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Objective: Over the years, scientific investigations have proven the importance of selenium as an essential element for mammals, emphasizing its activity against many diseases and even its prophylactic effects. It is also established now that a malconsumption of selenium can be harmful. Therefore, the nature and the concentration of selenium and its derivatives found in the diet, the body, and even in the environment, for example, in the soil, should be determined carefully. Methods: In this review, analytical methods for speciation and determination of selenium concentrations in biological samples are summarized. Results: Methods ranging from routine to cutting-edge are explored, focusing on their analytical characteristics, such as specificity for discrete selenium species, sensitivity, accuracy, reproducibility, and skills required. Conclusion: There are already numerous studies regarding the analysis of selenium species. Beyond the method employed for actual measurements, we propose to review the preanalytic steps for sample handling in biological matrices, which directly affect results that will be more accurate with careful pretreatment. Furthermore, to reach better outcomes in terms of the identification of selenium species, different combinations of techniques might be the answer. We highlight here the last and the cutting-edge methods to identify and quantify selenium such as, high-performance liquid chromatography combined to inductively coupled plasma mass spectrometry (HPLC-ICP-MS), hydride generation atomic absorption spectrometry (HG-AAS), hydride-generation combined to atomic fluorescence spectrometry (HG-AFS), or to inductively coupled plasma optical emission spectrometry (HG-ICP-OES). This review emphasizes the importance of such investigations and the need to achieve reliable, safe, and effective quantification and methods of determination.

Endothelial dysfunction predisposing to cardiovascular diseases is defined as an imbalance in the production of vasodilating factors, such as nitric oxide (NO), and vasoconstrictive factors. To insure its physiological role, NO, a radical with very short half-life, requires to be stored and transported to its action site. S-nitrosothiols (RSNOs) like S-nitrosoglutathione (GSNO) represent the main form of NO storage within the vasculature. The NO store formed by RSNOs is still bioavailable to trigger vasorelaxation. In this way, RSNOs are an emerging class of NO donors with a potential to restore NO bioavailability within cardiovascular disorders. The aim of this study was to compare S-nitrosothiols ability, formed of peptide (glutathione) like the physiologic GSNO or derived from amino acids (cysteine, valine) like the synthetics S-nitroso-N-acetylcysteine (NACNO) and S-nitroso-N-acetylpenicillamine (SNAP), respectively, to produce a vascular store of NO either in endothelium-intact or endothelium-removed aortae in order to evaluate whether RSNOs can be used as therapeutics to compensate endothelial dysfunction. Sodium nitroprusside (SNP), a marketed drug already in clinics, was used as a non-RSNO NO-donor. Endothelium-intact or endothelium-removed aortae, isolated from normotensive Wistar rats, were exposed to RSNOs or SNP. Then, NO-derived (NOx) species, representing the NO store inside the vascular wall, were quantified using the diaminonaphthalene probe coupled to mercuric ions. The bioavailability of the NO store and its ability to induce vasodilation was tested using N-acetylcysteine, then its ability to counteract vasoconstriction was challenged using phenylephrine (PHE). All the studied RSNOs were able to generate a NO store materialized by a three to five times increase in NOx species inside aortae. NACNO was the most potent RSNO to produce a vascular NO store bioavailable for vasorelaxation and the most efficient to induce vascular hyporeactivity to PHE in endothelium-removed aortae. GSNO and SNAP were equivalent and more efficient than SNP. In endothelium-intact aortae, the NO store was also formed whereas it seemed less available for vasorelaxation and did not influence PHE-induced vasoconstriction. In conclusion, RSNOs - NACNO in a better extent - are able to restore NO bioavailability as a functional NO store within the vessel wall, especially when the endothelium is removed. This was associated with a hyporeactivity to the vasoconstrictive agent phenylephrine. Treatment with RSNOs could present a benefit to restore NO-dependent functions in pathological states associated with injured endothelium.

