Many cardiovascular diseases are associated with a decreased endothelial-dependent bioavailability of nitric oxide (NO), leading to impaired vasodilation, pro-inflammatory/oxidative, pro-proliferative, and pro-thrombotic status (Vanhoutte and Boulanger, 1995; Le Brocq et al., 2008). Atherosclerosis, for example, which mainly concerns large conductance arteries where vasoactive functions depend specifically on the bioavailability of NO (Luksha et al., 2009) leads to the use of several NO donors (organic nitrates) for therapeutics. However, these treatments are known to provide fast NO release concomitant with induction of oxidative stress and tolerance (Bauer and Fung, 1991; Parker and Gori, 2001). New NO-donors, such as S-nitrosothiols have been proposed as interesting therapeutic alternatives as they do not present the drawbacks of organic nitrates.

S-nitrosoglutathione (GSNO), the nitrosated form of glutathione, is currently investigated as NO- donor to restore NO homeostasis, and is an interesting candidate for therapeutics as it mimics endogenous GSNO-related functions. Endogenous cellular formation of GSNO involves either direct reaction of NO/nitrosating species with GSH, or previous formation of protein-based or low molecular weight S-nitrosothiols followed by subsequent transnitrosation to GSH (Al-Sa'doni and Ferro, 2004; Broniowska et al., 2013). The ability of GSNO to transfer its NO to cystein residue of endogenous peptides or proteins via dynamic processes of nitrosation/denitrosation (Gaucher et al., 2013) explains its ability to store and transport NO to sites of utilization in the body (Wu et al., 2016).

Gamma-glutamyl transferase (GGT) is one of the enzyme activity implicated in the release of NO from GSNO and its uptake into the cell (Hogg et al., 1997; Bramanti et al., 2009). GGT specifically catalyzes endogenous as exogenous GSNO breakdown producing cysteinylglycine and NO in endothelial cells. There, either NO diffuses to the smooth muscle cells to activate the soluble guanylyl cyclase/cyclic guanosine monophosphate pathway and induce vasorelaxation (Tullett et al., 2001; Alencar et al., 2003; Heikal et al., 2011), or it reacts with endothelial glutathione or proteins cysteine residue to form S-nitrosothiols. Those S-nitrosothiols may release NO through transnitrosation processes involving enzymes such as the system thioredoxin/thioredoxin reductase (Marozkina and Gaston, 2012; Gaucher et al., 2013). We have previously documented that endothelial GGT is critical for GSNO-dependent NO-delivery and vasorelaxation in aortic rings isolated from normotensive rats (Dahboul et al., 2012). In this context, GSNO and its cellular metabolism enzymes are likely to be involved in blood pressure regulation (Ishibashi et al., 2011).

In the present study, we evaluated GSNO-induced vasorelaxation in the Spontaneous Hypertensive Rat (SHR), with the hypothesis that hypertension may impair GGT activity of the vessel wall. Therefore, the bioactivity of exogenous treatment with GSNO would be modified. In the present study, we analyzed concentration-vasorelaxant response curves (maximal response E_max and pD₂ calculated as –log EC₅₀, the half maximal effective concentration) to carbachol and sodium nitroprusside in order to evaluate endothelial dependent and independent NO-induced vasodilation, and to GSNO (exogenous NO vasodilation depending from the endothelial GGT activity). As protein disulfide isomerase (PDI), a membrane enzyme from the redoxins family, has also been reported to be involved in the release of NO from GSNO (Heikal et al., 2011), we also evaluated whether the vasorelaxant effect of GSNO may be warranted through such alternative enzymatic activity.

Thoracic aortic activity of GGT and glutathione content of the aortic wall decreased both by 33% in SHR and by 45 and 53% in SHR-S (Figure 1).

Each individual concentration response curve to sodium nitroprusside, carbachol and GSNO fitted the Hill model. Responses (E_max and pD₂) to sodium nitroprusside were similar in all groups (Table 1). Neither E_max nor pD₂ for carbachol changed in SHR compared to WKY. E_max for carbachol decreased by 14% in high salt diet SHR (p < 0.05 vs. WKY-S) with no change in pD₂ (Figure 2).

Concentration response curves to GSNO did not show any decrease in E_max with hypertension alone nor with high salt diet as compared to their corresponding control WKY. The concentration response curves were even slightly shifted to lower concentrations of GSNO in SHR (pD₂ + 0.4) and SHR-S (pD₂ + 0.5) vs. WKY and WKY-S, respectively (Figure 3). In the presence of serine borate complex (an inhibitor of GGT), or in the presence of bacitracin (an inhibitor of PDI), the concentration response curves to GSNO were shifted to the right in all groups as shown by the significant decreases in pD₂ (−0.5 to −1.5 log) (Figure 4).

In SHR and SHR-S of the present study, muscle cells dilation capacities (responses to sodium nitroprusside) were maintained, while endothelial NO-dependent vasodilation started to decrease in SHR-S. Aortic GGT activity decreased in SHR and SHR-S, but this did not impair the capacity of GSNO to relax isolated aortic rings. The pD₂ values for GSNO even increased.

To our knowledge, this is one of the few study, with that of Yilmaz et al., to address the possibility that impairment of NO homeostasis might be related to alterations in plasma (Yilmaz et al., 2013) or in vessel wall GGT activity.

