Research ArticlePosttranslational Modifications

The coordination of S-sulfhydration, S-nitrosylation, and phosphorylation of endothelial nitric oxide synthase by hydrogen sulfide

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Science Signaling  09 Sep 2014:
Vol. 7, Issue 342, pp. ra87
DOI: 10.1126/scisignal.2005478

Abstract

The gasotransmitter hydrogen sulfide (H2S), which is generated by cystathionine γ-lyase (CSE), signals by modifying proteins through S-sulfhydration and potentially other mechanisms. A target protein for H2S is endothelial nitric oxide synthase (eNOS), an enzyme that generates nitric oxide (NO), which causes vasodilation. We investigated whether H2S-induced S-sulfhydration affected the S-nitrosylation and phosphorylation of eNOS and the functional effects of changes in these posttranslational modifications on eNOS activity. In vitro, different NO donors induced the S-nitrosylation of eNOS without affecting its S-sulfhydration, whereas the H2S donor sodium hydrosulfide (NaHS) decreased the S-nitrosylation of eNOS. Cys443 was the primary S-sulfhydration site in eNOS and was one site that could be S-nitrosylated. Phosphorylation increases eNOS activity. Although exposure of eNOS-expressing HEK-293 cells to NaHS or vascular endothelial growth factor (VEGF) triggered the phosphorylation of wild-type and C443G-eNOS, VEGF did not affect the S-sulfhydration of eNOS and a mutant of eNOS that could not be phosphorylated was still S-sulfhydrated. eNOS can be present in cells in monomeric or dimeric form, but only eNOS dimers produce NO. In wild-type mice, eNOS proteins were predominantly dimerized, whereas eNOS from CSE-knockout (KO) mice, S-nitrosylated eNOS, and heterologously expressed C443G-eNOS was mostly monomeric. Accordingly, basal production of NO was lower in CSE-KO endothelial cells than in wild-type endothelial cells. Our data suggest that H2S increases eNOS activity by inducing the S-sulfhydration of eNOS, promoting its phosphorylation, inhibiting its S-nitrosylation, and increasing eNOS dimerization, whereas NO decreases eNOS activity by promoting the formation of eNOS monomers.

INTRODUCTION

Hydrogen sulfide (H2S) and nitric oxide (NO) are gasotransmitters involved in the homeostatic regulation of vascular functions (13). In vascular walls, endothelial nitric oxide synthase (eNOS) is the predominant NOS isoform for NO production, and cystathionine γ-lyase (CSE) is mostly responsible for H2S production (1, 2). Both NO and H2S function as endothelium-derived relaxing factors (1, 4, 5), and H2S is also an endothelium-derived hyperpolarizing factor (6, 7). eNOS is subjected to posttranslational modifications such as phosphorylation and S-nitrosylation (8). Phosphorylation results from the transfer of the negatively charged phosphates (PO43−) by protein kinases onto hydroxyl groups (-OH) of the target amino acid (9). Protein S-nitrosylation occurs when NO covalently attaches to the thiol side chain (-SH) of cysteine residues to form S-nitrothiols (SNOs) (10, 11). S-Nitrosylation is reversed by several denitrosylating enzymes such as S-nitrosoglutathione reductase (GSNOR) and thioredoxin (Trx) (12). GSNOR removes SNO through metabolism of GSNO to glutathione hydroxysulfenamide (12, 13). Trx breaks disulfide bonds of its target protein and then binds with the protein (14). In protein S-sulfhydration, H2S covalently modifies cysteine residue to form hydropersulfides (-SSH) (15). S-Sulfhydration enhances the activity of the modified proteins, but S-nitrosylation most likely inhibits them. One percent to 2% of proteins in liver total proteins are S-nitrosylated under basal conditions, whereas 10 to 25% of proteins are S-sulfhydrated (15). H2S-induced S-sulfhydration of adenosine triphosphate (ATP)–sensitive potassium (KATP) channel activates this channel, leading to hyperpolarization of vascular smooth muscle cells and vasorelaxation (16). Intermediate (IKCa) and small conductance (SKCa) calcium-activated potassium channels in vascular endothelial cells are also activated by their S-sulfhydration (7). Moreover, S-sulfhydration of Kelch-like ECH-associated protein 1 attenuates oxidative stress and delays cellular senescence in mouse embryonic fibroblast cells (17). Both H2S and NO can modify protein cysteine residues. However, the interaction of S-nitrosylation and S-sulfhydration on the same cysteine residue under the same experimental conditions has not been elucidated adequately, let alone the structural and functional consequences of this interaction.

Here, we found that the same cysteine residue of eNOS was S-sulfhydrated and S-nitrosylated. The interaction of these two thiol-binding mechanisms determined the conformation and function of eNOS proteins. We also investigated whether phosphorylation of eNOS was affected by S-sulfhydration and/or S-nitrosylation.

