Figure 7

The effects of purified PKG on the functional activities of isolated heart mitochondria. (A) – mitochondrial state 2 and state 3 respiration rates, (B) – mitochondrial Ca²⁺ accumulation, (C) – mitochondrial swelling, (D) – release of cytochrome c from mitochondria. Isolated heart mitochondria were preincubated in hypotonic conditions with 40 U/ml PKG Iα as described in Methods. Then mitochondria (0.2–0.5 mg) were added to 1 ml incubation buffer B (plus 1 mM pyruvate and 1 mM malate as respiration substrates) and mitochondrial respiration and swelling were measured. Mitochondrial swelling was assayed by measuring a decrease in absorbance at 540 nm after addition of 250 μM CaCl₂. After 15 min incubation mitochondria were removed by centrifugation, the supernatants were used for spectrophotometric measurements of released of cytochrome c. Mitochondrial calcium accumulation was measured in buffer C fluorimetrically using 100 nM Calcium Green-5N (excitation at 506 nm, emission at 531 nm) as described in Methods. DT-3 – protein kinase G Iα inhibitor (from Calbiochem). * – statistically significant effect of PKG (or Ca²⁺) (p < 0.05, Tukey test) if compared to control, # – statistically significant effect of PKG inhibitor DT-3 (p < 0.05, Tukey test) if compared to mitochondria + PKG group, ^ – statistically significant effect of PKG (p < 0.05, Tukey test) if compared to mitochondria plus Ca²⁺. Means ± standard errors of 7 separate experiments are presented.