Accepted Manuscript Title: Role of cGMP in hydrogen sulfide signaling Author: Sofia-Iris Bibli, Guangdong Yang, Zongmin Zhou, Rui Wang, Stavros Topouzis, Andreas Papapetropoulos PII: DOI: Reference:

S1089-8603(14)00511-4 http://dx.doi.org/doi: 10.1016/j.niox.2014.12.004 YNIOX 1455

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Nitric Oxide

Please cite this article as: Sofia-Iris Bibli, Guangdong Yang, Zongmin Zhou, Rui Wang, Stavros Topouzis, Andreas Papapetropoulos, Role of cGMP in hydrogen sulfide signaling, Nitric Oxide (2015), http://dx.doi.org/doi: 10.1016/j.niox.2014.12.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Role of cGMP in hydrogen sulfide signaling

Sofia-Iris Bibli1, Guangdong Yang2, Zongmin Zhou3, Rui Wang4, Stavros Topouzis5, Andreas Papapetropoulos1,3

1

Faculty of Pharmacy, University of Athens, Athens, Greece; 2 School of Kinesiology,

Cardiovascular and Metabolic Research Unit (CMRU), Lakehead University, Thunder Bay, Ontario, Canada; 3”G. P. Livanos” Laboratory, First Department of Critical Care and Pulmonary Services, Evangelismos Hospital, University of Athens, Athens, Greece; 4Department of Biology Lakehead University, Thunder Bay, Ontario, Canada;

5

Laboratory of Molecular Pharmacology, Department of Pharmacy,

University of Patras, Patras, Greece

Address for correspondence: Andreas Papapetropoulos, Ph.D Faculty of Pharmacy, Panepistimiopolis, Zografou, Athens 15771, GREECE; tel: +30 210 7274786; fax: +30 210 7274747; e-mail: [email protected] 0

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Highlights 2S donors and endogenous H2S increase cGMP S is a non-selective PDE inhibitor 2S release determines the concentration of donor needed to increase cGMP ease rates are more likely to exert their effects through cGMP

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Graphical Abstract

Abstract The importance of hydrogen sulfide (H2S) in physiology and disease is being increasingly recognized in recent years. Unlike nitric oxide (NO) that signals mainly through soluble guanyl cyclase (sGC)/cGMP, H2S is more promiscuous, affecting multiple pathways. It interacts with ion channels, enzymes, transcription factors and receptors. It was originally reported that H2S does not alter the levels of cyclic nucleotides. More recent publications, however, have shown increases in intracellular cGMP following exposure of cells or tissues to exogenously administered or endogenously produced H2S. Herein, we discuss the evidence for the participation of cGMP in H2S signaling and reconcile the seemingly divergent results presented in the literature on the role of this cyclic nucleotide in the biological actions of H2S.

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Keywords: hydrogen sulfide, cGMP, relaxation, angiogenesis, PKG

1. Introduction Initial observations about the presence of H2S in mammalian tissues were overlooked as it was thought to be metabolic waste, rather than a molecule of biological significance[1]. It is now known that H2S is produced by two enzymes of the transulfuration pathway cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), as well as by the concerted action of cysteine aminotransferase (CAT) or Damino acid oxidase (DAO) and 3-mercaptopyruvate sulfurtransferase (MST) [2; 3]. Endogenously produced H2S is a signaling molecule which participates in the regulation of various physiological processes in the cardiovascular, nervous, endocrine, reproductive gastrointestinal and immune systems [1; 4; 5; 6; 7]. Deregulated H2S production is observed in many pathological conditions including atherosclerosis, hypertension, heart failure, diabetes, cirrhosis, inflammation, sepsis, 2

