Hydrogen Sulfide: A gasotransmitter of clinical relevance

Abstract

Though the existence of H₂S in biological tissues has been known for over 300 years, it is the most recently appreciated of the gasotransmitters as a physiologic messenger molecule. The enzymes cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS) had long been speculated to generate H2S, and inhibitors of these enzymes had been employed to characterize influences of H₂S in various organs. Definitive evidence that H₂S is a physiologic regulator came with the development of mice with targeted deletion of CSE and CBS. Best characterized is the role of H₂S, formed by CSE, as an endothelial derived relaxing factor that normally regulates blood pressure by acting through ATP-sensitive potassium channels. H₂S participates in various phases of the inflammatory process, predominantly exerting anti-inflammatory actions. Currently, the most advanced efforts to develop therapeutic agents involve the combination of H₂S donors with non-steroidal anti-inflammatory drugs (NSAIDs). The H₂S moiety provides cytoprotection to gastric mucosa normally adversely affected by NSAIDs, while the combination of H₂S and inhibition of prostaglandin synthesis may afford synergistic anti-inflammatory influences.

Introduction

Hydrogen sulfide (H₂S) is the most recently appreciated of the three gasotransmitters, joining nitric oxide (NO) and carbon monoxide (CO). Though only recently recognized as being physiologically formed in mammalian tissues, H₂S has been known to exist in animal tissues for many years. Like NO and CO, H₂S is toxic, about 5 times more so than CO [1]. Recently, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) have been established as the major physiologic sources of mammalian H₂S based on studies showing that their deletion or inhibition markedly diminishes mammalian H₂S levels[2, 3]. As with NO and CO, identification of the biosynthetic enzymes now provides a firm basis for elucidating how H₂S is produced, signals to intracellular targets, and affects diverse physiologic processes. In the interest of brevity, the review will be limited to a few areas of H₂S disposition: focusing on physiologic roles in the cardiovascular system and inflammation and a brief discussion of regulatory mechanisms and signaling modalities.

Understanding the disposition of H₂S can be facilitated by comparisons with NO and CO. NO was identified as endothelial derived relaxing factor and as regulating macrophage function years before the first NO synthase (NOS) was purified and cloned[4, 5]. NO is formed by a family of NO synthase isoforms. Cloning and characterization of neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS) greatly facilitated research in the field and led to a vast expansion of NO literature [6]. The NOS isoforms are all heme-requiring enzymes, a property they share with CBS. nNOS and eNOS are constitutive but activated in response to Ca2+/calmodulin[7], which also activates CSE[8].

NO relaxes blood vessels by binding to heme in the active site of guanylyl cyclase to facilitate formation of cyclic GMP which, via protein kinase G, relaxes blood vessels[9]. S-nitrosylation is a more prominent and ubiquitous physiological signaling mechanism for NO whereby NO reacts with the SH group of cysteines in target proteins to inhibit or activate them [10]. As described below, H₂S appears to signal predominantly by an analogous mechanism - sulfhydration of target proteins, whereas no major action via cyclic nucleotides has been reported for H₂S [11].

nNOS is highly localized to discrete neuronal systems in the brain and to autonomic nerves in the periphery [12]. eNOS occurs in the endothelial layer of blood vessels and the respiratory system, while iNOS occurs in all cells of the body, but is notably enriched in macrophages[4]. Localizations of CBS and CSE are less well characterized, though the enzymes are highly expressed in liver and kidney and at lower levels in pancreas, adipose tissue, small intestine and brain[13]. In the brain, CBS is largely glial, while CSE occurs in neurons and endothelial cells [14, 15].