International audience; S‐nitrosoglutathione (GSNO), which is involved in the transport and the storage of NO, induces vasorelaxation. It requires gamma‐glutamyl transferase (GGT), an enzyme present on the endothelium, to transfer NO into the cell. We evaluated whether aging and hypertension, which induce NO‐related dilating dysfunction, are associated with decreased vascular GGT activity and modify the vasorelaxant effect of GSNO. Thoracic aortic rings isolated from male spontaneous hypertensive rats (SHR) and Wistar‐Kyoto rats (WKY) aged 20–22 (adult) or 57–60 weeks (mature) were preconstricted with phenylephrine, then submitted to concentration‐vasorelaxant response curves (maximal response: Emax; pD2) to GSNO and carbachol (the latter to measure NO‐related dilating function). GGT activity was measured using chromogenic substrate. Both aging and hypertension lowered Emax values for carbachol (Emax −8% in adult SHR, −42% in mature SHR vs. age‐matched WKY, page and phypertension < 0.05) demonstrating NO‐related dilating dysfunction. Aortic GGT activity also decreased with aging and hypertension (−22% in adult and −75%, reaching 3 nmol/min/g of tissue, in mature SHR vs. 12 in age‐matched WKY and 23 in adult WKY, page and phypertension < 0.05). The pD2 values of GSNO were similar in mature SHR and WKY but higher in adult SHR (pinteraction < 0.05). Aging in hypertensive rats decreased NO‐related vasorelaxant function and vascular GGT activity, but did not lower the vasorelaxant response to GSNO. This opens perspectives for GSNO‐based therapeutics restoring nitric oxide bioavailability and vascular protection in a context of endothelial dysfunction.

International audience; Background and PurposeAngiotensin II (AngII) and NO regulate the cerebral circulation. AngII AT1 receptors exert ligand‐dependent and ligand‐independent (myogenic tone [MT]) vasoconstriction of cerebral vessels. NO induces post‐translational modifications of proteins such as S‐nitrosation (redox modification of cysteine residues). In cultured cells, S‐nitrosation decreases AngII's affinity for the AT1 receptor. The present work evaluated the functional consequences of S‐nitrosation on both AngII‐dependent and AngII‐independent cerebrovascular responses.Experimental ApproachS‐Nitrosation was induced in rat isolated middle cerebral arteries by pretreatment with the NO donors, S‐nitrosoglutathione (GSNO) or sodium nitroprusside (SNP). Agonist‐dependent activation of AT1 receptors was evaluated by obtaining concentration–response curves to AngII. Ligand‐independent activation of AT1 receptors was evaluated by calculating MT (active vs. passive diameter) at pressures ranging from 20 to 200 mmHg in the presence or not of a selective AT1 receptor inverse agonist.Key ResultsGSNO or SNP completely abolished the AngII‐dependent AT1 receptor‐mediated vasoconstriction of cerebral arteries. GSNO had no impact on responses to other vasoconstrictors sharing (phenylephrine, U46619) or not (5‐HT) the same signalling pathway. MT was reduced by GSNO, and the addition of losartan did not further decrease MT, suggesting that GSNO blocks AT1 receptor‐dependent MT. Ascorbate (which reduces S‐nitrosated compounds) restored the response to AngII but not the soluble GC inhibitor ODQ, suggesting that these effects are mediated by S‐nitrosation rather than by S‐nitrosylation.Conclusions and ImplicationsIn rat middle cerebral arteries, GSNO pretreatment specifically affects the AT1 receptor and reduces both AngII‐dependent and AngII‐independent activation, most likely through AT1 receptor S‐nitrosation

