Hydrogen sulphide (H2S), nitric oxide (NO), carbon monoxide (CO)
Nitric oxide (NO), carbon monoxide (CO) and hydrogen sulphide (H2S) are gases which have always been present in the earth's atmosphere (Li et al. 2009). All three are colourless and flammable gases; NO and CO are odourless, whereas H2S gives off a characteristic smell of rotten eggs (Szabo et al. 2007). Each gas has natural origins; NO is formed by lightening in the upper layers of the atmosphere, while CO and H2S are found in high concentrations in volcanic discharges (Li et al. 2009). H2S is also a major component of natural gas and the hydrolysis of sulphide in hot springs and geysers leads to its generation (Li et al. 2009). All three gases can be formed during various industrial processes. Petroleum refineries generate NO, CO and H2S. The liberation of sulphur and H2S from petroleum occurs via the "hydrosulphurisation" process (Guidotti et al. 1996). H2S can also be produced by other industrial processes, such as natural gas plants, coke oven plants and tanneries.
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NO, CO and H2S are all known to be toxic gases and environmental pollutants, with the nervous system being most affected (Nicholls et al. 1982). There have been many cases where death has been the result of either accidental or deliberate exposure to CO and H2S. Hence, CO has been labelled as the 'silent killer' as it is odourless, non-irritant and highly toxic when inhaled (Krenzelok et al. 1996). H2S has also been known to cause human poisoning and death due to exposure in sewers, wells and septic tanks (Chaturvedi et al. 2001). The mechanism of toxicity is the same for each gas; they act by binding to the haem aa3 site of cytochrome c oxidase. This enzyme oxidises the terminal acceptor of the electron transport chain in mitochondria, ferrocytochrome c (Li et al. 2009). Therefore, inhibition of cytochrome c oxidase diminishes mitochondrial oxidative phosphorylation and cell metabolism is altered (Kimura. 2010). NO reversibly binds to the haem aa3 site, whereas H2S and CO bind in an irreversible manner (Nicholls et al. 1982).
Biosynthesis of H2S, NO and CO
"Gasotransmitter" is the term used to refer to a gaseous messenger molecule involved in any signalling process (Mustafa et al. 2009). Nitric oxide (NO) and carbon monoxide (CO) are endogenously produced gaseous mediators, and both are involved in normal physiology and disease (Bhatia. 2005). NO was the first gasotransmitter found to exert a number of physiological and pathophysiological roles within the body. NO is formed via the oxidation of a guanidino nitrogen of L-arginine and citrulline is produced as a co-product (Bredt et al. 1994). NO synthase (NOS) is the enzyme which catalyses the formation of NO, and it exists in three isoforms; neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). nNOS is localised in the cytoplasm and mitochondria, whereas eNOS is expressed in the plasma membrane and cytoplasm (Jobgen et al. 2006). They are constitutively produced and release low levels of NO. In contrast, iNOS generates a large amount of NO when induced by stimuli in the cytoplasm (Li et al. 2009) Endogenously produced NO causes vasorelaxation of vascular smooth muscle (Furchgott et al. 1991). Cyclic GMP is generated when NO binds to heme in the active site of soluble guanylyl cyclase (sGC). Cyclic GMP then activates protein kinase which phosphorylates protein and decreases cytosolic calcium, before dephosphorylation of the myosin light chain (Gadalla et al. 2010). Therefore, leading to smooth muscle relaxation.
