Articles in PresS. Am J Physiol Lung Cell Mol Physiol (December 30, 2014). doi:10.1152/ajplung.00327.2014

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Working with Nitric oxide and Hydrogen sulfide in biological systems

2 Shuai Yuan1, Rakesh P. Patel2* and Christopher G Kevil1*

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1

Department of Pathology, Louisiana State University Health Sciences Center, Shreveport, LA

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2

Department of Pathology, University of Alabama at Birmingham, Birmingham, AL

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*contributed equally to this work

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Corresponding Author

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Rakesh P. Patel, PhD

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Department of Pathology

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BMR2, room 532

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University of Alabama at Birmingham

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Birmingham, AL 35226

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e-mail: [email protected]

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1 Copyright © 2014 by the American Physiological Society.

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Abstract

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Nitric oxide (NO) and hydrogen sulfide (H2S) are gasotransmitter molecules important in numerous physiological and pathological processes. While these molecules were first known as environmental toxicants, it is now evident that that they are intricately involved in diverse cellular functions with impact on numerous physiologic and pathogenic processes. NO and H2S share some common characteristics, but also have unique chemical properties which suggest potential complementary interactions between the two in affecting cellular biochemistry and metabolism. Central amongst these is the interactions between NO, H2S and thiols that constitute new ways to regulate protein function, signaling and cellular responses. In this review, we discuss fundamental biochemical principals, molecular functions, measurement methods, and the pathophysiological relevance of NO and H2S.

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Key words: thiol, oxidation, redox, pulmonary, cardiovascular

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Introduction

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Historically considered only as industrial pollutants, nitric oxide (NO) and hydrogen sulfide (H2S) are now appreciated as two key physiologically produced signaling molecules which control multiple cellular functions. Appreciation that dysfunction in how these solvated gases affect these functions, either through deficient formation or downstream reactivity has opened up new therapeutic avenues for a host of diseases in all major organ systems including the lung. Our understanding of NO-homeostasis mechanisms are relatively advanced compared to H2S, primarily due to the latter’s discovery as a biologically relevant entity being post-dated by a decade or more compared to NO. While chemically distinct, there are intriguing parallel mechanisms / concepts in NO and H2S biology that include overlapping functions, technical challenges in how each of these gases are measured in biological milieu, appreciation of the mechanisms and factors that modulate how metabolism of each of these is regulated and therapeutic potential for either inhibiting or repleting NO or H2S. In this article, and within the framework outlined above we provide a general overview of NO and H2S biology.

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Nitric oxide formation: enzymatic and non-enzymatic sources

45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67

Enzymatic sources: Nitric oxide is synthesized by one of three different isoforms of nitric oxide synthase (NOS) that differ in enzymatic activity, how they are regulated (transcriptional, translational and posttranslational mechanisms) and expressed / compartmentalized in tissues as well as the sub-cellular level. These are called NOS I, II and III, corresponding to the inducible (iNOS), neuronal (nNOS) and endothelial (eNOS) isoforms, typically with high, mid and low activities respectively. In the systemic and pulmonary vasculature, eNOS plays a key role in controlling blood flow and maintaining an antithrombotic and anti-inflammatory luminal surface. On the other hand, iNOS is induced by inflammatory stimuli in leukocytes, airway epithelial cells and alveolar macrophages to mediate pathogen killing. However, production of NO at higher concentrations in this setting is also associated with collateral tissue damage mediated by increased oxidation, nitrosation or nitration of biomolecules, which is emblematic of acute lung injury and ARDS. However, this is only a general paradigm. The pleiotropic effects of NO are regulated only in part by high vs low concentrations. It is evident that the cellular source (i.e. compartmentalization) of NO also plays a key role (97, 98, 123). For example iNOS expressed in polymorphonuclear leukocytes (PMNs) regulates sequestration of these cells in the lung vasculature during cecal ligation and puncture (CLP) induced sepsis, but limits PMN infiltration into the alveolar space; on the other hand NO derived from iNOS in lung parenchymal cells plays no role in this process(99). From a biochemical perspective, how iNOS-derived NO produced in PMN’s elicit distinct effects compared to iNOS-derived NO produced in pulmonary endothelial cells is not clear and underscores the importance of compartmentalization with regard to the NO source, and the local concentration of NO where produced. An additional consideration is that selective effects of NO may be mediated by distinct redox derivatives and associated products (e.g. nitrosation and nitrated epitopes), which can have distinct biological activities compared to NO per se. The parameters of concentration, localization and redox conversion are also emerging as critical in the biological actions of H2S.

