The objective of this study was to determine if prior exposure of rat hearts to S-nitrosocysteine (CysNO) was able to provide protection against reperfusion injury. We probed NO release using the extracellular NO scavenger oxyhemoglobin (oxyHb), and we examined the involvement of the amino acid transport system L (L-AT), a known transporter of CysNO, using the L-AT competitor, L-leucine (L-Leu). Isolated (9- to 12-week-old Wistar male) rat hearts (six to eight per group) were perfused with CysNO (10 microM) for 30 min with or without the L-AT competitor L-Leu (1 mM) before 30 min of ischemia. Cardiac function was assessed before, during, and after treatment and during 120 min of reperfusion after ischemia. Functional recovery (rate-pressure product) was significantly improved in the CysNO group compared to hearts in the CysNO+L-Leu group and the control group (p<0.05). Necrosis, measured by triphenyltetrazolium chloride staining, was significantly reduced in CysNO hearts (p<0.05) and this improvement was reversed by L-Leu. The NO scavenger oxyHb (20 microM) was perfused either concomitant with CysNO or just before ischemia. In neither case did oxyHb affect the cardioprotection afforded by CysNO. OxyHb alone, given in either time window, did not alter the course of ischemia-reperfusion injury. When nitrite was used in place of CysNO, no protective effects were observed. Perfusion with CysNO increased tissue S-nitrosothiol (RSNO) levels from an unmeasurable background to a value of about 15.7+/-4.1 pmol RSNO/mg protein, as measured by triiodide-based chemiluminescence in the presence and absence of mercury(II) chloride. In the presence of L-Leu, this value dropped to 0.4+/-0.3 pmol RSNO/mg protein. This study demonstrates that exposure to CysNO before ischemia increases tissue S-nitrosothiol levels, improves postischemic contractile dysfunction, and attenuates necrosis. The mechanism of cardioprotection requires the uptake of CysNO via the L-AT and does not seem to involve NO release either during CysNO exposure or during ischemia. This suggests that the protective effects of CysNO are mediated through the posttranslational modification of cellular proteins through an NO-independent transnitrosation mechanism.
[Show abstract][Hide abstract] ABSTRACT: L'S-nitrosilazione e' un processo di comunicazione cellulare ubiquitario nei sistemi biologici. Tuttavia, la ricerca sul signaling richiede metodi analitici per la determinazione delle specie dell'ossido nitrico (RSNOs) sensibili e specifici. La determinazione diretta delle RSNOs (metodi UV ed elettrochimici) e' limitata a concentrazioni micromolari. Approcci alternativi non rivelano RSNOs nella loro forma intatta, ma si basano sulla rivelazione indiretta dei loro metaboliti dopo fotolisi o riduzione chimica del legame S-NO: il radicale dell'ossido nitrico (NO), o il nitrito, suo prodotto di ossidazione e il prodotto tiolico. Il nitrito rilasciato e' stato rivelato mediante varie tecniche come la reazione di Griess, fluorimetria o rivelazione elettrochimica accoppiata o no a cromatografia liquida, o la gas cromatografia accoppiata a spettrometria di massa (GC-MS). Il radicale NO viene rivelato generalmente mediante chemiluminescenza o fluorimetria. Notoriamente, la misura di questi metaboliti in matrici biologiche e' difficile e richiede procedure complicate di manipolazione del campione, che coinvolgono l'interazione dei metaboliti degli RSNOs con i reattivi impiegati e con i costituenti della matrice biologica. Il tipo di matrice, la scelta della procedura di preparazione del campione, del sistema impiegato per la rottura del legame S-NO (fotolisi, HgCl2, HgCl2/V(III), KI/I2, Cys/KI/Cu(I), Cu(I)/Cys, Cu(I)/KI/I2, CO/Cu(I)/Cys, DTT), dell'analita rivelato (NO, nitrito o tioli), del reagente per la rivelazione (cromoforo, fluoroforo o ozono), della tecnica di rivelazione, possono pesantemente influenzare i risultati dell'analisi. Altri saggi per gli RSNOs ragionevolmente sensibili e accurati sono quelli basati sull'immunostaining. Ciascun saggio, tuttavia, ha dei limiti e dovrebbe essere complementato in maniera quantitativa da altri