RESULTS

S-Sulfhydration of eNOS

We incubated lysates of endothelial cells isolated from the aortae of wild-type mice with the H2S donor sodium hydrosulfide (NaHS). The S-sulfhydration of eNOS was significantly increased after 15 min of incubation and reached its peak after 60 min (Fig. 1A). When wild-type endothelial cell lysates were incubated with the NO donor GSNO, eNOS was S-nitrosylated maximally after 15 min of incubation, and this S-nitrosylation declined to basal amounts within 150 min (Fig. 1A). Under basal conditions, about 30% of total eNOS proteins was S-nitrosylated, whereas 21% was S-sulfhydrated (Fig. 1A). We next injected wild-type and CSE-knockout (KO) mice with NaHS or phosphate-buffered saline (PBS). In PBS-injected mice, about 5% of total eNOS was S-sulfhydrated in wild-type aortic tissue, whereas eNOS was not S-sulfhydrated in CSE-KO aortic tissue (Fig. 1B). The latter result is in line with the absence of CSE in CSE-KO endothelial cells (Fig. 1C). In NaHS-injected mice, 13% of total eNOS were S-sulfhydrated in wild-type tissues compared to 6% in CSE-KO tissues (Fig. 1B) and NO content was increased in both wild-type and CSE-KO aortic tissue (Fig. 1D). NO content in aortic walls (Fig. 1E) and plasma (Fig. 1D) was lower in CSE-KO than in wild-type mice. Incubation of endothelial cell lysates with the CSE substrate l-cysteine at different concentrations increased S-sulfhydration of eNOS in wild-type endothelial cells, but not in CSE-KO endothelial cells (Fig. 1, F and G).

Fig. 1 S-Sulfhydration and S-nitrosylation of eNOS from wild-type and CSE-KO vascular tissues.

(A) Lysates of endothelial cells isolated from the aortae of wild-type (WT) mice were prepared treated with NaHS or GSNO, which were then subjected to S-sulfhydration or S-nitrosylation assay. S-Nitrosylated and S-sulfhydrated eNOS were quantified and normalized against total eNOS protein. n = 3 independent batches of endothelial cells, and each batch was from 21 animals. *P < 0.05 compared to time 0. (B) S-Sulfhydration of aortic eNOS isolated from CSE-KO and WT mice injected with NaHS or PBS. *P < 0.05 versus WT mice without NaHS injection; #P < 0.05 versus CSE-KO mice without NaHS injection. n = 3 independent batches of aortic tissue, and each batch was from 12 mice. (C) CSE protein abundance in WT and CSE-KO endothelial cells. Western blot shows two replicates and is representative of three independent experiments. (D) Plasma NOx concentration measured by Griess assay in WT and CSE-KO mice injected or not with NaHS. n = 3 independent experiments, and each experiment was pooled from 20 CSE-KO and WT mice. *P < 0.05 compared to PBS-treated WT mice. (E) Vascular NO production in aortic segments from WT and CSE-KO mice with or without NaHS injection, as measured by DAF-FM fluorescence. Images are representative of four independent experiments. (F and G) Effects of l-cysteine treatment on eNOS S-sulfhydration. The Western blot is representative of three independent experiments.

Reciprocal effects of H2S and NO on the S-nitrosylation and S-sulfhydration of eNOS

Treatment of wild-type endothelial cell lysates with GSNO, but not with NaHS, increased S-nitrosylation of eNOS (fig. S1A). GSNO treatment did not affect S-sulfhydration of eNOS (fig. S1B). Treatment of wild-type endothelial cell lysates with both NaHS and GSNO decreased the GSNO-induced S-nitrosylation of eNOS (fig. S1A). GSNO treatment alone did not decrease NaHS-induced S-sulfhydration of eNOS (fig. S1B). Treatment of wild-type endothelial cell lysates with the NO donor sodium nitroprusside (SNP) or NaHS induced concentration-dependent S-nitrosylation (Fig. 2A) or S-sulfhydration (Fig. 2B) of eNOS, respectively. NaHS and SNP at the concentrations used did not have cytotoxic effects on wild-type endothelial cells, as indicated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays (fig. S2, A and B). We subsequently examined the competitive interaction between S-sulfhydration and S-nitrosylation of eNOS by first incubating wild-type endothelial cell lysates with SNP at different concentrations, then adding NaHS for 30 min (Fig. 2C). SNP-induced concentration-dependent S-nitrosylation of eNOS was abolished by NaHS, but NaHS-induced S-sulfhydration of eNOS was not affected by SNP (Fig. 2C). In another set of experiments, wild-type endothelial cells were treated with SNP at different concentrations first, then SNP was washed out and NaHS was added into the culture medium for 30 min to avoid direct chemical interactions between NO and H2S molecules. Under these conditions, SNP-induced S-nitrosylation of eNOS was still abolished by NaHS (fig. S2C). We also treated wild-type endothelial cell lysates with various concentrations of NaHS first and then with one concentration of SNP for 30 min (Fig. 2D). Thus, NaHS-induced concentration-dependent S-sulfhydration of eNOS was not affected by SNP, and SNP did not induce eNOS S-nitrosylation in the presence of NaHS at different concentrations (Fig. 2, E and F).

Fig. 2 Competitive interaction between S-sulfhydration and S-nitrosylation of eNOS.

