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None of the data obtained from our studies was found to be statistically significant. This leads us to believe that the mechanism of H2S vasorelaxation that we observed does not involve K+ channels, be they non-specific or ATP-dependant, and that neither NO or the endothelium have any involvement in relaxing vascular smooth muscle. In addition to this our data shows that L-cysteine cannot be converted into H2S, suggesting a lack of CSE in porcine coronary artery.
H2S considerably relaxes vascular tissues after endothelium removal and there is no significant difference in the effect of H2S in the thoracic aorta with and without endothelial cells, suggesting that the contribution of the endothelium to the relaxatory effect of exogenously applied H2S must be low (Hosoki et al., 1997; Zhao and Wang 2002). This is consistent with our findings since there was no significant difference in response to NaHS between endothelium denuded and endothelium intact vessels, even when the gassing was changed.
On the contrary data indicates that H2S might act as a hyperpolarizing factor, of which the effect is amplified by the endothelium (Zhao et al., 2001) and that part of the relaxation caused by H2S is endothelium dependant and mediated by the release of NO and EDHF from the endothelium (Zhao et al., 2001; Cheng et al., 2004; Zhao and Wang 2002). Alternatively, endothelium-derived vasorelaxant factors may also be released by H2S as L-NAME was reported to inhibit the effect of endothelium-derived hyperpolarizing factor and reduced H2S induced vasorelaxation (Cheng et al., 2004; Doughty et al., 1999). Also, the presence of an intact endothelium might retain H2S in the blood vessel wall so that its vasorelaxant effect can be potentiated and prolonged (Zhao and Wang 2002).
With regards to the involvement of K+ channels we showed that when inhibiting non-selective K+ channels with TEA and ATP-sensitive K+ channels with glibenclamide there was no significant difference in response to NaHS compared to the control. Numerous articles account for the involvement of ATP-sensitive K+ channels with the relaxation of H2S (Zhao et al., 2001; Zhao and Wang 2002; Wang 2002 and 2004) and have proved this by reducing the relaxant effects of H2S by adding glibenclamide (Kubo et al., 2010). In addition to this H2S induced vasorelaxation is inhibited by high concentrations of TEA, and relaxation occurs mostly by opening ATP-sensitive K+ channels (Zhao et al., 2001) and different blockers for KCa or Kv channels fail to affect the vascular effects of H2S (Nelson and Quayle, 1995). Therefore we would have expected to see a decrease in the relaxatory response in the presence of these inhibitors, particularly glibenclamide.
The inhibition of NO using L-NAME showed no significant difference in response to NaHS when compared to the control; we would have expected to see a decreased relaxatory response for a number of reasons. Firstly, NO regulates the endogenous levels of H2S in vascular tissues by directly increasing CSE activity (Zhao et al., 2001), CSE contains 12 cysteine residues that are potential targets for S-nitrosation, which may enhance the activity of CSE (Koenitzer et al., 2007). In addition, NO up regulates the expression of CSE and by increasing the activity of cGMP dependant protein kinases can in turn stimulate CSE (Zhao et al., 2001).
Furthermore H2S induces vasorelaxation which is partially attenuated by blockade of NO synthase (Kimura 2010) and by direct inhibition of NO using L-NAME (Kubo et al., 2010; Zhao and Wang 2002). Although the relaxation effect of H2S alone is weak, there is a synergy between NO and H2S on vascular smooth muscle relaxation (Hosoki et al., 1997). Our studies failed to show an interaction between NO and H2S despite all literature on the topic indicating that a significant difference should have been observed.
Exogenously applied L-cysteine caused a concentration dependent relaxation of strips of human corpus cavernosum. L-cysteine induced relaxation is suppressed by a CSE inhibitor, PPG (Kimura, 2010). Our tissue preparations were unable to produce H2S from L-cysteine. This observation is peculiar since L-cysteine is the only known precursor to H2S and CSE is the only known H2S producing enzyme in the periphery (Zhao et al., 2001; Szabo, 2007). As a result the expectation would have been a response in the control and AOA experiments (CBS inhibitor). However, in a study conducted on rat coronary artery there was no statistically significant effect in response to the addition of NaHS or L-cysteine (Johansen et al., 2006).