International audience; Alginate/chitosan nanocomposite particles (GSNO-acNCPs), i.e. S-nitrosoglutathione (GSNO) loaded polymeric nanoparticles incorporated into an alginate and chitosan matrix, were developed to increase the effective GSNO loading capacity, a nitric oxide (NO) donor, and to sustain its release from the intestine following oral administration. Compared with free GSNO and GSNO loaded nanoparticles, GSNO-acNCPs promoted 2.7-fold GSNO permeation through a model of intestinal barrier (Caco-2 cells). After oral administration to Wistar rats, GSNO-acNCPs promoted NO storage into the aorta during at least 17 h, as highlighted by (i) a long-lasting hyporeactivity to phenylephrine (decrease in maximum vasoconstrictive effect of aortic rings) and (ii) N-acetylcysteine (a thiol which can displace NO from tissues)-induced vasodilation of aortic rings preconstricted with phenylephrine. In conclusion, GSNO-acNCPs enhance GSNO intestinal absorption and promote the formation of releasable NO stores into the rat aorta. GSNO-acNCPs are promising carriers for chronic oral application devoted to the treatment of cardiovascular diseases. In the cardiovascular system, deficiency of endogenous nitric oxide (NO) is the consequence of either insufficient synthesis (endothelium dysfunction) 1,2 or excessive NO degradation 3,4 (increased oxidative or nitrosative stresses, decreased antioxidant enzyme activity). NO depletion is one of the key factors in the initiation and progress of many diseases, such as atherosclerosis, 5 pulmonary hypertension, 6 thrombosis, 7 ischemia 8 and cardiac arrhythmia. 9 To maintain an appropriate level of NO and treat NO deficiency, several NO-related therapeutics have been developed such as nitrosamines, organic nitrates, metal–NO complexes, N-diazeniumdiolates. However, all act at very short term and lead to tolerance phenomena. S-nitrosothiols (RSNOs) present the advantage of a longer half-life, with no tolerance nor oxidative stress induction. Under physiological conditions, S-nitrosoglutathione (GSNO), a major endogenous RSNO, is one of the main storage forms of NO in tissues. 10 GSNO has been investigated for its powerful antiplatelet activity, 11,12 arterio/venous selective vasodilator effects, 13,14 antimicrobial 15 and antithrombotic effects. 16 Despite such therapeutic potencies, GSNO pharmaceutical forms are still lacking. This may be related to the fast and often unpredictable rate of decomposition of GSNO. In vitro, because of pH-, light-and temperature-dependent sensitivities , GSNO is susceptible to many degradation processes

BACKGROUND AND PURPOSE: Angiotensin II (AngII) and NO regulate the cerebral circulation. AngII AT(1) receptors exert ligand‐dependent and ligand‐independent (myogenic tone [MT]) vasoconstriction of cerebral vessels. NO induces post‐translational modifications of proteins such as S‐nitrosation (redox modification of cysteine residues). In cultured cells, S‐nitrosation decreases AngII's affinity for the AT(1) receptor. The present work evaluated the functional consequences of S‐nitrosation on both AngII‐dependent and AngII‐independent cerebrovascular responses. EXPERIMENTAL APPROACH: S‐Nitrosation was induced in rat isolated middle cerebral arteries by pretreatment with the NO donors, S‐nitrosoglutathione (GSNO) or sodium nitroprusside (SNP). Agonist‐dependent activation of AT(1) receptors was evaluated by obtaining concentration–response curves to AngII. Ligand‐independent activation of AT(1) receptors was evaluated by calculating MT (active vs. passive diameter) at pressures ranging from 20 to 200 mmHg in the presence or not of a selective AT(1) receptor inverse agonist. KEY RESULTS: GSNO or SNP completely abolished the AngII‐dependent AT(1) receptor‐mediated vasoconstriction of cerebral arteries. GSNO had no impact on responses to other vasoconstrictors sharing (phenylephrine, U46619) or not (5‐HT) the same signalling pathway. MT was reduced by GSNO, and the addition of losartan did not further decrease MT, suggesting that GSNO blocks AT(1) receptor‐dependent MT. Ascorbate (which reduces S‐nitrosated compounds) restored the response to AngII but not the soluble GC inhibitor ODQ, suggesting that these effects are mediated by S‐nitrosation rather than by S‐nitrosylation. CONCLUSIONS AND IMPLICATIONS: In rat middle cerebral arteries, GSNO pretreatment specifically affects the AT(1) receptor and reduces both AngII‐dependent and AngII‐independent activation, most likely through AT(1) receptor S‐nitrosation.