(A) SNP-induced S-nitrosylation of eNOS in WT endothelial cells. (B) NaHS-induced S-sulfhydration of eNOS in WT endothelial cells. (C) S-Nitrosylation and S-sulfhydration of eNOS from WT endothelial cells induced by SNP at different concentrations in the absence or presence of NaHS. (D) S-Sulfhydration and S-nitrosylation of eNOS from WT endothelial cells induced by NaHS at different concentrations in the absence or presence of SNP. (E) Summary of the effects of SNP alone or its interaction with NaHS on eNOS S-nitrosylation and S-sulfhydration. Red circles indicate SNO-eNOS formation and summarize representative data in (A), and blue triangles and red squares summarize representative data in (C). n = 3 independent experiments using three batches of endothelial cells isolated from 21 mice. *P < 0.05 compared to other groups; #P < 0.05 compared to SNP alone. (F) Summary of the effects of NaHS alone or its interaction with SNP on eNOS S-sulfhydration and S-nitrosylation. Blue diamonds indicate SSH-eNOS formation and summarize representative data in (B), and blue triangles and red squares summarize representative data in (D). The S-nitrosylation or S-sulfhydration of eNOS without the addition of SNP or NaHS is defined as 100%. “SNO” and “SSH” in (E) and (F) represent S-nitrosylation and S-sulfhydration of eNOS, respectively. n = 3 independent experiments using three batches of endothelial cells isolated from 21 mice. *P < 0.05 compared to SNP + NaHS group.

Role of oxidative stress in eNOS S-sulfhydration and S-nitrosylation

To determine whether H2S-induced NO production and eNOS S-sulfhydration are due to altered amounts of endogenous O2, we pretreated wild-type endothelial cells with polyethylene glycol–superoxide dismutase (PEG-SOD) before treating them with NaHS. SNP-induced eNOS S-nitrosylation, but not NaHS-induced S-sulfhydration, of eNOS was decreased by PEG-SOD pretreatment (Fig. 3, A and B). Pretreatment of wild-type and CSE-KO endothelial cells with PEG-SOD did not affect NaHS-increased NO production (Fig. 3C). Blockade of endogenous NO generation with the NOS inhibitor NG-nitro-l-arginine-methyl ester (l-NAME) decreased S-nitrosylation of eNOS in wild-type endothelial cells, and the subsequent application of SNP increased S-nitrosylation of eNOS (Fig. 3D). In addition, treatment with l-NAME, but not its inactive enantiomer NG-nitro-d-arginine-methyl ester (d-NAME), decreased basal NO amounts and NaHS-induced NO production (fig. S3).

Fig. 3 Effects of SOD on eNOS S-sulfhydration and S-nitrosylation.

(A and B) WT endothelial cells were pretreated with PEG-SOD, followed by NaHS (100 μM) or SNP (100 μM), and then subjected to S-nitrosylation assay (A) or S-sulfhydration assay (B). (C) NO production was measured in WT and CSE-KO endothelial cells (passages 4 to 7) using DAF-FM fluorescence. n = 4 independent experiments using four batches of endothelial cells isolated from 16 mice. *P < 0.05 compared to WT endothelial cells without any treatment; #P < 0.05 compared to CSE-KO endothelial cells without any treatment. (D) WT endothelial cells were pretreated with the NOS inhibitor l-NAME and then treated with SNP (200 μM). SNO-eNOS, S-nitrosylated eNOS; SSH-eNOS, S-sulfhydrated eNOS. The Western blots in (A), (B), and (D) are representative of three independent experiments.

Role of Cys443 and Ser1179 in the S-sulfhydration and S-nitrosylation of eNOS

NaHS treatment of lysates of human embryonic kidney (HEK) 293 cells expressing wild-type eNOS or eNOS mutated at Cys443 or Cys689 resulted in an increase in the S-sulfhydration of wild-type eNOS and C689S-eNOS but not that of C443G-eNOS or C443S-eNOS (Fig. 4A). We also explored the influence of these mutations on eNOS S-nitrosylation. Treatment of HEK-293 cell lysates with GSNO increased the S-nitrosylation of wild-type eNOS to a greater extent than that of C443G-eNOS (Fig. 4B). NaHS treatment also increased the S-sulfhydration of S1179A-eNOS (Fig. 4C). There was no difference in the basal phosphorylation of C443G-eNOS and wild-type eNOS (Fig. 4D). Treatment of transfected HEK-293 cells with NaHS or VEGF (vascular endothelial growth factor), but not with GSNO, increased the phosphorylation of wild-type or C443G-eNOS (Fig. 4D). Neither NaHS nor VEGF induced the phosphorylation of S1179A-eNOS (Fig. 4D). Treatment of wild-type endothelial cells with VEGF or bradykinin for 30 and 60 min did not alter the S-sulfhydration of eNOS (Fig. 4E).

Fig. 4 S-Sulfhydration and S-nitrosylation of eNOS mutated at Cys443, Cys689, and Ser1179, respectively.

HEK-293 cells were transfected with plasmids encoding WT-eNOS, C443G-eNOS, C443S-eNOS, C689S-eNOS, or S1179A-eNOS. (A) Effects of NaHS (100 μM) on S-sulfhydration of WT-eNOS, C443G-eNOS, C443S-eNOS, and C689S-eNOS. The Western blots are representative of three independent experiments. (B) Effects of GSNO (200 μM) on S-nitrosylation of WT-eNOS and C443G-eNOS. The Western blots are representative of three independent experiments. *P < 0.05 compared to WT-eNOS without any treatment; #P < 0.05 compared to GSNO-treated WT-eNOS group. (C) Effect of NaHS treatment on S-sulfhydration of WT-eNOS and S1179A-eNOS. The Western blots are representative of three independent experiments. *P < 0.05 compared to WT-eNOS without any treatment. (D) Effects of NaHS, VEGF (20 ng/ml), and GSNO (200 μM) on the phosphorylation of WT-eNOS, C443G-eNOS, and S1179A-eNOS. The signal for phosphorylated eNOS was normalized to that for total eNOS. The Western blots are representative of three independent experiments. *P < 0.05 compared to WT-eNOS. (E) Effects of VEGF or bradykinin (5 nM) incubation on the S-sulfhydration of eNOS from WT endothelial cells. The S-sulfhydration or S-nitrosylation of eNOS without the addition of NaHS or GSNO is defined as 100%. Mock represents the transfection of HEK-293 with empty pcDNA3.1 plasmid. The Western blots are the representative of three independent experiments.

