title: Nitric oxide protects the heart from ischemia-induced apoptosis and mitochondrial damage via protein kinase G mediated blockage of permeability transition and cytochrome c release.
abstract: BACKGROUND
  Heart ischemia can rapidly induce apoptosis and mitochondrial dysfunction via mitochondrial permeability transition-induced cytochrome c release. We tested whether nitric oxide (NO) can block this damage in isolated rat heart, and, if so, by what mechanisms.
  
  
  METHODS
  Hearts were perfused with 50 microM DETA/NO (NO donor), then subjected to 30 min stop-flow ischemia or ischemia/reperfusion. Isolated heart mitochondria were used to measure the rate of mitochondrial oxygen consumption and membrane potential using oxygen and tetraphenylphosphonium-selective electrodes. Mitochondrial and cytosolic cytochrome c levels were measured spectrophotometrically and by ELISA. The calcium retention capacity of isolated mitochondria was measured using the fluorescent dye Calcium Green-5N. Apoptosis and necrosis were evaluated by measuring the activity of caspase-3 in cytosolic extracts and the activity of lactate dehydrogenase in perfusate, respectively.
  
  
  RESULTS
  30 min ischemia caused release of mitochondrial cytochrome c to the cytoplasm, inhibition of the mitochondrial respiratory chain, and stimulation of mitochondrial proton permeability. 3 min perfusion with 50 microM DETA/NO of hearts prior to ischemia decreased this mitochondrial damage. The DETA/NO-induced blockage of mitochondrial cytochrome c release was reversed by a protein kinase G (PKG) inhibitor KT5823, or soluble guanylate cyclase inhibitor ODQ or protein kinase C inhibitors (Ro 32-0432 and Ro 31-8220). Ischemia also stimulated caspase-3-like activity, and this was substantially reduced by pre-perfusion with DETA/NO. Reperfusion after 30 min of ischemia caused no further caspase activation, but was accompanied by necrosis, which was completely prevented by DETA/NO, and this protection was blocked by the PKG inhibitor. Incubation of isolated heart mitochondria with activated PKG blocked calcium-induced mitochondrial permeability transition and cytochrome c release. Perfusion of non-ischemic heart with DETA/NO also made the subsequently isolated mitochondria resistant to calcium-induced permeabilisation, and this protection was blocked by the PKG inhibitor.
  
  
  CONCLUSIONS
  The results indicate that NO rapidly protects the ischemic heart from apoptosis and mitochondrial dysfunction via PKG-mediated blockage of mitochondrial permeability transition and cytochrome c release.
authors: Institute for Biomedical Research, Kaunas University of Medicine, Kaunas, Lithuania. vilbor@vector.kmu.lt
authors: Vilmante
authors: V
authors: Borutaite
authors: Ramune
authors: R
authors: Morkuniene
authors: Odeta
authors: O
authors: Arandarcikaite
authors: Aiste
authors: A
authors: Jekabsone
authors: Jurgita
authors: J
authors: Barauskaite
authors: Guy C
authors: GC
authors: Brown
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002227:Carbazoles / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D045304:Cytochromes c / Q000378:metabolism*; D007211:Indoles / Q000494:pharmacology; D008297:Male; D008929:Mitochondria, Heart / Q000187:drug effects* / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000187:drug effects*; D000083162:Mitochondrial Permeability Transition Pore; D017202:Myocardial Ischemia / Q000473:pathology*; D009569:Nitric Oxide / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D014226:Triazenes / Q000494:pharmacology*
chemical_list: C105351:1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene; D002227:Carbazoles; D007211:Indoles; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D020030:Nitric Oxide Donors; D047428:Protein Kinase Inhibitors; D011758:Pyrroles; D014226:Triazenes; C073601:KT 5823; C085989:Ro 32-0432; D009569:Nitric Oxide; D045304:Cytochromes c; D017869:Cyclic GMP-Dependent Protein Kinases; C064758:Ro 31-8220