Role of hydrogen sulphide in airways

Abstract

The toxicity of hydrogen sulfide (H₂S) has been known for a long time, as it is prevalent in the atmosphere. However accumulative data suggest that H₂S is also endogenously produced in mammals, including man, and is the third important gas signaling molecule, besides nitric oxide and carbon monoxide. H₂S can be produced via non enzymatic pathways, but is mainly synthesized from L-cysteine by the enzymes cystathionine-γ-lyase, cystathionine-β-synthetase, cysteine amino transferase and 3-mercaptopyruvate sulfurtransferase (3MTS). The formation of H₂S from D-cysteine via the enzyme D-amino acid oxidase and 3MTS has also been described. Endogenous H₂S not only participates in the regulation of physiological functions of the respiratory system, but also seems to contribute to the pathophysiology of airway diseases such as chronic obstructive pulmonary disease, asthma and pulmonary fibrosis, as well as in inflammation, suggesting its possible use as a biomarker for these diseases. This review summarizes the different implications of hydrogen sulfide in the physiology of airways and the pathophysiology of airway diseases.

Key Words: Hydrogen sulfide; Airways; Asthma; Chronic obstructive pulmonary disease; Inflammation

Core tip: Hydrogen sulfide (H₂S) is a metabolite produced in mammalian organisms both in physiological and in pathological conditions. The measured levels appear differentiated in inflammatory airway diseases, showing the need to acknowledge H₂S not only as a metabolic mediator but as a signaling biomarker as well. This could be of clinical importance since H₂S levels could be used in order to access staging or treatment efficiency in patients suffering from airway diseases.

INTRODUCTION

Hydrogen sulfide (H₂S) is prevalent in the atmosphere since it is generated from sources both manmade and natural. Therefore organisms may need to either protect themselves from H₂S or respond to it, but not particularly in a true signaling way. Although its production in mammalian tissues has been long known, H₂S was largely ignored as a metabolic waste. Its toxicity seems to depend mainly on the concentration and considerably less on the duration of H₂S exposure[1]. Importantly, accumulating data suggest that H₂S is indeed endogenously produced in mammals, including man, and it represents the third important gas-signaling molecule, besides nitric oxide (NO) and carbon monoxide (CO). Even more, a possible interaction between gas-signaling molecules, especially NO and H₂S, has been described[2,3]. Endogenously produced H₂S affects many biological processes in most human systems, including gastrointestinal, cardiovascular, nervous, endocrine system and kidneys[4-6]. Available data suggest that main cellular targets of H₂S are ion channels, such as ATP-sensitive potassium channels (K_ATP) and transient receptor potential vanilloid channels (TRPV)[7], transcription factors, such as heme oxygenase-1 (HO-1) and nuclear factor kappa B (NF-κB)[8,9], as well as kinases like mitogen-activated protein kinases (MAPK)[10]. These biological effects of H₂S have led to the study of its implication in many diseases and the development of H₂S-donating drugs with a possible clinical potential[6,11]. The involvement of H₂S in the early stages as well as in the development of inflammatory diseases of the respiratory system makes it important to identify it as a biomarker that could be helpful in the prediction or the treatment of such pathological conditions. This review focuses on the effects of H₂S on the respiratory system and its implication in airway diseases.

H₂S METABOLISM

The metabolic pathways of H₂S production in mammals, have been extensively described and are summarized elsewhere[11-13]. Briefly, H₂S can be synthesized from L-cysteine, a sulfur-containing amino acid derived from alimentary sources, synthesized from L-methionine through the so-called “trans-sulfuration pathway” with homocysteine as an intermediate, or released from endogenous proteins[1] (Figure 1). H₂S is synthesized from L-cysteine by the enzymes cystathionine-γ-lyase (CSE) and cystathionine-β-synthetase (CBS). These enzymes are responsible for the majority of the endogenous production of H₂S and their expression appears to be tissue specific[14]. Furthermore, the enzyme cysteine amino transferase (CAT) catalyzes the formation of 3-mercaptopyruvate from L-cysteine that is converted to H₂S by the enzyme 3-mercaptopyruvate sulfurtransferase (3MTS). H₂S can also be synthesized from D-cysteine via the enzyme D-amino acidoxidase (DAO) that converts D-cysteine to 3-mercaptopyruvate, followed by its conversion to H₂S by 3MTS[13]. H₂S can also be produced via non enzymatic pathways but these pathways account only for a small portion of the total H₂S production[5].