Despite these apparent disappointments the general trend we observed in the response to cumulative addition of NaHS is consistent with current literature. At low concentrations there is little effect, and if anything a slight contraction, but at higher concentrations there is a large relaxation preceded by a small contraction. JOURNAL THAT CONFIRMS THIS!
The reason for the delay in response until higher concentrations may due to the sensitivity of smooth muscle cells to H2S, and possibly there are several binding sites that must be occupied to elicit a relaxatory response (Zhao and Wang 2002).
The small contraction prior to the large relaxation is not something uncommon. (Zhoa et al., 2001; Cheng et al., 2004) report this contraction which may be the result of H2S inhibiting the NOS enzymes that produce NO. The large relaxation we observe may be the result of toxicity; this is plausible because the normal range of H2S in vascular tissues is (BLA BLA BLA.). Indeed the relaxatory effect of H2S is mainly due to the direct effect on ATP-sensitive K+ channels however as mentioned before H2S is toxic and can inhibit metabolism via inhibition of cytochrome c oxidase (Nicholls and Kim, 1982). It can therefore reduce the intracellular concentration of ATP which is needed for actin-myosin cross-bridge cycling and ultimately smooth muscle contraction (Beauchamp et al., 1984). In addition, GSH commonly acts as an antioxidant to remove ROS but it in the presence of H2S there is an excess of ROS which is a natural by product of metabolism, this leads to damage of DNA, oxidation of specific enzymes by oxidation of co-factors and lipid peroxidation (Truong et al., 2006).
The relaxation at higher concentrations of NaHS is also transient and attenuates near to its basal level before addition of the final NaHS aliquot. The reason for this short duration of relaxation could be attributed to the scavenging of H2S by metalloproteins, disulfide-containing proteins, thio-S-methyl-transferase and haem compounds (Zhao et al., 2001). Besides the direct effect of H2S on ATP-sensitive K+ channels causing relaxation it may also be the result of its toxicity, since the reduction in ATP may be the cause of the apparent increase in ATP-sensitive K+ channel currents produced by H2S. This is supported by the fact that metabolic inhibitors produce a glibenclamide-sensitive vasodilation in guinea pig coronary arteries (Daut et al., 1990).
Although there was no significant difference in response to NaHS in 95% air compared to 95% O2, a p value of 0.0931 for the peak contractions indicates a definite trend for the tissues to contract more in a less oxygenated environment until the highest concentration is added where they behave similarly. One reason for this is that H2S catalyzes the release of NO from S-nitrosoglutathione in an O2 dependent manner (Koenitzer et al. 2007), so in low O2 levels this process is not a pronounced giving a slight contraction. It is possible that maybe with more experiments we would uncover a significant difference between the responses in 95% air compared to 95% O2. The fact that the vessels responded similarly to the final addition of NaHS (peak relaxation of -33 ± 9% and 28 ± 20% in 95% air and 95% O2 respectively) is further evidence that the relaxation is mainly due to the toxic effects of H2S.
In conclusion what we have demonstrated is that H2S is an important endogenous factor, adding to existing data that groups it with the established gaseous vasorelaxant factors NO and CO (Wang, 1998). According to our data H2S causes a slight contraction at low concentrations by inhibiting the NOS enzymes and preventing by the release of NO from S-nitrosoglutathione, at higher concentrations it causes a transient large relaxation which is the result of the toxicity of H2S. We are lead to this conclusion because there was no statistically significant data to suggest the involvement of K+ channels, NO or the endothelium.
The literature on H2S in the vasculature suggests otherwise and states that the relaxation induced by H2S comprises a minor endothelium dependant effect and major direct effect on smooth muscle, and that the effect is mediated by the opening of ATP-sensitive K+ channels. In addition to this the presence of CSE but not CBS acting on L-cysteine has been shown in the vasculature, and NO has been found to play a role by increasing CSE expression and stimulating it (Zhoa et al., 2001; Cheng et al., 2004; Zhao and Wang 2002; Johansen et al., 2006; Szabo, 2007).
However, all but two of the journals mentioned in this dissertation actually experimented on coronary arteries and of the two that did (Johansen et al., 2006; Daut et al., 1990) neither were porcine. As a result our findings in the porcine coronary artery are plausible.