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neurodegenerative disease, erectile dysfunction, and asthma[1; 8; 9; 10; 11; 12]. In these conditions, H2S either participates in the pathogenesis or affects the course of the disease. To exert its many effects in physiology and disease, H2S utilizes a variety of signaling pathways. H2S affects many cellular redox processes and alters the activity of ion channels (potassium, calcium and chloride), kinases, phosphatases and transcription factors[1; 5; 6; 7; 11; 13]. Many of these effects do not require the participation of a second messenger, but rather result from S-sulfhydration [6]. For example, H2S has been shown to increase KATP channel activity by sulfhydrating both SUR1 and Kir6.1 (the pore-forming subunit)[14; 15]. In addition, H2S activates nuclear factor erythroid 2-related factor 2 (Nrf-2) by sulfhydrating Kelch-like ECH-associated protein 1 (Keap1) and disrupting the Nrf-2/Keap1 complex; Keap1 binding to Nrf-2 sequesters the transcription factor in an inactive form in the cytosol[16]. H2S also activates nuclear factor-κB (NF-κB) though sulfhydration of the p65 subunit; this enhances the binding to ribosomal protein S3, which increases p65 transcriptional activity in the nucleus[17]. In contrast to the above well-described pathways, the contribution of cyclic nucleotides to H2S signaling remains controversial. Herein, we will review the literature on the participation of cGMP in H2S signaling and biological activity and reconcile some of the divergent results that have appeared in recent publications.

2. Regulation of cGMP levels Resting cGMP levels are about a tenth of those of cAMP in cells [18]; the difference in the amounts of the two second messengers was partly responsible for the delay in the growth of the cGMP field, which expanded substantially when it became apparent 3

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that it mediates nitric oxide (NO)-stimulated vasorelaxation[19]. cGMP levels inside cells are dependent on the relative rate of synthesis vs degradation. cGMP is synthesized by guanyl cyclases (GC) and degraded by phosphodiesterases [19; 20]. GC are divided in two families: the soluble GC family [21] and the particulate GC that encompasses the natriuretic peptide receptors along with a number of orphan membrane-bound receptors [22] For cGMP levels to rise, either the synthesis of cGMP from GTP needs to be increased or its degradation reduced. sGC is an obligate heterodimer of a large α subunit and a smaller β subunit that carries a prosthetic heme group [23]. Two isoforms of the α subunit exist (α1 and α2), with different tissue distribution, while only a single β subunit (β1) contributes to functional sGC heterodimers [21]. The α1/β1 heterodimer is the predominant isoform in all tissues with the exception of the brain where the α1/β1 isoform is present in similar levels to the α2/β1. Basal activity of sGC is low and can be stimulated up to 400-fold when the enzyme is exposed to NO[19]. NO binds to the reduced iron of the heme prosthetic group; His-105 at the N-terminus of the β1 subunit serves as the axial ligand for heme[24]. NO activates sGC by binding to the sixth position of the heme ring, breaking the axial bond between histidine and iron to form a 5-coordinated ring with NO in the fifth position[25]. These molecular events are transmitted through the PAS (Per – period circadian protein; Arnt – aryl hydrocarbon receptor nuclear translocator protein; Sim – single-minded protein) and coil-coiled domains to the C-terminal catalytic domain of sGC, by a poorly understood mechanism, to stimulate its activity. Unlike sGC, membrane-bound GC are homodimers and expressing characteristic tissue distribution with several of them still being classified as orphan receptors[22]. Atrial and brain natriuretic peptides (NP) bind GC-A (also called NPR-A), while Ctype NP binds GC-B (also called NPR-B), while guanylin, uroguanylin and heat4

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stable enterotoxin activate GC-C. Activation of GC-D, GC-E and GC-F is not as well characterized[26]. Phosphodiesterases (PDE) regulate cyclic nucleotide levels by controlling their degradation. Eleven PDE families are known, with each having several different isoforms and splice variants. PDE 5, -6 and -9 are cGMP-specific, while PDE 1, -2, 3, -10 and -11 can hydrolyze cGMP, but also cAMP[20]. Finally, in some cells cyclic nucleotides are eliminated from the intracellular compartment, through multidrug resistant protein 4/5/8-dependent efflux[27]. Based on the above, any changes in cGMP levels brought about by H2S could be due to increased synthesis, decreased degradation, reduced efflux or a combination of these possible mechanisms. Most of the existing evidence that we will review below, suggests that H2S promotes cGMP accumulation by either directly inhibiting PDE activity or by increasing NO production/bioavailability.