Effect of H2S on eNOS coupling in vascular tissues

Next, we determined whether S-sulfhydration or S-nitrosylation correlated with the monomeric or dimeric structure of eNOS proteins; only dimeric eNOS is functional and produces NO. The dimer/monomer ratio of eNOS in wild-type mouse aortic tissue was sevenfold of that in CSE-KO mice (Fig. 5A). Treatment of wild-type endothelial cells with GSNO, but not with l-NAME, decreased the dimer/monomer ratio of eNOS (Fig. 5B). Treatment of wild-type endothelial cells with SNP or DeNONOate, another NO donor, at varying concentrations also increased eNOS monomer formation (fig. S4). Treatment of wild-type endothelial cells with NaHS maintained eNOS in dimeric form (Fig. 5B). Furthermore, NaHS cotreatment prevented the destabilizing effect of GSNO on eNOS dimers (Fig. 5B).

Fig. 5 Effects of H2S on the dimerization of eNOS and the relationship between eNOS dimerization and functionality.

(A) Relative abundance of eNOS monomers and dimers in aortic tissues from WT and CSE-KO mice. n = 4 independent experiments, and each individual experiment used endothelial cells pooled from four WT or CSE-KO mice. *P < 0.05. (B) Effects of NaHS, GSNO, and l-NAME on eNOS dimer stability in WT endothelial cells. n = 3 independent experiments using three batches of endothelial cells isolated from 12 mice. *P < 0.05 compared to the control without any treatment; #P < 0.05 compared to GSNO alone–treated group. d-eNOS, dimeric form of eNOS; M-eNOS, monomeric form of eNOS. (C) Dimer stability of WT-eNOS and C443G-eNOS expressed in HEK-293 cells, with or without GSNO and NaHS treatment. The Western blots are representative of three different experiments using LT-PAGE. (D) NO production of WT-eNOS and C443G-eNOS, detected using modified Griess assay. n = 4 independent experiments. *P < 0.05 compared to WT-eNOS without any treatment. (E and F) WT and CSE-KO endothelial cells were treated with NaHS (100 μM) for 1 hour at 37°C, and then superoxide (E) and NO (F) content was detected. n = 5 independent experiments using five batches of endothelial cells isolated from 15 mice. P < 0.05 compared to WT endothelial cells without any treatment. #P < 0.05 compared to CSE-KO endothelial cells without any treatment.

To determine whether the sulfhydrated cysteine was required for eNOS dimer assembly, we examined the dimer/monomer ratio of C443G-eNOS expressed in HEK-293 cells. C443G-eNOS migrated as monomers in low-temperature polyacrylamide gel electrophoresis (LT-PAGE), whereas the heterologously expressed wild-type eNOS migrated as both monomers and dimers. Treatment of the transfected HEK-293 cells with NaHS increased dimerization of wild-type eNOS but did not affect C443G-eNOS monomers (Fig. 5C). GSNO treatment, on the other hand, resulted in more wild-type eNOS monomers without affecting C443G-eNOS monomers (Fig. 5C). Pretreatment with NaHS partially prevented the destabilizing effect of GSNO on wild-type eNOS dimers (Fig. 5C). NaHS treatment of HEK cells increased NO production from wild-type eNOS, but not from C443G-eNOS. l-NAME decreased NO production from both wild-type eNOS and C443G-eNOS (Fig. 5D).

Effects of H2S on superoxide and NO production from mouse aortic endothelial cells

To investigate the implications of eNOS dimer uncoupling on NO and superoxide production, we measured NO and superoxide production in wild-type and CSE-KO mouse aortic endothelial cells. Superoxide production in CSE-KO endothelial cells was double that of wild-type endothelial cells (Fig. 5E), but NO production in CSE-KO endothelial cells was only half of that in wild-type endothelial cells (Fig. 5F). Superoxide content in isolated CSE-KO endothelial cells, but not wild-type endothelial cells, was significantly decreased by NaHS treatment (Fig. 5E). NaHS treatment increased NO content in both CSE-KO and wild-type endothelial cells (Fig. 5F).

DISCUSSION

S-Nitrosylation and S-sulfhydration are two posttranslational modification processes that target cysteine residues (18). S-Nitrosylation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at Cys150 abolishes its catalytic activity (19), whereas S-sulfhydration of GAPDH at the same residue augments its activity (15). Similarly, S-nitrosylation of p65 at Cys38 inhibits nuclear factor κB–dependent gene transcription (20), but S-sulfhydration of the same residue decreases cell apoptosis (21). We investigated whether S-sulfhydration and S-nitrosylation of the same or different cysteine residues of the same proteins affect each other and found that S-sulfhydration and S-nitrosylation competitively modified the same cysteine residue of eNOS, Cys443. We also demonstrated that S-sulfhydration of eNOS decreased its S-nitrosylation, whereas S-nitrosylation of eNOS did not affect its S-sulfhydration (Fig. 2). NaHS pretreatment decreased NO-induced concentration-dependent S-nitrosylation of eNOS. In contrast, SNP pretreatment did not affect NaHS-induced concentration-dependent S-sulfhydration of eNOS (Fig. 2). Acknowledging that these conclusions are based mostly on biotin switch assays performed on whole cell lysates, the same assay performed on living endothelial cells also supports these conclusions (fig. S3).

