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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; Cardiovascular diseases are associated with oxidative stress and a reduced bioavailability of nitric oxide (NO). To counteract both processes, the administration of S-nitrosoglutathione (GSNO) can be envisaged. GSNO is able to induce protein S-nitrosation (Pr-SNO), which is a post-translational modification of proteins, participating in the storage of NO in tissues, and protect thiol functions from oxidation. However, GSNO antioxidant power is poorly studied, which is probably linked to its low stability. This low stability can be addressed by nanotechnologies that will increase GSNO protection and provide a sustained release of the drug.

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

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.

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.