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Non-enzymatic sources: More recently, non-enzymatic sources of NO, via reduction of nitrite have emerged as viable NO-production processes in vivo. Widely used as a stable end-product for assessing NO-formation, it is now clear that nitrite is an integral player in NO-homeostasis pathways. Several mechanisms have been identified that operate under physiologic and pathophysiologic conditions that

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result in the 1-electron reduction of nitrite to NO. Common features of these mechanisms include acceleration of nitrite-reduction as a function of decreasing pH and oxygen tension(30). Intriguingly, in the lung nitrite can elicit signaling responses at low biological (nM-µM) concentrations under normoxia as well. For example, airway epithelial cell repair was stimulated by nitrite via a mechanism that appeared to involve stimulation of oxidative signaling(122). Figure 1 shows a scheme generalizing NO-formation pathways.

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NO and nitrite therapeutics for airway and pulmonary vascular diseases

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Unfortunately, the absence of a selective nitrite scavenger, or a way to selectively decrease nitrite levels in vivo, precludes definitive assessment of endogenous nitrite in NO-homeostasis pathways. However, data from exogenous or therapeutic nitrite clearly demonstrates this possibility. In the lung, inhaled nitrite reversed pulmonary hypertension and was suggested to be more effective compared to inhaled NO. In pre-clinical models, inhaled nitrite did not lead to rebound hypertension once nitrite inhalation was stopped, which is a frequently observed complication associated with acute termination of inhaled NO gas(42). Putative mechanisms of nitrite action involve activation of pulmonary vascular smooth muscle soluble guanylate cyclase (sGC) and the canonical NO-signaling cascade(10). Moreover, nitrite therapy also protects against pulmonary and vascular smooth muscle remodeling after injury and improves lung function after transplantation(4, 88, 111, 139). Additionally, nitrite therapy protects against acute lung injury induced by mechanical ventilation and inhaled chemical toxicants, in the latter of which the risk of developing reactive airways is also reduced(40, 96, 101, 132). Improvement in both inflammatory and permeability components of ALI has been documented, suggesting that nitrite therapy prevents early events in the disease. Interestingly, inhaled NO, which to date is a primary therapeutic for treatment of pulmonary hypertension in the new born, has also been shown to prevent ischemia-reperfusion injury in distal extrapulmonary tissues(26, 64, 65, 84, 117). In this case, the model proposed is that iNO increases the level of circulating nitrite (and perhaps other NO-adducts e.g. S-nitrosothiols) which then stimulate NO-signaling in other tissues. In fact, NO-repletion by nitrite or iNO has proven successful in multiple pre-clinical and clinical studies in different settings of acute and chronic inflammatory tissue injury and supports the model that biological pools of NO-signaling equivalents exist in the form of nitrite in tissues that can be manipulated therapeutically.

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H2S formation: enzymatic and non-enzymatic pathways