3. Debating the role of cGMP in H2S responses Early studies used a functional approach to test the contribution of cGMP to the relaxing effects of NaHS. This approach yielded mixed results. In one study, the sGC inhibitors ODQ and NS-2028 did not inhibit H2S-induced relaxation in rat aorta; instead relaxations to H2S were potentiated in the presence of both agents, leading the authors to the conclusion that H2S-induced relaxation occurs independently of the cGMP pathway[28]. In addition, Cheang et al., demonstrated that in rat coronary arteries ODQ did not affect the vasodilation caused by H2S [29]. In contrast, Dombkowski et al concluded that the sGC/cGMP pathway plays a role in the relaxation process in trout bronchial arteries[30]. In a different system, the effects of 5

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NaHS on Mercenaria gills brachial muscles were reduced by sGC/PKG inhibition[31]. Adding to the controversy, relaxation to H2S in the mouse gastric fundus and inhibition of platelet aggregation were reported to occur independently of cGMP[32; 33]. To the best of our knowledge the first study to measure cGMP levels in NaHS-treated cells was by Cai et al, who showed that exposure of monkey retinal RF/6A endothelial cells to 10-200μM NaHS did not increase cGMP levels[34]. In line with this observation, NaHS did not alter the levels of cGMP in cardiomyocytes isolated from Wistar Kyoto and spontaneously hypertensive rats [35]. In contrast, in our hands exposure of rat aortic smooth muscle cells to the same concentration range of NaHS, lead to a time and concentration-dependent increase in cGMP levels [36]. Apart from species and cell-type differences that might explain the conflicting results, we noticed that IBMX was used to inhibit PDE and boost the cGMP signal in the former studies. Interestingly, when we repeated our experiments using IBMX, the cGMP-enhancing effect of NaHS was lost, suggesting that PDE inhibition is responsible for the elevation in cGMP in cells treated with NaHS.

3.1. Effects of H2S on sGC H2S interacts with many heme-containing proteins such as cytochrome c oxidase, hemoglobin, and myoglobin[37]. Studying the interactions of H2S with hemoproteins from vertebrate and invertebrate species has led to the following conclusions regarding the interaction of H2S with these proteins[38]. H2S can coordinate to the Fe+3 heme without inducing reduction or sulfheme production, or can bind to the Fe+3 with subsequent reduction of the heme. Incorporation of H2S into one of the pyrrole 6

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rings of the heme, generating the sulfheme derivative can also occur. Finally, H2S can bind to alternate sites on hemoproteins, such as cysteine, copper, and zinc ions. H2S is slowly released from the Fe+3 heme in proteins having nonpolar or hydrogen bond donating residues in their heme binding site, without involvement of redox chemistry. In contrast, proteins with hydrogen bond acceptor groups near the heme, promote reduction of the heme iron by a second H2S molecule with the concomitant formation of polysulfides and/or S°. Subsequent to heme reduction, hemoproteins having histidine residues in their active site can also form the sulfheme complex in the presence of O2 and a slight H2S excess. In cytochrome c oxidase, H2S modifies enzyme activity by binding and reducing the metal centers, whereas in myoglobin and hemoglobin, H2S reacts with the oxy-form of the proteins (Fe+2-O2) generating the sulfheme derivative [38]. Since H2S affects the function of heme proteins, we performed experiments to test the possibility that NaHS activates sGC. When purified α1/β1sGC was incubated with NaHS we observed no increase in cGMP formation[39]. In addition, NaHS did not affect NO-induced cGMP generation. These observations provide evidence that the observed rise in cGMP levels in cells after exposure to H2S, does not result from increased cGMP synthesis through sGC.

