Role of hydrogen sulfide in the pathogenesis of parkinsons disease

An inflammatory process in CNS, often defined as neuroinflammation, is recently revealed to play an important role in the cascades of events leading to DA neuronal loss, and therefore gr

HYDROGEN SULFIDE: A NOVEL NEUROPROTECTIVE

AGENT TO TREAT PARKINSON’S DISEASE

HU LI-FANG

(MD, M.Sci, Nanjing Medical University, China)

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

DEPARTMENT OF PHARMACOLOGY NATIONAL UNIVERSITY OF SINGAPORE

2010

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ACKNOWLEDGEMENT

I would like to express my deepest gratitude to my supervisor, Prof Bian Jin-Song, for giving me the opportunity to work on this research project as a part-time postgraduate student I would thank my supervisor for his invaluable comments, enlightening ideas and continuous encouragement Without his great support, I would not have made great progress on my thesis

I would also thank Prof Gavin S Dawe for his guidance in the behaviour study I would express my special thanks to Mr Lu Ming for his kindly help and collaboration in the animal work

Sincere appreciation to my colleagues, Neo Kay Li, Pan Tingting, Yong Qian Chen,

Wu Zhiyuan, Ester Khin Sandar Win, Tan Choon Ping, Liu Yihong, Tiong Chi Xin, Xie

Li, and other friends in Prof Bian‘s lab for their technical support and help in various aspects over the four years I would also thank all my friends in Prof Gavin S Dawe‘s lab for their support on my animal work

I would also extend my deep gratitude to my husband for supporting and inspiring me

to reach my full potential

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TABLE OF CONTENT

PUBLICATIONS vii

ABBREVIATIONS viii

SUMMARY x

Chapter I Introduction 1

1.1 General overview 1

1.2 H 2 S 2

1.2.1 Chemical properties of H 2 S 2

1.2.2 H 2 S toxicity 3

1.2.3 H 2 S biosynthesis and metabolism in mammals 4

1.2.4 Biological roles of H 2 S 10

1.2.4.1 Roles of H 2 S in inflammation 10

1.2.4.2 Role of H 2 S in CNS and CNS diseases 14

1.2.4.3 Roles of H 2 S in cardiovascular system 25

1.2.4.4 Roles of H 2 S in gastrointestinal tract 28

1.2.4.5 Others 30

1.3 PD 31

1.3.1 Epidemiology 32

1.3.2 Risk factors 32

1.3.3 Pathology and pathogenesis 33

1.3.3.1 Pathology 33

1.3.3.2 Pathogenesis 34

1.3.4 Clinical features and diagnosis 38

1.3.5 Treatment 38

1.3.6 Experimental models 39

1.3.6.1 6-OHDA model 40

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1.3.6.2 MPTP/MPP+ model 42

1.3.6.3 Rotenone model 45

1.3.6.4 LPS model 46

1.3.6.5 Other models 49

1.4 Research rational and objectives 52

1.4.1 Rational 52

1.4.2 Objectives 53

Chapter II Anti-inflammatory effects of H 2 S on LPS-stimulated microglia 55

2.1 Introduction 55

2.2 Materials and methods 56

2.2.1 Chemicals and reagents 56

2.2.2 Cell culture 56

2.2.2.1 Microglia cell line culture 56

2.2.2.2 Primary cultured rat cortical microglia and astrocytes preparation 57

2.2.3 NO measurement 58

2.2.4 TNF-α measurement 58

2.2.5 Reverse transcription polymerase chain reactions (RT-PCR) 59

2.2.6 Transfection of CBS and CSE into BV2 cells 59

2.2.7 Western blot analysis 60

2.2.8 Statistical analysis 61

2.3 Results 61

2.3.1 Exogenous H 2 S suppresses LPS-stimulated NO production in microglia 61

2.3.2 The anti-inflammatory effect of H 2 S involves p38 MAPK 63

2.3.3 H 2 S inhibits TNF- secretion in BV2 cells 65

2.3.4 Endogenous H 2 S regulates NO production in BV2 cells 66

2.3.5 Over-expression of H 2 S synthesis enzyme suppresses NO production in microglia 68

2.3.6 H 2 S suppresses LPS-stimulated NO generation in astrocyte 68

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2.4 Discussion 69

Chapter III Anti-inflammatory effects of H 2 S on rotenone-stimulated microglia 73 3.1 Introduction 73

3.2 Materials and methods 74

3.2.1 Chemicals and reagents 74

3.2.2 Cell culture 74

3.2.3 Immunocytochemistry 74

3.2.4 Western blot assays 75

3.2.5 Intracellular ROS assay 75

3.2.6 Extracellular superoxide measurement 75

3.2.7 Microglia-mediated neurotoxicity assay 76

3.2.8 NF-κB activation assay 76

3.2.9 Statistical analysis 77

3.3 Results 77

3.3.1 NaHS inhibits rotenone-stimulated microglia activation 77

3.3.2 NaHS suppresses rotenone-induced intracellular ROS accumulation 79

3.3.3 NaHS inhibits rotenone-induced superoxide release from microglia 80

3.3.4 NaHS attenuates microglia-mediated neurotoxicity 81

3.3.5 NaHS inhibits rotenone-induced p38 MAPK/NF-κB activation 82

3.4 Discussion 86

Chapter IV Anti-apoptotic effect of H 2 S on SH-SY5Y cells 90

4.1 Introduction 90

4.2 Materials and methods 90

4.2.1 Chemicals and reagents 90

4.2.2 Cell culture and treatment 92

4.2.3 Total sulfide measurement 93

4.2.4 Cell viability assay 93

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4.2.5 Apoptosis quantification 93

4.2.6 Assessment of mitochondrial membrane potential (ΔΨm) loss 94

4.2.7 Western blot analysis 94

4.2.8 Analysis of cytosolic cytochrome c accumulation 94

4.2.9 Caspase-9 activity assay 95

4.2.10 Statistical analysis 95

4.3 Results 95

4.3.1 H 2 S suppresses rotenone-induced cytotoxicity and apoptosis 95

4.3.2 H 2 S inhibits rotenone-induced ΔΨm loss and cytochrome c release 98

4.3.3 H 2 S regulates Bax/ Bcl-2 proteins in SH-SY5Y cells 100

4.3.4 H 2 S suppresses caspase-9/3 activation and PARP cleavage 101

4.3.5 mitoK ATP channels contributes to the protective effects of H 2 S 103

4.3.6 Rotenone induces p38/JNK MAPK activation 105

4.3.7 H 2 S inhibits rotenone-induced p38/JNK MAPK activation 107

4.4 Discussion 108

Chapter V Therapeutic effect of H 2 S in rotenone-induced PD model rats 112

5.1 Introduction 112

5.2 Materials and methods 113

5.2.1 Chemicals 113

5.2.2 Animals 113

5.2.3 Behavioural test 113

5.2.4 H 2 S measurement 114

5.2.5 H 2 S-producing activity assay 115

5.2.6 Immunohistochemistry staining 115

5.2.7 NO assay 116

5.2.8 TNF-α assay 116

5.2.9 Western blot analysis 117

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5.2.10 Statistical analysis 117