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Enzymatic sources: For nearly a decade, H2S has been discussed as the third biological gasotransmitter along with NO and CO (126). Three distinct H2S generating enzymes have been identified in mammalian systems (Figure 2). The best-characterized H2S producing enzyme, cystathionine β-synthase (CBS), is a pyridoxal-phosphate (PLP) dependent enzyme that catalyzes the condensation between homocysteine and serine, giving rise to cystathionine Owing to the similarity of structures, CBS also uses cysteine, instead of serine, to generate H2S (47). Another PLP dependent enzyme, cysthathionine γ-lyase (CSE), catalyzes the conversion of cystathionine to cysteine, but also utilizes homocysteine and cysteine to generate H2S (16, 138). Additionally, CSE and CBS are crucial hubs in transsulfuration pathways which pass sulfur between various sulfur containing amino acids(50). The third pathway comprises two enzymes found within the mitochondrion. Cysteine aminotransferase (CAT) first converts cysteine to 3-mercaptopyruvate (3-MP), from which H2S is released by 3-mercaptopyruvate sulfurtransferase (3-MST). Importantly, this

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process involves two equivalents of reactive thiols or a reduced thioredoxin, with byproducts of a disulfide or an oxidized thioredoxin.

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All three enzymes, CBS, CSE and 3-MST, have been documented to be expressed in the lungs, with possible variations between species and cell types (5, 83, 94, 124, 131). The contribution of these enzymes on H2S production is affected by their expression levels and the availability of substrates. For example, CSE is ~60 fold higher than CBS in the mouse liver and accounts >97% H2S production. In comparison, CBS is the predominant H2S producing enzyme in the brain where its expression is higher than CSE (51). Although cysteine and homocysteine are the substrates of both CSE and CBS, CSE is the primary source of H2S in homocysteinemia, while in the kidney where the cysteine level is ~10 fold higher than in the liver or the brain, CBS may predominate. Classically considered as a cytosolic enzyme, CBS is also found to localize in other compartments such as mitochondria(7, 41, 112) and nuclei (1, 52). Its nuclear translocation is related to post-translational modification by small ubiquitin-like modifier (SUMO) which inhibits its activity (49). Moreover, CBS activity is regulated by other small molecules, including its activation by S-adenosylmethionine (SAM) and glutathione (GSH) (46, 86) and inhibition by NO and carbon monoxide (CO) (28, 49, 115). Relatively less is known about regulation of CSE and 3MST. Considered to be the major source of H2S in the cardiovascular system, CSE is expressed in both mouse lung endothelial cells and smooth muscle cells, with its effects on lung-function is poorly explored. 3-MST and CAT are found in most cell types but less abundant than CBS and CSE. Although found in both cytosol and mitochondria (78, 83), their activity may be greater in mitochondria due to higher concentrations of cysteine, the substrate for CAT (49). H2S has been proposed to be an endothelium-derived hyperpolarizing factor (EDHF), based on the deficiency of acetylcholine-induced vasorelaxation in CSE knockout mice (82, 125). However, the proposed mechanism is through KATP sulfhydration via CSE derived H2S, which does not support a direct role of H2S as an EDHF (22).

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Non-enzymatic sources: Similar to NO, alternate pathways for H2S formation may exist in the absence of CBS, CSE or 3-MST. An early study showed human erythrocytes reduce elemental sulfur to H2S, with reducing equivalents provided by glutathione, NADH and NADPH(102) (Equation 1). Other forms of sulfane sulfur, where sulfur binding to another sulfur as in thiosulfate (S 2O32-), polysulfide (RSnR) and persulfide (RSSH), may also be reduced to free H2S by low molecular weight reductants independent of enzymatic pathways (49, 70). The yield of H2S via this mechanism may be relatively low but could serve as storage depots of H2S equivalents, as sulfane sulfur can be generated by H2S autoxidation (Equation 2) (70, 73) and by serial oxidation processes in the mitochondria(49). Figure 2 summarizes the transsulfuration pathway and H2S metabolism. 𝐸𝑞𝑢𝑎𝑡𝑖𝑜𝑛 1: 𝑆8 + 16 𝐺𝑆𝐻 → 8 𝐻2 𝑆 + 8 𝐺𝑆𝑆𝐺 𝐺𝑙𝑢𝑡𝑎𝑡ℎ𝑖𝑜𝑛𝑒 𝑅𝑒𝑑𝑢𝑐𝑡𝑎𝑠𝑒