CO also displays some analogies to NO. It is generated by two isoforms of heme oxygenase (HO) with HO1 being inducible, similar to iNOS, while HO2 is constitutive. Like nNOS and eNOS, HO2 is activated by calcium/calmodulin [16]. HO2 is highly localized to neurons in the brain and the periphery and fulfills many characteristics of a neurotransmitter[17]. In the intestine HO2 and nNOS are co-localized in myenteric neurons where both appear to serve as neurotransmitters of non-adrenergic-non-cholinergic neurotransmission[18, 19]. As related below, there is some evidence for myenteric localizations of CSE which might fulfill similar functions as the other two gasotransmitters in the gut. A conjunction of all three gasotransmitters occurs in the carotid body, where nNOS, HO2, and CSE colocalize in glomus cells and regulate firing of carotid body efferents in response to hypoxia [20].

H₂S metabolism

There has been much controversy over endogenous, mammalian levels of H₂S and the extent to which the gas derives from exogenous sources or reflects influences of biosynthetic enzymes. Estimates for H₂S concentrations have ranged from the high micromolar to the low nanomolar with recent appreciation that physiologic levels are probably relatively low [21]. A major confounding factor in H₂S measurement has been the large endogenous stores of sulfane sulfur, which is artifactually reduced to H₂S during assays [22, 23]. Another difficulty relates to sensitivity and specificity of the various techniques employed to measure H₂S. Recently several groups have developed fluorescent probes which may be substantially more sensitive and may permit imaging of H₂S in intact cells [24, 25].

CBS and CSE, the enzymes generally acknowledged as the principal sources of physiologic mammalian H₂S, were both first known as participants in metabolism of cystathionine, which is formed by CBS via the condensation of homocysteine with serine to generate cystathionine as a thiol ether. The markedly elevated levels of homocysteine in patients with homocystinemia, a genetic deletion of CBS, lead to substantial cardiovascular disability[26]. CBS forms H₂S from cysteine or homocysteine with a combination of the two substrates providing maximal yields in vitro [27]. Inhibitors of CBS, such as hydroxylamine or amino-oxyacetate impair the generation of H₂S from cysteine in the brain, but they are nonspecific, affecting all pyridoxal phosphate enzymes. Moreover, because the K_m of CBS for cysteine and homocysteine is 3 – 7mM, high concentrations of these amino acids are employed in studies of H₂S formation, whereas physiologic levels are less than 10% of the K_m values [27].

The heme in CBS binds CO with high affinity, at least 100 times that of NO [28]. Hence, CO appears to be a physiologic inhibitor of CBS, which, as described below, may account for vasodilation of the cerebral circulation. CBS is also activated by S-adenosyl methionine, whose function is unclear but might reflect some relationship between signaling by H₂S and biologic methylation [29].

CSE was first characterized as cystathionase, responsible for the pyridoxal phosphate dependent hydrolytic degradation of cystathionine [30]. CSE was proposed as a physiologic generator of H₂S in peripheral tissues such as the liver, because inhibitors, such as propargylglycine (PAG) and β-cyanoalanine (BCA), diminish H₂S formation. While these inhibitors are relatively non-selective, more recent studies of CSE deleted mice have definitively established that CSE is the predominant source of H₂S in peripheral tissues [8]. Evidence supporting CSE as generating H₂S for signaling purposes comes from the finding that CSE, like nNOS, eNOS, and HO2, is activated by calcium/calmodulin [8].

Less well characterized than CBS and CSE as a source of H₂S in mammalian tissues is the enzyme 3-mercaptyopyruvate sulfotransferase (3-MST). Kimura and associates [23] developed evidence that 3-MST acts in conjunction with cysteine aminotransferase (CAT) to produce H₂S from cysteine in the presence of α-ketoglutarate. The combination of 3-MST and CAT might be responsible for the generation of H₂S in brain preparations from CBS deleted mice. Because 3-MST is maximally active at very high pH levels, it is not clear to what extent it is responsible for mammalian formation of H₂S.

CBS, CSE and 3-MST appear to be highly conserved, with the sequences of bacterial forms of these enzymes fairly similar to mammalian isoforms. Very recently, Nudler and associates [31] have discovered that H₂S is critical for the survival of bacteria and that a wide range of antibiotics, whose initial targets vary markedly, all act via H₂S as a final, common pathway. Thus, bacteria with deletion of the H₂S forming enzymes are markedly more sensitive to antibiotic killing. This discovery may portend a new class of antibiotic-sensitizing drugs that lower the bactericidal concentrations of antibiotics.

