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. Author manuscript; available in PMC: 2009 Sep 12.
Published in final edited form as: Circ Res. 2008 Jul 31;103(6):606–614. doi: 10.1161/CIRCRESAHA.108.175133

Nitroglycerin-induced S-nitrosylation and desensitization of soluble guanylyl cyclase contribute to nitrate tolerance

Nazish Sayed 1, David D Kim 1, Xavier Fioramonti 1, Toru Iwahashi 1, Walter N Durán 1, Annie Beuve 1
PMCID: PMC2737267  NIHMSID: NIHMS127131  PMID: 18669924

Abstract

Nitrates such as nitroglycerin (GTN) and NO-donors such as S-nitrosothiols are clinically vasoactive via stimulation of soluble guanylyl cyclase (sGC), which produces the second messenger cGMP. Development of nitrate tolerance, after exposure to GTN for several hours, is a major-draw back to a widely used cardiovascular therapy. We recently showed that exposure to NO and to S-nitrosothiols causes S-nitrosylation of sGC, which directly desensitizes sGC to stimulation by NO. Here we test the hypothesis that desensitization of sGC by S-nitrosylation is a mechanism of nitrate tolerance. Our results established that vascular tolerance to nitrates can be recapitulated in vivo by S-nitrosylation via exposure to cell membrane permeable S-nitrosothiols and that sGC is S-nitrosylated and desensitized in the tolerant, treated-tissues. We next determined that a) GTN treatment of primary aortic smooth muscle cells induces S-nitrosylation of sGC and its desensitization as a function of GTN concentration; b) S-nitrosylation and desensitization are prevented by treatment with N-acetyl-cysteine, a precursor of glutathione, used clinically to prevent development of nitrate tolerance; c) S-nitrosylation and desensitization are reversed by cessation of GTN treatment. Finally, we demonstrated that in vivo development of nitrate tolerance and cross-tolerance by 3-day chronic GTN treatment correlates with S-nitrosylation and desensitization of sGC in tolerant tissues. These results suggest that in vivo nitrate tolerance is mediated, in part, by desensitization of sGC via GTN-dependent S-nitrosylation.

Keywords: nitric oxide, cGMP, nitrosation, vascular tolerance, S-nitrosothiols

Introduction

Since the 19th century, nitroglycerin (glyceryl trinitrate: GTN) has been used in clinical medicine to treat angina pectoris and more recently, congestive heart failure, acute myocardial infarction and other cardiovascular diseases 1. It is used because of its excellent therapeutic profile even though it induces nitrate tolerance. Nitrate tolerance is defined as the attenuation or loss of vascular responsiveness to nitrate (e.g. GTN) after continuous exposure to GTN or other organic nitrates 2. Cross-tolerance corresponds to the lack of vascular responsiveness to NO and NO-donors (e.g. nitrosothiols) and is characterized by an increase in reactive oxygen species (ROS) 3. NO and nitrates exert their vasorelaxing effects via stimulation of soluble guanylyl cyclase (sGC), which produces cGMP 4. At the molecular level, tolerance corresponds to an absence of increased vascular cGMP production in response to NO or nitrate, highlighting the central role of sGC activity in vascular tolerance. The mechanism of development of nitrate tolerance remains a mystery; the prevalent actual model is impairment of GTN bioconversion via inhibition of mitochondrial aldehyde dehydrogenase (MitALDH2), thereby reducing the release of NO or derivatives 5,6. Other frequently cited mechanisms are a) depletion of thiols, which are thought to be required for GTN conversion to NO and derivatives7; b) increased phosphodiesterase activity, which breaks down cGMP8; c) reduced NO bioavailability by affecting the endogenous NO production 9 and d) desensitization of sGC 10.

