An individual to develop cardiovascular diseases

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Diagnosis of the metabolic syndrome can enhance the likelihood of an individual to develop cardiovascular diseases, such as hypertension and diabetes. Methylglyoxal (MG) is a reactive glucose metabolite and a known causative factor for hypertension and diabetes. Hydrogen sulfide (H2S), on the other hand, is a gasotransmitter with multifaceted physiological functions, including anti-oxidant and vasodilatory properties. The present study demonstrates that MG and H2S can interact with and modulate each others' physiological functions. Upon in vitro incubations, we found that MG and H2S can directly scavenge one another, resulting in the formation of three possible MG-H2S adducts. Furthermore, the endogenous production level of MG or H2S was significantly reduced in a concentration-dependent manner in rat vascular smooth muscle cells (A-10 cells) treated with NaHS, a H2S donor, or MG, respectively. Indeed, MG-treated A-10 cells exhibited a concentration-dependent down-regulation of the protein and activity level of cystathionine γ-lyase (CSE), the main H2S-generating enzyme in the vasculature. Moreover, H2S can induce the inhibition of MG-generated ROS production in a concentration-dependent manner in A-10 cells. In hypertensive male mice lacking the CSE gene (CSE-/-), and thus lower levels of vascular H2S, MG levels were significantly elevated in plasma and renal extracts from 6-22 week-old CSE-/- mice. Renal triosephosphates were also significantly increased in these mice. To identify the source of the elevated renal MG levels, we found that the activity of fructose-1,6-bisphosphatase (FBPase) was significantly down-regulated, along

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with lower levels of its product and higher levels of its substrate in the kidney of 6-22 week-old CSE-/- mice. We have also observed lower levels of the gluconeogenic regulator, peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, and its down-stream targets, FBPase-1 and -2, phosphoenolpyruvate carboxykinase (PEPCK), and estrogen-related receptor (ERR)α mRNA expression levels in renal extracts from 6-22 week-old CSE-/- mice. Indeed, administration of 30 and 50 µM NaHS induced a significant concentration-dependent increase in FBPase-1 and PGC-1α in rat A-10 cells. We have also observed a significant up-regulation of PEPCK and ERRα mRNA expression levels in 50 µM NaHS-treated A-10 cells, further confirming the involvement of H2S in regulating of the rate of gluconeogenesis and MG formation. Overall, this unique study demonstrates the existence of a negative correlation between MG and H2S in the vasculature. Further elucidation of this cross-talk phenomenon between MG and H2S could lead to more elaborate and effective therapeutic regimens to combat metabolic syndrome and its related health complications.

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1.0 Metabolic syndrome

Metabolic syndrome, also known as syndrome X, represents a cluster of risk factors that can increase an individual's chance of developing type 2 diabetes mellitus (T2DM) and cardiovascular diseases (Ford et al. 2002; Alberti et al. 2005). These collection of risk factors include abdominal obesity (a waist circumference of greater than 102 cm in men and 88 cm in women), high BP (130/85 mm Hg or higher), elevated fasting blood glucose levels (> 5.6 mM or 100 mg/dl), low high-density lipoprotein (HDL) cholesterol (< 1.03 mM or 40 mg/dl in men and 1.29 mM or 50 mg/dl in women), and high triglycerides (> 1.7 mM or 150 mg/dl) (Alberti et al. 2005). If an individual exhibits three or more of the above described conditions, then he/she is considered to have metabolic syndrome. A study done by Ford et al (2002) demonstrated that the prevalence of metabolic syndrome increases with age.

It is still of great debate on what is the exact cause of metabolic syndrome. Most studies have focused on insulin resistance such as the effects of insulin on glucose metabolism, lipid metabolism, protein synthesis, as well as cell-cycle control and proliferation (Bernal-Mizrachi and Semenkovich 2006). Genetics, older age and lifestyle, including a high-fat diet, and inactivity, also appears to play a role. Currently, there is a rise in metabolic syndrome, particularly due to growing rates of abdominal obesity and high BP (Ford et al. 2002). The rise in the diagnostic metabolic syndrome is of great concern, because metabolic syndrome is a substantial risk factor for cardiovascular diseases, primarily hypertension and T2DM (Bernal-Mizrachi and Semenkovich 2006).

1.1 Hypertension

Hypertension, or high blood pressure (BP), is a medical condition seen in individuals with chronically elevated arterial BP. The optimal BP reading from a healthy individual should be < 120 mm Hg, systolic, and < 80 mm Hg, diasystolic (Carretero and Oparil 2000). However, individuals with BP at or above 140/90 mm Hg are strongly recommended for immediate drug treatment (Chobanian et al. 2003).

There is a strong correlation between high BP and the risk of developing cardiovascular diseases, such as stroke, myocardial infarction, and heart failure (Carretero and Oparil 2000). Indeed, hypertension is the leading risk factor for stroke (O'Donnell 2010). Cardiovascular diseases are responsible for approximately 30% of all deaths worldwide (Murray and Lopez 1997; WHO 2002). In 2000, it was estimated that approximately 1 billion people were hypertensive (Kearney et al. 2005). If there are no serious interventions, that number is predicted to increase by 29%, to nearly 1.56 billion people in the year 2025 (Kearney et al. 2005).