5.3 Results 117

5.3.1 Endogenous H 2 S is reduced in the SN of rotenone-treated rats 117

5.3.2 NaHS alleviates rotenone-induced parkinsonian symptoms in rats 118

5.3.3 H 2 S attenuates rotenone-induced DA neuron loss in the SN 121

5.3.4 NaHS inhibits microglia activation and the subsequent release of inflammatory factors in the rotenone-induced PD model rats 122

5.4 Discussion 123

Chapter VI General discussion and conclusion 127

6.1 General discussion 127

6.2 Conclusion and perspectives 134

Bibliography 137

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PUBLICATIONS

1 Hu LF, Lu M, Tiong CX, Dawe GS, Hu G, Bian JS Neuroprotective effects of

hydrogen sulfide in Parkinson‘s disease rat models Aging Cell 2009; 9(2):135-46

2 Hu LF, Lu M, Wu ZY, Wong PT, Bian J Hydrogen sulfide inhibits rotenone-induced

apoptosis via preservation of mitochondrial function Mol Pharmacol 2009;

75(1):27-34

3 Hu LF, Wong PT, Moore PK, Bian JS Hydrogen sulfide attenuates

lipopolysaccharide-induced inflammation by inhibition of p38 mitogen-activated

protein kinase in microglia J Neurochem 2007; 100(4):1121-8

4 Hu LF, Pan TT, Neo KL, Yong QC, Bian JS Cyclooxygenase-2 mediates the

delayed cardioprotection induced by hydrogen sulfide preconditioning in isolated rat cardiomyocytes Pflugers Arch 2008 Mar; 455(6):971-8

5 Hu LF, Wong PT, Bian J. Hydrogen sulphide: neurophysiology and neuropathology Review Antioxid Redox Signal 2010 Sep 2

6 Hu LF, Wong PT, Bian J Hydrogen sulfide attenuates rotenone-induced

neuroinflammatory responses through down-regulation of NADPH oxidase/ROS signaling pathways in microglia (in preparation)

7 Tay AS, Hu LF, Lu M, Wong PT, Bian JS Hydrogen sulfide protects neurons against

hypoxic injury via stimulation of ATP-sensitive potassium channel/protein kinase C/extracellular signal-regulated kinase/heat shock protein90 pathway Neuroscience

2010 Feb 8

8 Lu M, Choo CH, Hu LF, Tan BH, Hu G, Bian JS Hydrogen sulfide regulates

intracellular pH in rat primary cultured glia cells Neurosci Res 2010; 66(1):92-8

9 Lu M, Hu LF, Hu G, Bian JS Hydrogen sulfide protects astrocytes against

H(2)O(2)-induced neural injury via enhancing glutamate uptake Free Radic Biol Med 2008;

45(12):1705-13

10 Lee SW, Hu YS, Hu LF, Lu Q, Dawe GS, Moore PK, Wong PT, Bian JS Hydrogen

sulphide regulates calcium homeostasis in microglial cells Glia 2006; 54(2):116-24

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ABBREVIATIONS

Apaf-1 apoptotic protease activating factor-1

iNOS inducible nitric oxide synthase

NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells

PPAR-γ peroxisome proliferator-activated receptor-γ

STAT signal transducers and activators of transcription

TNF-α tumor necrosis factor-α

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SUMMARY

H2S is a novel endogenous gaseous mediator alongside nitric oxide and carbon monoxide

It serves as an important neuromodulator in regulation of brain functions PD, characterized by the progressive loss of DA neurons in midbrain, is the second most common neurodegenerative disorder among old population In this thesis, the therapeutic effect of H2S on neurodegeneration and the underlying mechanisms were investigated in

both in vitro and in vivo studies

Neuroinflammation is one of the main pathological causes/features of PD In this thesis, the effect of H2S on neuroinflammation was first examined in glial cells It was found that both endogenous and exogenous application of H2S ameliorated LPS-stimulated production of nitric oxide and TNF-α, two important pro-inflammatory factors,

in primary cultured microglia and BV2 cells Similar results were also observed in primary cultured astrocytes NaHS, an H2S donor, also attenuated rotenone-induced intracellular reactive oxygen species and extracellular superoxide accumulation in microglia This implies that H2S plays an anti-inflammatory role in central nervous system The underlying mechanisms for the anti-neuroinflammatory role of H2S were demonstrated to be associated with its inhibitory effect on p38 MAPK and NF-κB signaling pathway In addition, H2S was also found to alleviate inflammation-mediated neurotoxicity on SH-SY5Y cells These data suggest that H2S may produce neuroprotective effects via its anti-neuroinflammatory action

Apart from the indirect neuroprotection, the direct effect of H2S on neuronal cells was also investigated It was found that H2S concentration-dependently suppressed rotenone-induced cellular injury and apoptotic cell death NaHS also prevented rotenone-induced

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p38- and JNK- MAPK phosphorylation and changes in Bcl-2/Bax levels, ΔΨm dissipation,

cytochrome c release, caspase-9/3 activation as well as PARP cleavage This effect was

mediated by mitoKATP channels Therefore, H2S may protect neuronal cells against rotenone-induced apoptosis through preservation of mitochondrial integrity and inhibition

on mitochondrial apoptotic pathways

The potential neuroprotection of H2S was further confirmed in vivo with the induced rat PD model It was found that both endogenous H2S level and its biosynthesis activity were reduced in rotenone-induced PD rats, implying that the impaired endogenous H2S production may contribute to the development of PD Interestingly, NaHS treatment significantly alleviated rotenone-induced behavioral deficits, DA neuronal loss in substantia nigra, microglial activation as well as elevation of nitric oxide and TNF-α content in the nigrostriatal tract in rat These data clearly suggest that H2S has the potential to be developed as a new agent to treat neurodegenerative diseases

rotenone-In summary, the present study demonstrates for the first time that H2S may serve as a neuroprotectant to treat and prevent neurotoxin-induced neurodegeneration via anti-inflammatory and anti-apoptotic mechanisms, and therefore has the potential therapeutic value for PD treatment

Parkinson‘s disease (PD) is a movement disorder characterized by the progressive loss of dopamine (DA) neurons in the substantia nigra (SN) Its etiology remains elusive and the mechanisms for initiating and aggravating neuronal death are yet to be defined, despite years of intensive research An inflammatory process in CNS, often defined as neuroinflammation, is recently revealed to play an important role in the cascades of events leading to DA neuronal loss, and therefore greatly contributes to the pathological progression of PD Hence, it is presumed that any strategy aimed at suppressing the neuroinflammatory process would be potentially effective for PD therapy

