺ from intracellular stores in CytC-loaded cells compared with their controls, indicating more complete store emptying. Mitochondrial Ca²⁺ uptake was larger in cells containing CytC (mitochondrial Ca²⁺ concentration, [Ca²⁺]_mito, was measured with RhodFF). E, pretreatment with Xestospongin-C (XeC; 10 μm, 1 h), U73122 (10 μm, 1 h), and thapsigargin (Thaps; 2 μm, 10 min) reduced the number of oscillating cells triggered by BK. F, cells were loaded with CT9 or with vehicle only (PI, similar to panel A). G, representative traces of loaded and control cells. H, CT9 peptide and CT9ΔI peptide significantly reduced the percentage of oscillating cells, in contrast to CT9^Rev. Significant differences are indicated as explained in panel C. I, Western blot analysis for Cx43-specific phosphorylated Ser-368 and total levels of Cx43 in control cells and cells treated with Tat-CT or Tat-CT^Rev (100 μm, 30 min) in the presence or absence of BK (0.5 μm). β-Tubulin was used as a loading control. The bar chart summarizes data derived from different Western blots.