Figure 1 Schematic presentation of pathways for hydrogen sulfide synthesis from L- and D-cysteine via the enzymes cystathionine-γ-lyase, cystathionine-β-synthetase, cysteine amino transferase, D-amino acid oxidase and 3-mercaptopyruvate sulfurtransferase. H₂S: Hydrogen sulfide; CSE: Cystathionine-γ-lyase; CBS: Cystathionine-β-synthetase; CAT: Cysteine amino transferase; DAO: D-amino acidoxidase; 3MTS: 3-mercaptopyruvate sulfurtransferase.

H₂S, once produced in mammalian cells, can be stored as bound sulfane sulphur and released later in response to a physiological stimulus[12]. H₂S is removed quickly from the cellular environment via three main catabolic pathways: (1) H₂S oxidation, which takes place mainly in mitochondria, initially to thiosulfate, followed by its conversion to sulfite and sulfate; (2) H₂S methylation by thiol S-methyltransferase (TSMT) to methanethiol and dimethylsulfide; and (3) sulfhemoglobin formation by H₂S binding to methemoglobin[7] (Figure 2).

Figure 2 Main catabolic pathways of hydrogen sulfide. H₂S: Hydrogen sulfide.

H₂S EFFECT ON AIRWAYS PHYSIOLOGY

Many different methods have been used in order to estimate H₂S physiological levels in the plasma or validate its use as a biomarker for a variety of pathophysiological conditions. This effort is not always easy, due to artifacts, which often lead to inconsistent and contradicting measurements[15]. However, there are studies showing that in healthy adults between the age of 56.6 to 75.0 years, the median H₂S serum concentration is approximately 35 μmol/L[16], while H₂S plasma concentration seems to be higher in 6-12 years old children (Table 1)[17]. H₂S concentration in exhaled air of healthy adult subjects was found to be 8-16 ppb[18]. On the other hand, H₂S concentration in lungs, at least in rat, is approximately 30 μmol/L[19]. H₂S levels appear altered in some pathological conditions of the airways, like asthma, Chronic Obstructive Pulmonary Disease (COPD) and pneumonia (Table 1) suggesting that H₂S is probably involved in the pathophysiology of some airways diseases. Therefore, like exhaled NO, H₂S may be a possible biomarker for pulmonary diseases and/or a potential target for new therapeutic approaches for these diseases. Recent studies suggest that H₂S participates in the relaxation of airway smooth muscle (Table 2). As far as the ability of airways to produce H₂S is concerned, it has only been showed in porcine airways that H₂S can be produced endogenously and that the H₂S precursor, L-cysteine caused a concentration-dependent relaxation in peripheral bronchioles[20]. Most of the studies concerning the effect of H₂S on airways are focused on the effect of exogenous H₂S, using H₂S donors. Contractility studies regarding the effect of the rapidly releasing H₂S donor, sodium hydrosulfide (NaHS) demonstrated that H₂S caused a concentration-dependent relaxation in porcine[20], mouse and guinea pig bronchi[21], as well as in rat trachea[22]. This relaxant effect did not depend on epithelium integrity, K_ATP channels opening or NO release[22]. On the other hand, in guinea pig bronchi[23], as well as mice lung[24], H₂S seems to induce the release of sensory neuropeptides, due to the activation of TRPV1 receptors, resulting to the contraction of these bronchi. Therefore, when sensory nerves were desensitized by capsaicin treatment, H₂S induced a slight relaxation[23]. Deviations from these findings emerged from the study of Kubo et al[21], which showed that NaHS did not cause contraction in guinea pig bronchi, but a slight relaxation. Recently, an in vivo study revealed that NaHS treatment inhibited the ozone-induced bronchial hyperresponsiveness in mice[25]. Studies regarding the mechanisms involved in H₂S-induced relaxation of airways showed that H₂S exerts its effect mainly by decreasing intracellular calcium levels. This is due both to reduced calcium influx[26] and to inhibition of Ca²⁺ release from intracellular stores through InsP₃ receptors[27].

Table 1 Hydrogen sulfide concentration in healthy subjects and patients with asthma, chronic obstructive pulmonary disease or pneumonia.