CO was known to be formed physiologically long before attention was devoted to its function (Krenzelok et al. 1996). CO is synthesised via the catabolism of heme to CO with iron and biliverdin produced as by-products (Leffler et al. 2007). Haem oxygenase (HO) catalyses this reaction, and it exists in the three isoforms; HO-1, HO-2 and HO-3. HO-1 is an inducible enzyme localised in the brain, it acts by cleaving the heme ring in red blood cells to the straight-chain biliverdin (Mustafa et al. 2009). This process leads to the degradation of heme and the release of CO. HO-2 is constitutively expressed, with highest amounts of the enzyme present in the brain (Maines et al. 2005). HO-3 has been identified in the brain and produces relatively modest amounts of CO, its specific role is still unknown (Li et al. 2007). It was thought that guanylyl cyclase was responsible for CO-induced vasodilation. However, extensive evidence suggests CO-induced dilation is mediated by activation of Ca2+-activated-K+-channels (BKCa channels) (Naik et al. 2003). CO binds to BKCa channel-bound heme which increases BKCa channel Ca2+ sensitivity. This decreases the activity of voltage-dependent Ca2+ channels (VDCC), causing membrane hyperpolarisation of smooth muscle cells (Leffler et al. 2006).
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H2S is now recognised as an important signalling molecule in the cardiovascular and nervous systems (Szabo et al. 2007) L-cysteine is the substrate responsible for endogenously synthesising H2S in mammalian cells via the activity of two pyridoxal-5'-phosphate-dependent enzymes, cystathionine ß-synthase (CBS) and/or cystathionine ?-lyase (CSE) (Wang. 2009). Firstly, CBS and CSE are involved in the formation of L-cysteine via the "transsulfuration pathway" (Fig 1). Methionine-derived homocysteine is catabolised to generate cyasthionine during a condensation reaction catalysed by CBS (Elsey et al. 2010). Cyasthionine is then hydrolysed by CSE to form L-cysteine, along with ammonia and an a-ketobutyrate (Gadalla et al. 2010). CBS and CSE are the enzymes responsible for catalysing the condensation reaction of homocysteine with L-cysteine, and the subsequent generation of H2S (Chen et al. 2004). Production of H2S by CBS is affected by the extent of allosteric activation of S-adenosylmethionine, and the enzyme's activity is reduced under hyperhomocysteinemic conditions (Singh et al. 2009).
The expression and activity of CBS and CSE varies greatly among different vascular beds (Zhao et al. 2003). CBS is a haem protein which is highly expressed in the mammalian brain, most notably the hippocampus and cerebellum (Kery et al. 1994). CBS is the only enzyme that produces H2S in the brain (Eto et al., 2002). Whereas CSE synthesises H2S in the vasculature (Li et al. 2009); especially smooth muscle cells and endothelial cells (Yang et al. 2008). The liver contains high levels of CBS and CSE enzyme expression (Whiteman et al. 2008).
A third H2S-producing enzyme is thought to exist as H2S was not depleted in CBS knockout mouse brain (Ishigami et al .2009). The enzyme is 3-Mercaptopyruvate sulfurtransferase (3MPST) which works with cysteine aminotransferase (CAT) to synthesise H2S from L-cysteine in the presence of a-ketoglutarate (fig 1) (Frendo et al. 1997). CAT is identical to another enzyme, aspartate aminotransferase (AAT). These enzymes then catalyse the reaction of L-cysteine with a-ketoglutarate to form 3-mercaptopyruvate, which is desulphurated to form H2S (Li et al. 2009). 3MPST is localised in various tissues within the body, including the liver, heart and brain (Kimura. 2010).
Catabolism of the gasotransmitters
In biological systems, each gas is rapidly broken down (Li et al. 2009). NO is catabolised to form NOx, a mixture of nitrite (NO2-) and nitrate (NO3-), which is found in low concentrations in plasma (Li et al. 2009). Low amounts of NO can be detected in expired air (Trolin et al. 1994). Most endogenously produced CO is also released from the body in expired air (Li et al. 2009).