The effect of H2S is now a well documented area, particularly in the vasculature and brain, but at present the information that exists has been carried out on a number of different animals such as rats, mice, cows and fish. Of these animals the effect in different species and vascular beds has been investigated, and as such it is a challenge to fully categorise the effect of H2S in the vasculature with the data coming from such a variety of sources.
Hydrogen sulphide or hydrogen sulfide (H2S) is a colourless and flammable gas, it has a high solubility in water and possesses the characteristic smell of rotten eggs. H2S is a major component of natural gas and although it is found in small quantities in normal air it occurs to a much greater extent in volcanoes, hot springs and hydrothermal vents, which are areas on the sea bed home to numerous bacteria that utilise H2S and therefore support a multitude of organisms. It is also encountered in sewage treatment plants, swine containment, and manure-handling operations and in any contained spaces in which organic material has decayed or in which inorganic sulphides exist under reducing conditions. By far the largest industrial route to H2S occurs in petroleum refineries via the hydrodesulphurization process which liberates sulphur from petroleum by the action of hydrogen (Guidotti, 1996). Besides being a by-product to industrial and agricultural processes H2S also has valuable applications in industry. Production of thioorganic compounds and alkali metal sulphides, which can be used to degrade polymers still depend on H2S. Also, the gas had importance in analytical chemistry for over a century although it has been superseded by thioacetamide as a source of sulphide ions in small scale analysis.
H2S is a highly toxic compound with numerous fatalities due to exposure, both accidental and premeditated. It exerts a number of adverse effects on body systems, indeed inhalation of up to 10 ppm H2S has little or no metabolic effect on human volunteers when resting or exercising (Bhambhani et al., 1996 & 1997). However, higher concentrations of inhaled H2S (up to 30 ppm) can cause nausea, vomiting, headache and breathlessness whilst even greater exposures (150-250 ppm) elicit respiratory tract irritation and pulmonary oedema (Oesterhelweg and Puschel, 2008).
H2S has been recognised as a potent reducing agent with reports in totally unrelated areas of science confirming this property. For example H2S reacts with and quenches the superoxide anion (O2-) as well as other reactive oxygen species (ROS) (Chang et al., 2008) and sulphide is capable of reducing cytochrome a3 as well as cytochromes c and a (Nicholls and Kim, 1982). H2S toxicity arises from binding to cytochrome oxidase enzymes in a complex mechanism which results in a final inhibitory effect resembling that of cyanide. Inhibition of the cytochrome c oxidase enzyme involves an initial rapid reaction of sulphide oxidation and oxygen uptake with a subsequent step that reduces cytochrome a. In the final inhibitory step sulphide binds reversibly to the haem aa3 site of cytochrome c oxidase (Nicholls and Kim, 1982) consequently inhibiting mitochondrial oxidative phosphorylation and blocking the capacity for adenosine triphosphate (ATP) production. Ultimately this disrupts cell respiration and metabolism since ATP holds a fundamental role in metabolic reactions as well as other energy dependant processes such as active transport and DNA synthesis. The treatment of hydrogen cyanide (HCN) poisoning is successful using methemoglobin, however this is not effective in H2S poisoning despite the ferric haem group of methemoglobin scavenging H2S. This gives rise to other mechanisms of H2S toxicity that involves depletion of GSH (glutathione) and activation of oxygen to form reactive oxygen species that can lead to deleterious effects (Truong et al., 2006).
Nitric oxide and carbon monoxide - established gasotransmitters
Nitric oxide (NO) and carbon monoxide (CO) are gases that also show toxicity in humans, both are found naturally in the atmosphere and also from anthropogenic sources. CO reversibly binds the haem aa3 site of cytochrome c oxidase like H2S to irreversibly inhibit mitochondrial oxidative phosphorylation. CO also exhibits toxicity by binding other haem-proteins such as cytochrome P450 and haemoglobin thereby capacity of O2 carriage (Piantadosi, 2002). NO also affects mitochondrial oxidative phosphorylation in the same way however it does so reversibly (Li et al., 2009).