saggi. Il progresso continuo nell'ambito dei metodi analitici per il dosaggio degli RSNOs, del controllo delle procedure di campionamento e della fase preanalitica e' fondamentale per la comprensione del ruolo fisio-patologico degli RSNOs. Parole chiave: Ossido nitrico; S-nitrosotioli; Metodi Methods of assay of nitric oxide and its derivates. A review. S-Nitrosylation is a ubiquitous signaling process in biological systems. However, research regarding this signaling requires sensitive and specific analytical methods for the determination of nitric oxide species (RSNOs). Their direct quantitative UV or electrochemical detection has been limited to micromolar concentrations. Alternative approaches do not detect RSNOs in their intact form, but are based on indirect detection of their metabolites after photolysis or chemical reduction of S-NO bond: nitric oxide (NO) radical or its end oxidation product nitrite (NO2 - ) and the thiolic product. Releasable nitrite has been detected by various techniques such as the Griess reaction, fluorimetry and electrochemical detection, coupled or not with liquid chromatography, or gas chromatography-mass spectrometry (GC-MS). NO radical is generally detected by chemiluminescence or fluorimetry. Notoriously, the measurement of these metabolites in biological matrices is difficult and it requires complex sample handling, involving the interaction of these metabolites with various reactants and biological constituents. The sample matrix, the choice of sample preparation, of the system employed for the cleavage of S-NO bond (photolysis, HgCl2, HgCl2/V(III), KI/I2, Cys/KI/Cu(I), Cu(I)/Cys, Cu(I)/KI/I2, CO/Cu(I)/Cys, DTT), of the detected analyte (NO, nitrite, or thiol), of the detection reactant (chromophore, fluorophore or ozone), of the detection technique may strongly affect the results of the analysis. Other assays that provide reasonably sensitive and accurate data regarding biological S-nitrosothiols include immunostaining. Each assay, however, has limitations and should be quantitatively complemented by separate assays. Continued improvement in assays, in the control of sampling and pre-analytical procedures is fundamental to understand RSNO physio-pathological role.
[Show abstract][Hide abstract] ABSTRACT: Nitrite has emerged as an endogenous signaling molecule with potential therapeutic implications for cardiovascular disease. Steady-state levels of nitrite are derived in part from dietary sources; therefore, we investigated the effects of dietary nitrite and nitrate supplementation and deficiency on NO homeostasis and on the severity of myocardial ischemia-reperfusion (MI/R) injury. Mice fed a standard diet with supplementation of nitrite (50 mg/liter) in their drinking water for 7 days exhibited significantly higher plasma levels of nitrite, exhibited significantly higher myocardial levels of nitrite, nitroso, and nitrosyl-heme, and displayed a 48% reduction in infarct size (Inf) after MI/R. Supplemental nitrate (1 g/liter) in the drinking water for 7 days also increased blood and tissue NO products and significantly reduced Inf. A time course of ischemia-reperfusion revealed that nitrite was consumed during the ischemic phase, with an increase in nitroso/nitrosyl products in the heart. Mice fed a diet deficient in nitrite and nitrate for 7 days exhibited significantly diminished plasma and heart levels of nitrite and NO metabolites and a 59% increase in Inf after MI/R. Supplementation of nitrite in the drinking water for 7 days reversed the effects of nitrite deficiency. These data demonstrate the significant influence of dietary nitrite and nitrate intake on the maintenance of steady-state tissue nitrite/nitroso levels and illustrate the consequences of nitrite deficiency on the pathophysiology of MI/R injury. Therefore, nitrite and nitrate may serve as essential nutrients for optimal cardiovascular health and may provide a treatment modality for cardiovascular disease.
Proceedings of the National Academy of Sciences 12/2007; 104(48):19144-9. DOI:10.1073/pnas.0706579104 · 9.67 Impact Factor
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