3.2. H2S is a PDE inhibitor Having ruled out the possibility that increased cGMP levels in smooth muscle cells after H2S exposure result from sGC activation, efforts focused on unraveling the contribution of PDE in cGMP elevation. In a semi-purified PDE preparation in vitro [36] , low nM concentrations of NaHS inhibited PDE activity, reducing both AMP 7

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and GMP formation from cAMP and cGMP, respectively. The effect of H2S on specific PDE enzymes, was then tested. NaHS inhibited purified PDE5 activity as effectively as sildenafil with an IC50 of 1.55μM[39]. PDE2A which uses both cAMP and cGMP as substrates , was also inhibited by NaHS, indicating that H2S is a nonselective PDE inhibitor. The IC50 for PDE2A was >50μM when cAMP was used as a substrate [40] and >500μM when cGMP was used (Panopoulos and Papapetropoulos, unpublished observations). These observations taken together suggest that H2S has i) different potency against different PDE isoforms and ii) it differentially affects cGMP vs cAMP breakdown by a single mixed-specificity PDE.

3.3. Mechanism of PDE inhibition by H2S From a theoretical standpoint, there are three possible mechanisms through which H2S can inhibit PDE: 1) S-sulfhydration, 2) binding to Zn and 3) changes in disulfide bridge formation. Since S-sulfhydration has been shown to alter enzyme activity of multiple proteins, we sought to determine if PDE5 becomes S-sulfhydrated after exposure to NaHS. Mouse aortic smooth muscle cells, as well as human umbilical vein endothelial cells (HUVECs) were incubated with 100 µM NaHS for 2 hours. The cells were then collected for biotin switch assay, and the biotinylated proteins were eluted by SDS-PAGE gel and subjected to Western blotting analysis using anti-PDE5 antibody (Cell Signaling Technology) or anti-β-actin (Sigma). S-sulfhydrated β-actin served as a positive control. H2S had no effect on PDE5 S-sulfhydration in either cell type (Fig. 1A&B). Furthermore, HEK-293 cells were transfected with PDE5 cDNA; 48 hours later, cells were treated with NaHS (100 µM) for 2 hours. No PDE5 Ssulfhydration could be detected under these conditions either (Fig.1C). 8

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H2S has a very high affinity for zinc and inhibits several Zn-containing enzymes, such as the angiotensin converting enzyme, for example[41]. While we have not tested experimentally the possibility that H2S inhibits PDE by binding to zinc that is required for its catalytic activity, the kinetics of cGMP accumulation in cells are not in favor of this hypothesis[36]. Binding of zinc to H2S is irreversible and would be expected to permanently inhibit PDE resulting in chronically elevated cGMP in cells, similarly to what is observed in cells treated with IBMX. In contrast, the effect of NaHS on cGMP levels is transient, lasting only a few minutes. Thus, the possibility that H2S affects the formation of disulfide bonds that are important for PDE activity remains the only viable hypothesis, but needs to be confirmed by identifying and mutating the cysteine residue(s) involved.

3.4. Exposure of cells to slow H2S releasing donors After establishing that NaHS increases cGMP content in smooth muscle cells, the ability of other H2S producing compounds to exhibit a similar behavior was determined. Thioglycine and thiovaline, two newer H2S donors that liberate H2S at a slower rate[42] also increased cGMP levels in smooth muscle cells in the absence of PDE inhibition. Interestingly, both amino acids caused a greater increase in cGMP levels compared to NaHS[42]. Typically, NaHS causes a maximal increase of about 2-fold, while thioglycine increased cGMP by more than10-fold that did not reach a plateau even at 300μΜ. The increase in cGMP triggered by thiolysine was even greater (Fig.2). One possible explanation for the fact that thioaminoacids increase cGMP to higher levels than NaHS is that the thioaminoacids are taken up by transporters on the membrane; being more stable than NaHS they release H2S 9

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intracellularly, closer to their target (PDE). If this were the case, excess glycine (that fails to enhance cGMP levels itself) should lower cGMP content of cells exposed simultaneously to glycine and thioglycine. This was, however, found not to be the case (Fig.3). Yu et al employed a different donor, S-allyl cysteine, and found that it significantly increased cGMP in the trophoblast cell line TEV-1[43], while diallyldisulfide analogues were shown to inhibit the decline of cGMP levels in tissues, in an animal model of L-NAME-induced hypertension[44].