In the peripheral system, H2S was recently demonstrated to regulate the inflammatory process (120, 151, 231) However, its role in neuroinflammation remains unknown Given that H2S regulates inflammatory processes in various diseases and that H2S exists

in brain at relatively high levels, it is hypothesized that H2S may regulate

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neuroinflammatory process, and thus the pathogenesis of PD Therefore, this thesis was designed to investigate the potential role of H2S in neuroinflammation and DA neuronal injury The therapeutic effect of H2S on PD was also tested in an animal PD model

1.2 H 2 S

Similar to NO and CO, H2S has historically been known as an industrial pollutant and environmental toxin; however, it has recently been shown to be an important gaseous transmitter modulating many physiological and pathological processes Specifically, H2S exerts important functions in the cardiovascular system and the CNS It was found to serve as a vasodilator and a novel neuromodulator H2S is also an endogenous regulator

of inflammatory response, playing both pro- and anti- inflammatory roles in different systems and situations Several lines of evidence suggest that H2S is an important biological molecule in mammalian tissues

1.2.1 Chemical properties of H 2 S

H2S is a colourless, flammable gas with a molecular weight of 34.08 It is responsible for the foul odour of rotten eggs and flatulence Its structural formula is illustrated as H-S-H, similar to that of water (H2O) It is soluble in water with solubility of 1 g in 242 ml water

at 20 °C H2S is weakly acidic because it easily dissociates into H+ and HS- in solution It

is noteworthy that both pH and temperature of solution affect H2S concentration According to a standard Henderson-Hasselbach calculation at 20°C, in solution with pH

at 7.4, H2S exists as ~30-33% (one-third) H2S and 67-70% (two-thirds) HS-, with negligible S2- due to the high pKa2 (11.96) However, it is not true when temperature reaches 37°C because pKa1 at 37°C is 6.755, rather than 7.04 for standard conditions at 20°C Accordingly, in saline at 37°C and pH 7.4, less than one-fifth of H2S exists as the undissociated form (H2S) (82, 166, 192) Because it is unlikely to determine which form

1.2.2 H 2 S toxicity

H2S has been known as a highly toxic gas for almost 300 years H2S poisoning is a serious issue due to its widespread environmental and occupational exposure derived from industrial activities, such as paper pulp mills, petroleum refinery and urban sewers H2S is a broad-spectrum poison affecting different systems, among which the nervous system is the most often intoxicated Acute inhalation of H2S (500-1000 ppm) causes neurotoxic effects such as headache, dizziness, amnesia and even unconsciousness (‗knockdown‘), due to the direct toxic effect of H2S on the brain These toxic effects are

usually reversible because the sufferers can completely recover from the acute intoxication if the exposure is rapidly removed However, if exposure is more pronounced (over 1000 ppm) or prolonged, fatal respiratory paralysis may occur and even lead to death, as a result of hypoxia secondary to H2S-induced respiratory insufficiency Death may come instantly, just like a vivid description ―death comes on like a stroke of lightening‖, when the concentration of H2S is higher than 5000 ppm (150, 163) In a few

cases, acute but nonfatal H2S intoxication is followed by brain damage featured by permanent neurological sequelae

H2S toxicity has been attributed to its ability to inhibit cytochrome c oxidase in a similar manner to hydrogen cyanide poisoning (197) It suppresses oxygen utilization and

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results in central respiratory paralysis Warenycia et al believed that its inhibition on

monoamine oxidase (MAO) also contributes to the loss of central respiratory drive after fatal intoxication with H2S (208) Recent studies found that generation of excessive ROS via a CYP450-dependent mechanism also contributed to H2S-induced cytotoxicity (50)

Of note, human nose is very sensitive to H2S and can detect its unpleasant smell as low as 0.02 ppm (estimated level of H2S in normal atmosphere is approximately 0.0001 ppm) However, it appears to be true only at low concentrations At higher concentrations (up to

100 ppm), the rotten-egg smell of H2S disappears and it emerges as an odourless gas, greatly increasing the risk of H2S poisoning This implies that disappearance of unpleasant smell could be a sign for increasing concentrations of H2S at sites where potential exposure of this poison exists

1.2.3 H 2 S biosynthesis and metabolism in mammals

The desulfhydration of Cys is proposed to be the major source of H2S in mammals This process is catalyzed via two pyridoxal-5‘-phosphate (PLP)-dependent enzymes CBS and CSE In the transsulfuration pathway, Cys is derived from Hcy with CBS catalyzing the β-replacement reaction of Hcy to yield cystathionine which is then lyzed by CSE into Cys and α-ketobutyrate (Figure 1.1) It has been shown that CBS can efficiently produce H2S via a β-replacement reaction in which Cys is condensed with Hcy to form cystathionine

and H2S and this reaction is far more efficient when compared to β-elimination of Cys (33) Detailed kinetic analysis performed by Banerjee‘s group demonstrated that CBS produces H2S overwhelmingly from Cys+Hcy (96%) under simulated physiological conditions, while Cys and Cys+Cys accounts for only 1-3 % (71) Therefore, cysteine and homocysteine are the preferred substrates of CBS for H2S biosynthesis On the other hand, CSE produces H2S from Cys (70%) or Hcy (γ-elimination, 29%) under normal conditions

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(10 µM Hcy) Interestingly, when the concentration of Hcy was increased from 10 to 40 and 200 µM to simulate mild and severe hyperhomocysteinemia, the contribution from Hcy increased from 29% to 63% and 90%, respectively, while contribution from Cys decreased correspondingly to 37% and 10%, respectively (13) As Vmax for the γ-elimination of Hcy is twice that for the β-elimination of Cys, this shift may represent a

marked increase in the generation of H2S under hyperhomocysteinemic conditions (13) Therefore, H2S production derived from CSE is sensitive to homocysteine (35) Basically, under normal conditions (10 µM Hcy) CSE represents ~32% of the H2S generation by the transsulfuration pathway, but it increases to ~45% and ~74% under moderate and severe hyperhomocysteinemia conditions (182) In contrast, CBS is not sensitive to Hcy concentrations with Cys+Hcy as the predominant substrates (71) For this reason, in homocystinurics with CBS deficiency, CSE may be the major enzyme to produce H2S Moreover, the level of H2S produced by CSE is predicted to be higher due to the enhanced accumulation of Hcy (35)

