During myocardial ischemia and reperfusion, nitric oxide ((.)NO) was shown to exert either beneficial or detrimental effects. Uncoupled (.)NO synthases (NOS) can generate superoxide anion under suboptimal concentrations of substrate and cofactors. The aim of our study was to investigate the role of NOS modulation on 1) the evolution of functional parameters and 2) the amount of free radicals released during an ischemia-reperfusion sequence. Isolated perfused rat hearts underwent 30 min of total ischemia, followed by 30 min of reperfusion in the presence of N(G)-nitro-D- or L-arginine methyl ester (NAME, 100 microM) or of D- or L-arginine (3 mM). Functional parameters were recorded and coronary effluents were analyzed with electron spin resonance to identify and quantify the amount of alpha-phenyl-N-tert-butylnitrone spin adducts produced during reperfusion. The antioxidant capacities of the compounds were determined with the oxygen radical absorbance capacity test. L-NAME-treated hearts showed a reduction of coronary flow and contractile performance, although neither L-NAME nor L-arginine improved the recovery of coronary flow, left end diastolic ventricular pressure, rate pressure product, and duration of reperfusion arrhythmia, compared with their D-specific enantiomers. A large and long-lasting release of alkyl/alkoxyl radicals was detected upon reperfusion, but no differences of free radical release were observed between D- and L-NAME or D- and L-arginine treatment. These results may indicate that, in our experimental conditions, cardiac NOS might not be a major factor implicated in the oxidative burst that follows a global myocardial ischemia.
[Show abstract][Hide abstract] ABSTRACT: Redox reactions play key roles in intra- and inter-cellular signaling, and in adaptative processes of tissues towards stress. Among the major free radicals with essential functions in cells are reactive oxygen species (ROS) including superoxide anion (O2 (•-)), hydroxyl radical ((•)OH) and reactive nitrogen species (RNS) such as nitric oxide ((•)NO). In this article, we review the forgotten and new radicals with potential relevance to cardiovascular pathophysiology. Approximately 0.3% of O2 (•-) present in cytosol exists in its protonated form: hydroperoxyl radical (HO2 (•)). Water (H2O) can be split into two free radicals: (•)OH and hydrogen radical (H(•)). Several free radicals, including thiyl radicals (RS(•)) and nitrogen dioxide (NO2 (•)) are known to isomerize double bonds. In the omega-6 series of poly-unsaturated fatty acids (PUFAs), cis-trans isomerization of γ-linolenate and arachidonate catalyzed by RS(•) has been investigated. Evidence is emerging that hydrogen disulphide (H2S) is a signaling molecule in vivo which can be a source of free radicals. The Cu-Zn superoxide dismutase (SOD) enzyme can oxidize the ionized form of H2S to hydro-sulphide radical: HS(•). Recent studies suggest that H2S plays an important function in cardiovascular functions. Carbonate radical, which can be formed when (•)OH reacts with carbonate or bicarbonate ions, is also involved in the activity of Cu-Zn-SOD. Recently, it has been reported that carbonate anion were potentially relevant oxidants of nucleic acids in physiological environments. In conclusion, there is solid evidence supporting the formation of many free radicals by cells leading which may play an important role in their homeostasis.