𝐺𝑆𝑆𝐺 + 𝑁𝐴𝐷(𝑃)𝐻 + 𝐻 + →

2 𝐺𝑆𝐻 + 𝑁𝐴𝐷(𝑃)+

𝐸𝑞𝑢𝑎𝑡𝑖𝑜𝑛 2: 𝐻2 𝑆 + 𝑂2 → 𝑆0 + 𝐻2 𝑂2 𝑆0

𝑆0

𝑆0

𝑆0

𝑅𝑆𝐻 → 𝑅𝑆𝑆𝐻 → 𝑅𝑆3 𝐻 → … → 𝑅𝑆𝑛 𝐻 144 145

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NO and H2S Donors

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NO and H2S donors are widely used experimental tools, and potential therapeutics to model and interrogate biological functions. Donors allow introduction of defined amounts with defined rates of release of test compound. For NO, many different NO-donors are commercially available that can expose biological systems to low levels of NO for long periods of time, to high levels of NO for short periods, and variations in between. In addition, NO-donors display pH dependence, sensitivity to oxidants and reductants, which influence what NO-derivatives are produced, and in the most recent generation also offer organelle targeting for NO-release (56). These aspects of NO-donors have been reviewed many times elsewhere. Therefore, we will focus our discussion on H2S donors, which is a rapidly evolving area.

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To better understand the pros and cons of available sulfide donors, it is helpful to review the chemical properties of H2S. H2S exists in gaseous form at ambient temperature and pressure. At equilibrium between gas and aqueous phases, its solubility at 37℃ is 80 mM (50). In aqueous solution, H2S dissolves into HS- and S2-, with the pKa of 7 and 12.2-15 respectively at room temperature (87, 133) (Equation 3). Accordingly, it is estimated that two thirds of H2S dissolved in water is HS-, and one third H2S, with a negligible amount of S2- . 𝐸𝑞𝑢𝑎𝑡𝑖𝑜𝑛 3: 𝐻2 𝑆 (𝑔) ⇔

pKa1 =7

𝐻2 𝑆 (𝑎𝑞) ⇔

pKa1 =12−15

𝐻𝑆 − + 𝐻+ ⇔

𝑆 2− + 2𝐻 +

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Equation 3 underscores the reversibility between H2S, HS- and S2-. Importantly then in an open system, the equilibrium will be pulled to the left leading to rapid decreases in solvated H2S levels ; note this is also the case with NO. An additional and important consideration is the permeability of H2S into lipid bilayers. While H2S freely diffuses through cell membrane (75), HS- does not. This may affect the availability of H2S in the cytoplasm or other cellular compartments such as mitochondria, although these diffusion limitations may be altered by changes in pH resulting in conversion of HS- to H2S. Therefore, when using sulfide donors, it’s important to consider its cellular accessibility versus its volatility. Currently, available H2S donors fall into three categories: sulfide salts, natural compounds and synthetic compounds.

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Sulfide Salts: Sulfide salts, such as Na2S and NaHS, are the simplest H2S generating compounds and most widely used in experimental studies. The release of H2S from Na2S and NaHS is instantaneous upon hydration and dissociation in aqueous solution. H2S release does not reach steady state however due to volatilization of H2S into the gas phase, and thus H2S exposure is transient. The half-life of H2S in plasma is ~20 minutes in humans and ~ 5 minutes in mice(8) after a single bolus injection of Na2S, while the half-life in a cell culture model is also 5 minutes (89). Apart from volatility, H2S is also vulnerable to oxidation. It is recommended that stock solutions be made fresh, sealed in air-tight container with minimal head space, and the concentration confirmed by validated methods. Due to these disadvantages, therapeutic potential of sulfide salts are highly limited.