CBS is highly expressed in the brain and thus believed to be the primary physiologic source of H2S in the CNS (1), although both CBS and CSE activities were detected in different brain regions (9, 201) CBS is a cytoplasm PLP-dependent enzyme Human CBS has a complex structure and regulatory mechanisms (134) It contains the N-terminal heme-binding domain, the catalytic domain, and the C-terminal regulatory domain Two other gaseous transmitters, CO and NO, can bind to the heme-binding domain and result

in the inhibition of CBS activity (190, 191) Moreover, the S-adenosyl-L-methionine, which may bind to the C-terminal domain, can instantaneously activate CBS At the transcriptional level, glucocorticoids can stimulate the CBS gene expression whereas insulin can inhibit it (162)

transsulfuration pathway GSH synthesis pathway

Figure 1.1 Endogenous sources of H 2 S in mammalian H2S is endogenously produced by the action of CBS and CSE in the transsulfuration pathway By kinetic simulation, it is found that CBS generates H2S most efficiently from Cys+Hcy, with cystathionine as a byproduct This reaction contributes >95% of the net H2S production by CBS On the contrary, the preferred substrates for CSE are Cys and Hcy Together they contribute well over 90% of the net H2S production by CSE In addition, the CAT and 3-MST are components of the Cys catabolism pathway CAT catalyzes the transamination of Cys to yield 3- mercaptopyruvate, a substrate of 3-MST to produce pyruvate and sulfane sulfur, which may liberate H2S in the presence of reductants such as dithiothreitol (DTT) and GSH The transsulfuration pathway is critical for creating Cys from the essential amino acid methionine, which is first converted to Hcy by demethylation CBS condenses serine and Hcy to produce cystathionine, which is converted to Cys and α-ketobutyrate by CSE The synthesis of glutathione (GSH) is regulated at the substrate level by Cys Thus, the transsulfuration pathway also links to the GSH homeostasis in brain

The cellular localization of CBS is still controversial Using immunohistochemical techniques, Robert et al showed that CBS protein has a predominantly neuronal localization in most areas of the brain, especially in hippocampus and cerebellum (166)

In contrast, Enokido et al later demonstrated that CBS is preferentially expressed in astrocytes rather than neurons, which is verified by combined biochemical and histological examination, as well as in situ hybridization (51) This fits with recent

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findings that CBS mainly localizes to astrocytes (112) Lee et al demonstrated that the basal H2S level in unstimulated human astrocytes is about 3.0 µmol/g protein, which is 7.9 fold higher than in cultured microglia More importantly, only astrocytes, instead of microglia, are strongly immunostained for CBS (112) However, Vitvitsky and his colleagues showed the incorporation of radiolabel from methionine into glutathione (GSH)

in both cultured human astrocytes and neurons (201) Another group also showed that inhibition of CSE leads to a significant loss of GSH in adult brain slices (48) Since the only known route for the transfer of radiolabelled methionine to GSH is via the transsulfuration pathway involving CBS and CSE, these experiments indirectly justify the existence of CBS in both astrocytes and neurons Nevertheless, studies consistently identified the temporal expression of CBS in developing and adult mouse CNS During the embryonic period, CBS protein level is generally low, but it dramatically increases from late prenatal to early postnatal period (51, 166)

In biomedical studies, small molecule inhibitors, such as hydroxylamine and aminooxyacetate acid (AOAA), have been used to determine the significance of the endogenously generated H2S These agents are able to inhibit the biosynthesis of H2S from Cys but they are general inhibitors of all PLP-dependent enzymes and are used quite

to liberate bound PLP for quantitation (92, 184) In addition to heme and PLP, hydroxylamine also reacts with non-heme iron proteins, e.g ribonucleotide reductase and

is used to inhibit cell growth Hence, caution should be taken when interpreting results obtained from work involving these inhibitors

In addition to CBS, there is also report showing that CSE plays an important role in human brain, despite its predominant localization in the cardiovasculature In fact, CSE is critical for maintaining GSH homeostasis in brain, which in turn preserves mitochondrial

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function (48) CSE is the rate-limiting enzyme in the transsulfuration pathway for the sulfur transfer methionine to Cys, which is a limiting reagent in the synthesis of GSH Moreover, CSE mRNA is localized in brain and found to be predominantly present in neurons by in situ hybridization The CSE activity in mouse brain was as low as 1% of the hepatic activity However, in human brain the activity was 100 times more than that in mouse brain Furthermore, an intact transsulfuration pathway in the brain mediated by both CBS and CSE links to GSH homeostasis, which greatly contributes to the redox-buffering capacity in brain (201) Nevertheless, the general consensus is that CSE is the primarily physiological source of H2S generation in the peripheral tissues There is definitive evidence that CSE knockout mice developed hypertension, which establishes that H2S as a major physiologic signalling molecule regulating vascular tone in mammals

So far, there is little knowledge about the physiologic relevance of CSE relative to CBS

in brain, in addition to its role in transsulfuration pathway linking to GSH homeostasis This issue merits further investigation

Recently, Kimura‘s group reported another source of H2S in the brain homogenates of

CBS-knockout mice (177) They show 3-mercaptopyruvate sulfurtransferase (3-MST) in combination with Cys aminotransferase (CAT) produces H2S from Cys Like CBS and CSE, CAT is also a PLP-dependent enzyme, which catalyzes the metabolism of Cys and α-ketoglutarate to yield 3-mercaptopyruvate as the substrate for 3-MST 3-MST is

localized to mitochondria and nerve endings As its name implies, it belongs to the family

of sulfurtransferases, which catalyze the transfer of sulfane sulfur from persulfide or thiosulfate or mercaptopyruvate to an acceptor, and liberates H2S under certain conditions Thus, 3-MST does not produce H2S by itself Instead, it produces sulfane sulphur (or bound sulphur), which in the presence of reductants like dithiothrietol , liberates H2S (92)

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Bound sulfur may be a source of H2S in brain and it can immediately release H2S in response to physiologic stimulation (90) This may explain why H2S was not depleted in the brain homogenates of CBS knock mice Presumably, H2S is derived from this pool of sulfane sulfur under reducing conditions However, the physiological significance of H2S derived from this source is yet to be determined with 3-MST knockout mice or other techniques With respect to development, 3-MST protein expression in the mouse brain is maintained from embryonic day 16 (E16) to postnatal day 14 (P14) but downregulated between P28 and P52, and then increased slightly thereafter up to P156 (177)

However, the contributions and differences of CBS and 3-MST with respect to H2S generation under physiological and pathological conditions are still not clearly understood As these two enzymes have different cell-type specific expression profiles in the brain, it is possible that they may have different functions in various pathophysiological situations It may be speculated that CBS may relate closely to the anti-neuroinflammatory role of H2S whilst 3-MST may contribute more to the anti-oxidant action due to their different cellular localization

In addition to biosynthesis, there are two forms of sulfur stores in mammals, labile sulfur and bound sulfane sulfur (90) The former store, mainly localized to the iron-sulfur center of enzymes in mitochondria, releases H2S under acidic conditions whilst the latter store, primarily localized to the cytoplasm, releases H2S under reducing conditions The physiological importance of H2S released by bound sulphur remains unclear However, the general consensus is that acid-labile sulfur is not a source of H2S under physiological conditions Although H2S is proposed to undergo various chemical reactions for its catabolism in mammals, such as oxidation to sulphate, methylation to