"This condition can be created in reperfused tissue after onset of ischemia. Low L-arginine concentration, high « NO formation and a shift of the NOS activity from synthesis of « NO to NADPH oxidase activity yielding O 2 «¡ , leads to a mixture of « NO and O 2 «¡ species which subsequently can react with each other at a rate limited by diVusion (6.7 £ 10 9 M ¡1 s ¡1 ), to produce ONOO ¡ . There are many other sources of O «¡ , but during I/R 70% of O «¡ is produced by NOS . "
[Show abstract][Hide abstract] ABSTRACT: The involvement of nitric oxide (*NO) in oxidative stress in the rat gastrocnemius muscle subjected to ischemia/reperfusion injury was investigated using a specific and sensitive chemiluminescence (CL) method for measurement of both membrane lipid peroxide and total tissue antioxidant capacity (TRAP). In addition, inhibitors of nitric oxide synthase enzymes were used. The CL time-course curve increased dramatically after 1, 2, and 4 h of reperfusion, reaching values about 12 times higher than those of both control and ischemic rats. Initial velocity (V0) increased from 13.6 cpm mg protein(-1) min(-1) in the ischemic group, to 7341-8524 cpm mg protein(-1) min(-1) following reperfusion. The administration of L-NAME prior to reperfusion significantly reduced (p<0.007) the time-course of the CL curve, decreasing the V(0) value by 51% and preventing antioxidant consumption for 1h following reperfusion. No significant change in CL time-course curve and TRAP values were observed with aminoguanidine treatment. On contrary, after 4h following reperfusion, pre treatment with aminoguanidine led to a significant decrease (p < 0.0001) in the time-course of the CL curve, where V0 decreased by 75% and TRAP returned to control levels. No significant change in CL time-course curve and TRAP values were observed with L-NAME treatment. When RT-PCR was carried out with an iNOS-specific primer, a single band was detected in RNA extracted from muscle tissue of only the 4 h ischemia/4 h reperfusion group. No bands were found in either the control, 4 h ischemia or 4 h ischemia/1 h reperfusion groups. Based on these results, we conclude that *NO plays an important role in oxidative stress injury, possibly via -ONOO, in skeletal muscle subjected to ischemia/reperfusion. Our results also show that cNOS isoenzymes are preferentially involved in *NO generation at the beginning of reperfusion and that iNOS isoenzyme plays an important role in reperfusion injury producing *NO later in the process.
"Quantitative ESR spectroscopy determines values for an unknown sample quantitatively from the spectral ratio of each of the obtained adducts by measuring the unknown sample and the primary standard such as TEMPOL or DPPH (1,1-diphenyl-2-picrylhydrazyl) at a known concentration under the same conditions . Indeed, using ESR spectroscopy and estimating spin concentrations are widely utilized in ESR applications (Zweier, 1988; Shoji et al., 1999; Vergely et al., 2002). The observed ESR signals due to NO-DTCS-Fe 2 + and TEMPOL used in this study as primary standards are due to signal from the same nitroxide radical, and these exhibit similar ESR behaviors. "
[Show abstract][Hide abstract] ABSTRACT: Using a Langendorff-perfused rat heart preparation and selective electrodes, we determined nitric oxide (NO) and oxygen levels in cardiac tissue. An NO-selective electrode that was calibrated by electron spin resonance (ESR) spectroscopy was inserted into the middle of the myocardium in the left ventricle. Simultaneously, we used an O2-selective electrode to measure the partial pressure of oxygen (pO2) in the perfusate, Krebs-Henseleit (K-H) solution, that was ejected from the heart. After 30 min of aerobic control perfusion, hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion. Under ischemic conditions, with a gradually decreasing pO2, NO detected by an NO-sensitive electrode within the myocardium was gradually increased. The maximum concentration increases in NO and decreases in pO2 during global ischemia were +10.200 +/- 1.223 microM and -58.608 +/- 4.123 mmHg, respectively. NO and pO2 levels both recovered to pre-ischemia baseline values when perfusion was restarted after global ischemia (reperfusion). The presence of Nomega-nitro-L-arginine methyl ester (L-NAME, 10 mM), a NOS inhibitor, prevented ischemia/reperfusion-induced changes in NO. This study shows that an NO-selective electrode that is calibrated by ESR can provide accurate, real-time monitoring of cardiac NO in normal and ischemic myocardium.
Life Sciences 11/2003; 74(1):75-85. DOI:10.1016/j.lfs.2003.06.027 · 2.70 Impact Factor
Anna Solini, Chiara Rossi, Emiliano Duranti, Stefano Taddei, Andrea Natali, Agostino Virdis
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