Cytoprotective effects of nitrite

Toxic effects of nitrite anions if used in unphysiologically high concentrations are known since nearly hundred years. Recently it has been realized that low concentrations of nitrite are cytoprotective against ischemic damage in a wide range of tissues. The mechanism of cytoprotection is poorly understood, but it is due to reduction of nitrite to NO. Two major properties of NO have been suggested to mediate beneficial effects of nitrite. The first one (vasodilation) is the regulation of systemic circulation and local tissue perfusion and the second one is the modulation of intracellular enzymatic activities, first of all mitochondrial function. The reduction of nitrite may occur in both blood and tissue. In the blood the reduction of nitrite is catalysed by hemoglobin (Hb) in red blood cells. Our recent publication (Mol Med) and preliminary data suggest that the portion of NO, which is formed from nitrite directly in red blood cells via reaction with hemoglobin can only modestly contribute to vasodilation. In contrast intracellular mechanisms of nitrite reduction including myoglobin, mitochondria, xanthine oxidase, and eNOS are likely the candidates for intracellular bioactivation of nitrite. Therefore the aim of this project is to identify the mechanism(s) of nitrite bioactivation inside of cells contributing to cytoprotective effects of nitrite. In the frame of this project we plan to investigate the nitrite reduction using tissue homogenates, isolated mitochondria, and cell culture. The NO production from nitrite will be detected using EPR method, NO-electrode, and confocal microscopy combined with corresponding fluorescent dyes. Bioactivation of nitrite will be estimated by determination of guanylyl cyclase activity (cGMP) and mitochondrial function (respiratory activities) using electrochemical methods and confocal microscopy. To characterize the impact of mitochondria, myoglobin, xanthine oxidase, and NOS to nitrite reduction, the NO production will be measured in the presence of specific inhibitors and antibodies. In addition we will use tissues/cells with (heart/cardiomyocites, aortic tissue/smooth muscle cell) and without myoglobin (liver/hepatocytes (RH). Myoglobin has been shown to reduce nitrite much more efficient than haemoglobin. The results of this project will extend our knowledge about the mechanisms of NO production from nitrite, clarify the impact of different nitrite reduction mechanisms to cytoprotective effect of nitrite, help the interpretation of different experimental data previously obtained in ischemia-reperfusion studies and provide theoretical basis for possible therapeutic application of nitrite.

This project aimed to combat cardiovascular diseases. One of the potent regulators of cardiovascular functions is nitric oxide (NO) synthesised by a special enzyme, NO-synthase under normoxic conditions. This enzyme, however, is oxygen-dependent and not sufficient under hypoxic conditions. Recently it has been shown that nitrite, occurring at low concentrations in the body and considered earlier as an end product of NO oxidation, may serve as NO source under hypoxic conditions and regulate blood supply to hypoxic organs. In addition nitrite exerts cytoprotective effects against ischemic injury. The mechanisms of NO release from nitrite (bioactivation of nitrite) are poorly understood; consequently beneficial effects of nitrite cannot be well controlled and predicted. This project aimed at the understanding of mechanism(s) of nitrite-bioactivation in the body in order to better control and predict its beneficial effects. In the frame of this project we have determined the predominant pathway of nitrite reduction delivering NO for the regulation of cardiovascular functions and a mechanism of cytoprotection operating in single cells. We have shown that the heart rate is regulated by nitrite in a red blood cell (RBC)- dependent manner, suggesting that hemoglobin is responsible for nitrite bioactivation regulating heart rate. In contrast, nitrite mediated changes in circulatory parameters, such as blood pressure, myocardial contractility, peripheral resistance, and arterial stiffness are regulated by nitrite reduction inside functional organ cells, like cardiomyocytes or hepatocytes. Further experiments in cardiomyocytes revealed that the release of NO and consequent synthesis of the cGMP, a molecule regulating myocardial contractility, are mitochondria-dependent. Moreover, the addition of RBC to the culture of cardiomyocytes decreased the rate of cGMP formation. Similar mechanisms of nitrite reduction were found in hepatocytes. Following metabolic fate of NO formed inside cardiomyocytes we found that it was able to diffuse into the medium and neighbours cells suggesting that it diffuses to blood vessels within a tissue to regulate vascular tonus in the body. Another important finding of this study was the understanding of the cytoprotective action of nitrite in a single cell. It is known that hypoxia or ischemia and following reoxygenation cause mitochondrial dysfunction due to the damage of mitochondrial complex I and the release of cytochrome c. The latter is known to induce programmed cell death (apoptosis) if released in cytoplasm. We have shown that nitrite prevents the release of cytochrome c due to inhibition of the oxidative damage of the outer mitochondrial membrane induced by transition metals releasing during hypoxic phase and damaging mitochondrial membrane during reoxygenation phase. Generally our data show that effects of nitrite are mitochondria and RBC-dependent. Consequently, acquired and inborn defects of mitochondria may diminish beneficial effects of nitrite; while high hematocrit may facilitate effects of nitrite on the heart rate, but diminish other cardiovascular effects of nitrite. An additional observation that we made is that immortalised cell lines are much more resistant to hypoxia than normal tissues and that beneficial effects of nitrite do not occur in those cell lines. Currently we are trying to understand why cell lines are more resistant to ischemia and nitrite treatment.