Figure 4. RuRed-and Ru360-dependent changes in calcium signalling in intact and permeabilized cells.

Cells cultures were transfected with mitochondria targeted inverse pericam (24h) and subsequently, pretreated with RuRed (Fluka, ≥ 85% purity, red) or Ru360 (Calbiochem, cyan) for 12-18h in serum-free medium: RBL-2H3, RuRed 5μM (A); HepG2, RuRed 10μM (B); H9c2, RuRed or Ru360 10μM each. Subsequently, cells were loaded with fura2/AM and incubated in a Ca²⁺ free extracellular medium. For the RuRed/Ru360 treated cells the drug was also present during the fura2 loading and the recording. Fluorescence [Ca²⁺]_c and [Ca²⁺]_m imaging was carried out as described before [3, 20]. Traces represent the means for 3-7 experiments for each condition.

(A) The RBL-2H3 cells were co-transfected with pericam and M1 muscarinic receptor. Cells were treated with carbachol, CCH of 100μM, thapsigargin, Tg of 2μM and CaCl₂, Ca of 2mM as indicated by the arrows.

(B) HepG2 cells were treated with ATP 100μM, Tg of 2μM and CaCl₂ of 5mM.

(C) H9c2 cells were treated with vasopressin, VP of 100nM, Tg of 2μM and CaCl₂ of 10mM (i). Bar charts show the size of the VP- and Ca-induced [Ca²⁺]_m and [Ca²⁺]_m rise calculated in single cells (ii). The rates for the Ca-induced [Ca²⁺]_c rise were 31.3±4.1 nM/s; 29.4±2.7 nM/s; 23.6±4 nM/s and for the [Ca²⁺]_m rise were 2.4±0.4 %/s; 2.6±0.5 %/s; 2.6±0.3 %/s in control, RuRed-and Ru360-pretreated cells, respectively (n=34, 30 and 32 cells).

(D) RuRed-and Ru360 induced suppression of the mitochondrial Ca²⁺ uptake in permeabilized H9c2 cells

(i) Pericam-transfected adherent H9c2 cells were permeabilized with saponin and incubated in an intracellular medium containing fura2 5μM. [Ca²⁺]_c and [Ca²⁺]_m were followed in single cells with CCD imaging of fura2 and pericam fluorescence, respectively. To evoke a Ca²⁺ release from the ER IP₃ 7.5μM was added. RuRed of 5μM was added 3min before IP₃ (red trace). Traces are means of three experiments.

(ii,iii) Suspensions of H9c2 cells (1mg protein/ml) were incubated in an intracellular medium and were permeabilized by digitonin. [Ca²⁺]_c was monitored in a fluorometer using fura2FF (0.5μM) added to the bathing medium. CaCl₂ 30μM and FCCP of 5μM was added as indicated by the arrows. RuRed (red) and Ru360 (blue) 1μM each was added 1min before Ca²⁺ addition. Representative traces for the fluorometer records (ii) and bar charts showing the RuRed/Ru360 sensitive fraction of the decay in [Ca²⁺]_c (iii).