Caecal ligation and puncture-induced sepsis and LPS-induced endotoxemia Hui et al

first reported that vascular H2S markedly elevated in rats with septic and endotoxic shock

(89) Similarly, Zhang et al found that induction of sepsis by caecal ligation and puncture

(CLP) resulted in substantial upregulation of CSE mRNA in liver, associated with increased plasma H2S level and liver H2S production (236) Furthermore, injection of NaHS upregulated the production of proinflammatory mediators such as interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α) and macrophage inflammatory protein (MIP)-2 in lung and liver (234), elevated substance P (SP) generation in lung (233), and promoted the leukocyte activation and trafficking (235) Administration of PAG (a CSE inhibitor) produced the opposite effects These observations strongly suggest that H2S could aggravate the sepsis-associated systemic inflammatory response and thus H2S has a

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pro-inflammatory role in this sepsis model Supportively, in another systemic inflammation model LPS-induced endotoxemia, plasma H2S level and CSE mRNA as well as its activity in liver and kidney were also significantly increased compared to that

in control group Intriguingly, plasma H2S concentration was found to be substantially increased (~3.4 fold) in patients with septic shock compared to healthy controls NaHS injection resulted in marked inflammatory damage in lung, as evidenced by a considerable increase in plasma TNF-α level and myeloperoxidase (MPO) activity in both lung and liver Inhibition of endogenous H2S by PAG administration alleviated multiorgan injury caused by endotoxemia (39, 120) More importantly, it appears that there is an interplay between NO and H2S in the pathogenesis of LPS-induced endotoxemia because NO-releasing flurbiprofen could reduce the H2S production and thus attenuate LPS-induced plasma accumulation of various cytokines (39) All these data clearly suggest that H2S plays a pro-inflammatory role in systemic inflammation, at least

in these two septic shock models

Acute pancreatitis Acute pancreatitis is a sudden inflammation of pancreas,

commonly occurs in clinical practices It is often associated with severe complications and high mortality in severe cases Both CBS and CSE, two H2S -forming enzymes were found to be highly expressed in pancreatic acinar cells; thus the possible role of H2S was

intensively explored in this disease with the mice model induced by caerulein Bhatia et

al first noted that blockade of H2S formation by PAG, prophylactic as well as therapeutic treatment, significantly alleviated the caerulein-induced pancreatitis severity and its associated lung injury (15) Subsequently, researchers from the same group found that caerulein increased the levels of H2S and CSE mRNA but decreased CBS mRNA in isolated mouse pancreatic acinar cells (187) In addition, PAG treatment not only

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inhibited endogenous H2S formation, but also suppressed the up-regulation of SP and its receptor neurokinin-1 (NK-1R) as well as the gene expression of preprotachykinin-A, a precursor of SP, in the caerulein-induced acini Furthermore, NaHS resulted in a significant increase of SP and its receptors These data suggest that H2S also exerts a pro-inflammatory action in acute pancreatitis, possibly mediated by SP-NK-1R related

pathway Collectively, these in vivo and in vitro findings consistently indicate the

pro-inflammatory effects of H2S in acute pancreatitis

Carregeenan-induced hindpaw edema The possible role of H2S in another inflammatory situation, carrageenan-induced hindpaw oedema was also investigated (14)

An increase of H2S synthesis enzyme activity and MPO activity was observed in inflamed hindpaw Pretreatment with PAG dose-dependently reduced carrageenan-induced hindpaw oedema, in which the activities of both H2S synthesis enzyme and MPO were also decreased in a dose-dependent manner These observations also support a pro-inflammatory role of H2S in this model

Neurogenic inflammation Neurogenic inflammation is defined as an inflammation

caused by an injurious stimulus of afferent neurons resulting in release of neuropeptide such as SP and NK A, which may affect vascular permeability and help initiate pro-inflammatory responses at the site of injury Since H2S is reported to be able to regulate

SP release in several diseases, it is possible that H2S may play an important part in the

pathogenesis of neurogenic inflammation Trevisani et al reported that NaHS induced the

airway contraction and increased neuropeptide release (196) These effects could be abolished by co-pretreatment with tachykinin receptor antagonists, and attenuated by the transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine These data suggest that H2S evoked tachykinin-mediated inflammatory reaction in airways, resulting

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from the stimulation of TRPV1 receptor on afferent nerve endings Similar effects of H2S

on TRPV1-mediated neurogenic inflammation are recently reported in polymicrobial sepsis (5) These two studies consistently demonstrate a regulatory role of H2S in neurogenic inflammation

Anti-inflammation

A great body of evidence from both in vivo and in vitro studies mentioned above strongly

supports the pro-inflammatory actions of H2S However, lots of studies also note an inflammatory role of H2S

anti-H2S is demonstrated to alleviate the inflammatory hallmarks including swelling and pain Tissue swelling often results from plasma exudation (or ‗edema formation‘) and neutrophil infiltration, as well as adherence of leukocytes to vascular endothelium H2S-releasing drug S-diclofenac was shown to inhibit the carrageenan-evoked hindpaw swelling and neutrophil infiltration, as determined by the decrease of hindpaw MPO activity (179) Moreover, S-diclofenac produced these effects more potently than its parental moiety diclofenac Hence, the enhanced anti-inflammatory effect of S-diclofenac was proposed to be related to its ability to release H2S at the inflamed site H2S donors were also displayed to induce neutrophil apoptosis and suppress endothelial adhesion molecules expression With intravital microscopy, both NaHS and Na2S were found to inhibit aspirin-evoked leukocyte adherence in mesenteric venules via activation of KATP

channels (231) In addition, in vitro and in vivo studies have shown that H2S inhibits the

generation and release of pro-inflammatory mediators For example, in cultured murine RAW264.7 macrophages, H2S donors (NaHS and GYY4137, a novel slow-releasing H2S donor) were shown to concentration-dependently inhibit LPS-stimulated release of pro-inflammatory mediators such as IL-1β, IL-6, TNF-α, NO and PGE2 but enhance the

14

production of anti-inflammatory chemokine IL-10 (151, 213) GYY4137 was also shown

to protect against endotoxic shock in the rats (121) H2S-promoted gastric ulcer healing effects were also related to its anti-inflammatory actions (202) Hence, all these data consolidate the notion that H2S also has anti-inflammatory effects