International audience; Purpose: In a previous work, we have synthetized a new dinitrosothiol, i.e. S,S′-dinitrosobucillamine BUC(NO)2 combining S-nitroso-N-acetylpenicillamine (SNAP) and S-nitroso-N-acetylcysteine (NACNO) in its structure. When exposed to isolated aorta, we observed a 1.5-fold increase of •NO content and a more potent vasorelaxation (1 log higher pD2) compared to NACNO and SNAP alone or combined (Dahboul et al., 2014). In the present study, we analyzed the thermodynamics and kinetics for the release of •NO through computational modeling techniques and correlated it to plasma assays. Then BUC(NO)2 was administered in vivo to rats, assuming it will induce higher and/or longer hypotensive effects than its two constitutive S-mononitrosothiols.Methods: Free energies for the release of •NO entities have been computed at the density functional theory level assuming an implicit model for the aqueous environment. Degradation products of BUC(NO)2 were evaluated in vitro under heating and oxidizing conditions using HPLC coupled with tandem mass spectrometry (MS/MS). Plasma from rats were spiked with RSNO and kinetics of RSNO degradation was measured using the classical Griess-Saville method. Blood pressure was measured in awake male Wistar rats using telemetry (n = 5, each as its own control, 48 h wash-out periods between subcutaneous injections under transient isoflurane anesthesia, random order: 7 mL/kg vehicle, 3.5, 7, 14 μmol/kg SNAP, NACNO, BUC(NO)2 and an equimolar mixture of SNAP + NACNO in order to mimic the number of •NO contained in BUC(NO)2). Variations of mean (ΔMAP, reflecting arterial dilation) and pulse arterial pressures (ΔPAP, indirectly reflecting venodilation, used to determine effect duration) vs. baseline were recorded for 4 h.Results: Computational modeling highlights the fact that the release of the first •NO radical in BUC(NO)2 requires a free energy which is intermediate between the values obtained for SNAP and NACNO. However, the release of the second •NO radical is significantly favored by the concerted formation of an intramolecular disulfide bond. The corresponding oxidized compound was also characterized as related substance obtained under degradation conditions. The in vitro degradation rate of BUC(NO)2 was significantly greater than for the other RSNO. For equivalent low and medium •NO-load, BUC(NO)2 produced a hypotension identical to NACNO, SNAP and the equimolar mixture of SNAP + NACNO, but its effect was greater at higher doses (-62 ± 8 and -47 ± 14 mmHg, maximum ΔMAP for BUC(NO)2 and SNAP + NACNO, respectively). Its duration of effect on PAP (-50%) lasted from 35 to 95 min, i.e. shorter than for the other RSNO (from 90 to 135 min for the mixture SNAP + NACNO).Conclusion: A faster metabolism explains the abilities of BUC(NO)2 to release higher amounts of •NO and to induce larger hypotension but shorter-lasting effects than those induced by the SNAP + NACNO mixture, despite an equivalent •NO-load.

In this study, we evaluated the impact of the calcium concentration present in the perfusion medium (1.2-3 mM) on contractile performance, lactate dehydrogenase (LDH) release and secondary free radical production during post-ischaemic reperfusion of isolated rat hearts. The impact of calcium concentration on post-ischaemic free radical release was investigated using the Electron Paramagnetic Resonance (EPR) technique and spin trapping with the lipophilic spin trap alpha-phenyl N-tert-butylnitrone (PBN). The evolution of left ventricular end diastolic pressure (LVEDP) in both groups followed the same pattern, but we observed that ischaemic and post-ischaemic contracture was more severe in the group of hearts perfused with 3 mM of calcium as compared with those perfused with 1.2 mM of calcium. A large release of alkyl/alkoxyl species occurred in all hearts from the onset of reperfusion and remained at a high level during the 30 min of reperfusion with no return to basal values. The kinetics and intensity of these releases were the same in both groups. In conclusion, in a range of extracellular calcium levels (1.2-3 mM), the release of alkyl/alkoxyls radicals does not seem to be calcium-dependent. Due to the protective actions of PBN itself, the results of simultaneous investigations of the effects of radical scavengers on isolated heart function may be limited. However, since many pharmacological properties (antioxidant, cellular protector, NO precursor ...) are attributed to PBN, studies investigating oxidative stress with such a multi-faceted tool make interpretation difficult.

Spin traps might exert antioxidant cardioprotective effects during myocardial ischaemia-reperfusion where free radicals are thought to be responsible for the occurrence of reperfusion injury. The aim of our study was to investigate the effects of two new alpha-phenyl N-tert-butylnitrone (PBN)-derived beta-phosphorylated nitrones: 2-N-oxy-N-[benzylidène amino] diéthyl propyl-2-phosphate (PPN) and 1-diethoxyphosphoryl-1-methyl-N-[(1-oxido-pyridin-1-ium-4-yl) methylidene] ethylamine N-oxide (4-PyOPN) compared with PBN on (1) the evolution of cardiovascular parameters and (2) the postischaemic recovery. Anaesthetized rats were injected with 120 micro mol/kg of the nitrones or 14 micro mol/kg of amiodarone, used as a reference antidysrhythmic drug. Ischaemia was induced in vivo through ligation of the left anterior descending coronary artery for 5 min followed by 15 min of reperfusion after release. Cardiovascular parameters and occurrence of ventricular premature beats (VPB), ventricular tachycardia (VT) and fibrillation (VF) were recorded throughout the experiment. Under nonischaemic conditions, none of the three spin traps was shown to modify cardiovascular parameters during the 25-min measurement period. Solvent-treated (NaCl 0.9%) animals challenged with ischaemia-reperfusion exhibited 39 +/- 10 VPB, 156 +/- 39 s of VT and 60% mortality caused by sustained VF. Nitrones improved slightly postischaemic recovery, reducing the occurrence of VF and mortality to 33% whereas amiodarone injection totally suppressed rhythm disturbances and mortality. Our study has shown only limited antidysrhythmic cardioprotective effects of PBN-derived beta-phosphorylated nitrones during reperfusion after a regional myocardial ischaemia but also minor antioxidant properties of these spin trapping agents.