1.1.1 Types of hypertension

To date, hypertension is classified into two distinct categories: primary (essential) hypertension and secondary hypertension. Essential hypertension represents about 90-95% of cases for which medical cause is not clear. The remaining 5-10% of cases, referring to the secondary hypertension pathology, are caused by conditions that affects the kidneys, arteries, heart, or endocrine system.

1.1.2 Pathogenesis of essential hypertension

Despite the fact that no clear cause is known in the pathophysiological development of essential hypertension, numerous functional abnormalities have been identified (Figure 1-1). These pathophysiologic factors include, but are not limited to, enhanced sympathetic nervous system activity, overproduction of sodium-retaining hormones and vasoconstrictors, inappropriate rennin secretion, increased production of angiotension II and aldosterone, abnormalities of resistance vessels, as well as increased oxidative stress, endothelial dysfunction, and vascular remodelling (Oparil et al. 2003). The diagnostic of cardiovascular complications such as diabetes mellitus, insulin resistance, and obesity are also linked to the pathogenesis of essential hypertension (Oparil et al. 2003).

Figure 1-1: Proposed schemes of the pathophysiological development of essential hypertension. CNS: central nervous system. (Modified from Ann. Intern. Med. 139:761-776, 2003)

1.2 Diabetes

Diabetes mellitus is a chronic, heterogeneous metabolic illness in which a person has high blood sugar either because the body does not produce enough insulin (type 1 diabetes mellitus; T1DM), or because cells do not respond to the insulin that is produced (T2DM) (Canadian Diabetes Association 2008). In 2000, 171 million people around the world were diagnosed with diabetes, which is projected to increase to 366 million by 2030 (Wild et al. 2004). The impact of diabetes is also felt in Canada, where approximately 5.5% of the population was diagnosed with diabetes in 2005 (Landolt et al. 2005).

All forms of diabetes are characterized by chronic hyperglycemia, where the fasting plasma glucose level are ≥ 7.0 mM (126 mg/dl) and ≥ 11.1 mM (200 mg/dl) from the glucose tolerance test (WHO 2006). Obesity is strongly associated with an increased risk for the development of T2DM (Mokdad et al. 2001) by contributing to the increased in endogenous glucose production (Roden et al. 2000; Staehr et al. 2003). Prolong exposure to hyperglycemia could increase the risk of developing diabetic-specific microvascular complications, such as microvascular damage to the eyes (retinopathy), kidney (nephropathy), and nerves (neuropathy) (Brownlee 2001), as well as macrovascular complications, including ischaemic heart disease, stroke and peripheral vascular disease (WHO 2006).

1.2.1 Hyperglycemia

Despite wide variations in daily food intake and physical activity, plasma glucose levels are tightly maintained in the range of approximately 70 to 160 mg/dl (Gerich 2000). This complex homeostatic-regulating system involves the precise balancing of glucose production, its reabsorption, as well as its use in the peripheral tissues, which is achieved through a network of hormones, neural pathways, and glucose transport proteins (Marsenic 2009). However, if these mechanisms fail in a way that allows glucose to rise to abnormal levels, hyperglycemia is the result. Hyperglycemia, or high blood sugar, is a chronic elevation in the plasma glucose levels that are higher than 10 mM (180 mg/dl), which could lead to organ damage (Brownlee 2001). Hyperglycemia is commonly associated with T2DM, due to the defects of T2DM in both insulin secretion and tissue sensitivity to insulin (Brownlee 2001), as well as obesity (Mokdad et al. 2001; Roden et al. 2000; Staehr et al. 2003).

1.2.2. Obesity

Obesity is a medical condition that has the potential to reduce life expectancy and/or increased cardiovascular diseases, such as T2DM and hypertension (Haslam and James 2005). Obesity is the 6th most important risk factor contributing to the overall burden of disease worldwide (Ezzati et al. 2002), and alarmingly, it is estimated that 1.1 billion adults, including 312 million whom are obese, and 10% of children are classified as overweight or obese (Haslam and James 2005). However, with the new Asian BMI of 23.0 kg/m2 classified as overweight, this number is projected to be about 1.7 billion (James et al. 2004). WHO recognizes if an individual is overweight if the body mass index (BMI), a measurement which compares weight and height of an individual, is 25.0 kg/m2 and obese if at 30.0 kg/m2 or higher (WHO 2000). However, the risks of developing hypertension, diabetes, and dyslipidaemia increase from a BMI of approximately 21.0 kg/m2 (James et al. 2004).

An increased in fat deposit can lead to an increase in the secretion of products, such as cytokines and tumour necrosis factor α, can potentially lead to insulin resistance (Haslam and James 2005). Indeed, the tumour necrosis factor α has a paracrine suppressive effect on adiponectin secretion, a powerful insulin sensitizer. Thus, with an expanded adipocyte mass, less adiponectin will be secreted (Haslam and James 2005). Additionally, increased fat deposit in the pancreatic islet cells can decrease the islets' capacity to maintain the increased insulin output in the insulin resistant state (Haslam and James 2005). Dieting and physical exercise are the mainstays of treatment for obesity, however, better management and prevention is needed to combat the increasingly grow epidemic of obesity.

2.0 Hydrogen sulfide (H2S)

Throughout the decades, hydrogen sulfide (H2S) has been considered mainly as a toxic gas and an environmental hazard. However, this all changed upon the discovery that this "gas of rotten eggs" was actually being produced in mammals including humans (Wang 2002). In fact, H2S is the most recent addition to the endogenous gasotransmitter family that includes nitric oxide (NO) and carbon monoxide (CO). This endogenous physiological regulator/modulator has emerged as a major player in the immune system, as well as the peripheral and central nervous system (PNS, CNS, respectively) (Szabá½¹ 2007).