Subjects	Age(yr)	H2S concentration in serum/plasma (μmol/L)	Ref.
Healthy
	71-80	35.7 ± 1.2	[16]
	61-70	34.0 ± 0.9	[16]
	50-60	36.4 ± 1.1	[16]
	64.1 ± 8.7	35.4 ± 5.3	[43]
	9.22 ± 1.80	52.60 ± 5.56	[17]
Patients with bronchial asthma
Bronchial asthma	6-12	44.17 ± 10.95	[17]
Neutrophilic group	53.0 ± 13.9	8.8 ± 4.7	[46]
Paucigranulocytic group	45.5 ± 15.7	6.9 ± 2.0	[46]
	9.03 ± 1.84	44.17 ± 10.95	[17]
Patients with stable COPD
Patients with acute exacerbations of COPD
	73.9 ± 8.3	33.8 ± 18.6	[43]
Patients in stage I to II	65.6 ± 1.6	40.5 ± 6.3	[16]
Patients in stage III		33.4 ± 2.9	[16]
Patients in stage IV		27.6 ± 1.6	[16]
Patients with pneumonia	57.6 ± 20.4	22.7 ± 14.6	[43]
		H2S concentration in exhaled air (ppb)
Healthy	52.86 ± 19.81	8.0-16.0	[18]
Patients with bronchial asthma
Eosinophilic group	46.0 ± 15.2	7.7 ± 4.2	[46]
Paucigranulocytic group	45.5 ± 15.7	11.1 ± 4.6	[46]
Patients with COPD
Acute exacerbations	67.5 ± 11.47	8.0-13.0	[18]
Stable COPD	64.11 ± 8.79	9.0-12.0	[18]

H₂S: Hydrogen sulfide; COPD: Chronic Obstructive Pulmonary Disease.

Table 2 The effect of hydrogen sulfide on airway smooth muscle function.

Tissue	H2S effects	Involved mechanism	Ref.
Porcine peripheral bronchiols	Relaxation	Alteration in K+ channels activity	[20]
Guinea pig main bronchus	Slight relaxation		[21]
Guinea pig airways	Neurogenic inflammatory responses	Stimulation of TRPV1 receptors on sensory nerves endings	[23]
Mouse main bronchus	Relaxation	Independent of NK1/NK2 tachykinin receptors, KATP channels, production of NO, cGMP and prostaglandins	[21]
Mouse lung	Neurogenic inflammation	Stimulation of NK1 and Substance P release	[24]
Mouse small intrapulmonary airways	Relaxation	Inhibition of Ca2+ release from intracellular stores through InsP3 receptors	[27]
Mouse tracheal smooth muscle cells	Relaxation	Activation of BKCa channels	[26]
Rat trachea	Relaxation	Independent of KATP channels, β-adrenoceptors, epithelium and production of NO, cGMP and prostaglandins	[22]
Human ASMCs	Relaxation	Opening of KATP channels	[29]
Isolated human airway smooth muscle cells	Relaxation	Inhibition of ERK-1/2 and p38 MAPK phosphorylation	[30]
	Decrease of cell proliferation and IL-8 release

H₂S: Hydrogen sulfide; TRPV: Transient receptor potential vanilloid channels.

It has also been shown, that H₂S is involved in the relaxation of different smooth muscle types, by affecting a variety of ion channels. For example, in vessels, H₂S induces smooth muscle relaxation via its effect on K_ATP channels located on vascular smooth muscle cells, or on small to medium conductance K⁺ channels located on vascular endothelial cells, which results to membrane hyperpolarization and smooth muscle relaxation[28]. Similarly, in airways, evidence suggests the implication of K⁺ channels in the relaxant effect of H₂S[20]. Moreover, in primary cultured mouse tracheal smooth muscle cells NaHS seems to activate large conductance calcium activated potassium channels (BK_Ca) causing an increase in potassium outward currents, cell hyperpolarization and inhibition of Ca²⁺ influx[26]. Furthermore, H₂S caused relaxation by opening K_ATP channels in isolated human airway smooth muscle cells[29].

Finally, both endogenous and exogenous H₂S decreased human airway smooth muscle cell proliferation and interleukin (IL)-8 release induced by FCS, via the inhibition of the phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 and p38 MAPK[30]. The effects of H₂S donors that have been described were not affected by the inhibition of CSE, the blockade of K_ATP channels or NO production.