International audience; Angiotensin II (Ang II) induces constriction (AT(1)) and dilation (AT(2) receptors) of cerebral arterioles. High sodium intake induces changes in receptors expression and loss of AT(2)-mediated vasodilation in extracerebral vessels. We investigated whether high salt modifies the AT(2)-mediated response of cerebral arterioles.Three-month-old male Wistar rats received drinking water supplemented or not with 1% NaCl. We measured at day 4 or 30 plasma aldosterone concentration, AT receptors expression (brain microvessels, western blot, RT-qPCR), internal diameter of pial arterioles (cranial window) following suffusion with Ang II (10(-6) mol/l, or 10(-8) mol/l + losartan 10(-5) mol/l), serotonin (5-HT, 10(-6) mol/l), sodium nitroprusside (10(-5) mol/l) and adenosine diphosphate (ADP, 10(-4) mol/l).High salt did not modify arterial pressure, baseline arteriolar diameter, vasoconstriction to Ang II or 5-HT, nor vasodilation to SNP. High salt lowered plasma aldosterone concentration (d4 138 ± 71 not significant vs. control 338 ± 73; d30 150 ± 21 P < 0.05 vs. control 517 ± 79 μmol/l). AT receptors mRNA did not change while protein level of AT(2) receptors decreased at d4 (64 ± 9% of control, P < 0.05). AT(2)-mediated vasodilation (control d4; d30 8 ± 2; 5 ± 2%) was abolished at d4 (-2 ± 2%, P < 0.05) and reversed to vasoconstriction at d30 (-7 ± 2%, P < 0.05). ADP-induced vasodilation is abolished at d30 (2 ± 2, P < 0.05 vs. control 19 ± 4%).High salt specifically abolishes AT(2)-mediated vasodilation, immediately, via decreased level of AT(2) receptor protein, and after 30 days, in association with abolition of endothelial vasodilation. Such loss of AT(2)-mediated vasodilation may be deleterious in case of stroke.

Several reports suggest that consumption of red wine is associated with a lower risk of stroke. We investigated the chronic effect of red wine polyphenols (RWP) on the functional and structural characteristics of cerebral arterioles in chronic hypertension, which is an important risk factor of stroke. Spontaneous hypertensive rats (SHR) were treated with RWP extract (100 mg/kg/day in drinking water) for 10 weeks. We measured the effect of agonist- and hypotension-induced changes in internal diameter of cerebral arterioles using an open cranial window technique. Wall mechanical parameters were determined in deactivated cerebral arterioles. The activity of antioxidant enzymes in plasma was determined. Adenosine diphosphate-induced vasodilatation was decreased by 48% in SHR and normalized in SHR treated with RWP. RWP had no effect on hypotension-induced dilatation. RWP decreased the wall thickness/external diameter ratio by 13% and significantly shifted the stress-strain relationship of the arteriole wall to the left. There was a decrease in glutathione-S-transferase and glutathione peroxidase after treatment of RWP in SHR. In summary, chronic oral administration of RWP to SHR improved endothelium-dependent dilatation, normalized wall stress and diameter, and altered the systemic antioxidant state. These effects of RWP could be useful in the prevention of stroke in hypertensive patients.