H2S can dissociate to hydrosulfide (HS) at pH 7.4 (2). The latter reacts with RS-NO (S-nitrosylated cysteine) and releases NO to produce RS-SH (S-sulfhydrated cysteine) in the following reaction: HS + RS-NO → NO + RS-SH. This would explain why S-sulfhydration can reverse S-nitrosylation but not the other way around because NO cannot interact with the cysteine residue once a stable RS-SH is formed.

NO-induced S-nitrosylation of eNOS peaked at 15 min and declined after. H2S-induced S-sulfhydration of eNOS peaked at 60 min and stabilized after. It appears that formation of hydropersulfide is slower than that of SNOs but much more stable. The S-nitrosylated bond (12 to 20 kcal/mol) is weaker than the S-sulfhydrated one (60 kcal/mol) (22). Moreover, nitroso-thiol is exceptionally labile (23) and reacts readily with other thiols by trans-nitrosylation or by disulfide formation. This may explain why S-nitrosylated cysteine was less stable than the S-sulfhydrated one.

We were interested in the interaction between S-sulfhydration and other posttranslational modifications of eNOS and found that H2S increased the phosphorylation of wild-type eNOS and C443G-eNOS, increased the S-sulfhydration of wild-type eNOS, but did not S-sulfhydrate C443G-eNOS. eNOS with a mutation in the phosphorylation site Ser1179 could still be S-sulfhydrated (Fig. 4). These results imply that H2S-induced S-sulfhydration and phosphorylation of eNOS can be independently regulated by the same gasotransmitter. Phosphorylation of Ser1179 activates eNOS by inducing a conformational change in eNOS that enhances the electron transfer rate (1, 24) or by inhibiting calmodulin dissociation from activated eNOS when calcium concentrations are low (25), thereby increasing NO production (26). H2S stimulates the activity of several upstream kinases such as p38 and Akt, and p38 and Akt phosphorylate eNOS (27). VEGF and bradykinin stimulate phosphorylation, but not S-sulfhydration, of eNOS, indicating that phosphorylation is not directly caused by S-sulfhydration.

Not all cysteine residues in proteins are accessible for posttranslational modification (28, 29). The presence of metal ions (Mg2+ or Ca2+), local pH, and the acid base motifs affect thiol reactivity to S-nitrosylation (11, 3032), and possibly to S-sulfhydration as well. There are 28 cysteine residues in eNOS protein. Cys443 is located between acid (glutamine) and base (arginine) amino acids (fig. S5) and is the last residue close to the C terminus of the oxygenase domain in the dimer interface of eNOS. Thus, this cysteine is a good target for S-sulfhydration and is in a position in the protein to potentially affect dimer stability. We showed that S-nitrosylation of wild-type eNOS was increased after treatment with different NO donors. S-Nitrosylation of eNOS with the mutation C443G was decreased, but not completely abolished, implying that Cys443 is only one of multiple cysteine residues that can be S-nitrosylated. In contrast, S-sulfhydration of eNOS was completely eliminated after Cys443 was mutated, indicating that S-sulfhydration of eNOS occurs only at Cys-443.

eNOS proteins must dimerize to produce NO (24). eNOS uncoupling into monomers occurs in various forms of endothelial dysfunction such as hypertension (33), type 2 diabetes (34), and age-related erectile dysfunction (35). Uncoupling of eNOS dimers results in a higher superoxide and lower NO bioavailability (36). In addition, the NO donor SPRENO inhibits eNOS activity in bovine endothelial cells (37). Here, we demonstrated that H2S or S-sulfhydration is crucial for eNOS dimer stability. The aortic tissues isolated from CSE-KO mice had a significantly lower dimer/monomer ratio of eNOS than that from wild-type mice. CSE deficiency–associated eNOS instability is further substantiated by our observation that exogenous H2S stabilized eNOS dimer and prevented NO-induced eNOS monomer formation. In CSE-KO endothelial cells, which lack endogenous H2S, decreased S-sulfhydration and dimerization of eNOS resulted in decreased NO production (Fig. 5F). In the presence of exogenous H2S, which occurs in NaHS-treated wild-type endothelial cells, both the amount of eNOS dimer (Fig. 5B) and NO production (Fig. 5F) were increased. Similarly, superoxide content was higher in CSE-KO endothelial cells than in wild-type endothelial cells, which was suppressed by NaHS treatment (Fig. 5E).