Like NO and CO, H2S is also detectable in human expired air (Li et al. 2009), however it can also be degraded by various chemical and enzymatic processes. The exact concentration of H2S in the plasma is unknown, although values of 20-80 µl has been suggested (Whiteman et al. 2008). These values also include the hydrosulphide anion (HS-) and the sulphide anion (S2-), so the concentration of free H2S gas is probably much lower (Furne et al. 2008). H2S can react with methaemoglobin to form green sulphaemoglobin, which may act as a metabolic sink for circulating H2S (Lowicka et al. 2007). Haemoglobin could also act as a sink for NO and CO, and saturation with one gas would affect the binding of the other two (Wang. 2002). For example, the saturation of the haemoglobin sink with CO leads to a significant increase in endogenous H2S concentration (Searcey et al. 1998). In cells, mitochondria are responsible for the oxidation of H2S to thiosulphate and then converted into sulphate (Hildebrandt et al. 2008). Thiosulfate sulfurtransferase and thiosulfate reductase are the two enzymes which act upon thiosulphate during this process (Li et al. 2009) Thiosulfate sulfurtransferase is predominantly expressed in the liver, it is also present in the kidney but much lower levels are found in other organs (Kamoun. 2004). Thiosulfate reductase is expressed in equal amounts in the liver and kidney, with the brain, heart and testis also containing significant amounts (Kamoun. 2004). It is not possible to use sulphate as a 'bio-marker' of H2S synthesis in biological systems as it is also formed via oxidation of cysteine by cysteine dioxygenase (Li et al. 2009).
There are other mechanisms to degrade H2S in the body, although they not seen to be as important. Methylation by thiol-S-methyltransferase breaks down H2S to form methanethiol and dimethylsulphide as products (Levitt et al. 1999). Finally, the strong reducing activities of H2S could account for its consumption by hydrogen peroxide, an endogenous oxidant species, in the vasculature (Geng et al. 2004).
Endogenous effects of H2S
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H2S can be produced enzymatically in vascular tissues and causes vasorelaxation both in vivo and in vitro (Hosoki et al. 1997). Examples of blood vessels relaxed by H2S in vitro include the isolated rat aorta, portal vein, mesenteric and hepatic, but not the coronary (Li et al. 2009), vascular beds. The effect that H2S has on the vasculature is dependent upon the vascular bed. H2S can cause vasorelaxation or constriction, or both in non-mammalian vertebrates (Dombkowski et al. 2005). Bovine pulmonary arteries contract to H2S, whereas rat pulmonary arteries undergo a complex contraction-relaxation-contraction response (Li et al. 2007). H2S causes both vasorelaxation and vasoconstriction in different arteries from a range of vertebrates including shark, hagfish, sea lamprey, toad, alligator, duck and rat; suggesting that H2S is a versatile vasoregulatory molecule that can be used to suit both organ-specific and species-specific requirements (Dombkowski et al. 2005).
The mechanism by which H2S causes vasorelaxation involves the direct stimulation of ATP-sensitive K+ channels (KATP) in vascular smooth muscle cells (Tang et al. 2005). The opening of KATP channels hyperpolarises the cell membrane, thereby inactivating voltage-dependent L-type Ca2+ channels which reduces intracellular free Ca2+ and leads to cell relaxation and vasorelaxation (Nelson et al. 1995).
Some studies have found the relaxation effect of H2S alone to be weak, but an interaction between NO and H2S occurs which has a relaxant effect on vascular smooth muscle (Hosoki et al. 1997). There is thought to be involvement of additional KATP-dependent mechanisms due to glibenclamide only partially blocking the vascular effects as H2S-induced vasorelaxation (Webb et al. 2008). Studies have found that the removal of the endothelium, blockade of NO synthase or the Ca2+ dependent K+ channel blockers all attenuate H2S-induced vasorelaxation. This suggests that H2S may influence the release of endothelium-derived vasorelaxant factors, including NO, that facilitate the relaxation of smooth muscle (Zhao et al. 2002). Some studies have found H2S-induced vasorelaxation to occur via a non-ATP-associated increased conductance of the KATP channel (Koenitzer et al. 2007). It is likely that the vasodilator effect of H2S on blood vessels involves multiple cellular targets and mediators, and that the size of the blood vessels is also important, as is the concentration of endogenous H2S achieved locally (Li et al. 2006).