Despite their toxicity NO and CO are relevant mediators in physiological processes and disease states and are produced endogenously, like H2S. NO synthesis takes place in the vascular endothelium with L-arginine acting as a substrate for NO synthases (NOS), of which two isoforms exist. Endothelial (e) NOS is constitutively expressed in endothelial cells and produces NO in response to physiological stimuli and stress, whereas inducible (i) NOS generates larger amounts of NO in response to immunological stimuli and is transcriptionally regulated (Palmer and Moncada, 1989). NO produced in the endothelium has a relaxatory effect on vascular smooth muscle by activating soluble guanylate cyclase (sGC) which increases intracellular levels of cyclic GMP and protein kinase G, ultimately dephosphorylating myosin light chain kinase and relaxing vascular smooth muscle to cause vasodilation (Surks, 2007).
The endothelium affects smooth muscle and maintains the balance between vasodilation and vasoconstriction as well as inhibition and stimulation of smooth muscle cell proliferation and migration, and thrombogenesis and fibrinolysis. Endothelial dysfunction leads to the disruption of this balance and causes damage to the arterial wall, in the vasculature this is an early indicator to many cardiovascular diseases such as atherosclerosis, myocardial ischemia and coronary artery disease (Herman and Moncada, 2005). Despite numerous other endothelial functions, endothelial dysfunction has become synonymous with reduced biological activity of NO (Yetik-Anacak and Catravas, 2006), therefore NO is an essential biological mediator that besides controlling vascular tone also prevents cardiovascular disease.
The synthesis of CO is mediated by heme oxygenase (HO) of which three isoforms exist, HO-1, HO-2 and HO-3 (Wu and Wang 2005). The HO enzymes catalyse the degradation of heme yielding biliverdin, CO, and iron as the final products (Kikuchi et al., 2005). HO-1 is an inducible enzyme whereas HO-2 is a constitutively expressed enzyme expressed highly in the brain (Leffler et al., 2006), the role of HO-3 is not as clearly defined as the other two isoforms. CO is a dilatory mediator that works by binding to heme which is bound to calcium (Ca2+)-activated-potassium (K+)-channels (BKCa channels), elevating the Ca2+-channel sensitivity opening BKCa channels and hyperpolarising the smooth muscle cell. Also, CO can either inhibit or accentuate vascular cell proliferation and apoptosis, depending on the specific apoptotic signal and cell type (Leffler et al., 2006). CO also has potential as a therapeutic agent in systemic inflammation, lung disease and cardiovascular disease giving evidence of potential treatment of conditions such as diabetes, obesity and asthma (Foresti et al., 2008).
Hydrogen sulphide synthesis
Although toxic, in recent decades H2S has been found to be produced endogenously in different tissues with its relevance as a biologically active gas becoming apparent. In mammalian tissues the process of H2S production is reliant upon two pyridoxal 5-phosphate (PLP)-dependent enzymes, cystathionine gamma-lyase (CSE) and cystathionine beta-synthase (CBS), with other enzymes playing a role. L-cysteine is the only known precursor of H2S in mammals, CSE and CBS are not only responsible for its conversion into H2S, they also play a role in the production of L-cysteine (Fig1). CBS catalyzes the condensation of homocysteine (Hcy) and serine forming cystathionine which is then hydrolysed by CSE to form L-cysteine, which now contains the sulphur atom from Hcy, and Î±-ketobutyrate (Stipanuk and Ueki, 2009).
Fig 1. Transsulfuration pathway for homocysteine degradation and cysteine synthesis.
