![H2S on astrocytes
• H2S donors elicit calcium waves in astrocytes and
increase intracellular levels of calcium.
• The increased [Ca]ᵢ occurs rapidly following H2S
exposure and decays slowly, whereas the
oscillations of calcium decay rapidly.
• The [Ca]ᵢ in astrocytes after H2S administration
reflects calcium entry, reduced in Ca-free media
and is associated with a direct influx of calcium
similar to that elicited by Ca ionophores.
• The type of calcium channel involved has not yet
been established.](https://image.slidesharecdn.com/hydrogensulphideasagasotransmitterloading-150119074748-conversion-gate01/75/Hydrogen-sulphide-as-a-gasotransmitter-loading-30-2048.jpg)
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Hydrogen sulphide as a gasotransmitter loading
- 1. Hydrogen sulphide asa Gasotransmitter ! CREDIT SEMINAR ON
- 2. Nitric oxide(NO) is a physiologic vasodilator and mediates the tumoricidal/bactericidal actions of macrophages (Moncada et al. 1991). Subsequently, NO was established as a neurotransmitter/neuromodulator in the brain and peripheral nervous system (Bredt & Snyder 1989) Carbon monoxide (CO) physiologically generated and mediating (NANC) neurotransmission in the intestine as well as neural activity in the brain (Boehning et al. 2004). Both are well accepted as gasotransmitters; a term which,does not necessarily imply that the gaseous molecule is a neurotransmitter but rather that it transmits information between cells in various parts of the body.
- 4. THE ORIGIN ANDTYPES : nNOS and eNOS are constitutive enzymes activated by Ca- CD response to depolarizing events (Bredt & Snyder 1989 NO, three isoforms of nitric oxide synthase (NOS), derived from three distinct genes, convert arginine to NO and citrulline, By contrast, iNOS is inducible, response to inflammatory stimulation, and is not notably influenced by calcium CO by two isoforms of heme oxygenase (HO) which derive from distinct genes (Maines 1988). HO-1 an inducible enzyme whose formation; stimulated by diverse stressors, including heme, and is high in liver, kidney and spleen; (Poss and Tonegawa 1997). By contrast, HO-2, localized to neurons in the brain and the endothelial layer of blood vessels, activated by calcium-calmodulin, ( Boehning et al. 2004).
- 5. It wasreferred as aer hepaticus (hepatic air) by alchemists (Myers 2007). In 1777 Carl Wilhelm Scheele was the first chemist; characterized H2S, described as “sulfuretted hydrogen,” H2S is odoriferous at concentrations less than 1 ppm, causes headaches at 4 ppm and is lethal at high levels (Reiffenstein et al., 1992).
- 6. It isabout 5 times more potent as a toxin than CO, acting largely by inhibiting cytochrome C oxidase (Lloyd 2006). There are abundant levels of H2S in our gut derived bacteria which form H2S by the reduction of sulfate . With the decomposition of sulfur containing amino acids such as cysteine and methionine, sulfated polysaccharides and sulfur containing lipids.
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- 10. Serine homcoystine The twoprincipal enzymes : physiologic sources of H2S both metabolize cystathionine : namely (CBS) cystathionine β- synthase and cystathionine-gamma- lyase (CSE) Cystathionine as an intermediate in various cycles involving sulfur-containing amino acids . In human and rat CBS exists primarily as a homotetramer with M.V of 63 kDa. Each subunit also binds the cofactors pyridoxal 5′-phosphate (PLP), S- adenosyl methionine (SAM) and heme . The heme appears to be a redox sensor, while SAM is an allosteric activator of the enzyme. Cystathionine cystathionine β- synthase (CBS) CBS
- 11. CYSTATHIONINE-GAMMA-LYASE (CSE) CSE formsH2S from cyst(e)ine by hydrolyzing cystathionine into cysteine The enzyme converts cystine to thiocysteine, pyruvate and NH3, ( a β- disulphide elimination recn), with the thiocysteine then reacts with cysteine or other thiols to produce H2S and cystine or the corresponding disulfide . CSE inhibitors in generating H2S physiologically. The two principal inhibitors utilized are DL- propargylglycine (PAG) and β-cyano-L-alanine (β-CNA). It is of interest that PAG and β-CNA do suppress H2S production by the liver and kidney but not by the brain; that CBS is the predominant source of H2S in brain tissue (Abe and Kimura 1996).
