Protective role of nitric oxide synthase against ischemia-reperfusion injury in guinea pig myocardial mitochondria.
In guinea-pig myocardial mitochondria preparation, lowering the Ca2+ concentration or pH level in the perfusate rapidly elevated the fura-2 Ca2+ signal ([Ca2+]m). Pretreatment with 10(-4) M L-Arg inhibited the rapid [Ca2+]m influx, whereas administration of 10(-4) M L-NAME did not, suggesting some association between nitric oxide (NO*) synthase (NOS) activation and Ca2+ kinetics in mitochondria. Immunoblotting analysis showed that endothelial (e)-NOS was present in mitochondria, but not inducible (i)-NOS or brain (b)-NOS. Electron microscopy observations revealed that the e-NOS antibody-reactive site in the mitochondria was the inner cristae. The production of reactive oxygen species and NO* in isolated mitochondria was detected by the spin trapping technique with electron paramagnetic resonance (EPR) spectrometry. Pretreatment with 10(-5) M S-nitroso-N-acetyl-DL-penicillamine (SNAP) and 10(-5) M 3-[2-Hydroxy-1-(1-methylethyl)-2-nitrosohydrazino]-1-propananin e (NOC 5), which spontaneously generate NO*, completely inhibited the [Ca2+]m uptake. In addition, N-morpholino sydnonimine hydrochloride (SIN-1) (10(-5) M), which simultaneously generates NO* as well as *O2- and peroxynitrite anion (ONOO-), inhibited the increase in [Ca2+]m. ONOO- (3 x 10(-4) M) itself also inhibited this increase. Pretreatment with the *O2(-)-scavenger manganese superoxide dismutase or catalase (200 units/ml) completely inhibited the increase in [Ca2+]m caused by lowering of either the Ca2+ concentration or the pH in the perfusate. These results suggested that the formation of reactive oxygen species promoted the [Ca2+]m influx. The agents that inhibited the [Ca2+]m influx improved contractility even in Langendorff preparations after ischemia. Based on these findings, we concluded that e-NOS exists in mitochondria and that NO* may play an important protective role in reperfusion cardiac injury after ischemia, by inhibiting the Ca2+ influx into mitochondria which are otherwise damaged by *O2-.
Hotta
Y
Y
Department of Pharmacology, Aichi Medical University, Japan. i3hotta@amugw.aichi-med-u.ac.jp
Otsuka-Murakami
H
H
Fujita
M
M
Nakagawa
J
J
Yajima
M
M
Liu
W
W
Ishikawa
N
N
Kawai
N
N
Masumizu
T
T
Kohno
M
M
D000818:Animals; D001120:Arginine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000493:pharmacokinetics; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D016166:Free Radical Scavengers / Q000494:pharmacology; D005609:Free Radicals / Q000378:metabolism / Q000494:pharmacology; D006168:Guinea Pigs; D006321:Heart / Q000187:drug effects; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007150:Immunohistochemistry; D008297:Male; D008929:Mitochondria, Heart / Q000201:enzymology* / Q000378:metabolism / Q000648:ultrastructure; D015428:Myocardial Reperfusion Injury / Q000201:enzymology / Q000517:prevention & control*; D009206:Myocardium / Q000166:cytology / Q000201:enzymology / Q000473:pathology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009566:Nitrates / Q000737:chemistry; D009569:Nitric Oxide / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000502:physiology* / Q000648:ultrastructure; D052250:Nitric Oxide Synthase Type III; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D019097:Spin Trapping; D013482:Superoxide Dismutase / Q000494:pharmacology
D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D005609:Free Radicals; D009566:Nitrates; D020030:Nitric Oxide Donors; D017382:Reactive Oxygen Species; C110881:S-nitro-N-acetylpenicillamine; C039642:peroxynitric acid; D009569:Nitric Oxide; D001120:Arginine; D006861:Hydrogen Peroxide; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D013482:Superoxide Dismutase; D010396:Penicillamine; D002118:Calcium; D019331:NG-Nitroarginine Methyl Ester