2.1 Formation of H2S

2.1.1 Enzymatic synthesis of H2S

2.1.1.1 The pyridoxal-5´-phosphate-dependent enzymes

In mammalian cells, H2S is synthesized endogenously by the pyridoxal-5´-phosphate-dependent enzymes including cystathionine γ-lyase (CSE; EC 4.4.1.1) and cystathionine β-synthase (CBS; EC 4.2.1.22) (Wang 2002). The substrate of CSE and CBS, L-cysteine, can be made available from alimentary sources, or endogenous proteins (Szabό 2007). This important amino acid can also be synthesized from L-methionine through the trans-sulfuration pathway, which uses homocysteine as an intermediate (Szabό 2007) (Figure 1-2). Indeed, these enzymes are expressed in a tissue-specific manner. For example, CSE is mainly expressed in the liver and kidney (Ishii et al. 2004; Tripatara et al. 2009), pancreas (Wu et al. 2009), as well as in vascular smooth muscle cells (VSMCs) (Chang et al. 2010; d'Edmmanuele et al. 2009). CBS, on the other hand, is the predominant H2S-producing enzyme in the CNS (Tan et al. 2010).

Figure 1-2: Endogenous synthesis of H2S in mammalian cells. B6: Vitamin B6; CBS: cystathionine β-synthase; CSE: cystathionine γ-lyase; GCS: γ-glutamyl cysteine synthase; GS: glutathionine synthase. (Modified from Kid. Int. 76:700-704, 2002)

2.1.1.2 3-mercaptopyruvate sulfurtransferase

Recently, Kimura and colleagues have identified a new H2S-generating enzyme, 3-mercaptopyruvate sulfurtransferase (3MST), which can endogenously produce H2S in the brain (Shibuya et al. 2009a) and in the endothelium (Shibuya et al. 2009b). This unique mechanism inquires 3-mercaptopyruvate, which is produced by cysteine aminotransferase from cysteine and α-ketoglutarate, as a precursor for 3MST-induced H2S generation. This discovery introduces the possibility that production and release of H2S from the endothelium could act as a smooth muscle relaxant. Although, more research in order to sufficiently identify endogenous regulators, modulators, as well as possible other locations that 3MST may be found in the mammalian system.

2.1.2 Non-enzymatic synthesis of H2S

The non-enzymatic generation of H2S produces a less significant source of this endogenous physiologic regulator (Searcy and Lee 1998). It is thought that this unique pathway generates H2S from the reduction of elemental sulfur produced from the reducing equivalents of oxidized glucose, which occurs during glycolysis (Searcy and Lee 1998). In fact, this is the major source of non-enzymatic production of H2S (Searcy and Lee 1998). To a lesser extent of H2S production, the phosphogluconate pathway was also as described as a non-enzymatic mechanism in erythrocytes (Searcy and Lee 1998). However, more research is needed in order to better understand the production of H2S through these non-enzymatic pathways.

2.2 Metabolism of H2S

In order to maintain a proper physiological balance of H2S, the mammalian cell must be able to metabolize excess amounts of H2S. This endogenous gasotransmitter can either be oxidized to sulfate, with GSH acting as an intermediate, in the mitochondria, or methylated to CH3SCH3 in the cytosol (Wang 2002). Furthermore, H2S can be scavenged by methemoglobin or oxidized glutathione (GSSG) (Wang 2002). In fact, haemoglobin acts as a "sink" for H2S in the blood stream, and may compete with other gasotransmitters, such as NO and CO, for binding (Wang 2002). It is thought that H2S binds to haemoglobin through the attractive forces of the iron molecule (Szabá½¹ 2007). Indeed, the binding of one gasotransmitter could affect the binding probability of another gas, thus alternating their bioavailability (Wang 2002).

2.3 H2S concentration and its various effects

Throughout the years, many groups have reported on the paradox effects of H2S on mammalian cells. In fact, it can be speculated that this contradiction could reside in the amounts of H2S, which could lead to this endogenous gasotransmitter producing cytoprotective or cytotoxic effects.

2.3.1 Low concentrations: cytoprotective effects

2.3.1.1 Anti-oxidant properties

Many reports have shown that low concentrations of exogenously applied H2S, approximately 10-100 µM, can have anti-oxidant capabilities (Chang et al. 2010; Yan et al. 2006; Kimura and Kimura 2004; Kimura et al. 2006; Whitman et al. 2004; Ali et al. 2006). Indeed, H2S can significantly increase intracellular levels of the potent anti-oxidant, GSH, in rat thoracic aortic vascular smooth muscle cells (A-10 cells) (Chang et al. 2010), in rat primary cortical neurons (Kimura and Kimura 2004), and in HT22 immortalized hippocampal cells (Kimura et al. 2006). Perhaps, increased GSH upon H2S administration could be due to H2S-induced enhancement of the activity of γ-glutamylcysteine synthetase (γ-GC), resulting in increased γ-GC expression levels, which is the precursor for L-cysteine production and alternatively GSH production (Kimura et al. 2004; Kimura et al. 2006). Furthermore, Kimura et al (2004) also demonstrated that H2S can enhance cysteine/glutamate antiporter xc-, thus increasing available cysteine for glutamate production. Lastly, due to its reducing abilities, H2S also can act as an oxidant scavenger, including peroxynitrite (ONOO-) in SH-SY5Y human neuroblastoma cells (Whitman et al. 2004) and in A-10 rat VSMCs (Yan et al. 2006), NO in rat aortic rings (Ali et al. 2006), and homocysteine- (Yan et al. 2006) MG-induced hydrogen peroxide (H2O2) and ONOO- production in A-10 cells (Chang et al. 2010).