Using S-nitrosothiols, we recently demonstrated that S-nitrosylation of sGC is a mechanism of desensitization to NO in primary aortic smooth muscle cells 11. Interestingly, it was shown that in vivo GTN treatment induces an increase in erythrocyte S-nitrosothiol content 12. Earlier, GTN treatment was proposed to increase intracellular concentration of S-nitrosothiols such as Snitrosoglutathione (GSNO) and S-nitrosocysteine (CSNO) 7,13, and in fact these S-nitrosothiols were thought to mediate GTN vasorelaxing effects via sGC activation. However, we now know that GSNO and CSNO are also capable of S-nitrosylation, probably via transnitrosylation 14. Together, these findings led us to hypothesize that GTN treatment could S-nitrosylate sGC, leading to its desensitization, which in turn would participate in development of nitrate tolerance. If this hypothesis is correct, then (a) inducing S-nitrosylation in vivo should mimic development of tolerance, (b) GTN treatment should induce S-nitrosylation and desensitization of sGC and (c) cross-tolerance to NO-donors that bypass GTN conversion or endothelial dysfunction by acting directly on sGC should be observed. Herein, we provide evidence that desensitization of sGC via S-nitrosylation could be an additional mechanism for GTN-induced nitrate tolerance.

MATERIALS AND METHODS

Materials and reagents

Fetal bovine serum was from GIBCO (Carlsbad, CA). All other cell culture reagents were from ATCC (Manassas, VA). Other materials are described in Supplemental Material and Methods. L-cysteine and CSNO solutions were prepared as previously described 11.

Hamster cheek pouch preparation and arteriolar diameter measurements

The hamster left cheek pouch was prepared for intravital microscopy as described 15. Under acute conditions, CSNO or GTN was applied to the cheek pouch via a side port into the suffusate bicarbonate buffer line. For 3-day treatment, GTN or solvent (propylene glycol) was infused via osmotic pump at the rate of 10µg. min−1. kg−1. GTN or solvent were then topically applied to the cheek pouch to measure arterial vasodilation (see Supplemental Methods). All procedures were approved by the Institutional Animal Care and Use Committee of New Jersey Medical School.

Preparation of cytosols from tissues and cells

see Supplemental Methods.

sGC activity assay

sGC activity was determined by formation of [α-32P]cGMP from [α-32P]GTP, as described 16. 40µg of cell cytosol or 50µg of tissues were used in each assay reaction.

Measurement of cGMP Production

cGMP production was measured by radioimmunoassay (RIA) in the presence of 1mM IBMX as described in 16 and see Supplemental Methods.

IP with anti-SNO and Biotin switch assay to detect S-nitrosylation

IP is detailed in Supplemental Materials and Methods. Biotin-Switch assay was performed using the NitroGlo™ Kit from Perkin Elmer 17 on the cytosols of cells or tissues (300µg) as described in 11.

Statistical analysis

is described in Material and Methods supplement.

RESULTS

CSNO treatment induces vascular tolerance in vivo

To show that S-nitrosylation could induce vascular tolerance, we used the hamster cheek pouch preparation, in which vasodilation of arterioles is measured by intravital microscopy in response to topical application of vasoactive agents 18(see Methods and Supplement). S-nitrosocysteine (CSNO) was used because it a) induces vasorelaxation (probably via release of NO19), b) is a S-nitrosylating agent that causes S-nitrosylation and desensitization of sGC in cells11 and c) accumulates during GTN treatment 13. L-Cysteine (L-Cys), as control, and CSNO at 10µM were applied for 5min, vasodilation was measured, and after 10min washout, 10µM of the NO-donor SNAP was applied for 5 min and vasodilation assessed. As shown in Fig. 1A, following initial application of 10µM L-Cys, SNAP produced immediate vasodilation of the arteriole. Application of 10µM CSNO induced vasodilation (as expected for a NO-donor), yet after return toward the baseline value, arterioles failed to vasodilate in response to SNAP (Fig. 1B). This result shows that the S-nitrosothiol CSNO causes vascular tolerance in an in vivo system under acute conditions.

Figure 1.

