Hypoxia/Reoxygenation of isolated rat heart mitochondria causes cytochrome c release and oxidative stress; evidence for involvement of mitochondrial nitric oxide synthase

Abstract

The objective of the present study was to delineate the molecular mechanisms for mitochondrial contribution to oxidative stress induced by hypoxia and reoxygenation in the heart. The present study introduces a novel model allowing real-time studying mitochondria under hypoxia and reoxygenation, and describes the significance of intramitochondrial calcium homeostasis and mitochondrial nitric oxide synthase (mtNOS) for oxidative stress. The present study shows that incubating isolated rat heart mitochondria under hypoxia followed by reoxygenation, but not hypoxia per se, causes cytochrome c release from the mitochondria, oxidative modification of mitochondrial lipids and proteins, and inactivation of mitochondrial enzymes susceptible to inactivation by peroxynitrite. Those alterations were prevented when mtNOS was inhibited or mitochondria were supplemented with antioxidant peroxynitrite scavengers. The present study shows mitochondria independent of other cellular components respond to hypoxia/reoxygenation by elevating intramitochondrial ionized calcium and stimulating mtNOS. The present study proposes a crucial role for heart mitochondrial calcium homeostasis and mtNOS in oxidative stress induced by hypoxia/ reoxygenation.

Mitochondria-dependent signalling pathway are involved in the early process of radiation-induced bystander effects

“…However, this hypothesis is very difficult to explain, as NO production can be significantly decreased by preventing mitochondrial Ca 2 þ uptake. Recently, Ghafourifar and his colleagues have reported that hypoxia/ reoxygenation and tamoxifen can elevate [Ca 2 þ ] m by increasing mitochondrial calcium uptake and the release of [Ca 2 þ ] m from the granules in mitochondria, and stimulate mtNOS to generate NO Zenebe et al, 2007). By inhibiting mitochondrial calcium uptake with RR, we found that the relative fluorescence intensity of NO significantly decreased in postirradiated r þ A L cells ( Figure 3B).…”

Nitric oxide irreversibly inhibits cytochrome oxidase at low oxygen concentrations: Evidence for inverse oxygen concentration‐dependent peroxynitrite formation

“…Peroxynitrite is a powerful oxidative NO congener that irreversibly inhibits the activity of various mitochondrial proteins including COX (7,17). Uric acid is a well-known peroxynitrite scavenger (17) that protects against peroxynitrite-induced damaging mitochondrial components (10,18). As shown in Figure 2A, uric acid dose-dependently protected the irreversible COX inactivation induced by NO.…”

“…Those include mitochondrial superoxide dismutase that removes superoxide, one of the two precursors of peroxynitrite. Additionally, mitochondria contain large quantities of reduced glutathione that neutralizes peroxynitrite to S-nitrosating species that exert reversible interaction with mitochondrial components (10). Thus, those protective mechanisms preserve the reversibility of COX inhibition by NO via decreasing the probability of peroxynitrite formation or neutralizing the peroxynitrite formed.…”

“…Such conditions may occur under pathological state including ischemia in which oxygen concentration is lowered, NO formation is increased, and mitochondrial superoxide dismutase and reduced glutathione are hindered (10).…”

“…Figure 1A is the representative original trace showing irreversible inhibition of COX by NO at various oxygen concentrations. The trace marked NO shows that the NO signal detected by the NO-sensitive microsensor mounted in the chamber (10). This figure shows that the inhibition of COX activity was not fully recovered even after NO was fully metabolized.…”