2.3.1.2 Apoptotic affects

The reducing ability of H2S allows it to regulate cellular signal transduction pathways, leading to the alternation of various genes and gene products expression (Szabó 2007). For instance, H2S has been shown to induce DNA fragmentation (apoptotic phenotype) due to caspase activation, specifically caspase-3, in human aortic SMCs (Yang et al. 2006). H2S-induced DNA fragmentation mechanism most likely occurs via the activation of extracellular signal-regulated kinases (ERK) and p38 mitogen-activated protein kinases (MAPK) pathways (Yang et al. 2006). To note, MAPK represents important signal transduction machinery and can influence cell growth, differentiation, and apoptosis (Yang et al. 2004). Additionally, H2S was also shown to inhibit cell proliferation through the increase activation of ERK and p21Cip/WAK-1 activity in human embryonic kidney (HEK)-293 cells (Yang et al. 2004) and increase other apoptotic signaling proteins, such as p53, as well as the translocation of Bax and cytochrome c (in human pulmonary fibroblasts) (Baskar et al. 2007). Moreover, H2S can indirectly inhibit the activation of nuclear factor-кB (NF-кB) pathway (Oh et al. 2006). Activation of NF-κB has been reported to be essential for proliferation in VSMCs (Bellas et al. 1995). In fact, due to its ability to induce cell apoptosis or inhibit proliferation, H2S can be used to improve the hypertrophy/hyperplasia state of VSMCs, as well as decrease aortic ring thickening seen in spontaneous hypertensive rats (SHRs) (Shi et al. 2007; Zhao et al. 2001). Overall, this suggests the importance of H2S in order to maintain vascular integrity.

2.3.1.3 Physiologic vasodilator

Interestingly, unlike CO and NO, H2S can function as a vasorelaxant through the activation of K+-dependent-ATP (KATP ) channels in VSMCs (Yang et al. 2005; Zhao et al. 2001, 2003), as well as in pancreatic β-cells (Cook et al. 1988; Yang et al. 2005; Ali et al. 2007) (the latter will be discussed in more detail in section 2.3.3.3.1). Generally, upon the opening of the KATP channels in VSMCs, the membrane hyperpolarizes which causes the voltage-dependent Ca2+ channels to close and reduces intracellular Ca2+ levels (Szabό 2007). Ca2+ plays an important role in the contractile responses of VSMCs, where low a level of intracellular Ca2+ results in vasodilation (Szabό 2007).

Zhao et al (2001) were the first group to demonstrate that intravenous injection of H2S (2.8 and 14 mM/kg) significantly decreased BP in Sprague Dawley (SD) rats, which was successfully attenuated by a KATP channel inhibitor glibenclamide. These authors also showed that by opening the KATP channels, H2S induced relaxation in isolated rat aortic rings (Zhao et al. 2001). To note, the hypotensive responses of H2S had no effect on heart rate in the SD rats (Zhao et al. 2001). However, the exact mechanism by which H2S activates KATP channels in VSMCs is unclear.

2.3.1.3.1 Hypertensive in genetic knockdown of CSE in mice

Additionally, the administration of DL-propargylglycine (PGG), a CSE inhibitor, significantly increased the BP in SD rats (Zhao et al. 2003). Indeed, genetic knockdown of CSE in mice displayed impaired endothelium-dependent vasorelaxation, age-related increased BP, among other findings (Yang et al. 2008). At 7 weeks of age, both male and female CSE-/- mice exhibited higher BP than their wild type counterparts, which increased in an age-related fashion, until at 12 weeks of age, the male CSE-/- had a BP reading of 18 mm Hg higher than the control mice (Yang et al. 2008). Indeed, upon intravenous bolus injections of NaHS, a H2S donor, the systolic BP significantly decreased in both CSE-/- and CSE+/+ mice, where the magnitude decreased was greater in the CSE-/- mice, specifying the enhanced sensitive of H2S stimulation in the CSE-/- mice (Yang et al. 2008). Therefore, H2S is a vital physiologic vasodilator and regulator of BP, and quite possibly, it could an endothelium-derived relaxing factor (EDRF), or an endothelium-derived hyperpolarizing factor (EDHF), which was proposed by Rui Wang (Wang 2009).

2.3.1.4 Anti-inflammatory agent

Apart from the previously mentioned H2S mechanisms, H2S can also play an important role in inflammation. H2S was found to be generated at sites of inflammation and can influence the ability of neutrophils to cause tissue injury (Whiteman et al. 2004). Because of this, H2S was shown to exert protective effects in animal models of inflammation and inflammation-related pain (Whiteman et al. 2004). Indeed, Moore and colleagues (2007) demonstrated that sulfide-releasing diclofenac derivative reduced tissue neutrophil infiltration and interleukin (IL)-1β levels, up-regulated IL-10 levels and attenuated the activation of NK-κB in an endotoxin-induced lung and liver inflammation model. Additionally, H2S-releasing modified anti-inflammatory compound exerted therapeutic effects in rodent models of inflammation (Distrutti et al. 2006; Baskar et al. 2008). Furthermore, H2S-induced anti-inflammatory actions decreased leukocyte rolling velocity (Zanardo et al. 2006) and can also suppress expression of some leukocyte and endothelial adhesion molecules (Zanardo et al. 2006). This mechanism likely occurs through the activation of KATP channels, since pre-treatment with glibenclamide (KATP channel antagonist) reversed the effects of H2S donors and mimicked by pinacidil (KATP channel agonist) (Zanardo et al 2006).