It has also been found that H2S reacts with NO to form a nitrosothiol moiety, therefore causing a contractile response (Whiteman et al. 2008). Some studies have suggested that low concentrations of H2S are able to quench NO, resulting in constriction (Elsey et al. 2010). Studies performed in mouse aortic rings and in human internal mammary artery showed a dual vasodilator and vasoconstrictor effect (Kimura. 2010). It is therefore important to discover whether or not the H2S-derived vasoconstriction is potentially a species, strain and /or vascular bed-sensitive process (O'sullivan. 2006).
Low concentrations of H2S in the nervous system do not affect excitatory postsynaptic potentials (Kimura. 2010). However, H2S does enhance NMDA receptor-mediated currents, which is required for the induction of hippocampal long-term potentiation (LTP)(Teague et al. 2002). LTP may be related to associative learning, whereby two inputs simultaneously entered into the same H2S neuron produce a synergistic effect (Kimura. 2010). Endogenous H2S in the brain enhances NMDA receptor-induced currents, thus initiating the induction of LTP (Chen et al. 2007). The induction of LTP occurs as H2S increases cAMP levels in neuronal and glial cell lines, which activates protein kinase A and NMDA-receptor-mediated excitatory currents (Wang et al. 2002). Aside from its role in signal transduction, H2S protects neuron cells from oxidative stress by increasing levels of the antioxidant glutathione and by activating KATP and Cl- channels (Chen et al. 2007).
Endogenously-produced H2S in the brain has been associated with disease. Some studies suggest that H2S causes neuronal toxicity by inhibiting cytochrome c oxidase and/or overstimulating of NMDA receptors (Lowicka et al. 2007). This leads to a progressive mental retardation in patients with 21 trisomy (Lowicka et al. 2007); a genetic abnormality in which there are three copies of a particular chromosome, instead of two. Studies show that patients with Down syndrome have an overproduction of H2S in the brain. This is due to the location of the CBS gene chromosome 21 in humans (Chen et al. 2007). However in some diseases, such as Alzheimer's disease, there is a deficiency of H2S and CBS activity is dramatically decreased (Eto et al. 2002).
Most studies carried out in vivo models suggest that H2S is a pro-inflammatory mediator (Elsey et al. 2010). An early stage in inflammation involves the adherence of leukocytes to vascular endothelium, they can then migrate into underlying tissue (Gadalla et al. 2010). Experiements on lipopolysaccharide-treated mice have shown an increase in the plasma H2S concentration and CSE expression (Li et al. 2009). These findings suggest H2S increases the inflammatory response and the damage to organs associated with sepsis (Elsey et al. 2010).
In contrast, some studies indicate that endogenous H2S is anti-inflammatory (Gadallaf et al. 2010). The CSE inhibitor ß-CNA causes a large increase in the aherance of leukocytes to the endothelium as well as carrageenan-induced leukocyte infiltration and paw oedema (Zanardo et al. 2006). Whereas H2S-releasing derivatives of diclofenac have been shown exert anti-inflammatory activity in endotoxic shock and against carrageenan-induced hindpaw swelling (Li et al. 2009). H2S appears to have varying roles although a small increase in H2S production enhances in inflammation; non-specific host defences are enhanced by small increase in H2S production, overproduction may lead to inflammation and tissue damage (Elsey et al. 2010).
H2S exhibits negative ionotropic activity both in vitro and in vivo (Li et al. 2009), but still protects cardiac muscle from ischemic injury (Kimura. 2010). Activation of KATP channels provides protection and CFTR Cl- channels stabilise membrane potential, the same mechanism is involved in the protection of neurons from oxidative stress (Kimura et al. 2010). A long-term cardiprotective effect of H2S has been suggested as the treatment of spontaneously hypertensive rats with NaHS reduced the hypertrophy of intra-myocardial arterioles and ventricular fibrosis (Li et al. 2007). H2S induces in vitro and in vivo angiogenesis (perhaps by Akt), promoting endothelial cell proliferation, adhesion and migration, whereas higher but still non-toxic concentrations yield no such effects (Hoefer. 2007).