H₂S Signaling

Unlike NO and CO, H₂S does not appear to stimulate guanylyl cyclase, even though it can bind with reasonably high affinity to heme, which occurs in the active site of the enzyme [32]. H₂S has been shown to signal via a mechanism analogous to nitrosylation whereby it forms a covalent linkage to the SH of cysteines, a process designated sulfhydration [11]. Sulfhydration was first detected by the biotin switch assay employed to monitor nitrosylation. In this procedure free thiols are blocked by methyl methane thiosulfonate (MMTS). The SH groups of nitrosylated cysteines (-SNO) can then be exposed by treatment with ascorbate and subsequently labeled and identified [33]. Even in the absence of ascorbate, some proteins are labeled by the biotin switch technique, which provided a clue to the existence of sulfhydration. Accordingly, sulfhydration can be detected in a modification of the biotin switch procedure with omission of the ascorbate step [11].

Recently, sulfhdyration has been monitored by a new technique which overcomes concerns that some free thiols might not be blocked by MMTS [34]. The newer procedure employs a fluorescent maleimide derivative, which interacts selectively with sulfhydryl groups of cysteines, both sulfhdyrated and non-sulfhydrated. Treatment of samples with dithiothreitol (DTT) selectively cleaves disulfide bonds, detaching the fluorescent signal from sulfhydrated but not non-sulfhydrated proteins and leading to decreased fluorescence [34]. This technique can be modified to simultaneously detect nitrosylation using a differently colored fluorescent maleimide after treatment with ascorbate to remove NO from nitrosylated cysteines, exposing previously nitrosylated SH groups [34].

Sulfhydration appears to be substantially more prevalent than nitrosylation. Whereas nitrosylation typically affects only about 1-5% of most proteins, 10-25% of endogenous GAPDH, β-tubulin and actin are basally sulfhydrated [11].

Sulfhydration can influence protein function differently than nitrosylation. Nitrosylation provides an NO ‘cap’ to reactive SH groups of cysteines typically inactivating proteins, though in some instances it has been shown to have an activating effect [35]. By contrast, in sulfhydration, an SH is converted to SSH which, with its lower pKa, is more reactive chemically than SH and may have greater exposure to the cellular environment. This notion is substantiated by the finding that sulfhydration of GAPDH increases catalytic activity 700%, and sulfhdyration of actin similarly augments biologic activity. Activation of GAPDH by sulfhdyration is physiologically relevant, as total GAPDH activity of liver extracts is reduced about 25 – 30% in CSE deleted mice despite normal levels of GAPDH protein [11]. It appears that, as with nitrosylation, many, if not most, proteins are sulfhdyrated.

Physiologic actions of H₂S

Cardiovascular system

Like NO and CO, H₂S dilates blood vessels. Studies with exogenous H₂S largely report vascular relaxation, though under some conditions, such as high oxygen concentration, vasoconstriction is evident. NO was first elucidated as endothelial derived relaxing factor (EDRF). Investigations employing eNOS knock-out mice and NOS inhibitors reveal only a partial reduction of EDRF activity in certain vascular beds [8, 36, 37]). EDRF activity in HO2 knockout mice has not yet been reported. Studies of H₂S in CSE knockout mice indicate a major contribution to EDRF activity [8]. Immunohistochemical analysis shows that CSE is highly localized to the endothelial layer of blood vessels. Cholinergic relaxation of the mesenteric artery is reduced by about 75 – 80% in homozygous CSE deleted mice and about 50% in heterozygotes. This cholinergic relaxation reflects EDRF activity being abolished by removal of the endothelium. CSE knockout mice develop age-dependent hypertension with maximal increases in blood pressure of about 20 mm Hg, similar to levels of hypertension in eNOS knockouts [8].