Although, it must be noted some contradicting reports regarding the role of H2S and inflammation. Zhang et al (2007) demonstrated that H2S injection up-regulated leukocyte attachment and rolling in blood vessels and also increased the intercellular adhesion molecule-1 levels in sepsis mice, which was attenuated by PPG (Zhang et al. 2007). Additionally, Zhi et al (2007) showed that H2S administration increased the generation of pro-inflammatory cytokines, tumor necrosis factor (TNF)-α, IL-1β, and IL-6, through activation of the ERK-NF-κB signaling pathway, in human monocytes. Overall, more research is needed in order to clearly identify H2S as an anti- or pro-inflammatory agent.

2.3.1.5 Suspended animation

However, due to H2S affinity for cytochrome c oxidase, it has been pharmacological linked to the phenomena of H2S-induced suspended animation (Blackstone et al. 2005). To explain this, mice were exposed to 81 ppm H2S in the air, which resulted in a decrease in their breathing rate from 120 to 10 breaths per minute, along with decreased carbon dioxide production and oxygen consumption, and their temperature lowered from 37 °C to 2 °C (Blackstone et al. 2005). Interestingly, these mice survived this exposure to H2S for 6 hours and afterwards showed no negative health consequences (Blackstone et al. 2005).

So what could this mean for us? Due to the fact that H2S can slow metabolic rate and induce a hibernation-like state, this could be used as a life saving tool in emergency-related situations (Szabá½¹ 2007). For example, H2S could provide emergency personnel more precious time to transport victims suffering from trauma, to the hospital, instead of rushing the victim as fast as they can to the hospital and hope for the best (Szabá½¹ 2007). By slowing the metabolic rate of the victim, such as respiration, temperature, etc, this could slow the biological chain reaction of events which occurs in trauma-related situations. If researchers are able to safely manipulate the side effects of H2S poisoning, it is possible that this hazardous gas could be used as a life saving tool.

2.3.2 H2S-releasing drugs

Currently, research regarding the role of H2S in various aspects of the metabolic syndrome, including the conditions of hypertension, obesity, and diabetes, is still in its infancy. However, there is promising potential in some novel H2S-releasing drugs that could be used as preventive agents/treatments for the metabolic syndrome (Figure 1-3). These include a new H2S-releasing compound that has antihypertensive and vasodilator properties known as [morpholin-4-ium 4 methoxyphenyl (morpholino) phosphinodithioate (GYY4137)] (Li et al. 2008), as well as an H2S-releasing phosphodiesterase compound, which is selective inhibitor of cyclic guanosine monophosphate (cGMP) phosphodiesterase type-V, used in conditions of endothelial dysfunction (Sparatore and Wallace 2006). Also, a new H2S-releasing moiety to the statin, simvastatin, which can reduce platelet aggregation and increase platelet cyclic adenosine monophosphate (cAMP), shows potential (Wallace et al. 2009). Additionally, a series of H2S-releasing compounds are being tested to determine their effectiveness as an anti-inflammatory agent (Scherrer and Sparatore 2006; Sparatore et al. 2009; Li et al. 2007; Distrutti et al. 2006; Baskar et al. 2008). Inflammation underlies the metabolic syndrome, including diabetes, obesity, and hyperlipidemia (Desai et al. 2011). Unfortunately, there is much uncertainty regarding the therapeutic usefulness of H2S in inflammation, since the idea of H2S having anti-inflammatory properties is controversial. More research is needed in order to clarify whether this physiological regular has pro- or anti-inflammatory properties, or if it this would be a concentration-related manner.

2.3.3 High concentrations: cytotoxic effects

For about 300 years, H2S has been chemically known to humans, and throughout most of its known existence, it has been identified was an extremely hazardous gas (Reiffenstein et al. 1992). Indeed, significant H2S poisoning usually occurs by inhalation (Reiffenstein et al. 1992). This section will outline the main mechanism of action regarding fatal H2S poisoning.

Figure 1-3: The components of the metabolic syndrome and the therapeutic potential of hydrogen sulfide (H2S)-releasing drugs. (Modified from Rev. Clin. Pharmacol. 4:63-73, 2011)

2.3.3.1 Inhibition of cellular respiration

In respect to the high chemical reactivity of H2S, it has a strong affinity for cytochrome c oxidase (Szabό 2007). Indeed, it can be argued that H2S is far more potent than cyanide poisoning (Reiffenstein et al. 1992). The main mechanism of H2S toxicity is due to its high affinity for cytochrome c oxidase, thereby in an effect similar to cyanide toxicity.  (Lowicka and Beltowski 2007). Briefly, cytochrome c oxidase is a key factor in the electron transport chain within the mitochondria, which regulates cellular respiration (Lowicka and Beltowski 2007). If the activity of cytochrome c oxidase is inhibited, it would arrest aerobic metabolism (Lowicka and Beltowski 2007). H2S can inhibit cellular respiration by binding the copper centre of the cytochrome c oxidase, thus blocking the regulator of cellular oxygen consumption (Hill et al. 1984).