Figure 1

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S-nitrosocysteine (CSNO) treatment induces vascular tolerance that correlates with sGC S-nitrosylation and desensitization. (A) The relative luminal diameter increases by 2.0 ± 0.2 fold in response to SNAP with L-Cys treatment in the hamster cheek pouch. (B) No further vasodilation was observed in response to 10µM of SNAP after treatment with 10µM CSNO for 5min; n=4. 10µM CSNO was used as we did not observe vasodilation in the presence of 1µM CSNO and observed persistent vasodilation if 1mM CSNO was applied. (C) Western blots (WB) with anti-sGC following IP with anti-SNO show that S-nitrosylated sGC is pulled down from the CSNO-treated pouches but not from L-Cys-treated pouches or naïve controls. Inputs represent 30% of the precleared cytosols and indicate similar levels of sGC in the various samples. No sGC was pulled down with IgG indicating specificity of the anti-SNO antibodies. (D) WB with anti-sGC of biotin-avidin assay before (middle panel) and after IP with anti-sGC (right panel) indicates that sGC S-nitrosylation increased in CSNO-treated tissues. Tissues treated with 1µM CSNO did not have any detectable S-nitrosylated sGC (not shown). The sGC contains α1 and β1 subunits with a molecular weight (M.W) of 80 and 72 kDa, indicated by arrows. (D) Basal and NO-stimulated activity of the same cytosols showed that sGC from CSNO-treated tissues did not respond to NO stimulation compared to L-Cys and naïve controls. SNAP was used at 1mM. Experiments were repeated 4 times with each measurement done in duplicate and expressed in pmol/min. mg ± SE. *, P< 0.05 CSNO treated vs. naïve or L-Cys.

CSNO-induced tolerance is associated with S-nitrosylation and desensitization of sGC

After 5min of 10µM C S N O o r L-Cys treatment (with verification that CSNO induced vasodilation as above), the treated pouches and the untreated contralateral pouch (naïve control Cys or CSNO) were collected, instead of being treated with SNAP, to determine sGC S-nitrosylation and activity. S-nitrosylation was assayed by three methods: immunoprecipitation (IP) with anti-SNO antibodies20 (Fig. 1C), the biotin switch assay followed by avidin purification 21, and IP with anti-sGC followed by biotin/avidin switch assay (Fig. 1D). Western blots with anti-sGC showed that sGC was strongly S-nitrosylated in the CSNO-treated tissues that exhibited tolerance in comparison to L-Cys-treated pouches or in the contralateral pouches (Fig. 1C and B). The same cytosols were assayed for sGC activity. Fig. 1E showed that the basal sGC activity was similar between the L-Cys, CSNO treated tissues and their naive controls but the sGC in the CSNO-treated pouches lost significant sensitivity to SNAP, in comparison to controls. These results indicate that the CSNO-treated pouches that exhibit tolerance, i.e. lack of relaxation of their arterioles in response to NO stimulation, contain high levels of S-nitrosylated sGC and displayed a significant decrease in NO-stimulated sGC activity.

Treatment with GSNO does not induce tolerance and does not lead to detectable S-nitrosylation

In vivo treatment with more commonly used S-nitrosothiols such as S-nitrosoglutathione (GSNO) or SNAP do not lead to tolerance 22,23. Interestingly, we and others have observed that GSNO does not readily S-nitrosylate intact cells or tissues unlike CSNO 20,24. To investigate further the correlation between S-nitrosylation of sGC and vascular tolerance, the hamster cheek pouch was treated with GSNO under the same conditions as above (5min, 10µM GSNO) and after washout, 10µM SNAP was applied, as in Fig.1. Fig. 2A is a control showing vasorelaxation in response to SNAP. As shown in Fig. 2B, GSNO application induces vasorelaxation, as expected for a NO-donor, but in contrast to CSNO, there was complete vasorelaxation in response to a subsequent SNAP application indicating that GSNO did not lead to tolerance. Moreover, no S-nitrosylation could be detected in the treated tissues (Fig. 2C).

Figure 2.