The EDRF activity associated with NO is most evident in large vessels such as the aorta, while in the resistance vessels that are the primary determinants of blood pressure, actions of NO are less prominent. In the mesenteric artery, a resistance vessel, H₂S is predominant [8]. Relative roles of H₂S, NO, and CO in various vascular beds may be elucidated by systematic comparison of mice with deletion of HO2, eNOS and CSE.

NO and H₂S differ markedly in mechanisms whereby they influence blood vessels. NO and CO stimulate cyclic GMP levels while recent studies indicate that H₂S vasodilation largely reflects hyperpolarization elicited by opening ATP-sensitive potassium channels (K_ATP) [38-40]. While vasorelaxation by exogenous H₂S has long been known to involve such channels, recent work establishes that physiologic vasorelaxation is mediated by H₂S. Thus, glibenclamide, a potent and selective inhibitor of the K_ATP channel, reduces effects of H₂S and diminishes cholinergic hyperpolarization of mesenteric arteries smooth muscles by about 70% while not affecting relaxation elicited by NO donors [40].

H₂S stimulates K_ATP channels by sulfhydrating them at cysteine-43. These channels are activated physiologically when bound by phosphatidylinositol(4,5)bisphosphate (PIP2). The binding of PIP2 to K_ATP channels is abolished in cells devoid of CSE or containing a catalytically inactive form of the enzyme. Moreover, H₂S donors substantially enhance the binding of PIP2 to K_ATP channels, and PIP2 binding occurs at the sulfhydrated cysteine-43 [40].

The observation that H₂S physiologically acts by sulfhydrating and activating the K_ATP channel supports the notion that H₂S is a major if not predominant mediator of EDRF activity. Numerous investigators have found much if not most EDRF activity involves cGMP independent blood vessel hyperpolarization [37] implying that EDRF is primarily dependent upon an endothelial-derived hyperpolarizing factor (EDHF) whose activity is largely attributable to H₂S.

The major role of H₂S in regulating the peripheral circulation suggests that it may be the principal vasoactive gasotransmitter, implying therapeutic relevance. This notion is supported by the limited success of studies. devoted to inhibiting or enhancing NO formation respectively to combat endotoxic shock or to treat hypertension [41].

H₂S may also impact the cerebral circulation. Hypoxia is well known to stimulate cerebral blood flow, but underlying molecular mechanisms have been elusive. Very recently CSE has been identified as a major regulatory factor for cerebral arteriolar vasodilation, acting in conjunction with CO formed by HO2 [42, 43]. HO2 is an established physiologic O₂ sensor, especially in the carotid body where it is exquisitely sensitive to activation by oxygen and is inhibited by hypoxia in a precisely graded fashion [44]. At physiologic concentrations, CO inhibits CBS, the predominant generator of H₂S in the cerebral circulation [29]. Thus, by inhibiting HO2, hypoxia would lead to activation of CBS and generation of H₂S as a vasorelaxant.

Before endogenous H₂S was shown to regulate blood vessels, exogenous H₂S had been shown to exert beneficial cardiovascular actions. Many studies have dealt with myocardial ischemia, which is substantially diminished by administration of H₂S donors during ischemia/reperfusion of the heart [45-47]. Numerous mechanisms had been proposed for these cardioprotective actions[48, 49]. Particularly promising is evidence that H₂S acts by inhibiting apoptosis, as H₂S donors reproducibly diminish poly(ADP-ribose) polymerase (PARP) cleavage, as well as cleavage of caspase-3 [50]. H₂S also preserves mitochondrial structure and function in response to myocardial ischemia. H₂S may also be cardioprotective by decreasing the “work” of the heart, analogous to beta-blockers, through diminishing contractility of cardiac myocytes, largely by inhibiting L-calcium channels [51].

Because of the promising cardiovascular actions of H₂S a variety of drugs have been developed based on this gasotransmitter. Some are simple H₂S donors, such as GYY4137, while others combine an H₂S donating structure with an anti-inflammatory drug such as diclofenac or a classical vasodilator such as sildenafil[52-54].