All the above-mentioned findings suggest one possibility that H2S may act as a

―double edged sword‖, and thus plays an important role in modulating inflammation This

is similar to two other gaseous transmitters, NO and CO In addition, there is an interaction among these three gaseous molecules involved in regulating inflammatory responses Contradictory observations on the role of H2S in inflammation may be attributed to several factors Different H2S donors (NaHS, Na2S, GYY4137, H2S-releasing non-steroidal anti-inflammatory drugs (NSAIDs)) may produce conflicting results due to their dissimilar pharmacokinetic and pharmacodynamic properties Different regimen (dose and time of drug administration) may also contribute to the diverse effects of H2S in different disease conditions Hence, so far it is still in an early stage to make a definitive conclusion whether H2S is a pro- or anti- inflammatory molecule It appears that physiological concentrations of H2S produce anti-inflammatory effects, whilst higher (pathological) concentrations, which could be endogenously produced in already inflamed tissues, can exert pro-inflammatory effects Collectively, its biology in inflammation is proving to be complex and difficult to unmask, and merits further intensive examination

1.2.4.2 Role of H 2 S in CNS and CNS diseases

H2S serves as a neuromodulator that potentiates or inhibits the transmission of nerve pulses in neurons A typical example is its regulation on long-term potentiation (LTP) (as

15

shown in Figure 1.2) It selectively stimulates N-methyl-D-aspartic acid (NMDA) receptor-mediated currents and thus facilitates LTP induction in the presence of a weak titanic stimulation, via activating adenylyl cyclase (AC) and the subsequent cAMP/PKA cascades (1, 100) However, H2S alone does not induce LTP, implying H2S mainly modulates LTP in active synapses (1) Our recent study also found that H2S could promote the astrocytic glutamate uptake (128), which plays an important part in clearing excessive glutamate in synaptic cleft and maintaining normal neurotransmission among

neurons In addition, Kombian et al found that H2S reversibly inhibited both fast and

slow synaptic responses in dorsal raphe serotonergic neurons (109) These observations offer important and direct evidence for the modulatory role of H2S in CNS

Intracellular calcium ([Ca2+]i) homeostasis is crucial for regulation of synaptic activity and plasticity, as well as signal transmission between neuron and glial cells The modulatory role of H2S on [Ca2+]i in both neuron and glial cells has been intensively investigated We and other groups found that H2S regulates [Ca2+]i in neurons, astrocytes, and microglia (115, 145, 227) The increase in [Ca2+]i triggered by H2S mainly involves calcium influx through calcium channels located in plasma membrane and calcium release from intracellular calcium store, as represented in Figure 1.3 H2S-induced [Ca2+]iincrease appears to the consequence of the stimulatory effects of H2S on multiple targets The Ca2+ influx is related to the activating effects of H2S on L-/T- type Ca2+ channels and NMDA receptor Besides, we also found that both protein kinase A (PKA) and phospholipase C (PLC)/protein kinase C (PKC) pathways mediate the action of H2S on [Ca2+]i (115, 227) In light of the reciprocal interactions between glia and neuron, H2S may herein regulate synaptic activity by modulating the activities of both neurons and glia (145) In addition, H2S also evokes neurite outgrowth in NG108-15 cells through

16

activating T-type Ca2+ channels (146) Furthermore, it was reported that differentiated astrocytes acquire sensitivity to H2S which is diminished by the transformation into reactive astrocytes (198) More importantly, inflammatory stimulation of microglia and astrocytes causes down-regulation of CBS and H2S synthesis (112) Thus, these findings consolidate the neuromodulatory role of H2S

Apart from [Ca2+]i, our laboratory recently found that H2S also regulates intracellular

pH (pHi) in rat primary cultured glial cells through regulating the activities of Cl-/HCO3- exchanger and Na+/H+ exchanger (127) pHi homeostasis is essential for the maintenance

of normal cell function via changes of ion channel conductance, synaptic transmission as well as gap junctions pHi disturbance is an early event that occurs in brain under pathophysioligical conditions, such as hypoxia and ischemia Gathering evidence shows acid-base transporters contribute to the pHi regulation in CNS (Figure 1.4) The regulatory effects of H2S on pHi via these transporters also support the notion that H2S serves as a novel neuromodulator, not only under physiological conditions, but also in pathological situations

presynaptic terminal

by its generating enzyme, presumably CBS (its neuronal localization is still controversial), and the H2S liberated by sulfane sulfur under reducing conditions may activate adenyl cyclase (AC) and its downstream PKA pathway to modulate NMDA receptor activity and thus facilitates NMDA receptor-mediated LTP formation in the hippocampus

cAMP/PKA but not PLC/PKC/IP3 pathway involved

both PKA and PLC/PKC pathways involved

as cAMP/PKA and PLC/PKC, are involved in regulation of intracellular Ca2+ in glia and neuronal cells

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Cl HCO 3 -

Figure 1.4 Schematic illustration of the acid-base membrane transporters involved in intracellular

pH regulation in glial cells According to their functions, these transporters can be categorized into two

groups: acid extruders and acid loaders The acid extruders include Na+/H+ exchanger (NHE), Na+ dependent Cl-/HCO3- exchanger, Na+/HCO3- cotransporter, as well as H+ proton pump Na+-independent

-Cl-/HCO3- exchanger is the main acid loader in glia All these major acid-base regulators play pivotal roles

in the maintenance of pHi under physiologic and pathologic conditions

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cystine and cysteine transport as well as glutamate uptake (103, 104, 128) (Figure 1.5) Cysteine is a rate-limiting factor in GSH synthesis Extracellular cysteine is easily oxidized to cystine The transport of cystine into cells, mainly mediated by cystine/glutamate antiporter, is therefore essential in providing cells with cysteine as substrates for GSH synthesis The promotion of H2S on glutamate uptake, which lowers glutamate concentrations in synaptic cleft and in turn stimulates the activity of cystine/glutamate antiporter, enables the transportation of cystine into cells, and eventually results in the elevation of intracellular cysteine These results provide evidence for the powerful anti-oxidative action of H2S in CNS, and simultaneously offer evidence for its neuroprotective effects because excitotoxicity, mainly derived from excessive accumulation of glutamate in synaptic cleft, greatly contributes to the development of stroke, traumatic brain injury and neurodegenerative disorders as well In addition, mounting evidence shows that H2S has anti-apoptotic effects on neuronal cells and thereby might become a potential candidate for neuroprotection H2S inhibits β amyloid-induced PC12 cell damage (188) H2S protects hippocampal neurons against vascular

dementia-induced injury via its anti-apoptotic function (238) Additionally, Thomas et al

recently indentified the interaction between H2S and human neuroglobin, a protein associated with mitochondria and protecting neurons from apoptotic stress (24); however, the biological significance of this interaction remains to be defined in future