Background and Purpose— In light of the equivocal data on the cerebral vasoconstrictor and vasodilator actions of angiotensin II (Ang II) and the potential clinical importance of this, we investigated the effects of Ang II on rat pial arterioles. Methods— We determined the effect of Ang I (3.10 −6 mol/L) in the absence and presence of the converting enzyme inhibitor, captopril (10 −5 mol/L) in cerebral arterioles of male Wistar rats (open-skull preparation), and those of Ang II (3.10 −12 to 3.10 −6 mol/L) in the absence and presence of the Ang II receptor (AT 1 ) antagonist, telmisartan (10 −5 mol/L) or the AT 2 antagonist, PD123319 (10 −5 mol/L). We examined the effect of PD123319 (10 −5 mol/L) and the Ca 2+ -activated K + (BK Ca ) channel blocker, tetraethylammonium (10 −4 mol/L) on the Ang II responses in the presence of telmisartan (10 −5 mol/L). Results— Ang II-induced dose-dependent constriction with a maximum decrease of −20.1±1.0% at 10 −6 mol/L. Captopril significantly decreased Ang I-induced vasoconstriction (−4.0±0.9 versus −21.3±2.5%; n=4). Telmisartan reversed Ang II-induced vasoconstriction (9.5±2.5 versus −20.1±1% at 10 −6 mol/L; n=5). PD123319 significantly increased Ang II-induced vasoconstriction (−12.9±0.8 versus −10.2±0.4% at 10 −6 mol/L; n=5). PD123319 abolished (−2.6±0.7 versus 9.3±1.1% at 10 −6 mol/L; n=5) whereas tetraethylammonium reversed (−12.1±1.6 versus 9.9±1.0% at 10 −6 mol/L; n=4) Ang II-induced vasodilatation in the presence of telmisartan. Conclusion— Angiotensin is converted locally into Ang II; the overall effect of Ang II is vasoconstrictor following stimulation of the AT 1 receptor, but a vasodilator response can be evoked following stimulation of the AT 2 receptor and activation of BK Ca .

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.

The aim of our work was to evaluate the enzymatic pathways involved in the release of nitric oxide and in the vasorelaxant effect of S-nitrosothiols (RSNO). We were interested in two enzymes: the gamma-glutamyltransferase (GGT) and the protein disulfide isomerase (PDI), because they play an important role in RSNO denitrosation. Two RSNO were studied: S-nitrosoglutathione (GSNO), an endogenous mononitrosothiol, and S,S'-dinitrosobucillamine (BUC(NO)2), a new dinitrosothiol. We synthesized RSNO and we structurally characterized these products. The resulting data are consistent with the expected structure. Our products have a high purity (>97%) and a limited amount of impurities allowing their suitable use in biological experiments. The vasorelaxant effects of RSNO and the involvement of GGT and PDI were evaluated. The results indicate that purified GGT and PDI denitrosate GSNO in vitro. Furthermore, we demonstrated by using an ex vivo model consisting in an aortic ring isolated from Wistar rat that the vasorelaxant effect of GSNO (EC50=3,2±0,5.10-7 M) was dependent on the endothelium and GGT and PDI activities. As concerns BUC(NO)2, this dinitrosothiol catabolized in vitro by PDI, is more potent (EC50=2,2±0,2.10-8 M) than the most of nitrosothiols described in the literature. This vasorelaxation effect was dependent on PDI activity. In conclusion, our data led to a better understanding of the enzymatic mechanisms involved in the vascular effects of RSNO, which will permit, in physiopathological context, to optimize the choice of the best RSNO for use in a therapeutic purpose; L'objectif de notre travail a consisté en l'étude des mécanismes enzymatiques impliqués dans la libération de l'oxyde nitrique à partir des S-nitrosothiols (RSNO) et dans leurs effets vasorelaxants. Notre intérêt porte sur deux enzymes : la gamma-glutamyltransférase (GGT) et la protéine disulfure isomérase (PDI) car elles jouent un rôle important dans la dénitrosation des RSNO. Nous avons choisi d'étudier la dénitrosation de deux RSNO : le S-nitrosoglutathion (GSNO), un mononitrosothiol endogène et la S,S'-dinitrosobucillamine (BUC(NO)2), un nouveau dinitrosothiol. Nous avons synthétisé ces RSNO et nous avons vérifié la nature du produit obtenu par une caractérisation physico-chimique complète. Les analyses ont montré que ces RSNO présentent une pureté élevée (>97%) avec un niveau faible d'impuretés permettant leur utilisation dans des expérimentations biologiques. Les effets vasorelaxants des RSNO ainsi que l'implication des enzymes ont été évalués. Nos résultats montrent que la GGT et la PDI sont capables de dénitroser in vitro le GSNO. Le modèle ex vivo d'anneau aortique isolé de rat Wistar nous a permis de démontrer que l'effet vasorelaxant de GSNO (CE50=3,2±0,5.10-7 M) est dépendant de l'endothélium et de l'activité de la GGT et de la PDI. Concernant la BUC(NO)2, ce dinitrosothiol est catabolisé in vitro par la PDI, est un vasorelaxant plus puissant que la plupart des RSNO (CE50=2,2±0,2.10-8 M) et met en jeu l'activité de la PDI vasculaire. Nos travaux ont conduit à une meilleure compréhension des mécanismes enzymatiques impliqués dans les effets vasculaires des RSNO, ce qui permettra d'optimiser le choix de la meilleure RSNO à utiliser dans une finalité thérapeutique