H2S donors with slower H2S release rates than thioaminoacids (GYY-4137 and ATB337[45; 46]) have also been tested for their ability to enhance cGMP levels. GYY4137 used up to 100μM failed to increase cellular cGMP content[47]; higher concentrations of GYY4137 (300μM) lead to a 25% increase in cGMP levels that reached statistical significance; this probably has no biological relevance for the action of GYY4137. Similarly ATB-337 which consists of diclofenac linked to a hydrogen sulfide-releasing moiety, failed to increase cGMP when used up to 50μΜ (data not shown).

H2S donors that fail to increase cGMP levels are still able to exert a variety of pharmacological effects[1]. In addition, even donors capable of increasing cGMP, like NaHS and Na2S, have been shown to utilize several additional pathways[11; 48]. Especially in the cardiovascular system, H2S-releasing agents exert some of their effects by modifying ion channel activity[1; 5]. Although various K+ and Ca+2 channels have been implicated in H2S responses, KATP have received a lot of 10

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attention, as their inhibition attenuates the beneficial effects of H2S. Glibenclamide has been shown to reduce or blunt H2S-induced angiogenesis and vasorelaxation and to inhibit the cardioprotective actions of H2S after ischemia/reperfusion [28; 49; 50].

Having observed that not all H2S donors when used within the range that have pharmacological effects trigger increase cGMP levels one wonders whether endogenously produced H2S affects cGMP levels. Several lines of evidence suggest that indeed endogenous H2S modulates cGMP levels. First, pharmacological inhibition of CSE/CBS by PAG and AOAA reduces cGMP levels in rat aorta; similarly CSE silencing reduces cGMP in smooth muscle cells [36]. Second, adenovirus-mediated gene transfer of CSE, enhanced cGMP in cultured smooth muscle cells[36]. Finally, plasma cGMP levels are lower in CSE knockout (KO) mice compared to wild-type littermates[47]. Tissues levels of cGMP are lower in aorta and mesentery both under basal conditions and following stimulation with sodium nitroprusside. Assuming that expression of sGC, eNOS and PDE is unaltered in CSE KO mice, the differences in cGMP observed are the result of higher PDE activity in the CSE KO mice, due to the deficiency in H2S. Direct measurements of PDE activity in CSE KO, however, are needed to lend further credence to this hypothesis.

In summary, fast releasing H2S donors would lead to higher levels of H2S within a cell, while donors that liberate H2S more slowly would achieve lower concentrations. Obviously, even when using the same H2S donor, the amount of H2S released and the absolute cellular concentration of H2S observed will vary depending on conditions (redox environment, pH, hypoxia, etc) and might not be uniform throughout the cell, 11

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as for example in the case of mitochondrial-targeted H2S donors. Using higher concentrations of slow H2S releasers, like in the case of GYY-4137, will eventually be expected to produce enough H2S to inhibit PDE. To prove the contribution of cGMP in the biological activity of a given H2S donor, measurable increases in this cyclic nucleotide should be observed within the concentration range it produces its pharmacological effects.

3.5. Indirect effects of H2S on cGMP: modulation of eNOS and increased NO bioavailability Although direct inhibition of PDE can explain the rise in cGMP levels after H2S exposure, an additional mechanism of enhancing cGMP levels has been observed at least in cells expressing eNOS, such as endothelial cells and cardiomyocytes. It is known that exposure to NaHS activates Akt [50], perhaps through inhibition of PTEN[51], resulting in enhanced eNOS phosphorylation on Ser1179 [39; 52; 53; 54]. Exposure of endothelial cells to NaHS was also shown to reduce phosphorylation of the inhibitory site Thr495[39; 55]. In addition, H2S S-sulfhydrates Cys443, preventing the formation of monomeric, inactive eNOS[56]. Increased NO production from eNOS, will in turn stimulate sGC to synthesize cGMP. Enhanced eNOS phosphorylation has been observed in both cultured cells and tissues, but is speciesspecific as H2S increases eNOS phosphorylation in mouse tissues and human cells[39; 57], both not in the rabbit heart ([58] and unpublished observations). Both slow releasing H2S donors and H2S-generating salts have been shown to promote eNOS phosphorylation[39; 52; 57]. Additional means to eNOS activation for increasing NO levels after exposure to H2S 12

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have been reported. Bir el al, showed that H2S stimulates nitrite reduction to NO in hypoxic, but not normoxic, endothelial cells[59]. Interestingly, H2S enhanced eNOS phosphorylation of Ser1177 only in cells under normoxia. In addition, H2S was found to promote conversion of nitrite to NO and enhance NO bioavailability and tissue cGMP levels by activating xanthine oxidase.