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Natural Compounds: Garlic consumption has been associated with health benefits for a long time and there are a variety of garlic-based products available and marketed accordingly. Allicin (diallyl thiosulfinate), the best known active ingredient in garlic extracts, decomposes into diallyl disulfide (DADS) and diallyl trisulfide (DATS) (3). These compounds have at least one disulfide bond and an allyl group whose double bonded carbon is adjacent to the sulfur bonded carbon. In the presence of GSH or

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other reduced thiols, a nucleophilic displacement of the α-carbon of this allyl group can generate a hydrodisulfide that is further reduced by GSH to H2S (6, 70). The requirement for cellular metabolism and reducing equivalents is an important consideration in assessing how much H 2S is generated by these compounds and typically allows for slower and more sustained release of H2S compared to sulfide salts. Additionally, these natural products are lipophilic allowing H2S formation to occur in all cell compartments. This hydrophobicity however, is also a limitation as both DADS and DATS are immiscible in water, making delivery of these compounds challenging. Other natural compounds proposed to release H2S include isothiocyanates, such as sulforaphane from broccoli and allyl isothiocyanate, from wasabi (53). However, further understanding of their mechanisms of action and role of H2S in health promoting effects is required.

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Synthetic Compounds: Several efforts have been made to develop synthetic H2S donors. GYY4137 is a derivative of Lawesson’s reagent that releases H2S over several minutes via nucleophilic attack and after its dissociation in solution. GYY4137 is believed to release H2S in a prolonged manner, although some studies using amperometry have showed that the release rate was significantly reduced compared to NaHS (69). Characterization of GYY4137 dependent sulfide release in plasma has been reported(67, 69); however, the methylene blue method was used to measure H2S (89). This method for measuring H2S levels is questionable and have we have observed that GYY4137 no longer increased free sulfide 30 minute after application. Considering a high dosage of GYY4137 is required for its physiological effects (400 μmol/L for a plateau concentration of 4h, led to nitrogen dioxide concentrations of 2) to be lower than what we observed. This is likely due to the fact that our chemical work flow for reductant labile sulfide also includes measurement of thiosulfate levels that were not measured using LC-MS methods in the report by Ida et al. However, similar GSSH levels were reported by Lu and colleagues using MBB and LC-MS/MS(72). Together, comparison of these three studies suggests the existence of other reductant-labile sulfur in the tissue, such as protein persulfide/polysulfide and thiosulfate could be biologically relevant forms of sulfide (43, 90).

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Table 2: Levels of Persulfide/Polysulfide and Reductant Labile Sulfur

Tissue Measurement T Ida, et al. (nmol/mg tissue)

GSSH GSSSH CysSSH GSSSG GSSSSG CysSSSSCys CysSSSSSCys Total C Lu, et al. GSSH (nmol/mg protein) X Shen, et al. Reductant Labile Sulfur (nmol/mg protein)

Heart

Lung

0.27 0.02 0.0067 0.0008 0.0195 0.001 0.0005 0.001235 0.00075 0.00015 0.0043 0.0045 0.00525 0.0069 0.307 0.034585

Liver

Brain

0.3 0.002 0.005 0.00375 0.0003 0.006 ND 0.31705

0.77 0.0032 0.011 0.00035 0.00295 ND ND 0.7875

N/A

N/A

~0.2

~0.5

0.4398

1.4508

0.4719

1.392

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Electrochemistry: Similar to NO, H2S can be measured based on its redox properties. The sulfide-specific ion-selective electrodes (ISEs) are well documented but used mostly in non-biological samples. ISEs require an alkaline environment (pH>11) which favor the accumulation of sulfide ion (S2-). In biological samples, however, this could lead to artificially high readings due to hydroxyl replacement of the cysteine sulfur(89). Alternatively, Kraus and colleagues developed a polarographic electrode with a H2Spermeabile membrane, which allows the diffusion of H2S from biological samples at pH 7, into the electrode (pH 10)(21, 60) . The polarographic electrode has the advantage of high sensitivity (10-20nM H2S gas) and the capacity of real-time measurement(129). It may be possible to measurement different sulfide pools by altering sample pH. However, it is sensitive to temperature and pressure and requires frequent calibration.