Figure 2

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S-nitrosoglutathione (GSNO), a non-permeable S-nitrosothiol, does not induce vascular tolerance or S-nitrosylation of sGC in vivo. (A) SNAP (10µM, 5min) induces marked relaxation in the cheek pouch. (B) Similar relaxation with SNAP was observed after GSNO treatment (10µM, 5min). There was no significant difference in the arterioles luminal diameter in response to SNAP in control vs. GSNO. n=3. (C) Biotin-avidin assay followed by Western Blot with anti-sGC of 2 cheek pouches treated with GSNO and SNAP did not show S-nitrosylation. sGC was expressed in the cheek pouches (Input). Positive control of the assay indicates S-nitrosylation of WT after treatment with GSNO.

Nitroglycerin (GTN) induces S-nitrosylation of sGC and desensitization in primary rat aortic smooth muscle cells (SMC) in a concentration-dependent manner

To determine whether GTN treatment leads to S-nitrosylation and desensitization of sGC, we exposed primary SMC to 100µM of GTN for 1h. As shown in Fig. 3A, both biotin-avidin and immunoprecipitation show that GTN induces S-nitrosylation of sGC. No S-nitrosylated sGC could be detected when SMC were treated with methanol (vehicle). We next assayed whether GTN also induces desensitization of sGC by measuring by RIA the cGMP produced in response to SNAP after treatment with various concentrations of GTN. Fig. 3B shows that increasing concentrations of GTN (100 to 500µM) significantly decreased production of NO-stimulated cGMP by SMC. In parallel, biotin switch assay on the cytosols of GTN-treated SMC indicated that S-nitrosylated sGC levels increased with increasing concentration of GTN (inset, Fig. 3B). These results provide evidence that GTN treatment leads to S-nitrosylation of endogenous sGC thiols and induces its desensitization.

Figure 3.

Figure 3

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GTN treatment of primary aortic SMC induces S-nitrosylation and desensitization of endogenous sGC. (A) IP with anti-SNO (left panel) and biotin-avidin assay (right panel) indicate S-nitrosylation of sGC by GTN. Input (10% of cytosols) showed equal amount of sGC in the various cytosols. These blots are representative of 3 experiments with identical results. (B) Desensitization and S-nitrosylation of sGC is dose-dependent function of GTN. NO-stimulated cGMP production by SMC decreases with increased concentrations of GTN. Before SNAP application, the levels of cGMP were between 2.26 and 10.62 fmol/106cells for pretreatment with MeOH and 500µM GTN, respectively. These experiments were repeated 3 times with each RIA done in duplicate. SNAP: 100µM. Inset is a biotin-avidin assay showing that levels of S-nitrosylated sGC increased with increasing concentrations of GTN.

GTN-induced S-nitrosylation and desensitization of sGC in SMC are prevented by N-acetyl-cysteine (NAC) treatment and are reversible

S-nitrosylation is a reversible post-translational modification dependent on the redox state. Therefore, NAC, as a precursor of glutathione (GSH) synthesis can prevent S-nitrosylation 11,25. To confirm that GTN induces desensitization by S-nitrosylation of sGC, SMC were treated for 2h with 2.5mM NAC prior to exposure to GTN (100µM) or vehicle for 1h. Pretreatment with NAC not only drastically reduced the levels of S-nitrosylated sGC (Fig. 4A) but also partially restored sensitivity of sGC to NO stimulation (Fig. 4B). In parallel, SMC treated with 100µM GTN or vehicle were washed and incubated for 1h with medium only. Fig. 4C shows that free-nitrate incubation reversed S-nitrosylation (Western blot with anti-sGC), which correlated with resensitization to NO (51.4 vs. 5.0 % desensitization without or with washout, respectively). These results showing that NAC prevents GTN-induced desensitization and S-nitrosylation of sGC together with reversion of S-nitrosylation and resensitization by GTN-free incubation support the idea that GTN-dependent changes in redox state facilitate S-nitrosylation.

Figure 4.