The degree of severity of endothelium dependent NO release vasodilation is commonly evaluated by a decrease in the response to endothelial muscarinic receptor stimulated by carbachol or acetylcholine, in the absence of any modification of the endothelial independent NO vasodilation (no change in response to nitroprusside, as observed in the present study). The extremely abundant literature using such experimental approaches interprets endothelial NO-dependent dysfunction on the basis of a decrease in either pD₂ or E_max or both (Duarte et al., 2001; Demougeot et al., 2005; Bagnost et al., 2008; Kane et al., 2010; Isabelle et al., 2012). In the present study, as those of Isabelle et al. (2012) or Demougeot et al. (2005), 20-week-old adult SHR did not show any decrease in maximal effect for carbachol nor acetylcholine. In salt-treated SHR of the present study, endothelium dependent NO release vasodilation was weak with a −14% decrease in E_max for carbachol. It has to be noticed that global endothelial dysfunction in SHR not only involves decreased NO bioavailability but also decreased production of endothelium-hyperpolarizing factor and prostacyclin (Deanfield et al., 2005; Schini-Kerth et al., 2011), higher production of endothelium-dependent vasoconstrictive agents (Touyz and Schiffrin, 2004; Félétou et al., 2009) and possible long term structural changes (Touyz and Schiffrin, 2004; Lee and Griendling, 2008). For other cardiovascular diseases, such as atherosclerosis, the prominent feature for endothelial dysfunction lies on the reduction in NO bioavailability, which is regarded as a major factor in the pathogenesis of the disease (Mudau et al., 2012; Jensen and Mehta, 2016; Vanhoutte et al., 2016).

Several recent studies have implicated the GGT activity in cardiovascular diseases. In particular, increased levels of GGT in serum have been correlated with hypertension (Mason et al., 2010) as well as with impaired aortic elasticity in prehypertensive patients (Celik et al., 2010). As GGT is implicated in GSNO catabolism and NO release (Hogg et al., 1997; Bramanti et al., 2009), it is possible that increasing levels of serum GGT may reduce the bioavailability of GSNO in blood, thus hampering its peripheral utilization for regulation of vascular tone. We have previously shown that increasing levels of GGT activity in blood do correspond to increased rates of clearance of exogenously added GSNO (Bramanti et al., 2009). Another study conducted in patients with chronic kidney disease showed increased serum GGT activity in association with endothelial dysfunction (Yilmaz et al., 2013).

Besides the soluble enzyme found in serum, GGT is also present at cellular level in vascular tissues. Substantial levels of the enzyme are expressed in arterial endothelium (Cotgreave and Schuppe-Koistinen, 1994). We have previously shown that such GGT activity is involved in the utilization of GSNO, in that it promotes the local release of NO from GSNO thus mediating its vasorelaxant effect (Dahboul et al., 2012). In atherosclerotic lesions, GGT was found to accumulate in plaques, in association with cells of the macrophagic lineage as well as with the lipid core (Paolicchi et al., 2004; Franzini et al., 2009). This accumulation may derive both from insudation of circulating GGT and from cellular macrophages dependent release of GGT. Moreover, several GGT fractions may be involved (Franzini et al., 2008). Finally, the relationship between tissue and circulating GGT are complex, especially in a pathological environment associating inflammation (atherosclerosis) and/or oxidative stress (present).

A loss of GGT activity was described in endothelial cells submitted to oxidative stress (Muruganandam et al., 2011). Therefore, the pro-oxidant environment associated with hypertension, as witnessed by the 33–53% fall in aortic wall glutathione content in SHR and SHR-S, could participate in the decrease in aortic wall GGT activity we observed. However, this decrease in aortic wall GGT activity did not impair the vasorelaxant effect of GSNO. Furthermore, GSNO-mediated vasodilation was still dependent on GGT, as shown by our experiments under inhibition with serine-borate complex. This suggests that (i) even if it decreases, the GGT activity remains sufficient to maintain GSNO vasorelaxant effect and/or (ii) other pathway of GSNO bioactivation are substituting for GGT. Membrane PDI, a membrane enzyme from the redoxins family, which is involved in the release of NO from GSNO, and in GSNO vasorelaxant effect (Heikal et al., 2011), increases its expression under oxidative stress (Janiszewski et al., 2005; Gaucher et al., 2013). SHR of the present study developed oxidative stress (decreased GSH content, Figure 1) (e.g., through upregulation of NOX; Tabet et al., 2008; Montezano and Touyz, 2012). Moreover, inhibition of PDI with bacitracin decreased the pD₂ values of GSNO in SHR and SHR-S demonstrating that the vasorelaxant effect of GSNO was dependent on PDI. Therefore, the decreased GGT activity occurring during hypertension may be balanced by an oxidative stress-related increase in PDI expression and/or activity, slightly improving vasorelaxant effects of GSNO in SHR and SHR-S.

One limitation of this study relies on the use of bacitracin. Bacitracin is not a specific inhibitor of PDI, which displays several activities (oxidoreductase, chaperone, and isomerase, Khan and Mutus, 2014). However, most of those activities are involved in the release of NO from GSNO (Shah et al., 2007).

In conclusion, our data suggest that hypertension is accompanied by a decreased activity of aortic GGT, one of the enzymes catalyzing the release of bioactive NO from GSNO. Nevertheless, vasorelaxation induced by GSNO is slightly improved. We suggest that under hypertension and oxidative stress, GSNO effect is likely warranted by the concomitant action of PDI, the other major (oxidative stress-inducible) enzyme involved in NO release from GSNO.

MP and FD performed the experiments, acquired data and interpreted the results. CP designed the work, acquired data, interpreted the results and drafted the manuscript. IL designed the work, interpreted the data, drafted, and revised the manuscript. AP and PL designed the work and revised the manuscript. All authors gave final approval of the manuscript to be published.

This work was supported by the French Ministry of Education, Research and Technology (Paris, France, EA3452), Région Lorraine and Université de Lorraine (France, project AO186 “Rôles des enzymes impliquées dans la libération de NO à partir des S-nitrosothiols au cours de l'hypertension artérielle”).