Work regarding the possible roles of H2S in regulating endocrine function has centred on the well known role of KATP channels in controlling the function of insulin-secreting pancreatic ß-cells (Li et al. 2007). A reduced amount of glucose-induced insulin release from rat insulinoma cells was found with the application of exogenous H2S or transinfection with CSE, whereas the CSE inhibitor PAG increased release (Yang et al. 2005). This indicates that H2S generated in the pancreas may help to regulate insulin release, and that the insulin deficiency associated with diabetes may be caused by abnormally high H2S production (Li et al. 2007).
H2S is an important endogenous modulator of the hepatic circulation; its vasodilatory effects are not affected by NOS inhibitors or the presence of NO (Fiorucci et al. 2006). CBS and CSE are not expressed in the endothelium of the liver blood vessels, which indicates the vasorelaxant effects of H2S are endothelium-independent (Fiorucci et al. 2005).
Liver cirrhosis is associated with the alteration of methionine metabolism, and inhibition of the transsulfuration pathway causes impaired liver function; both lead to the accumulation of homocysteine in the plasma (Fiorucci et al. 2006). This increase in plasma concentrations of homocysteine occurs due to the impairment of the enzymes CBS and CSE, which are responsible for the metabolism of homocysteine (Horowitz et al. 1997). In cirrhotic hepatic stellate cells there is reduced CSE activity which causes contraction around the sinusoids, thereby contributing to increased intra-hepatic resistance and portal hypertension (Fiorucci et al. 2006).
Our study aimed to discover the effects that H2S exerts on the PCA and to determine the mechanisms involved. We also wanted to see if L-cysteine could lead to the generation of endogenous H2S.
We found H2S does not induce a vasorelaxant response via the activation of KATP channels or non-selective K+ channels. Our results indicated that NO and the endothelium do not affect vascular tone in VSMCs. Additionally, it would appear that CSE is not present in PCA tissue as L-cysteine cannot be converted into H2S.
During our study, the addition of low concentrations of NaHS had little effect on vascular tone; however a large vasorelaxation occurred at higher concentrations. This effect is consistent with the literature as H2S usually causes smooth muscle relaxation (Szabo et al. 2007). Some studies have also identified the high concentrations required to cause the vasorelaxation of blood vessels; these levels are commonly associated with sepsis, shock and inflammation (O'Sullivan. 2006).
Our study showed no difference in vascular tone in the presence of glibenclamide (KATP channel blocker) or TEA (non-selective K+ channel blocker) when compared with the control. This indicates that the relaxant effect on blood vessels caused by H2S is not due to the activation of KATP channels or non-selective K+ channels. The expression of different KATP channel subunits may be tissue-specific, so H2S may not stimulate KATP channels in the same way in all tissues (Bhatia. 2005). This theory could explain the different effects of H2S in different vascular beds; however it has not been widely reported. A study carried out in isolated rat cardiomyocytes found that H2S-mediated opening of KATP channels did not occur, which suggests the effects of H2S on KATP channels may be cell specific (Li et al. 2009). In contrast, some literature suggests that H2S-induced vasorelaxation of blood vessels is due to the opening of KATP channels in a non-ATP-associated manner (Webb et al. 2008). This conclusion has been based on the observation that KATP channel inhibitors, such as glibenclamide, abolish the vasorelaxant effect of H2S (Lowicka et al.2007). Studies have also shown that TEA also inhibited H2S-induced vasorelaxation, achieved by blocking many K+ channels in vascular SMCs, including KCa, Kv and KATP channels (Bhatia. 2005). Another theory for the large relaxant response seen at the highest concentration of NaHS addition could be due to toxicity via the inhibition of cytrochrome c oxidase (Mancardi et al. 2009). By inhibiting this enzyme mitochondrial metabolism is affected, ATP production is decreased and cellular metabolism is suppressed; thus affecting smooth muscle contraction (Szabo et al. 2007).