Figure 4

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GTN-induced S-nitrosylation and desensitization of sGC are prevented by NAC treatment in SMC. (A) WB with anti-sGC shows that the amount of sGC pulled down with anti-SNO is greatly reduced in the NAC + GTN cytosols compared to cells not pretreated with NAC. Cells treated with MeOH or MeOH +NAC did not contain detectable S-nitrosylated sGC. GTN was used at 100µM. Inputs indicate a similar amount of sGC in the precleared cytosols of the various treatments. (B) The cytosols pretreated with NAC are more responsive to NO-stimulation compared to the ones treated with GTN only. SNAP: 100µM. The basal sGC activity was similar for the various treatments. NAC had no effect on NO-stimulated activity for samples treated with MeOH. (C) WB of biotin-avidin assay indicating that washout of GTN for 1h decreases S-nitrosylation of sGC to level close to control. Conversely, washout resulted in a drop of desensitization (to 5%) in response to 100µM SNAP. Measurements of sGC activity were done in 3 independent experiments and expressed as mean ± SE. *, P< 0.05 GTN + NAC vs. GTN

Mutations of α1C243 and β1C122 significantly reduce the desensitization observed in the WT treated with GTN

We tested whether α1C243 and β1C122, whose S-nitrosylation is involved in desensitization of sGC by CSNO11, are also responsible for GTN-dependent desensitization. COS-7 cells, which do not have detectable sGC, were transfected for 48 h with wild-type (WT), α1C243A/β1 or α1/β1C122A mutants then treated for 1h with 100µM GTN or vehicle; NO-stimulated sGC activity of the various cytosols was assayed. As shown in Fig. 5, WT lost more than 50 % of the response to SNAP following GTN treatment (50.7 % ± 1.4 desensitization). Mutants C243A and C122A had a 30.1% ± 4.6 and 33.6% ± 4.5 decrease in NO-stimulated activity, respectively, which corresponds to elimination of ~ 40% of the desensitization seen in the WT. These results indicate a causal relationship between S-nitrosylation by GTN and desensitization of sGC in cells. We next tested if this causal relationship could explain nitrate tolerance.

Figure 5.

Figure 5

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Mutations α1C243A and β1C122A significantly alter GTN-dependent desensitization. (A) COS-7 cells expressing wild-type (WT) and mutants were treated for 1h with GTN 100µM or methanol (MeOH). NO-stimulated sGC activity was measured in the cytosols with 100µM SNAP. These experiments were repeated 4 times with 3 transfections; results are expressed as the percentage of NO-stimulated activity with GTN treatment vs. vehicle. *, P<0.05 (B) WB with anti-sGC indicates that expression of sGC and mutants is not altered by GTN.

GTN-induced nitrate tolerance correlates with S-nitrosylation and desensitization of sGC in vivo

Using a similar set-up than with CSNO, we first established nitrate tolerance in the hamster cheek pouch preparation under acute conditions (50min topical application of 1µM GTN). As shown in Online Figure I, after initial vasodilation, the arterioles failed to relax in response to a dose challenge of 10µM GTN; the nitrate tolerant tissues contain S-nitrosylated sGC (Fig. S1B) which was desensitized by GTN as shown by the significant decreased in cGMP production in response to 10µM GTN, 100µM GTN and 1mM SNAP (Online Figure I-C).

To study the sGC properties under conditions that mimic the development of nitrate tolerance in clinical settings, we used osmotic pumps to infuse continuously for 3 days low doses GTN (10µg. min−1. kg−1) or solvent (propylene glycol, PG), as previously described26. There was vasodilation in response to 10µM GTN in animals treated with the solvent (1.37 ± 0.13 fold increase) and no vasodilation in the 3-day GTN treated animals confirming nitrate tolerance development (Fig.6A). Following the 3-day infusion, the lungs and cheek pouches were collected. Both biotin-avidin assay (Fig. 6B) and IP with anti-SNO (Fig. 6C) show that sGC is S-nitrosylated following chronic exposure to GTN in the pouches and the lungs and that no or little S-nitrosylated sGC was detected in the animals infused with vehicle. Specificity of sGC S-nitrosylation was verified by pretreatment with 30mM ascorbate (Online Figure II). Chronic GTN treatment led to a marked increase in the amount of sGC in both pouches and lungs (input of Fig. 6B), as previously observed by others 27, yet the fraction that was S-nitrosylated remarkably increased as shown by the lower amount of sGC present in the unbound fraction (i.e. the un-nitrosylated fraction) of the GTN-treated animals (Fig. 6C, right panel).