H₂S and inflammation

The literature on NO, CO, and H₂S has been plagued with conflicting claims for their effects. Nowhere has this been most evident than with H₂S and inflammation. Prominent proinflammatory effects have been reported in association with increased formation of sulfide in neutrophils as well as activation of these cells [55]. Administration of H₂S donors has been reported to accentuate inflammatory factors associated with burns, while burn injuries were reduced by treatment by the CSE inhibitor propargylglycine [56]. Lung injury elicited by bacterial sepsis can be alleviated by treatment with propargylglycine and worsened with H₂S donors [55]. By contrast, there are numerous reports of anti-inflammatory effects for H₂S donors as described below. A consensus has emerged in recent years that the apparently contradictory findings largely reflect variations in dose-response relationships. At relatively low, physiologic concentrations H₂S appears to be anti-inflammatory, while high concentrations elicit inflammation, a pattern reminiscent of NO, which is anti-inflammatory in low concentrations and pro-inflammatory at high levels. CO, well known to be lethal in high doses, is also often beneficial when administered in low doses [57, 58].

What physiologic mechanisms underlie influences of H₂S on inflammation? One of the best characterized involves the disposition of leukocytes, especially their adherence to vascular endothelium as well as their extravasation. H₂S donors and sulfide salts diminish lymphocyte and neutrophil infiltration in models of inflammation, whereas inhibitors of H₂S biosynthesis increase leukocyte adherence[59]. H₂S donors diminish edema, presumably due to inhibition of plasma exudation, while CBS and CSE inhibitors increase the formation of edema in response to inflammatory stimuli[59]. A molecular mechanism underlying anti-inflammatory roles of H₂S may include its scavenging peroxynitrite, a toxic derivative of NO, as well as other oxidants.[60]

H₂S has been shown to exert beneficial influences in disorders of joints, including resolving synovitis in rodents [61] and alleviating the pathology of carageenen-associated arthritis [62]. H₂S donors also have been extensively explored in intestinal disorders, with beneficial effects in several models of colitis [63].

H₂S may participate in some actions of TNFα. While TNFα is regarded as proinflammatory, it does display anti-apoptotic actions mediated via NF-κB. The anti-apoptotic actions of NF-κB appear to be mediated by H₂S generated by CSE [34]. TNFα treatment triples H₂S generation by stimulating the binding of the transcription factor SP1 to the CSE promoter. The H₂S generated by CSE enhances the binding of NF-κB to promoters of downstream genes, whose signaling is markedly diminished in CSE knockout mice. H₂S acts by sulfhydrating the p65 subunit of NF-κB, which promotes its binding to the co-activator ribosomal protein S3 (RPS3). The anti-apoptotic influences of NF-κB are substantially reduced in CSE deleted mice [34].

The anti-inflammatory influences of H₂S have led to efforts to develop therapeutic agents. Classic non-steroidal anti-inflammatory drugs (NSAIDs) often cause gastric irritation by inhibiting the formation of prostaglandins, which are physiologic cytoprotectants of the gastric mucosa. H₂S, on the other hand, reduces mucosal inflammation, protects the gastrointestinal mucosa from injury and also augments tissue repair. In direct comparisons of naproxen and its H₂S -linked derivative, the latter exerted comparable therapeutic efficacy with reduced gastric damage [64, 65]. Several other NSAIDs have been combined with H₂S donors. Mechanistic studies have been conducted with some of these drugs, with particularly extensive investigations utilizing S-diclofenac [52, 66, 67]. S-diclofenac has been shown to inhibit cell proliferation [68, 69] and to protect against ischemia-reperfusion injury in perfused hearts [53].

Conclusions

Evidence for H₂S as a physiologic gasotransmitter has lagged behind CO and NO, but H₂S is rapidly catching up. Therapeutic applications may emerge in the not-too-distant future, especially in the area of anti-inflammatory drugs. Definitive understanding of how H₂S participates in inflammatory processes may come from studies of inflammation in mice with deletion of CSE and/or CBS. In the gastrointestinal system and liver, CSE levels greatly exceed those of CBS. Because many major proteins are physiologically sulfhydrated, it is possible that overall metabolic functions of the liver are determined in notable part by the actions of H₂S, as is evident by the substantial decrease in GAPDH activity in livers of CSE knockout mice, due to the loss of the activating influence of GAPDH sulfhydration [11].

