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.