Because H2S can inhibit cytochrome c oxidase, and cytochrome c oxidase is found virtually in all cell types, this classifies H2S as a broad-spectrum poison, meaning that it can poison several different systems in the body (Reiffenstein et al. 1992). For example, when fatally high levels of H2S is inhaled (500-1000 ppm), it will inhibit the cytochrome c oxidase in the brain, reduce oxygen uptake into cells, and inhibited the reuptake of L-glutamate, an excitatory neurotransmitter, thus quickly leading to death (Nicholson et al. 1998). In fact, approximately 500 ppm of H2S will cause consciousness; where between 500-1000 ppm will result in respiratory paralysis, neural paralysis, cardiac arrhythmias, eventually leading to death (Reiffenstein et al. 1992) (Table 1-1).

.3.2 Oxidative stress

Although low levels of H2S (approximately 10-100 µM) may have anti-oxidant capabilities, higher concentrations of H2S, in the millimolar range, tends to generate free radicals and oxidants. In the presence of peroxidase and H2O2, H2S yields the free radicals: SH. And S. (Wang 2002). These free radicals enable H2S to be a highly reactive molecule (Wang 2002). In fact, it was demonstrated that high amounts of exogenous H2S depleted GSH levels in rat primary hepatocytes (Truong et al. 2006). Interestingly, O'Brien and colleagues show that the H2S donor, NaHS, depleted cellular GSH levels more so under acidic conditions. Additionally, the authors founds that H2S also increased the formation of reactive oxygen species (ROS), presumably by the inhibition of the electron transport chain, through cytochrome c oxidase, as well as significantly decreasing available GSH levels.

2.3.3.3 Inhibition of insulin secretion

2.3.3.3.1 Activation of KATP channels

Amounting evidence in recent years have indicated that H2S can act as an endogenous modulator of insulin secretion from β-cells (Yang et al. 2005; Ali et al. 2007; Kaneko et al. 2006; Yang et al. 2007; Kaneko et al. 2009; Wu et al. 2009). This is largely, if only, due to H2S-induced activation the KATP channels in the insulin-secreting cell lines, including INS-1E at 100 µM NaHS (Yang et al. 2005) and HIT-T15 at 100 µM H2S (Ali et al. 2007). Briefly, when glucose enters the pancreatic β-cell, this increases the intracellular levels of ATP which will then inhibit KATP channels, depolarizing the plasma membrane, encouraging the opening of Ca2+ channels, with the final result of insulin secretion (Desai et al. 2011). However, upon H2S-induced KATP channel activation, the plasma membrane will not depolarize, the Ca2+-channels would not open, and insulin would not be release from pancreatic β-cells. Indeed, Ali et al (2007) demonstrated that upon administration of 10 µM glibenclamide, an anti-diabetic drug that inhibits the opening of KATP channels, to pancreatic β-cells, blocked 100 µM NaHS-induced inhibition of insulin secretion. Another group, also showed that L-cysteine (precursor for H2S formation) and NaHS reduced intracellular Ca2+ levels and ATP production, which also prevented insulin release in isolated mouse islets and MIN6 (mouse β-cell line) (Kaneko et al. 2006).

2.3.3.3.2 Induced apoptosis of pancreatic β-cells

To further suggest that H2S could be involved in the maladaptive role of insulin secretion, this KATP channel activator can also induce apoptosis in β-cells. In fact, by activating the p38 MAPK pathway and upregulating BiP and CHOP (indicators of endoplasmic reticulum stress), overexpression of CSE induced apoptosis of INS-IE cells (Yang et al. 2007). In agreement, it was shown that NaHS treatment can induce apoptosis in isolated pancreatic acinar cells (exocrine cells that assist in digestion), by causing phosphatidylserine externalization, which is an indicator of early stages of apoptosis (Cao et al. 2006). In fact, Cao et al (2006) also demonstrated that H2S can induce apoptosis in these cells by activating both mitochondrial and death receptor pathways.

However, H2S-induced apoptosis in pancreatic β-cells is controversial. On the contrary, Kaneko et al (2006) showed that exogenous 3 mM L-cysteine and 100 µM NaHS prevented 20 mM glucose-induced apoptosis in β-cells, and in fact, increase total glutathione levels. To note, β-cells are highly susceptible to glucotoxicity, because of their low anti-oxidant defense mechanisms (Desai et al. 2011). Indeed, this same group also demonstrated that 2 mM PPG (CSE inhibitor) blocked the protective effects of 3 mM L-cysteine against glucose-induced apoptosis in β-cells (Kaneko et al. 2006). Overall, the discrepancy between H2S-induced apoptosis in INS-IE cells and H2S anti-apoptotic affect in isolated mouse islets could be due to different induction methods of apoptosis. As well, the INS-IE cells were derived from a tumour cell line; thus introducing variable biological differences, such as the cell survival and apoptotic pathways. Therefore, further study is needed in order to determine the exact role H2S plays in the pancreatic β-cell survival mechanisms.

2.4 H2S and hormones

2.4.1 Insulin

Please see section 2.3.3.3.