One area not addressed in this review is the role of H₂S in the brain, discussed in a previous review[70]. Studies with mice lacking CBS and CSE suggest that the majority of H₂S in the brain derives from CBS rather than CSE. The limited immunohistochemical studies thus far performed reveal CBS predominantly in glia [14]. CSE may have neuronal as well as glial localizations so that even if it generates a smaller amount of H₂S, this enzyme might be the source of a neurotransmitter pool [15]. In the intestine there is evidence that CSE is localized to the myenteric plexus of neurons and may exert physiologic influences on intestinal motility [15, 71]. Conceivably, neuronal CSE in the gut occurs in the same neurons known to possess HO2 and nNOS, which are co-localized in neuronal populations [72].

Figure 1. Pathways of H₂S metabolism.

Cysteine metabolism from methionine and dietary cyst(e)ine, which enter cells via specific transporters, leads to H₂S production. H₂S is derived from cysteine, cystine, and 3-mercaptopyruvate (3MP). 3-Mercaptopyruvate sulfurtransferase (3MST) and 2-cysteine aminotransferase (CAT) produce H2S and pyruvate from 3MP, which is formed from cysteine and α-ketogluterate produced by CAT. Cystathionine-β-Synthase (CBS) catalyzes the β-replacement of cysteine with homocysteine (Hcy) to generate H₂S and the corresponding thiol ether (Hcy-S-Cys). Cystathionine-γ-lyase (CSE) catalyzes β-disulfide elimination on cystine, the product of which reacts with available thiols (Cys is shown) to generate H₂S and a disulfide (Cys-S-S-Cys).

Figure 2. Structures of Substances Involved in H2S Physiology.

Table 1.

Actions of H₂S agents with therapeutic relevance.

Model	Hydrogen Sulfide Donor/Inhibitor/KO Mouse	Ref.
Hypertension: CSE -/- mice	Age dependent hypertension observed beginning at 7 wks Cholinergic relaxation of mesenteric artery reduced 75-80% in CSE -/- mice	[8]
Ischemia Reperfusion Injury (Rat, Mouse, Pig)	NaHS and H2S donors reduced myocardial infarct size in rat, mouse, and pig models while PAG attenuated this effect and increased infarct size	[45-48, 50]
Ischemia reperfusion injury: perfused rabbit heart	H2S releasing NSAID S-Diclofena protected against ischemia-reperfusion injury in isolated rabbit heart	[53]
Burn Injury induced inflammation in mouse	Prophylactic and therapeutic administration of PAG reduced burn-associated systemic inflammation, while NaHS increased systemic burn-associated inflammation	[56]
LPS induced lung and liver inflammation in mouse	NaHS administration increased TNF levels, lung inflammation and MPO activity in the liver and lung, while PAG reduced lung and liver MPO activity and ameliorated lung and liver tissue damage	[55]
Mouse air pouch model	NaHS and other donors suppressed leukocyte infiltration, which was enhanced by H2S inhibitors	[59]
Carrageenan-induced paw edema: mouse	NaHS and other donors suppressed carrageenan-induced paw edema in the mouse to a level similar to KATP channel agonist.	[59, 62]
Carrageenan-induced joint synovitis model: rat	The H2S donor Lawesson's reagent attenuated pain response and inflammatory biochemical changes, whereas PAG potentiated synovial iNOS activity and enhanced macrophage infiltration	[61]
TNBSA induced mouse model of colitis	H2S donating mesalamine derivative ATB-429 reduced colitis severity and granulocyte infiltration by 70%.	[63]
NSAID induced gastropathy: Rat	NaHS and S-Diclofenac reduced NSAID induced gastric injury and decreased inflammatory mediators TNF, COX2, ICAM1, while H2S inhibitor BCA increased these mediators and enhanced gastric injury	[52, 65]