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Fluorescent/Chemiluminescent Probes: Sulfide specific fluorescent/chemiluminescent probes are a fastdeveloping tools in H2S research that generally provide the potential for real-time and subcellular measurements. These probes are usually designed according to the reducing and nucleophilic properties of sulfide (76). Current probes provide moderate to good specificity to sulfide compared to other biological thiols. To highlight a few, hydrosulfide naphthalimide-2 (HSN2) has an azido group which can be reduced by H2S into a fluorogenic amine (79). The fluorescence of HSN2 is increased 60 fold by 100 equivalents of H2S. Its specificity to H2S is maintained with the presence of 2,000 equivalents of GSH and cysteine. However, the activation of HSN2 takes 45 minutes and real-time measurement is not possible. Interestingly, Ai and Chen reported a genetically coded azide in GFP, which they used to image endogenous H2S production in HEK 293T cells (15). This theoretically allows sulfide probing at target proteins. Guo and co-workers also developed a ratiometric fluorescent probe, based on nucleophilic addition of H2S to the probe, which is membrane permeable and has little cytotoxicity, therefore allowing real-time H2S probing in living cells (71). Although azide based fluorescent probes usually provide 15

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excellent selectivity for H2S over other thiols, excitation with higher power or extended exposure photoactivate these probes. To meet this challenge, Pluth and Bailey developed a chemiluminescent probe, CLSS-2, to minimize noise due to photo-activation (76). It is worth noting that most probes were tested in different conditions (sample concentration, buffer pH, oxygen pressure, etc), which may alter biological sulfide pools. Selectivity and sensitivity of these probes need to be further investigated under physiological conditions and specific cell culture conditions.

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Tag-Switch Technique: Mounting evidence indicates that H2S elicits its biological effects by forming protein persulfides, termed sulfhydration or persulfidation (63, 81, 82, 103). However, due to similar reactivities of persulfides and thiols, it is challenging to identify protein persulfides specifically. The modified biotin switch assay developed by Snyder and colleagues uses methyl methanethiosulfonate (MMTS) to mask free thiols but not persulfide, followed by biotin-HPDP labeling of persulfide (81). However, it is unknown how MMTS spares persulfide specifically. Actually, it has been shown that MMTS may also react with small molecular thiols such as GSH (92). Another method uses iodoacetic acid to label both persulfides and free thiols, after which persulfide adducts are reduced by dithiothreitol (DTT) (63). But the selectivity of DTT reduction also remains unclear. Recently, a thiol blocker, methylsulfonyl benzothiazole (MSBT), developed by Xian et al. was applied in a tag-switch assay (135). MSBT reacts with protein persulfide and yields a highly activated disulfide, which distinguishes it from free thiol adducts and native protein disulfides, so that certain nucleophiles, such as cyanoacetate-biotin, can specifically label persulfides. Additionally, the MSBT based tag-switch technique also showed potential to provide spatial resolution in cell culture (135).

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Conclusion

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As a gasotransmitter, NO plays fundamental role in vascular function and redox biology. For decades, our understanding of NO biology has evolved through empirical medications, identification of sGC as a receptor that transduces NO-signaling in various pathways, bioavailable metabolites, effects on protein post-translational modifications and novel therapeutic approaches. Although many refined tools have been developed to study NO biology as discussed in this review, not all of them are readily available in each and every laboratory. More importantly, careless choice of tools or misinterpretation of the data often leads to convoluted and false conclusions, especially when various commercial user-friendly kits are employed inappropriately. By comparison, the field of H2S biology is considerably further behind. Our knowledge on H2S chemistry and biology is confusing and contradictory on several aspects. The field is still trying to resolve basic questions such as how are H2S producing enzymes regulated, what are physiological concentrations of H2S, and how does H2S elicit its various biological functions, all of which are limited by available tools and reagents for study. While it is dangerous to ignore flaws in models and methods, especially when identified, it is also unwise to be frightened by difficulty. Without a doubt, the future NO and H2S research will yield interesting and important new discoveries in the years to come. Hopefully this review provides a good introduction to investigators new to the field of NO and H2S that will help them deftly approach study of these molecules in the biological systems.