2.4.2 Corticotropin-releasing hormone

Aside the fact that H2S can regulate insulin secretion, as mentioned above, but it has also been implemented with other hormones. Interestingly, H2S was shown to decrease the release of corticotropin-releasing hormone (CRH) from the hypothalamus in a concentration-dependent manner (Russo et al. 2000). Briefly, CRH is secreted in response to biological stress, which then stimulates the synthesis and release of corticotropin, also known as adrenocorticotropic hormone (ACTH), from the anterior pituitary gland (Kimura 2002). ACTH then stimulates the production and release of glucocorticoids, which can affect carbohydrate metabolism, by enhancing gluconeogenesis and lipolysis, as well as immune function, due to its potent anti-inflammatory and immunosuppressive properties (Kimura 2002). Additionally, these authors also show that S-adenosyl-L-methionine (SAM; CBS activator) administration mimicked H2S effects by inhibiting the KCl-induced CRH release (Russo et al. 2000). Thus, these results suggest that H2S plays an important role in regulating the response of the hypothalamo-pituitary axis.

2.4.3 Testosterone

H2S has also been linked to testosterone. Eto and Kimura (2002) showed that testosterone can regulate the brain H2S level by enhancing the activity level of SAM. Endogenous H2S levels are significantly less in the female brain than in the male brain (Eto and Kimura 2002). As such, these authors showed that testosterone administration increased the production of H2S levels in the female brain, which was comparable to that in the male brain. Additionally, significantly less testosterone, SAM, and H2S levels were observed in brain matter from castrated male mice (Eto and Kimura 2002).

Indeed, it has been suggested by Cirino and associates (2009) that H2S could be the key player in testosterone-induced vasorelaxation in the vascular system (Bucci et al. 2009). These authors show that testosterone induced a concentration-dependent vasorelaxation in rat aortic rings, which was attenuated by PGG and β-cyanoalanine (BCA), both specific CSE inhibitors. Bucci et al (2009) also demonstrated that testosterone can increase the conversion of L-cysteine to H2S, which was significantly abrogated by PGG and BCA (Bucci et al. 2009). Overall, these results open a new window regarding the interconnected mechanisms between sexual reproduction hormones and H2S. However, it has yet to be determined if estrogen, or progesterone, the main female-dominant hormones, may also influence the production rate of H2S.

2.5 The association of H2S and diseases

The production of H2S is a tightly regulated mechanism, where any minuscule changes could have severe outcomes. Indeed, the pathologic implications of H2S have been linked to many diseases (Table 1-2). Due to its diverse, and potent, physiological actions, overproduction of H2S has been connected to the pathogenesis of septic shock, diabetes mellitus, ischemic stroke and Down syndrome, just to name a few. On the other hand, abnormally low levels of H2S have been blamed for hypertension, and may also be interconnected to Alzheimer's disease.

2.5.1 Overproduction of H2S

2.5.1.1 Septic shock

Due to its potent vasodilator properties, H2S, along with NO and CO, has been implemented in septic shock. Septic shock is characterized by severe vasodilatation and hypotension that is commonly caused by overwhelming infection of Gram-negative bacteria (Lowicka and Beltowski 2007). Indeed, upon lipopolysaccharide administration, CSE expression and activity is significantly up-regulated in the kidney and liver (Li et al. 2005), which likely contributes to the excessive H2S levels (Hui et al. 2003; Lyons et al. 2001). Furthermore, H2S has negative inotropic effects, such as decreasing heart rate, which could further contribute to this disease (Geng et al. 2004; Lowicka and Beltowski 2007).

2.5.1.2 Diabetes mellitus

There is strong correlation to high H2S levels and the deterioration of insulin release in pancreatic β-cells (Yang et al. 2005; Ali et al. 2007; Kaneko et al. 2006; Yang et al. 2007; Kaneko et al. 2009; Wu et al. 2009). This is mainly due to the association of H2S and the increased opening probability of KATP channels (Lowicka and Beltowski 2007). Indeed, streptozotocin-induced diabetic rats, rats that have no insulin-producing β-cells, exhibited increased mRNA and activities of CSE and CBS in the liver (Yusuf et al. 2005; Hargrove et al. 1989; Nieman et al. 2004; Jacobs et al. 1998) and pancreas (Yusuf et al. 2005). Yet, interestingly enough, insulin treatment (8 U/kg, s.c., for 5 d) attenuated the up-regulation of both CSE and CBS mRNA and activity levels in these type 1 diabetic rats (Yusuf et al. 2005). Additionally, Wu et al (2009) showed that Zucker diabetic fatty (ZDF) rats exhibited higher levels of pancreatic CSE and H2S production than their counterparts, Zucker fatty (ZF) and the Zucker lean (ZL) rats. Thus, it is suggestive to assume that inhibition of H2S in the pancreas could be therapeutic in diabetic conditions. However, more research is needed in order to better understand the relationship between H2S and hormones in diabetes mellitus.

2.5.1.3 Ischemic stoke

Ischemic stroke occurs when the blood supply to the brain is interrupted by a blood clot, either by thrombotic (formation of a clot in a narrow artery), or embolic (a clot that travels up to the brain to block a small artery) formation. In light of this, H2S is consisted to be a potent neuromodulator (Szabá½¹ 2007). This is mainly accredited to its ability to enhance the N-methyl-D-aspartate (NMDA) receptor function through the activation of adenylyl cyclase, which increases the production of cAMP and thus the activation of protein kinase A, leading to the phosphorylation and activation of the NMDA receptor-mediated excitatory postsynaptic current (Szabá½¹ 2007). Therefore, it is possible that H2S may augment NMDA receptor-mediated excitotoxicity of glutamate, thus enhancing the destructive effects of cerebral ischemia (Qu et al. 2006). Indeed, it was shown by Wong and associates (2006) that NaHS can increase the infarct volume after middle cerebral artery occlusion (MCAO), which was completely abrogated by dizolcilpine maleate (a NMDA, blocker). Additionally, after induction of the MCAO, the endogenous level of H2S and the activity of CBS significantly increased in the lesioned cortex (Qu et al. 2006). Thus, after a stroke, it is possible that H2S could be involved in cerebral ischemic damage. Therefore, drugs that antagonize the activity of CBS, the main H2S-producing enzyme in the brain, could be used for stroke therapy.