H₂S IN THE PATHOPHYSIOLOGY OF AIRWAY DISEASES

Endogenous H₂S participates in the regulation of physiological functions of the respiratory system (Table 3) and seems also to contribute in the pathophysiology of airway diseases such as COPD, asthma and pulmonary fibrosis (Table 3), suggesting its possible use as a biomarker for these diseases. Αpart from the specific features of the pathophysiology, inflammation is a common theme of these diseases. Over the past decade, research data support a key role for H₂S in acute or chronic inflammation in different clinical conditions[31] and suggest that H₂S has anti-inflammatory and cytoprotective effects that could be beneficial in lung diseases. Animal studies suggest that H₂S in the lung increases the anti-inflammatory cytokine, IL-10, while it decreases the pro-inflammatory cytokine, IL-1β in burn and smoke-induced acute lung injury murine models[32] or hyperoxia-induced acute lung injury models in mice[33]. Animal studies also revealed that treatment with H₂S attenuated lung injury and prolonged the subjects’ survival[32,33]. Similarly, inhalation of H₂S appears to be protective against ventilator-induced lung injury, in mice, by limiting cytokine release and neutrophil transmigration[34]. This protective role is associated with down-regulation of genes related to oxidative stress and inflammation and up-regulation of anti-apoptotic and anti-inflammatory genes[35]. Activating transcription factor 3 (Atf3), a protein that limits pro-inflammatory cytokine expression and controls the balance between proliferative and apoptotic signals[36,37], may have an important role in H₂S mediated lung protection, since H₂S inhalation up-regulated Atf3 gene[35]. Finally, in mice, NaHS treatment reduced the ozone-induced increase of the total cell number, including neutrophils and macrophages; the levels of cytokines, including tumor necrosis factor-α (TNF-α), chemokine ligand 1, IL-6 and IL-1β[25], as well as the increase of the bronchial alveolar lavage (BAL) fluid. On the other hand, inhalation of H₂S protects against ventilator-induced lung injury by preventing edema formation, apoptosis, proinflammatory cytokine production, neutrophil accumulation, and inhibits heme oxygenase-1 expression[34].

Table 3 Implication of hydrogen sulfide in the pathophysiology in human airway diseases - its use as a biomarker.

Disease
COPD	Higher serum H2S level in patients with COPD compared with healthy subjects[16]
	Acute exacerbation of COPD decreases serum H2S level compared to patients with stable COPD[16,42]
	Higher sputum H2S levels in patients with acute exacerbation of COPD compared to those with stable COPD[42]
	Higher sputum-to-serum ratio of H2S in COPD subjects with acute exacerbation comparative with those with stable disease[42]
	Lower serum H2S levels in patients with COPD who required antibiotics treatment[43]
Asthma	In children, serum H2S concentration was significantly decreased compared to healthy subjects and correlated positively with FEV1[17]
	In adults, exhaled H2S was lowest in eosinophilic asthma correlated positively with FEV1[46]
Pulmonary fibrosis	H2S suppress human fibroblast migration, proliferation and phenotype transform stimulated by fetal bovine serum and growth factors and inhibits the TGF-β1-induced differentiation of fibroblasts to myofibroblasts[53]

H₂S: Hydrogen sulfide; COPD: Chronic Obstructive Pulmonary Disease; FEV₁: Forced expiratory volume during first second; TGF-β₁: Transforming growth factor beta 1.

Although most of the studies suggest that H₂S has an anti-inflammatory role, some studies have showed that it may contribute to neurogenic inflammation in airways[38]. Thus, both in guinea pig[23] and mouse[24] H₂S induced the release of sensory neuropeptides, while only in mice, it also affected the level of substance P in the lungs, in sepsis-associated lung injury[39].

COPD

Despite inflammation, smoking is the main contributory factor for developing this disease. Animal studies suggest that H₂S is protective against smoking-induced lung injury. Namely, exposure of rats to cigarette smoke resulted to an increase in CSE levels and the subsequent H₂S administration reduced the number of inflammatory cells, as well as airway hyperresponsiveness[40]. Similar findings were reported in mice with tobacco smoke-induced emphysema[41]. Clinical studies showed evidence that H₂S may be implicated in the pathophysiology of COPD and alteration of its levels may be connected with the severity of the disease. In humans, H₂S serum levels were significantly higher in patients with COPD compared to healthy subjects and a positive correlation between the severity of COPD and H₂S serum levels has been shown. Namely, in patients with stable COPD, H₂S serum levels were lower in patients with stage III than in those with stage I obstruction. Additionally, in patients either with or without COPD H₂S correlated positively with the percentage of predicted forced expiratory volume (FEV₁)[16]. On the other hand, acute exacerbation of COPD decreases H₂S serum levels compared to those of patients with stable COPD[16,42]. On the contrary, H₂S sputum levels were significantly higher in patients with acute exacerbation of COPD compared to those with stable COPD, which resulted in a higher sputum-to-serum level ratio of H₂S in COPD subjects with acute exacerbation in comparison to those with stable disease[42]. As far as COPD treatment is concerned, measured H₂S serum levels were significantly lower in patients with COPD who required antibiotics treatment[43], while theophylline treatment did not alter significantly H₂S serum levels of COPD patients[44].