Figure 6.

Figure 6

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sGC is heavily S-nitrosylated and desensitized to NO and nitrates in a chronic model of nitrate tolerance. (A) 10µM GTN induces vasorelaxation in the cheek pouch of hamsters treated with propylene glycol (PG) while no relaxation is seen in the 3-day GTN-treated animals, n=3 175133/R2 23 (B) WB of biotin-avidin assays indicate high levels of S-nitrosylated sGC in the pouches (left panel) and lungs (right panel) of animals treated with GTN. (C)WB with anti-sGC of IP with anti-SNO confirmed that GTN-tolerant tissues have higher levels of S-nitrosylated sGC than PG-treated tissues. Correspondingly, less un-nitrosylated sGC is present in the unbound fraction in tolerant tissues. (D) cGMP-production in response to SNAP or GTN is significantly impaired in the lungs of GTN-treated animals. Similar results were obtained with the pouches (inset). The levels of cGMP were too low with 10µM GTN over basal levels to obtain significant stimulation-fold values in the pouches. n=4; *, P<0.05 sham vs. GTN-treated.

(E) 10µM SNAP induces vasorelaxation in the cheek pouch of hamsters treated with PG while significantly less relaxation was seen in the GTN-treated animals, n=5; P<0.05 Area Under the Curve PG vs. GTN.

We next assayed desensitization of sGC. In the animals chronically treated with GTN, the cGMP production in response to 10 and 100µM GTN was greatly reduced in comparison to the sham-treated animals in lungs (Fig 6D) and in cheek pouches at 100µM GTN (inset of Fig. 6D). Similarly, sGC desensitization was remarkable in response to 1mM SNAP, as chronic GTN treatment induced a 59% and 85% NO-dependent desensitization of sGC in lungs and pouches, respectively. The levels of cGMP produced in the lungs were also much higher than in the pouches (not shown), which could be due to the high levels of sGC present in the lungs (Fig. 6B, input).

Importantly, we showed that there was cross-tolerance: relaxation in response to 10µM SNAP was significantly reduced in the animals treated with GTN in comparison to the PG-treated animals (Fig. 6E). This decrease in SNAP-dependent relaxation, which was associated with decreased SNAP-dependent cGMP levels (Fig. 6D), suggests that sGC desensitization per se is, in part, responsible for the observed tolerance.

DISCUSSION

In these in vitro and in vivo studies, we provide evidence that nitroglycerin treatment, whether at low therapeutic doses in animals or at higher doses in cells, is associated with S-nitrosylation of sGC and its desensitization, which correlate with the development of nitrate tolerance and cross-tolerance in an in vivo model of hamster cheek pouch. This link between S-nitrosylation of sGC and vascular tolerance was supported by the observations that the S-nitrosylating compound CSNO induced vascular tolerance together with S-nitrosylation and desensitization of sGC. This correlation was strengthened by the fact that another S-nitrosothiol GSNO did not induce tolerance and did not S-nitrosylate of sGC, under the same conditions.