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Acknowledgements: This research was supported by the CounterACT Program, National Institutes of Health, Office of the Director, and the National Institute of Environmental Health Sciences, Grant Number 1U01ES023759 to RPP and by NIH HL11331 to C.G.K and a Malcolm Feist Cardiovascular Predoctoral Fellowship to S.Y.

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Conflicts of Interest:

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RPP and CGK are co-inventors on patents for use of nitrite salts for the treatment of cardiovascular conditions. C.G.K. has I.P. interest in hydrogen sulfide detection technology and is chief scientific officer and co-founder of Innolyzer LLC.

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121. Verdon CP, Burton BA, and Prior RL. Sample pretreatment with nitrate reductase and glucose-6phosphate dehydrogenase quantitatively reduces nitrate while avoiding interference by NADP+ when the Griess reaction is used to assay for nitrite. Analytical biochemistry 224: 502-508, 1995. 122. Wang L, Frizzell SA, Zhao X, and Gladwin MT. Normoxic cyclic GMP-independent oxidative signaling by nitrite enhances airway epithelial cell proliferation and wound healing. Nitric oxide : biology and chemistry / official journal of the Nitric Oxide Society 26: 203-210, 2012. 123. Wang le F, Patel M, Razavi HM, Weicker S, Joseph MG, McCormack DG, and Mehta S. Role of inducible nitric oxide synthase in pulmonary microvascular protein leak in murine sepsis. Am J Respir Crit Care Med 165: 1634-1639, 2002. 124. Wang P, Zhang G, Wondimu T, Ross B, and Wang R. Hydrogen sulfide and asthma. Exp Physiol 96: 847-852, 2011. 125. Wang R. Physiological implications of hydrogen sulfide: a whiff exploration that blossomed. Physiol Rev 92: 791-896, 2012. 126. Wang R. Two's company, three's a crowd: can H2S be the third endogenous gaseous transmitter? FASEB journal : official publication of the Federation of American Societies for Experimental Biology 16: 1792-1798, 2002. 127. Wang X, Bryan NS, MacArthur PH, Rodriguez J, Gladwin MT, and Feelisch M. Measurement of nitric oxide levels in the red cell: validation of tri-iodide-based chemiluminescence with acidsulfanilamide pretreatment. J Biol Chem 281: 26994-27002, 2006. 128. Whiteman M, Li L, Kostetski I, Chu SH, Siau JL, Bhatia M, and Moore PK. Evidence for the formation of a novel nitrosothiol from the gaseous mediators nitric oxide and hydrogen sulphide. Biochemical and biophysical research communications 343: 303-310, 2006. 129. Whitfield NL, Kreimier EL, Verdial FC, Skovgaard N, and Olson KR. Reappraisal of H2S/sulfide concentration in vertebrate blood and its potential significance in ischemic preconditioning and vascular signaling. American journal of physiology Regulatory, integrative and comparative physiology 294: R1930-1937, 2008. 130. Wintner EA, Deckwerth TL, Langston W, Bengtsson A, Leviten D, Hill P, Insko MA, Dumpit R, VandenEkart E, Toombs CF, and Szabo C. A monobromobimane-based assay to measure the pharmacokinetic profile of reactive sulphide species in blood. Br J Pharmacol 160: 941-957, 2010. 131. Ya-Hong C, Rui W, Bin G, Yong-Fen Q, Pei-Pei W, Wan-Zhen Y, and Chao-Shu T. Endogenous hydrogen sulfide reduces airway inflammation and remodeling in a rat model of asthma. Cytokine, 2009. 132. Yadav AK, Doran SF, Samal AA, Sharma R, Vedagiri K, Postlethwait EM, Squadrito GL, Fanucchi MV, Roberts LJ, 2nd, Patel RP, and Matalon S. Mitigation of chlorine gas lung injury in rats by postexposure administration of sodium nitrite. American journal of physiology Lung cellular and molecular physiology 300: L362-369, 2011. 133. Yadav PK, Yamada K, Chiku T, Koutmos M, and Banerjee R. Structure and kinetic analysis of H2S production by human mercaptopyruvate sulfurtransferase. J Biol Chem 288: 20002-20013, 2013. 134. Yong QC, Hu LF, Wang S, Huang D, and Bian JS. Hydrogen sulfide interacts with nitric oxide in the heart: possible involvement of nitroxyl. Cardiovascular research 88: 482-491, 2010. 135. Zhang D, Macinkovic I, Devarie-Baez NO, Pan J, Park CM, Carroll KS, Filipovic MR, and Xian M. Detection of protein S-sulfhydration by a tag-switch technique. Angewandte Chemie 53: 575-581, 2014. 136. Zhang J, Wang X, Chen Y, and Yao W. Correlation between levels of exhaled hydrogen sulfide and airway inflammatory phenotype in patients with chronic persistent asthma. Respirology 19: 11651169, 2014. 137. Zhang P, Li F, Wiegman CH, Zhang M, Hong Y, Gong J, Chang Y, Zhang JJ, Adcock I, Chung KF, and Zhou X. Inhibitory Effect of Hydrogen Sulfide on Ozone-induced Airway Inflammation, Oxidative Stress and Bronchial Hyperresponsiveness. Am J Respir Cell Mol Biol, 2014.