The same dehydration reaction that CBS catalyses to produce cystathionine is employed to produce H2S endogenously, the only difference being the replacement of serine with L-cysteine under physiological conditions. Other alternative reactions catalyzed by CBS appear to make a negligible contribution of L-cysteine to desulphuration (Singh et al., 2009). In humans CSE combines L-cysteine with water to produce serine, pyruvate, NH3 and H2S (Chen et al., 2004). H2S exerts a negative feedback effect on the activity of these enzymes by inhibiting CSE activity and also inhibiting the rate of gluconeogenesis from L-cysteine (Wang, 2002) and in addition CBS activity is mediated by Ca2+ and calmodulin, its activity is suppressed by calmodulin-specific inhibitors (Eto et al., 2002). CSE appears to be the prominent enzyme responsible for generating H2S in mammalian systemic vessels (Szabo, 2007), producing H2S in vascular smooth muscle cells but not in the endothelium (Zhao et al., 2001) Activity of CSE is also notable in the liver and kidney (Ishii et al., 2004). CBS mRNA is highly expressed in the brain, especially in the hippocampus, while CSE mRNA is not detectable (Abe and Kimura, 1996), in addition (Eto et al., 2002) states that CBS is the only enzyme that produces H2S in the brain and is also expressed in peripheral nerves. Brain homogenates of CBS-knockout mice produce H2S at levels similar to those of wild-type mice, suggesting the presence of another H2S-producing enzyme. 3-mercaptopyruvate sulphurtransferase (3MST) along with cysteine aminotransferase (CAT) are found to produce H2S in the brain from 3-mercaptopyruvate (3MP), which is synthesised from L-cysteine and Î±-ketoglutarate by CAT (Shibuya et al., 2009). In addition 3MST and CAT are localised to endothelial cells of the thoracic aorta, although only 3MST is present in vascular smooth muscle cells (Shibuya et al., 2009). Another less important endogenous source of H2S is the non-enzymatic reduction of elemental sulphur to H2S using reducing equivalents obtained from the oxidation of glucose (Searcy and Lee, 1998)
Endogenous effects of hydrogen sulphide
Brain and nervous system
At physiological concentrations the function of H2S in the brain is to modify long term potentiation (LTP) (Abe and Kimura, 1996), this is a long lasting enhancement in signal transmission between two neurons that results from stimulating them simultaneously and is widely considered one of the major cellular mechanisms that underlies learning and memory (Cooke and Bliss, 2006). In addition, H2S can be locally and transiently increased in response to neuronal excitation therefore suppressing excitatory postsynaptic potentials (EPSPs) (Abe and Kimura, 1996). H2S targets NMDA receptors in the brain to enhance NMDA receptor-mediated currents and facilitate the induction of hippocampal LTP. H2S modulates NMDA receptors and enhances the induction of LTP by increasing production of cAMP and activating protein kinase A resulting in the activation of NMDA-receptor-mediated excitatory postsynaptic currents in neuronal and glial cells as well as oocytes (Kimura, 1999; Wang, 2002).
The CBS gene is encoded on chromosome 21, a region associated with Down syndrome and as a result it has been proposed that over production H2S may be involved in the cognitive dysfunction associated with Down syndrome (Kamoun, 2001). In Alzheimer's disease subjects have abnormally low levels of H2S due to changes in the concentration of CBS (Kamoun, 2004). Combining this knowledge with the fact that polymorphism of the CBS gene is significantly underrepresented in children with high IQ compared with those with average IQ suggests a role for CBS and therefore H2S in cognitive function (Kimura, 2002).
Much is known about the role of ATP-regulated K+ channels in controlling the function of insulin secreting pancreatic Î² cells. Rat insulinoma cells transfected with CSE or given H2S exogenously both showed reduced insulin release whereas CSE inhibitors caused an increase in insulin release (Yang et al., 2005).
The effect of H2S in inflammation is contentious. Experiments on mice with cecal ligation and puncture (CLP)-induced sepsis found that H2S significantly aggravated sepsis-associated systemic inflammation whereas CSE inhibitors significantly reduced sepsis-associated systemic inflammation (Zhang et al., 2006). Furthermore mice injected with LPS (E. coli lipopolysaccharide), a marker of inflammation, that were then administered with H2S showed an increase in lung inflammation and raised plasma TNF-alpha concentration. Conversely in mice given a CSE inhibitor marked anti-inflammatory activity was observed. In separate experiments humans with septic shock showed higher levels of plasma H2S than normal (Li et al., 2005).
H2S donors reduce oedema formation and leukocyte adherence to the vascular endothelium and can increase the resistance of the gastric mucosa to injury and accelerate repair. In addition increased biosynthesis of H2S has been demonstrated in animal models of septic, endotoxic and haemorrhagic shock, pancreatitis and carrageenan-evoked hindpaw oedema in rats. In each case, pharmacological inhibition of H2S biosynthesis is anti-inflammatory (Wallace 2007; Li et al., 2006).