4. Implications for biological activity So far we have summarized the existing evidence that exposure of cells to physiological amounts of endogenous H2S or to ultra-rapid (NaHS) or fast-releasing H2S donors (thioaminoacids) increase intracellular cGMP levels. We will next review the literature on the biological role of cGMP in H2S-stimulated responses in vitro and in vivo. It should be mentioned that lack of inhibition of a H2S-triggered biological effect by ODQ does not exclude the participation of cGMP-regulated pathways in any given H2S effect. In the face of sGC inhibition, cGMP levels might still be increased if PDE is inhibited and NPR are basally active or stimulated by their cognate ligands. On the other hand, biological responses that result from elevated cGMP that are due to H2S-triggered eNOS activation and/or increased NO bioavailability should be blocked by sGC inhibition. The reader should also keep in mind that activation of the eNOS pathway in the vessel wall by H2S is relevant only for endothelial cells, while PDE-dependent rises in cGMP levels in response to H2S could occur in both endothelial and smooth muscle cells (Fig.4). We have shown that exposure of endothelial cells to NaHS increased cGMP levels[39]; the increase in cGMP accumulation leads to activation of cGMPdependent protein kinase (PKG), as suggested by the increased phosphorylation of 13

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VASP, a surrogate marker of its activity. Increased cGMP in response to NaHS resulted in enhanced migration, proliferation and in vitro angiogenesis that could by inhibited by the PKG-I inhibitor peptide DT-2, but not the control peptide (TAT)[39]. Vasorelaxation in endothelium-intact rat aortic rings elicited by NaHS could be reduced by genetic or pharmacological inhibition of eNOS and by the PKG-I inhibitor DT-2 [39; 47] , while DT-2 did not reduce the response to GYY4137, a result that is in line with the inability of this H2S donor to enhance cGMP levels[47]. NaHSinduced relaxations were reduced in phenylephrine precontracted mouse aortae from PKG-I KO mice, compared to wild-type controls[47]. Similarly, L-cysteine-induced relaxations in the same experimental setup were reduced in PKG-I KO mice, suggesting that endogenously produced H2S from CSE/CBS increases cGMP that in turn activates PKG-I to contribute towards relaxation. In line with our observations, Hu et al. [60]showed that blockade of PKG attenuated the NaHS-induced suppression of the sodium-hydrogen exchanger activity and limited the cardioprotection afforded by NaHS. Activation of PKG-I has also been shown to mediate the effects of NaHS in vivo[47]. Pretreatment of mice with DT-2 abolished the hypotensive response to NaHS. In more recent studies, we observed that the cardioprotective effect of NaHS was associated with enhanced cGMP levels and VASP phosphorylation in the ischemic myocardium. PKG-I inhibition by DT-2 reversed the infarct size-limiting effect of NaHS in rabbits[58]. We have also observed that the wound healing promoting effect of NaHS administration in rats could be reversed by LNAME treatment, presumably through inhibition of angiogenesis [39]. Finally, some of the actions of H2S have been shown to result from enhanced eNOS 14