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Figure 1 NO-formation pathways: NO is synthesized by one of three nitric oxide synthases (NOS) that use L-arginine, oxygen and NADPH (reducing equivalents) as substrates to produce NO and citrulline. Citrulline is recycled back to L-Arginine via arginosuccinate synthase (ASS) and Arginosuccinate lyase (ASL). Alterantively, nitrite can be reduced to NO in a hypoxia and pH dependent manner via heme- or metalloproteins that can couple lowering oxygen tesnions and pH with a 1 electron reduction to NO. Once formed, NO can facilitate specific biological responses by nitrosylated heme proteins, by undergoing oxidation to facilitate nitrosation reactions resulting in protein S-nitrosothiols (RSNO) or nitrosamine (XNO) or undergoing rapid reactions with other free radicals (e.g. superoxide) to form peroxynitrite (ONOO-), which can mediate oxidation and nitration reactions. Ultimately, NO can be oxidized to nitrate, potentially via nitrite. Nitrate is then excreted or reduced back to nitrite by commensal nitrate-reducing bacteria in the oral cavity.

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Figure 2 Sulfide metabolism and transsulfuration pathways CBS and CSE govern the flow of sulfur through homocysteine, cystathionine and cysteine, which is known as the transsulfuration pathway. These two enzymes are also the major catalytic pathways for generating H2S from cysteine and homocysteine in the cytosol. The third pathway via CAT and 3-MST may be more important in the mitochondrion and requires the transfer of sulfur from a persulfide (RSSH). On the other hand, persulfide can be a source of H2S at the expense of reductants such as glutathione (GSH) and thioredoxin (Trx(SH)2). In the mitochondrion, H2S reduces sulfide quinone oxidoreductase (SQR), generating a persulfide which is further transferred to an acceptor (RSH). This persulfide is then oxidized by persulfide dioxygenase (ETHE1) to SO32-, which is eventually converted to S2O32- and SO42- by rodanase and sulfite oxidase respectively.

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Figure 3 Potential mechanisms by which H2S regulates protein thiol post-translational modifications S-nitrosation is a post-translation modification. In the illustrated scheme, the formation of SNO on a reactive thiol converts the protein from a “closed” conformation to an “open” one. SNO may serve as a target for H2S, which restores the protein to the “closed” form, generating thionitrous acid (HSNO) and hydrogen disulfide (HSSH) plus nitroxyl (HNO) in succession. However, HSNO may also mediate transnitrosation or undergo homolysis to NO, H2S or nitrite (26). HSSH may further modify the active thiol to a persulfide resulting in a stabilized “open” or “closed” conformation, which may vary from case to case.

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