Asthma

Clinical studies indicate that H₂S serum levels were decreased in patients with either stable asthma or severe acute exacerbations. Even more the changes in H₂S serum[45] or exhaled air[25] levels correlated positively with FEV₁ and negatively with the count of sputum cells, neutrophils[46] or eosinophils[25]. Similarly in children with asthma, H₂S serum concentration was significantly decreased compared to healthy children and the concentration was positively correlated with lung function indices[17]. Whether the decrease of H₂S serum levels in patients suffering from asthma is the cause or the consequence is not yet clear. Therefore, it is not clear if H₂S levels could be used as a biomarker for the disease, like exhaled NO. However, Tian et al[17] proposed that decreased H₂S serum levels might be used to indicate decreasing lung function and Wang et al[45] suggested that nasal H₂S could be a way of accurately detecting H₂S metabolism in the respiratory system since its levels will not be affected by oral conditions.

Additional evidence for the possible implication of H₂S in the pathophysiology of asthma comes from animal studies. In the lungs of OVA-treated rats with asthma H₂S serum levels and H₂S production from the lungs were decreased in correlation with the decreased CSE expression level and CSE activity in lung tissues[47]. Even more the administration of NaHS or the CSE blocker, D,L-propargylglycine, alleviated or aggravated, respectively, airway hyper-responsiveness in both cigarette smoke exposure model and OVA-induced asthma rat models[40,47].

Pulmonary fibrosis

Pulmonary fibrosis is the final common pathway of a diverse group of lung disorders and is characterized by accumulation and abnormal activation of fibroblasts and myofibroblasts, resulting in excess extracellular matrix deposition and alveolar disruption[48]. Idiopathic pulmonary fibrosis (IPF) is caused by unknown reasons and its pathophysiology has not yet been clarified, while there is a controversy among researchers whether inflammation constitutes the initial stimulus. Nevertheless, the initial stage is quickly followed by abnormal wound healing[49] and the main protein involved in this process seems to be epithelial cell-derived transforming growth factor beta 1 (TGF-β₁)[50,51]. Evidence suggests that the endogenous CSE/H₂S pathway may participate in the pathogenetic process of pulmonary fibrosis. Myofibroblasts have a main role in the pathogenesis of this disease and although they are generally considered to be differentiated from existing interstitial fibroblasts or bone marrow-derived stem/progenitor cells, epithelial cells also seem to be an important source of myofibroblasts in pulmonary fibrosis[25]. H₂S seems to facilitate the maintenance of alveolar epithelial cell phenotype, since TGF-β₁ induces epithelial–mesenchymal transition and this effect is suppressed by H₂S through a decrease in Smad2/3 phosphorylation, in lungs[52]. Fang et al[53] reported that H₂S suppressed human fibroblast migration, proliferation and phenotype transform that was stimulated by fetal bovine serum and growth factors and more specifically inhibited the TGF-β₁-induced differentiation to myofibroblasts. These effects on pulmonary fibroblasts were partially mediated by decreased phosphorylation of ERK. Animal studies showed that NaHS administration ameliorated the bleomycin induced pulmonary fibrosis in rats[54,55]. This protective effect of H₂S is due, partly, to inhibition of NF-κB p65 expression and regulation of Th1/Th2 balance[55].

Last but not least it is important to point out the potential therapeutic use of H₂S. Studies show that both the metabolite itself and its donors could be potentially used in the treatment of various diseases. Specifically, the H₂S donor GYY4137 exhibits antihypertensive activity[1], while other donors have anti-inflammatory[56] and antioxidant properties[57]. As far as respiratory diseases are concerned, there are studies showing that the donor-induced elevated levels of H₂S are useful in the treatment of respiratory distress syndrome as well as other pathological conditions, since H₂S can reduce the oxidative stress that is present in such disorders[1].

CONCLUSION

H₂S appears to play a role both in the physiological function and the pathobiological conditions of the respiratory system. Its presence as a metabolite in inflammatory diseases, as well as the correlation that is found between H₂S and inflammation mediators such as cytokines or growth factors, support its use as a biomarker of pathological conditions in both the lungs and the airways. However, determining H₂S levels in body fluids is not an easy task, because H₂S levels are influenced by H₂S inhaled from atmospheric air. Such artifacts make its use as a biomarker difficult. Therefore, further studies are required in order to determine the physiological H₂S levels and their correlation with the phase, arising or deteriorating, of inflammatory diseases. Overall, it seems that H₂S is not a cell waste, but an important metabolite that has yet to receive the proper attention.

ACKNOWLEDGMENTS

We would like to thank Dr. Makris D and Paraskeva E for their critical reading of the manuscript.