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activation as a result of increased phosphorylation on S1177 (bovine S1179) in vivo. eNOS KO mice were not protected after H2S donor treatment, in contrast to wild-type controls in models of cardiac ischemia/reperfusion injury [53; 55] heart failure [61] and cardiac arrest[54]. Tissue cGMP levels were only measured in one of these studies [53] and were found to be elevated in response to H2S. However, it is expected that in all of the above cases, enhanced eNOS activation would lead to stimulation of sGC activity to increase cGMP levels to mediate the effects of H2S. It should be mentioned that H2S-stimulated responses involving NO in vivo are not dependent solely on eNOS. In a hind-limb ischemia model, H2S enhanced blood flow in both wild-type and eNOS KO, although the increase in blood flow in eNOS KO was 75% of that of seen in wild-type. The effect on flood flow was inhibited by the NO scavenger 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) in both eNOS KO and wild-type animals, suggesting an increase in NO bioavailability through NOS-independent mechanisms. The remaining 75% increase in blood flow seen in eNOS KO was blocked by febuxostat (XO inhibitor), confirming that in vivo, in addition to in vitro, XO can enhance the conversion of nitrite to NO to promote ischemic vascular remodeling.

A discussion on the effects of H2S on cGMP signaling would be incomplete, if the role of HS− in 8-nitro-cGMP were not mentioned. Protein S-guanylation caused by 8nitro-cGMP was markedly augmented after knockdown of CBS or CSE. The nucleophilic properties of HS− support a reaction with various electrophiles in cells via direct chemical sulfhydration; thus, HS− might react with 8-nitro-cGMP in cells to form 8SH-cGMP and limit its detrimental effects[62]. It should be noted that SH15

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cGMP still has cGMP activity, in that it effectively activates PKG. Treatment of mice with NaHS attenuated left ventricle dilation and dysfunction after left anterior descending coronary artery ligation. This improvement was attributed to inhibition of the action of 8-nitro-cGMP that was generated in excess in cardiac tissues after myocardial infarction; in particular, HS− suppressed myocardial cell S-guanylation– dependent activation of H-Ras and its downstream signaling pathways improving cardiac performance and preventing cardiomyocyte senescence.

5. Conclusions To summarize, not all H2S donors and treatment regimens have the ability to increase cGMP levels in cells. In order to access the contribution of cGMP to the effects of a particular H2S-releasing compound one should measure the levels of this cyclic nucleotide. From the available data, ultra-fast (salts) or fast H2S-releasing compounds (thioaminoacids) are more likely to exert their effects in a cGMP-dependent manner, compared to slow releasing H2S donors. The increase in cGMP levels can result from 1) PDE inhibition, 2) eNOS activation or 3) increased NO bioavailability, as has been described for the xanthine oxidase (XO)‐mediated nitrite reduction. The increase in cGMP levels, activates PKG-I to lead to vasorelaxation or angiogenesis. While this review was being written, NaHS-derived polysulfides were shown to activate PKG-I through the formation of an inter-protein disulfide [63]. Thus, NaHS can activate PKG both in a cGMP-dependent and independent manner. While a lot of the mechanistic details remain to be unraveled, the eNOS/cGMP/PKG axis is emerging as an important component of H2S signaling and biological activity.

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Acknowledgments This work has been co-financed by the European Union (European Social Fund – ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF) Research Funding Program: Thalis (MIS 380259); Investing in knowledge society through the European Social Fund and Aristeia 2011 (1436) to AP, by EU FP7 REGPOT CT-2011-285950 – “SEE-DRUG” and by the COST Action BM1005 (ENOG: European network on gasotransmitters).

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Figure Legends Figure 1: Lack of evidence for PDE5 S-sulfhydration. (A) Mouse smooth muscle cells (SMCs) or (B) human umbilical vein endothelial cells (HUVECs) were incubated with 100 µM NaHS for 2 hours. After that, the cells were collected for biotin switch assay, and the biotinylated proteins were eluted by SDS-PAGE gel and subjected to Western blotting analysis using anti-PDE5 antibody (Cell Signalling Technology) or anti-β-actin (Sigma). S-sulfhydrated β-actin was used as a positive control. This experiment was repeated four times with similar results. (C) HEK-293 cells were transfected with PDE5 cDNA for 48 hours, after that, the cells were treated with NaHS (100 µM) for 2 hours and processed as above. Figure 2: Thiolysine increases cGMP. Rat aortic smooth muscle cells were exposed to the indicated concentration of thiolysine (μM) for 5min; cGMP content was extracted using 0.1N HCl and measured by EIA as described in [42] ; n= 4;* P