For NO to be produced, the donated electrons from NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) must be transferred to the reductase domain of one monomer of eNOS through flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) redox carriers, and bind to heme group in the oxygenase domain of another monomer of eNOS to catalyze the transformation of l-arginine to NO (1). The heme-oxygen complex dissociates from uncoupled eNOS dimers, and electrons are diverted to molecular oxygen rather than to l-arginine, resulting in increased production of superoxide anion and decreased production of NO (1). The exact molecular mechanisms by which S-nitrosylation or S-sulfhydration modulates eNOS dimerization are not clear. Glutathione induces dose-dependent S-glutathionylation and dimer uncoupling of human eNOS, increasing superoxide and decreasing NO generation (38), which has been proposed to be due to oxidant stress (38). We found that H2S donors still induced S-sulfhydration of a form of eNOS with a mutation in the proposed S-glutathionylation site (Cys689) (Fig. 4A). Because mutation of Cys443 prevented S-sulfhydration and dimer coupling, S-sulfhydration and S-glutathionylation of eNOS occur at different cysteines with different mechanisms and are not interdependent. PEG-SOD treatment decreased S-nitrosylation induced by SNP, but this treatment did not decrease S-sulfhydration induced by NaHS. Superoxide therefore appears essential for S-nitrosylation but not for S-sulfhydration of eNOS. We propose that H2S-induced S-sulfhydration stabilizes eNOS dimers by preventing S-nitrosylation of eNOS, leading to increased NO production and decreased oxidative stress (Fig. 6).

Fig. 6 Schematic overview of the proposed H2S/NO interactions on the posttranslational modification of eNOS.

NO induces S-nitrosylation of eNOS by modifying several cysteine residues and uncouples eNOS dimers. The uncoupling of eNOS dimers decreases NO production and increases superoxide production in endothelial cells. H2S S-sulfhydrates eNOS at Cys443 and stabilizes eNOS dimers. This will lead to increased NO production and decreased superoxide generation, both of which are essential to maintain normal endothelial functions.

In summary, H2S and NO competitively modify the same cysteine residue of eNOS to induce S-sulfhydration and S-nitrosylation. S-Sulfhydration of eNOS decreases eNOS S-nitrosylation and increases eNOS dimer stability, resulting in increased NO bioavailability. H2S also increases the phosphorylation of eNOS. Thus, H2S is a pivotal gasotransmitter that can coordinate S-sulfhydration, S-nitrosylation, and phosphorylation of eNOS to finely tune endothelial function under physiological and pathophysiological conditions. This important coordinating role of H2S may also apply to other proteins in different cells, tissues, and systems in the body.

MATERIALS AND METHODS

HEK-293 cell transfection

Control and mutant plasmids were transfected into HEK-293 cells (American Type Culture Collection), using Lipofectamine 2000 reagent as described in the manufacturer’s protocol (Invitrogen). Transfected cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing geneticin (1.5 mg/ml) (G418), which was supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin (Sigma). After 35 to 45 days of transfection, individual geneticin-resistant colonies were picked with verified stable expression of eNOS and its mutants. The cells were cultured in DMEM containing G418 (0.5 mg/ml) for further passaging in an atmosphere of 95% O2 and 5% CO2.

Isolation of primary endothelial cells from the aortae of CSE-KO and wild-type mice

CSE-KO mice were bred as previously described (4). All animal experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85 23, revised 1996) and approved by the Animal Care Committee of Lakehead University, Canada. Male CSE-KO mice and wild-type littermates of ages 10 to 12 weeks were anesthetized. Abdomen aortae were perfused with PBS containing heparin (1000 U/ml) (Sigma). The aortae were then dissected out and immersed in DMEM containing heparin (1000 U/ml) and 20% (v/v) FBS. Fat and connective tissues were rapidly cleaned off, a surgical ligation clip was applied to one end of the aortae, aortae were filled with collagenase type II (2 mg/ml) dissolved in DMEM, and then the other end was closed. The aortae were incubated for 45 min at 37°C, and then aortic endothelial cells were released by flushing the aortae with 5 ml of DMEM. The outflows were centrifuged at 1200g for 5 min. Supernatants were discarded, and cell pellets obtained after centrifugation were resuspended in 2 ml of DMEM with 10% (v/v) FBS and transferred to 35-mm collagen type I–coated plates (Invitrogen). After 2 hours, the medium was removed, cells were washed with PBS, and new DMEM was added containing 20% (v/v) FBS, penicillin G (100 U/ml), streptomycin (100 μg/ml), 2 mM l-glutamine, 25 mM Hepes (pH 7.4), heparin (100 μg/ml), and endothelial cell growth supplement (100 μg/ml) (39, 40). The identity of aortic endothelial cells was confirmed by the presence of endothelium-specific markers CD31 (Santa Cruz Biotechnology) and eNOS (Cell Signaling Technology), and the absence of smooth muscle cell marker α-actin (Santa Cruz Biotechnology) by Western blotting (fig. S6). Culture medium was changed every 2 days, and cultured cells of passages 3 to 7 were used. To study the effect of H2S in vivo, wild-type and CSE-KO mice were injected intraperitoneally under aseptic conditions with NaHS (39 μmol/kg) or PBS for 14 days. The animals were kept in conventional animal facilities and received water and food until euthanized and used for experiments.