21

Figure 1.5 Schematic paradigms illustrating the mechanisms for the elevation of GSH induced by H 2 S

in brain cells H2S enhances the transportation of cystine and Cys into cells to provide substrates for GSH synthesis in neurons H2S also enhances glutamate uptake via glutamate transporter GLT-1 in astrocytes and thus clears the excessive glutamate in synaptic cleft This process may also relieve the inhibition by glutamate on cystine transportation and thus facilitates the cystine transport into the neuronal cell In addition, H2S enhances the activity of γ-glutamylcysteine synthetase (γ-GCS), a rate-limiting enzyme in GSH synthesis and facilitates the redistribution of GSH into mitochondria and protects against oxidative stress

Apart from its physiological effect (acting as a neuromodulator), the pathophysiological significance of H2S is now continuously emerging as a hot research topic among neuroscientists Great progress has been made in elucidating its effects in CNS diseases and findings suggest that, like another gaseous transmitter NO, H2S plays important roles

in the development and regulation of several CNS diseases, such as seizures, stroke and Down‘s syndrome (summarized in Table 1.1) In addition, in human the deficiency of CBS results in homocysteinuria, with increased plasma levels of homocysteine and methionine but decreased levels of cysteine The clinical phenotype of these patients

22

includes mental retardation, lens dislocation, skeletal abnormality This also demonstrates the biochemical and medical significance of H2S in health

Ischemic Stroke Ischemic stroke is the rapidly developing loss of brain functions due

to disturbance in the blood supply to the brain, commonly caused by thrombosis or embolism It is a medical emergency and the leading cause of adult disability in the

United States and Europe Wong et al found that high plasma cysteine level is correlated with poor clinical outcome in patients with acute stroke (217) Qu et al further

demonstrated that administration of cysteine, by either i.p or i.c.v route, dependently increased the infarct volume in rats after experimental stroke induced by middle cerebral artery occlusion (MCAO) This cysteine effect was mimicked by NaHS (161) but reversed by co-administration of the CBS inhibitor AOAA (217) Moreover, endogenous H2S as well as its synthesizing activity in the cerebral cortex were significantly increased by MCAO Interestingly, the increased infarct caused by either cysteine or NaHS was reversed by MK-801, a NMDA receptor blocker Notably, pre-administration of the inhibitors of CBS (AOAA and HA) and CSE (BCA and PAG) before MCAO could reduce the infarct volume Among these inhibitors, AOAA, at a dose

dose-of 0.05 mmol/kg, appears to be most effective in reducing MCAO-induced ischemic infarction However, AOAA at higher doses (up to 0.5 mmol/kg) was not effective in ameliorating ischemic infarction (161) These findings imply that cysteine, most likely via its conversion to H2S, can influence the outcome of stroke and that abnormal H2S formation may be involved in the pathogenesis of ischemic stroke (32)

Intriguingly, there is an in vitro study showing that NaHS protects cells against

CoCl2-induced hypoxia by suppressing oxidative stress and caspase-3 dependent cell

apoptosis (32) Seemingly, the in vitro findings are contradictory to the in vivo results

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obtained by Qu et al However, it is important to note that, compared to earlier studies, higher dose of NaHS (0.18 mmol/kg) was applied in the in vivo study, which may

produce H2S at levels beyond the physiological range and result in subsequent deleterious

effects According to Warenycia et al., the LD50 for NaHS is about 14.6 mg/kg (0.26

mmol/kg) (207) The dose used by Qu et al corresponds to the sublethal dose (0.66

*LD50) of NaHS and thus it may produce the toxic effects Furthermore, Qu‘s data also showed that excessive inhibition of H2S with higher doses of AOAA did not reduce but tended to exacerbate MCAO-induced cerebral ischemic infarction Also, it should be careful to distinguish between the causation and correlation because H2S production is possibly increased in compensatory response to ischemic insult Due to the debate on the actual value of H2S in CNS and the ensuing difficulty in the choice of H2S dose in application, the transgenic mice (knock in or knock down of endogenous H2S-producing enzymes) would be a suitable approach to unravel the role of H2S in the development of ischemic stroke and to address this discrepancy

Alzheimer’s disease (AD) AD is the most common age-related neurodegenerative

disorder Its etiology remains unclear but current evidence indicates the involvement of

amyloid and tau proteins In 1996, Morrison et al first reported that the brain levels of

S-adenosylmethionine, a CBS activator, are severely decreased in AD patients (142) The total serum level of hcy (a precursor of Cys when acted on by CBS followed by CSE) is accumulated and increased in AD patients (38) One possible explanation is that the transsulfuration pathway linking Hcy and GSH metabolism, mediated by CBS and CSE,

is disrupted Because CBS is an important biosynthetic source of H2S generation in brain, although the contribution of 3-MST to H2S formation is also reported in brain, the dysfunction of the transsulfuration pathway may lead to the reduced production of H2S in

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AD, in addition to GSH Indeed, several lines of evidence from both in vivo and in vitro

studies indicate that H2S treatment elicits neuroprotective effects against pathological progression of AD First of all, H2S is shown to scavenge the cytotoxic lipid oxidation product 4-hydroxynonenal (171), which is markedly increased in brains of severe AD patients Secondly, H2S was shown to ameliorate β amyloid-induced damage in PC12

cells through reducing the loss of mitochondrial membrane potential and attenuating the increase of intracellular reactive oxygen species (188) Thirdly, H2S-releasing compounds are capable of attenuating neuroinflammation (113), a contributing factor implicated in

AD pathogenesis Importantly, H2S attenuates LPS-induced cognitive impairment in rats via its anti-inflammatory action (67) Additionally, garlic extracts, mainly the organosulfur-containing compounds such as S-allylcysteine and di-allyl-disulfide, have been shown to reduce cerebral amyloid, inflammation and tau conformational changes in

AD transgenic model (30) Moreover, these garlic extracts (both fresh and boiled) not only inhibited β amyloid fibril formation but also was capable of defibrillating β amyloid

preformed fibrils, thus exhibiting an anti-amyloidogenic activity on amyloid-beta fibrillogenesis (73) H2S can be formed nonenzymatically from polysulfides in garlic (11) Based upon these findings, it is logical to assume that H2S would be beneficial for AD treatment However, more direct evidence for the potential benefits of H2S or its donors

in AD animal models is lacking at present

Other diseases Overproduction of endogenous H2S has been reported in Down syndrome patients and the ‗H2S hypothesis‘ has been proposed to be responsible for the mental retardation in Down syndrome (94, 95) The role of H2S in Huntington‘s disease (HD), another age-related neurodegenerative disorder that often gives arise to dementia,

is yet to be determined However, there is evidence that CBS interacts with Huntingtin,