2.5.1.4 Down syndrome

In addition to its involvement in ischemic stroke, H2S is also associated with Down syndrome. Down syndrome is a chromosomal disease caused by the presence of an extra chromosome, the 21st chromosome. Patients with Down syndrome have a significant overexpression of CBS by 166% in fibroblasts (Chadefaux et al. 1985) and by 1,200% in myeloblasts (Taub et al. 1999). In agreement, low concentrations of plasma homocysteine, the substrate of CBS, were detected in Down syndrome patients (Chadefaux et al. 1988). Indeed, Chadefaux-Vekemans and associates (2003) observed an abnormal level of thiosulfate, the main catabolite of H2S, in the urinary excretion from Down syndrome patients, thus supporting the notion at H2S is linked to Down syndrome.

Interestingly, it was pointed out by Chadefaux-Vekemans and associates (2003) that the biological and clinical signs of Down syndrome mimic chronic H2S poisoning. This includes, reduced sensory nerve conduction velocity, and impaired color vision and contrast sensitivity in workers exposed to carbone disulfide (Takebayashi et al. 1988; Raitta et al. 1981; Vanhoorne et al. 1996) and in Down syndrome patients (Christensen et al. 1988; Rocco et al. 1977; Perez-Carpinell et al. 1994). These authors suggested that low doses of sodium nitrite (or nitrate, which is a nitrite precursor) could be used to combat the overproduction of H2S in Down syndrome patients, since nitrite is used to treat acute H2S poisoning.

2.5.2 Underproduction of H2S

2.5.2.1 Hypertension

Approximately, 90-95% of hypertension is caused by unknown factors, known as essential hypertension. Throughout the decade, there has been indirect evidence suggesting H2S as a regulator of BP (Wang 2002; Zhao et al. 2001; Kimura 2002; Mok et al. 2004; Fiorucci et al. 2005). Indeed, the first physiological evidence that reports H2S to have vasorelaxant properties was demonstrated by Wang and associates (2001). These authors showed that injection of H2S at 2.8 and 14 mM/kg bodyweight induced a concentration-dependent decrease in the mean arterial BP in anaesthetized SD rats by 12.5 ± 2.1 and 29.8 ± 7.6 Hg mm, respectively (Zhao et al. 2001). Moreover, this depressive effect of H2S was mimicked by pinacidil (KATP channel opener) and attenuated by glibenclamide (KATP channel blocker) in SD rats (Zhao et al. 2001), which are consistent with findings from other groups (Cheng et al. 2004; Ali et al. 2006).

Given this, Rui Wang and associates (2008) generated mice with the targeted deletion of the gene encoding CSE, which resulted in these mice becoming hypertensive. Indeed, the CSE-/- mice also developed impaired endothelium-dependent vasorelaxation upon methacholine (vasorelaxant) administration in the mesenteric arteries that was preconstricted with phenylephrine (Yang et al., 2008). Interestingly, as young as 7 weeks old, both male and female CSE-/- mice exhibited significantly higher BP reading than the age-matched wild-type mice (Yang et al. 2008). Likewise, at 12 weeks of age, this age-dependent increase in BP in CSE-/- mice increased further to 135 Hg mm, which was about 18 mm Hg higher than the wild-type mice (Yang et al. 2008). The H2S/CSE system was likely responsible for the elevated BP readings in the CSE-/- mice because, no difference was observed regarding the H2S levels in the brain, endothelial NO synthase (eNOS) protein was unchanged, the kidney architecture was preserved, and administration of L-methionine, homocysteine precursor, did not increase BP (Yang et al. 2008). Thus, alternations of the CNS, impaired eNOS function, renal damage, or excess homocysteine levels were not causative factors for the observed high BP in the CSE-/- mice (Yang et al. 2008). This exciting discovery by Rui Wang and associates points to the possibility of H2S being the next EDRF in the cardiovasculature (Wang 2009). Indeed, pharmacological approaches that employ the usage of H2S-releasing drugs could be an excellent approach for the treatment of hypertension.

2.5.2.2 Alzheimer's disease

Alzheimer's disease is an age-related, degenerative disease that is the most common cause for dementia. It was demonstrated that SAM, a CBS activator, is significantly decreased (Morrison et al. 1996), whereas homocysteine levels in the serum of Alzheimer's disease are elevated (Clarke et al. 1998). Thus, these findings suggest decreased CBS activity and thus H2S in patients with Alzheimer's disease. Indeed, Kimura and associates (2002) demonstrated that endogenous levels of H2S, along with the enzymatic activity of CBS, are significantly lowered in Alzheimer's disease brains. However, the relationship between H2S and Alzheimer's disease is unclear and it is also not certain if lack of H2S may be involved in the prognosis of Alzheimer's. More research is needed in this field.

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