Measurement of NO in endothelial cells

Determination of the stable end products of NO, nitrite and nitrate (NOx), was conducted as a measure for NO production (41). Total nitrate/nitrite concentrations in cell culture medium were measured after conversion of nitrate to nitrite, using a modified Griess assay (41). Cells were seeded (104 per well) in 96-well plates and cultured until they became fully confluent. Cells were incubated with nitrate reductase (10 U/ml dissolved in 50 mM phosphate buffer, pH 7.5), NADPH (5 mM), and FAD (0.5 mM) for 1 hour at 37°C. Culture medium was collected and poured into 10K cutoff filters (Centraisart I) and centrifuged at 2000g for 5 min. Cell culture medium was tested by adding 1:1 ratio of 1% sulfanilamide for 10 min and then 0.3% N-1-naphthylethylenediamine dihydrochloride for another 10 min at room temperature in the dark. Nitrite was quantified at 540 nm with a spectrophotometer, using sodium nitrite as a standard (Promega).

For plasma NO measurements, wild-type and CSE-KO mouse blood samples were collected and centrifuged at 10,000g for 10 min to separate plasma. Plasma (100 μl) was added with 5 mM NADPH, 0.5 mM FAD, and nitrate reductase (10 U/ml) and incubated for 1 hour at 37°C. After incubation, plasma was collected carefully and poured into 10K cutoff filters (Centraisart I) and centrifuged at 1000g for 5 min. Plasma was diluted in 400 μl of Milli-Q ultrapure water. NOx was measured using a Griess assay.

The intracellular NO production was visualized in real time using the fluorescent NO probe 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM DA) (Invitrogen). Wild-type and CSE-KO aortae were incubated with DAF-FM (5 μM) at 37°C in Kreb’s buffer and then rapidly removed and frozen at −20°C. Aortic tissue samples were embedded in optimal cutting temperature compound until frozen, then sectioned using Leica CM1850 UV microtome-cryostat (Leica Biosystems). The tissue blocks were cut into 10-μm-thick sections and observed under a fluorescence microscope (Olympus IX71) (42).

Measurement of superoxide in endothelial cells

Dihydroethidium (DHE) is membrane-permeable and reacts with superoxide (O2) to form ethidium, which in turn intercalates with DNA to produce nuclear fluorescence. Aortic endothelial cells from wild-type and CSE-KO mice (passages 3 to 5) were used in these experiments. The tested cells were treated with DHE (10 μM) (Sigma) for 30 min at 37°C in the dark. The production of superoxide was detected using a fluorescence microplate reader (FLUOstar OPTIMA) with an excitation wavelength of 485 nm and an emission wavelength of 620 nm (43).

Site-directed mutagenesis

pcDNA3.1 plasmids encoding wild-type eNOS or S1179A-eNOS mutant (serine replaced with alanine) were purchased from Addgene (44). The eNOS mutants were generated using QuikChange Site-Directed Mutagenesis Kit (Stratagene) (17). The primer sequences of eNOS gene (GenBank M89952.1) used to produce these mutations are listed in table S1. Briefly, the polymerase chain reactions (PCRs) contained 10 ng of template DNA, 125 ng of each of the primers, 200 μM of each deoxynucleotide triphosphate, 1× Pfu DNA polymerase reaction buffer, and 2.5 U of DNA polymerase (Pfu). The PCR cycling parameters used for the reaction were 1 cycle at 95°C for 1 min, further 18 cycles of 95°C for 50 s each, 1 cycle at 60°C for 50 s, 1 cycle at 68°C for 10 min, and extension at 68°C for 7 min. Positive clones of eNOS mutants were identified by electrophoresis on 1% (w/v) agarose gel. The plasmid DNA templates from positive clones of eNOS mutants were prepared using the QIAprep Spin Miniprep Kit (Qiagen). DNA sequencing was performed at the Paleo-DNA Laboratory of Lakehead University, Ontario, Canada, to confirm that the correct mutations were generated.

S-Sulfhydration and S-nitrosylation assays

Endothelial cells or tissues were collected and washed twice with ice-cold PBS and then suspended in 250 μl of HEN buffer (250 mM Hepes-NaOH, 1 mM EDTA, and 0.1 mM neocuproine). For S-sulfhydration assay, but not for S-nitrosylation assay, 100 μM deferoxamine was added to HEN buffer. Cell suspensions were sonicated and centrifuged at 14,000g for 15 min at 4°C. The resultant cell lysates were then treated with SNP or NaHS. Four volumes of the blocking buffer [HEN buffer, 2.5% SDS, and 20 mM S-methyl methanethiosulfonate (MMTS)] were added at 50°C for 20 min with shaking in the dark to block the free thiol (-SH). MMTS was removed by acetone, and proteins were precipitated at −20°C for 20 min. After acetone removal, the proteins were centrifuged at 2000g at 4°C for 10 min, and protein pellets were resuspended in HENS buffer (HEN buffer adjusted to 1% SDS). The suspension was added to 40 mM biotin-HPDP without ascorbic acid and incubated for 3 hours at 25°C in the dark (15, 17). However, for the S-nitrosylation assay, 1 mM ascorbic acid was also added to the HEN buffer in this step. After incubation for 3 hours at 25°C, biotinylated proteins were precipitated by streptavidin-agarose beads (1 hour at 37°C). The beads were washed five times with PBS and spun down at 5000g for 15 s. The biotinylated proteins were eluted from the beads by SDS-PAGE sample buffer (3% SDS, 1% β-mercaptoethanol, 62.5 mM tris-base, and 0.005% bromophenol blue) at 37°C for 20 min with shaking. The biotinylated protein samples were heated at 95°C for 5 min and subjected to Western blotting analysis (15, 17, 45). Densitometric quantification was performed using Alpha Digi Doctor Software and NIH ImageJ software.