Vascular tolerance is obtained with the cell-permeable CSNO, which induces S-nitrosylation and desensitization of sGC

We showed that topical application of an S-nitrosylating agent, CSNO, is sufficient to induce vascular tolerance in vivo and that the tolerant tissues contain S-nitrosylated sGC, which is desensitized. This CSNO-induced tolerance, which we define as the lack of relaxation of arterioles in the hamster cheek pouch preparation in response to SNAP, is intriguing because to date, organic nitrates such as GTN were the only established tolerance inducers. Tolerance is not readily observed following treatment with NO-donor such as sodium nitroprusside 28 and S-nitrosothiols such as SNAP or GSNO 22,23. These NO-donors, unlike CSNO, remain in the extracellular compartment or in the cell membranes and release NO extracellularly. Interestingly, we have observed that CSNO strongly S-nitrosylated sGC in intact cells 11 but not the other S-nitrosothiols GSNO or SNAP (not shown). These observations were in support of results showing that S-nitrosylation and modification of intracellular thiols depend on the ability of CSNO to readily enter cells via L-cysteine transporters, and do not depend on the release of NO24. To strengthen the potential link between S-nitrosylation of sGC and tolerance, we repeated the topical application with GSNO under the same conditions than with CSNO and showed that the relaxation of arterioles in response to SNAP was maintained, indicating no development of tolerance. Moreover, the GSNO-treated tissues did not have detectable S-nitrosylated sGC unlike the CSNO-treated ones. Yet, we cannot rule out that part of this phenotype is due to reversion by GSNO reductase. These results suggest that CSNO induces tolerance because it S-nitrosylates sGC potentially through trans-nitrosylation following its transport into cells. It should be mentioned that in our study the concentrations of CSNO (and GSNO) required to induce arterioles vasorelaxation are higher than in other reports 29. One potential explanation for the decreased response to these NO-donors is the fact that the hamster cheek pouch preparation is different from conventional ex vivo models such as isolated aortic rings that are pre-contracted with norepinephrine.

Does GTN treatment induces S-nitrosylation of sGC thereby its desensitization thus contributing to tolerance?

This hypothesis is based on our result showing that vascular tolerance could be caused by CSNO-induced S-nitrosylation hence desensitization of sGC 11, and the recent report that GTN treatment leads to increased S-nitrosylation of tissues, as measured by RSNO formation 30. GTN and other nitrates vasorelaxing effects are mediated by stimulation of sGC, and many communications reported on desensitization of sGC by GTN treatment and its relationship to nitrate tolerance but without providing a mechanism of desensitization 6,27,31. We showed that GTN-treatment whether at low therapeutic doses in vivo or at various doses in SMC lead to dose-dependent S-nitrosylation of sGC. We previously showed in a purified sGC system using GSNO and by mutational analysis of the S-nitrosylated cysteines αC243 and βC122 that S-nitrosylation directly causes desensitization11. Thus, it was not surprising to observe that GTN-induced S-nitrosylation correlated with desensitization of sGC in cells. Moreover, replacement of αC243 and βC122 led to partial elimination of GTN-dependent desensitization, supporting a causal relationship between GTN treatment and sGC desensitization. Elimination of desensitization was not complete suggesting that other Cys could be involved in GTN-dependent desensitization of sGC. We observed the same association in our in vivo model of nitrate tolerance. We first use an “acute” model (50min GTN infusion in the cheek pouch) because the same tissue is assayed for tolerance (measurement of arteriole relaxation), sGC S-nitrosylation and desensitization (Online Figure I). However, nitrate tolerance develops clinically under prolonged exposure to low doses of GTN. Thus, to mimic development of nitrate tolerance in patients, we used osmotic pumps to infuse clinically relevant doses of GTN27 and showed that tolerant tissues contained S-nitrosylated sGC that was desensitized to GTN and to SNAP (cross tolerance). As reported by others27, we detected an increase in total sGC amount in 3-day GTN-treated animals; yet this increase was accompanied by an increase in the S-nitrosylated form of sGC, hence desensitized sGC, thus explaining the apparent discrepancy between higher sGC expression and decreased cGMP production.