H2S shows negative inotropic effects in rat myocardial tissue (Geng et al., 2004) but at the same time plays a cardioprotective role. H2S produced in the heart protected against damage following coronary artery litigation in rats (Zhu et al., 2007) and also has a role in angiogenesis, a process whereby new blood vessels are grown. The mechanism behind this process seems to be related to activation of Akt (Cai et al., 2007)
H2S is generally regarded as a smooth muscle relaxant that increases the calibre of blood vessels subsequently causing a decrease in blood pressure in isolated rat aorta, gastric artery and portal vein (Hosoki et al., 1997; Zhao et al., 2001) and also in perfused rat mesenteric (Cheng et al., 2004), but not coronary vascular beds (Johansen et al., 2006). The relaxatory effect is also reported in different species of rat, mice and also bovine tissue (Zhao et al., 2001; Cheng et al., 2004; Yang et al., 2008). In addition to this the same effect is seen in other invertebrates (Dombkowski et al., 2005). However there seems to be pronounced species and vascular bed differences in the response to H2S. For example H2S causes a relaxation in isolated rat pulmonary arteries (Wang et al., 2008) but in bovine pulmonary arteries it caused a contraction (Dombkowski et al., 2005). In addition a contraction response has been reported in rat aortic rings (Kubo et al., 2007; Koenitzer et al., 2007; Ali et al., 2006) and a dual vasodilator and vasoconstrictor effect of H2S has also been observed in the human internal mammary artery (Elsey et al., 2010).
The relaxatory response of H2S is mostly due to its opening of ATP-regulated K+ channels in vascular smooth muscle cells (Zhao et al., 2001) which causes hyperpolarisation of the cell membrane and closes voltage-gated Ca2+ channels, subsequently decreasing the concentration of Ca2+ within vascular smooth muscle cells (Wilson et al., 2005). This decreases the contractility of the smooth muscle cells which contract via protein kinase A and myosin light chain kinase (MLCK) activation in the presence of increased intracellular Ca2+.
The application of the NOS inhibitor L-NAME or the co-application of Ca2+-dependant K+ channel blockers and the removal of the endothelium attenuated the vasorelaxant effect of H2S (Zhao and Wang, 2002). The relaxation induced by H2S is partially endothelium dependant and mediated by the release of NO and any vasoconstricitve effect observed with H2S is the result of inhibiting the NOS enzyme involved in the pathway of NO production (Zhao et al., 2001; Cheng et al., 2004). This indicates that the mechanism of relaxation associated with H2S does not only involve ATP-regulated K+ channels, and the release of NO, EDHF (endothelial-derived hyperpolarising factor) or the effects of H2S on ATP production and cell pH might also contribute to its vasoactivity. The short duration of the hypotensive effect of H2S could be attributed to the scavenging of H2S by metalloproteins, disulfide-containing proteins, thio-S-methyl-transferase and haem compounds (Zhao et al., 2001).
Apart from affecting vessel calibre H2S may also prevent diseases in the vasculature. Its angiogenic and reducing properties coupled with its ability to inhibit hypochlorite-induced modification of LDL, an important step in atherogenesis, makes H2S a contributor to the prevention of atherosclerosis (Laggner et al., 2007).
In comparison to NO and CO H2S is not as greatly investigated as a gasotransmitter. Research concerning H2S effects on the vasculature is also limited, and of that which exists contradicting statements aren't uncommon, particularly in reference to the effect of H2S on vessel calibre and the role of the endothelium. Additionally, the studies are carried out on a range of vascular beds in various animals which may distort the overall characterisation of H2S effects in the vasculature.
This project aims to continue research exploring the effects of H2S in mammalian vasculature and in specific porcine vasculature since much of the current literature is based on rats and mice. We also aim to determine the mechanisms of its action, and also confirm endogenous production of H2S by CSE.
To fulfil this aim we will study the effects of endothelium removal on porcine coronary artery as well as NO inhibition, non-selective K+ channel blockade and ATP-sensitive K+ channel blockade in the presence of H2S. We will also look at the effect that L-cysteine, a precursor to H2S, has on porcine coronary artery and the influence that CSE inhibitors have on this.