Western blotting

Cells or aortic tissues were collected using cold PBS and incubated in a lysis buffer containing 0.5 M EDTA, 1 M tris-Cl (pH 7.4), 0.3 M sucrose, and a mixture of protease inhibitors (27). The cell extracts were sonicated three times (5 to 10 s per each) on ice using a cell sonicator (Sonic Dismembrator Model 100, Fisher Scientific). Cellular extracts were separated by centrifugation at 14,000g for 15 min at 4°C. The supernatants were collected, and protein concentrations were measured using Bradford assay. Equal amounts of proteins were loaded on 7.5% SDS-polyacrylamide gels and run at 120 V for 1.5 hours. Gels were transferred onto nitrocellulose membranes (Pall Corporation) at constant current of 200 mA for 3 hours using the Bio-Rad transfer system (Bio-Rad Laboratories Inc.). The membranes were incubated with primary antibodies at 4°C overnight. The dilution ratios for the antibodies were 1:1000 for anti–phospho-eNOS (Ser1179) (Cell Signaling Technology), anti-eNOS (Cell Signaling Technology), anti-CSE (Proteintech Group), and anti–platelet endothelial cell adhesion molecule (also known as CD31) (Santa Cruz Biotechnology), and 1:10,000 for anti–α-actin (Santa Cruz Biotechnology) and anti–β-actin (Sigma). The membranes were then incubated with appropriate conjugated secondary antibody for 2 hours at room temperature. After the incubation, the membranes were washed three times with 1× PBS and 1% Tween for 30 min, and two times with 1× PBS for 20 min. The membranes were visualized using an enhanced chemiluminescence Western blotting system (GE Healthcare).

Determination of eNOS dimer uncoupling

The monomer and dimer forms of eNOS (M-eNOS and d-eNOS) were detected by using LT-PAGE and Western blotting (43). Briefly, eNOS protein extracts were prepared from aortae or cultured cells on ice. Equal amounts of the isolated proteins were mixed with sample buffer [62.5 mM tris-HCl (pH 6.8), 40% glycerol, 0.01% bromophenol blue] and kept on ice. Electrophoresis was performed at a constant current of 35 mA at 4°C for 6 hours. To preserve the dimers, gels and buffers were prepared without SDS-polyacrylamide and preequilibrated to 4°C before electrophoresis, and the buffer tank was placed in an ice bath during electrophoresis to maintain gel temperature below 15°C. The gels were transferred onto a nitrocellulose membrane, and eNOS proteins were analyzed by Western blotting with anti-eNOS antibody (Cell Signaling Technology) for both dimers and monomers at 1:1000 dilution.

Endothelial cell viability and cytotoxicity assays

Endothelial cells were seeded in a 96-well plate at a density of 104 cells per well, allowed to adhere for 24 hours, and treated with NaHS and/or SNP (50, 100, or 200 μM) for 30 min. Twenty-five microliters of freshly prepared MTT solution (5 mg/ml) was added to the cell culture, and cells were incubated for 4 hours in 37°C in the dark. Actively respiring cells convert the water-soluble MTT to an insoluble purple formazan. After the incubation period with MTT solution, 200 μl of sterile dimethyl sulfoxide was added to the culture plate to solubilize formazan, and cells were covered with tinfoil and incubated for another 15 min with agitation. Formazan concentration was determined by optical density, using an enzyme-linked immunosorbent assay reader at 590 nm (FLUOstar OPTIMA, BMC Labtech).

Chemicals and statistical analysis

NaHS, SNP, DeNONOate, GSNO, VEGF, bradykinin, PEG-SOD, l-NAME, and d-NAME were purchased from Sigma-Aldrich. Unless otherwise specified, the treatment of cells or cell lysates with NaHS, SNP, or GSNO (100 μM) was of 30-min duration.

Statistical comparisons were made using Student’s t or two-way analysis of variance followed by Fisher’s and Tukey’s post hoc tests as applicable using Origin 8 software (OriginLab Corporation). Significance was set at P < 0.05. n values represent the number of independently derived biological replicates.

SUPPLEMENTARY MATERIALS

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Fig. S1. The competitive effects of NaHS and SNP on S-sulfhydration and S-nitrosylation of eNOS in endothelial cells.

Fig. S2. The concentration-dependent effects of NaHS and SNP on endothelial cell viability.

Fig. S3. The effects of d-NAME and l-NAME on NO production.

Fig. S4. The effects of different NO donors on eNOS dimer stability.

Fig. S5. The amino acids adjacent to Cys443 (the S-sulfhydration site) in eNOS.

Fig. S6. The identification of primary aortic endothelial cells isolated from wild-type and CSE-KO mice.

Table S1. List of primer sequences used for site-directed mutagenesis to generate different eNOS mutants.

REFERENCES AND NOTES

Funding: This study has been supported by an Operating Grant to R.W. from Canadian Institutes of Health Research. G.Y. was supported by a New Investigator award from the Heart and Stroke Foundation of Canada. Author contributions: Z.A., G.Y., Y.J., and R.W. designed the research; Z.A. and Y.J. performed the research; Z.A. and R.W. wrote the paper. Competing interests: The authors declare that they have no competing interests. Data and materials availability: R.W. requires a materials transfer agreement for the CSE-KO mice.
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