Thiols depletion and the sGC S-nitrosylation/desensitization model

The reversion of both S-nitrosylation and desensitization after 1h washout following GTN treatment and the fact that NAC prevents S-nitrosylation and desensitization of sGC in SMC fit well with the pharmacodynamics of nitrate tolerance. Reversibility of S-nitrosylation and desensitization could explain why nitrate sensitivity is progressively restored (4 to 12h) in patients after cessation of GTN treatment; this characteristic is used clinically in “intermittent nitrate therapy” to avoid development of nitrate tolerance32. Likewise, NAC, a precursor of glutathione synthesis is used to prevent the development of nitrate tolerance in patients treated with nitroglycerin33, based on the early Needleman’s model suggesting that nitrate tolerance is due to free thiol depletion 7 (yet, others have not observed thiol depletion 34). In this model, GTN produced nitrosothiols via reactions with free thiols 35,36, which in turn activate sGC, therefore a prolonged treatment leads to depletion of the thiols and cessation of activation. However, we observed that GTN-dependent reduced vasorelaxation and desensitization of sGC is seen in response to the NO-donor SNAP, which does not require free thiols to activate sGC. Thus, we speculate that NAC facilitates the reversion of S-nitrosylation due to thiol depletion, which has been associated with increased S-nitrosylation of protein thiols37. It is known that superoxide, generated by GTN treatment26, is responsible for the production of reactive nitrogen species in particular N2O3 38, which could directly S-nitrosylate thiol of proteins or convert GSH or L-Cys to form GSNO and CSNO39, respectively. This latter reaction not only will deplete the cells of free thiols but also generate S-nitrosothiols, known to be increased by GTN treatment 30, which could S-nitrosylate proteins via trans-nitrosylation14. We speculate that continuous GTN treatment by affecting the equilibrium between the ROS and RNS produced and intracellular GSH levels could favor S-nitrosylation.

How does sGC desensitization by S-nitrosylation fit in the mechanism-based, classical tolerance?

The development of nitrate tolerance is multi-factorial and the cGMP-dependent loss of vasorelaxation is not well understood3. Chronic GTN treatment leads to generation of superoxide 26 and this ROS production could induce thiol oxidation. In this study, we measured specifically S-nitrosylation and therefore we cannot rule out that the sGC could be desensitized by other thiol modifications including disulfide bond formation or glutathionylation. Upstream of sGC, the high production of ROS was shown to lead to endothelial dysfunction which is proposed to be a mechanism of nitrate tolerance; for example, chronic GTN treatment resulted in a loss of acetylcholine-induced relaxation40. Consequently, this reduced NO availability should decrease sGC activation. Recently, inhibition of mitALDH2, which is involved in GTN bioconversion has been identified as a major mechanism of nitrate tolerance6. This impairment in GTN bioconversion is reflected by a decreased stimulation of sGC because of reduced NO or derivatives3. Downstream of sGC, increased in cGMP degradation via increased PDE expression could also contribute to nitrate tolerance (in our experiments, this mechanism was eliminated by the use of IBMX an inhibitor of PDE) 8. We showed that chronic nitroglycerin treatment (or application of the S-nitrosylating agent CSNO) leads to a partial loss of arterioles relaxation and decreased cGMP production in response to SNAP, suggesting cross tolerance. This result indicates that sGC activity per se is affected by the nitroglycerin/CSNO treatment as SNAP spontaneously released NO, bypassing GTN bioconversion or endothelial NO availability, at least in the vascular system of hamster cheek pouch. Nonetheless, the response to SNAP was not as blunted as the response to GTN suggesting that desensitization of sGC is another site affected by nitroglycerin in addition to the main upstream site (MitALDH2 inhibition) for establishment of nitrate tolerance. Thus, nitroglycerin-induced tolerance could be the result of a double setback for sGC: impairment of GTN bioconversion deprived sGC from activation by NO derivatives and S-nitrosylation of sGC prevents any further activation.

Supplementary Material

1

ACKNOWLEDGMENTS

We thank Dr. Andrew Harris for critical reading of the manuscript.

SOURCES OF FUNDING: NIH GM067640, HL089771 (AB) and HL070634 (WND).

Footnotes

Subject codes

[138] Cell signaling/signal transduction, [95] Endothelium/vascular type/nitric oxide

DISCLOSURES: none

REFERENCES

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