25

mutants of which cause HD and that the plasma total homocysteine levels were also found to be elevated in HD patients (4, 23) As homocysteine is an important precursor for H2S biosynthesis, this implies that H2S may also be involved in the development of

this neurodegenerative disorder In addition, Han et al demonstrated that NaHS improved

the hippocampal damage induced by recurrent FS in rats whereas CBS inhibitor aggravated this damage (76) But they also reported the increased plasma level of H2S and CBS expression in hippocampus of febrile seizure (FS) rat model This increase of both H2S and CBS expression could only be explained as the result of self-protective mechanisms in FS Interestingly, H2S acts in synergy with CO in the pathogenesis of recurrent FS (75)

Ischemic stroke middle cerebral artery occlusion increased tissue H 2 S in cerebral cortex

Administration of cysteine and NaHS increases infarct size

Alzheimer‘s disease (AD) Total homocysteine are increased in AD brains and serum

H 2 S attenuated beta-amyloid induced damage in PC12 cells

Down syndrome Overaccumulation of H2S in brains of Down syndrome patients

Huntington‘s disease (HD) Plasma total homocysteine is increased in HD patients

CBS interacts with Huntingtin

Febrile seizure (FS) Increased plasma level of H 2 S and expressions of CBS in

hippo-campus of febrile seizure (FS) model rat NaHS improve s hippocampal damage induced by recurrent FS

Plasma homocysteine levels are elevated in PD patients treated with l-dopa medications

Parkinson‘s disease (PD)

161 217

38 188

76

94, 95

4 23

244

1.2.4.3 Roles of H 2 S in cardiovascular system

The regulatory effect of H2S on vascular structure was first demonstrated by Hosoki and her colleagues that NaHS relaxed norepinephrine induced-contraction of portal vein and

26

thoracic aorta in a dose-dependent manner (82) The vasodilatory effects of H2S were confirmed by a number of studies from other groups Furthermore, it was found that in spontaneous hypertensive rats (SHR), the expression and activity of CSE in thoracic aorta

as well as plasma H2S levels were significantly decreased (223) Administration of NaHS markedly reduced blood pressure in SHR but not in normal rats Similar effects were observed in L-NAME (an inhibitor of nitric oxide synthase)-induced hypertension Moreover, pronounced hypertension and reduced endothelium-dependent vasorelaxation were displayed in mutant mice lacking CSE where H2S levels in the serum, heart, aorta as well as other tissues were remarkably decreased (224) These findings provide abundant evidence for H2S as a vasodilator and regulator of blood pressure, and also suggest that H2S may be involved in the pathogenesis of hypertension and other related diseases For example, H2S was found to attenuate the elevation of pulmonary arterial pressure and lessen the pulmonary vascular structure remodeling during hypoxic pulmonary hypertension (36) Additionally, H2S is revealed to be an endogenous stimulator of angiogenesis The aortic rings isolated from CSE knockout mice exhibited markedly reduced microvessels formation in response to vascular endothelial growth factor (VEGF) (155) Besides vasorelaxation, H2S also produces vasoconstriction through down-regulation of cAMP/PKA pathway (122) It should be noted that in contrast to NO, the major cellular source of H2S is once thought to be vascular smooth muscle cell, rather than endothelial cell, based on lines of experimental observations On one hand, CSE is mostly found in liver, vascular and nonvascular smooth cells, but not in endothelium On the other hand, the vasorelaxation of H2S on aortic rings was not altered by the removal

of endothelium However, Zhao et al later demonstrated that although the H2S-induced

maximum relaxation of the pre-contracted tissues was not affected by the endothelium removal, the H2S concentration-response curve was shifted to right, implying that the

27

H2S-induced vasorelaxation might be facilitated by functional endothelium-mediated mechanisms (241) This could be explained by recent findings that 3-MST and CAT are localized to vascular endothelium and the thoracic aorta and produce H2S (176) Therefore, it is most likely that H2S derived from both smooth muscle cell and endothelium participates in regulation of vascular tone H2S also acts in synergy with NO, the previously identified endothelium-derived relaxing factor (EDRF) to modulate the contractile activity of vascular smooth muscle and thus regulates blood pressure Hence, H2S is recently referred to as a new EDRF (205)

With the abundant evidence for the vasorelaxation of H2S, the underlying mechanisms have also been intensively examined In addition to inducing the release of NO and endothelium-derived hyperpolarization factors (EDHF), other molecular targets and events have been identified to mediate its effects, such as activation of KATP channels (242), stimulation of Cl-/HCO3- exchanger and induction of intracellular acidification (114), as well as metabolic inhibition (105) Furthermore, it is reported that the vascular effect of H2S was dependent on the extracellular calcium entry but independent of the cGMP pathway activation (241)

In addition to vascular regulation, researchers also pay much attention to the functions

of H2S in heart Geng et al reported the detection of CSE mRNA and endogenous H2S in heart tissues (63) They found that perfusion with NaHS decreased myocardial contractility These observations imply that H2S plays a role in regulating heart function under physiological conditions The same group also found that H2S level was lowered in isoproterenol (ISO)-induced myocardial injury and exogenously applied H2S may protect

myocytes and contractile activity It was further demonstrated by Yong et al that H2S

could negatively modulate β-adrenoceptor function via inhibiting AC activity (229) This

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indicates that H2S participates in the pathogenesis of ischemia-induced cardiac injury Subsequently, H2S-induced cardioprotection was extensively and intensively investigated

by several groups Bian et al demonstrated for the first time the role of H2S in

cardioprotection caused by metabolic and ischemic preconditioning in isolated rat heart and cardiac myocytes (16, 153) In addition, H2S also contributes to ischemic postconditioning-induced cardioprotection against myocardial infarction (228) The H2S pre- and post- conditioning effects have been demonstrated to be mediated by a number

of molecules: PKC (154, 228), sarcolemmal KATP(153), extracellular signal-regulated kinase (ERK)1/2 (88), phosphoinositide 3-kinase (PI3K)/Akt (88, 228), cyclooxygenase-2 (COX-2) (85), NO(153), nuclear factor-like 2 (Nrf-2) (27) and so forth More importantly,

Pan et al found that a single bolus injection of NaHS at 0.1-10 µmol/kg one day before

myocardial infarction could produce a strong infarct-limiting effect lasting at least three days Administration of NaHS 1 µmol/kg/day for 3 consecutive days after myocardiac infarction also significantly reduced infarct size; however, the cardioprotection was lower than that offered by H2S pretreatment A combination of both pre- and post-treatment failed to provide additional benefits (152) It is of great clinical significance that preconditioning should be preferred in the clinic and that post-treatment is unnecessary if preconditioning has been achieved

1.2.4.4 Roles of H 2 S in gastrointestinal tract

H2S is in close contact with the gastrointestinal tract, especially the bacterially-derived H2S in flatus It can also be enzymatically produced in gastrointestinal tissues, although

no evidence indicates the regulation of H2S production in these tissues It is thus logically

to assume that H2S may also serve as an important signaling molecule to regulate the physiological and pathological functions of gastrointestinal tract H2S was reported to