The aim of our work was to evaluate the enzymatic pathways involved in the release of nitric oxide and in the vasorelaxant effect of S-nitrosothiols (RSNO). We were interested in two enzymes: the gamma-glutamyltransferase (GGT) and the protein disulfide isomerase (PDI), because they play an important role in RSNO denitrosation. Two RSNO were studied: S-nitrosoglutathione (GSNO), an endogenous mononitrosothiol, and S,S'-dinitrosobucillamine (BUC(NO)2), a new dinitrosothiol. We synthesized RSNO and we structurally characterized these products. The resulting data are consistent with the expected structure. Our products have a high purity (>97%) and a limited amount of impurities allowing their suitable use in biological experiments. The vasorelaxant effects of RSNO and the involvement of GGT and PDI were evaluated. The results indicate that purified GGT and PDI denitrosate GSNO in vitro. Furthermore, we demonstrated by using an ex vivo model consisting in an aortic ring isolated from Wistar rat that the vasorelaxant effect of GSNO (EC50=3,2±0,5.10-7 M) was dependent on the endothelium and GGT and PDI activities. As concerns BUC(NO)2, this dinitrosothiol catabolized in vitro by PDI, is more potent (EC50=2,2±0,2.10-8 M) than the most of nitrosothiols described in the literature. This vasorelaxation effect was dependent on PDI activity. In conclusion, our data led to a better understanding of the enzymatic mechanisms involved in the vascular effects of RSNO, which will permit, in physiopathological context, to optimize the choice of the best RSNO for use in a therapeutic purpose, L'objectif de notre travail a consisté en l'étude des mécanismes enzymatiques impliqués dans la libération de l'oxyde nitrique à partir des S-nitrosothiols (RSNO) et dans leurs effets vasorelaxants. Notre intérêt porte sur deux enzymes : la gamma-glutamyltransférase (GGT) et la protéine disulfure isomérase (PDI) car elles jouent un rôle important dans la dénitrosation des RSNO. Nous avons choisi d'étudier la dénitrosation de deux RSNO : le S-nitrosoglutathion (GSNO), un mononitrosothiol endogène et la S,S'-dinitrosobucillamine (BUC(NO)2), un nouveau dinitrosothiol. Nous avons synthétisé ces RSNO et nous avons vérifié la nature du produit obtenu par une caractérisation physico-chimique complète. Les analyses ont montré que ces RSNO présentent une pureté élevée (>97%) avec un niveau faible d'impuretés permettant leur utilisation dans des expérimentations biologiques. Les effets vasorelaxants des RSNO ainsi que l'implication des enzymes ont été évalués. Nos résultats montrent que la GGT et la PDI sont capables de dénitroser in vitro le GSNO. Le modèle ex vivo d'anneau aortique isolé de rat Wistar nous a permis de démontrer que l'effet vasorelaxant de GSNO (CE50=3,2±0,5.10-7 M) est dépendant de l'endothélium et de l'activité de la GGT et de la PDI. Concernant la BUC(NO)2, ce dinitrosothiol est catabolisé in vitro par la PDI, est un vasorelaxant plus puissant que la plupart des RSNO (CE50=2,2±0,2.10-8 M) et met en jeu l'activité de la PDI vasculaire. Nos travaux ont conduit à une meilleure compréhension des mécanismes enzymatiques impliqués dans les effets vasculaires des RSNO, ce qui permettra d'optimiser le choix de la meilleure RSNO à utiliser dans une finalité thérapeutique