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-propanaine (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 . O-2(-) and peroxynitrite anion (ONOO-), inhibited the increase in [Ca2+](m). ONOO- (3 X 10(-4) M) itself also inhibited this increase. Pretreatment with the . O-2(-)-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 . O-2(-). (C) 1999 Elsevier Science B.V. All rights reserved.