We found the PCA responded in the same way with denuded endothelium as with fully functional endothelium. This is consistent with some studies which found that the relaxation effect of H2S was not affected by the presence of endothelium (Kimura. 2010). The removal of the endothelium does not abolish the relaxant effect of H2S, implying that the endothelium has little effect on H2S-induced vasorelaxation (Hosoki et al. 1997). Studies have also found that CBS and CSE are not present in vascular endothelium, suggesting H2S is only generated in smooth muscle cells (Zhao et al. 2002). However, other studies show that the endothelium does have an effect on H2S-induced vasorelaxation. Some of the H2S-induced vasorelaxation can be attenuated by removal of the endothelium, which could be explained by the release of vasorelaxant factors from the endothelium upon stimulation by H2S (Bhatia. 2005). A study found that the removal of endothelium shifted the dose-response curve of H2S-induced vasorelaxation to the right, but the maximal response remained constant (Zhao et al. 2002). This indicates that a small portion of H2S-induced vasorelaxation is endothelium-dependent could be mediated by the release of NO and/or endothelium-derived hyperpolarising factor (EDHF); both of which cause hyperpolarisation of smooth muscle cells (Cheng et al. 2004).
Our study showed no significant differences between PCA responses in the presence and absnece of L-NAME (NOS inhibitor). This indicates that endothelium-derived NO does not have any effect on H2S-induced vasorelaxaton. In contrast, the literature shows that NO does enhance H2S-induced vasorelaxation via a synergistic effect (Kimura. 2010). This occurs due to an interaction between NO and H2S, resulting in the up-regulation of H2S synthesis (Oh et al. 2006). NO acts by up-regulating CSE activity, therefore increasing the amount of H2S produced by the enzyme (Zhao et al. 2001). This corresponds with a study which discovered the inhibition of CSE expression in the thoracic artery and mesenteric artery in rats by L-NAME (Whiteman et al. 2009).
We found that a slight contractile response occurred in the PCA before the large valorelaxation. H2S Experiments on rat aorta have also found these results, whereby low concentrations caused a contraction and higher concentrations lead to vasorelaxation (Ali et al. 2006). NOS inhibitors, such as L-NAME, have been found to cause contraction of blood vessels (Moore et al. 2000). This contractile effect could be due to H2S quenching NO to form a nitrosothiol, therefore inhibiting the vasorelaxant actions of NO (Bhatia. 2005).
There were contractile tendencies of PCA when gassed in air (95% air : 5% CO2), however this did not occur when gassed in Carbogen (95% O2 : 5% CO2). A p value for the peak contractions of PCA gassed in 95% air compared to 95% O2 was p=0.09; this was almost statistically significant. The contractile tendencies were abolished with the removal of the blood vessel endothelium. Studies have found that the contractile effect of H2S is an indirect effect involving endothelial cells, rather than direct action on vascular smooth muscle cells (Ali et al. 2006). This suggests the mechanism may involve H2S 'mopping up' endothelium-derived vasorelaxant factors; although this cannot be NO as L-NAME had no effect on PCA response. Another possible reason for the contractile tendencies of PCA could be due to the release of endothelin, an endothelium-derived constrictor (Elsey et al. 2010). This would also account for the abolishment of contractile tendencies when the endothelium is removed. A study confirmed this by stating that vasoconstrictive factors can be released from the endothelium under hypoxic conditions (Michiels. 2003).
Our study showed that it is not possible to generate H2S endogenously via the addition of the exogenous precursor L-cysteine. This was also found to be the case in a study which found no effect when NaHS or L-cysteine were added to rat coronary artery (Johansen et al. 2006). This contrasts to some studies which found that endogenous CSE can generate H2S from exogenous L-cysteine (Bhatia. 2005), and a vasorelaxant effect occurs. CSE is involved in the final step for the generation of H2S from L-cysteine (Elsely et al. 2010) and the vasorelaxant effect is inhibited by PPG (CSE inhibitor) (Kimura. 2010). Another study also found that left ventricles of hearts perfused with L-cysteine showed increased levels of endogenous H2S; CSE inhibitors inhibited the vasorelaxant effect of L-cysteine (Lowicka et al. 2007).