- 13. 1. H2S existsin an ionized form as HS−. 2. A characterized form of H2S which they refer to as “bound sulfur.”(Kimura et. al,.2014) 3. Bound sulphur arises when the sulfur of H2S is incorporated into proteins, bound to other sulfur atoms to form persulfides. 4. Presumably this bound sulfur releases H2S under reducing conditions. 5. The oxidation of H2S to thiosulfate and sulfate by sulfide quinone reductase terminates H2S signaling. Indeed, CSE is selectively activated by calcium-calmodulin similar to the activation of eNOS, nNOS and HO-2
- 15. SIGNALING MECHANISMS :NO AND CO NO and Co Act by stimulating sGC Stimulated cGMP activates Ca-CD Thus decrease in Cyt. Ca and dephosp.of myosin light chain
- 16. HOW does H2Ssignal? H2S also binds with high affinity to heme..However, it does not appear to physiologically stimulate sGC . The ability of H2S to relax blood vessels is not impaired in the presence of inhibitors of sGC If H2S does not act through sGC, how does it signal? •normally from NO is released due to an activity of S-Nitrosylation cysteines of proteins .Because both NO and the thiol groups of cysteines are chemically reactive. •. Demonstration of physiologic nitrosylation of proteins under basal conditions by end. generated NO by technique of biotin switch assay. •S-sulfhydration, attachment of an additional sulfur to the thiol (–SH) groups of cysteines yielding a hydropersulfide (–SSH) moiety. •S-thiolation blocks the protein thiol rendering it non-reactive, whereas S-sulfhydration yields a hydropersulfide (–SSH) moiety which has enhanced chemical reactivity
- 17. Numerous proteins, such as β-tubulin,actin, and GAPDH, are basally sulfhydrated. For most proteins, especially GAPDH in the liver, sulfhydration is substantially more prevalent than nitrosylation. Sulfhydration is abolished in CSE knockout mouse liver, but is unaffected in livers of nNOS, eNOS and iNOS knockouts. Sulfhydration occurs at physiologic levels of L- cysteine with maximal stimulation of GAPDH, β- tubulin and actin in the liver. Sulfhydration the majority, of proteins are basally sulfhydrated and that sulfhydration alters protein function, suggests that sulfhydration is an important physiologic signal.
- 19. HOW H2S SIGNALSTOPS • The mitochondrial enzyme sulfide quinone reductase contributes to catabolism in peripheral tissue. • The oxidation of H2S to thiosulfate and sulfate by sulfide quinone reductase terminates H2S signaling. • The overall tissue concentrations of H2S are maintained at low levels, preventing inhibition of cytochrome c preventing H2S induction of cytotoxicity.
- 20. HOW H2S SIGNALSTOPS • This catabolic pathway can be inhibited by stigmatellin, a mycobacteria-derived antibiotic. • Stigmatellin reduces H2S consumption in colonic musculature and potentiates fast nicotinic synaptic transmission in the peripheral sympathetic ganglion. • A second mechanism by which H2S-mediated signaling can be terminated is by binding of H2S to sulphane-sulfur pools and bound sulfate pools. • Application of exogenous H2S to brain, liver and heart homogenates of mouse generates bound sulfur rather than acid-labile sulfur ( Ishigami et.al ……………..)
- 21. (Circ J 2014)LarryA. Barr et.al...
- 22. PHYSIOLOGIC ROLE OFH2S (Circ J 2014)Larry A. Barr et.al...
- 23. PHYSIOLOGIC ROLE OFH2S ON BLOOD VESSELS : • CO also behaves like an EDRF. Like eNOS, HO-2 is localized to endothelial layer of blood vessels whose endothelial-dependent relaxation is blocked by HO inhibitors. • Thus, most EDRF activity of the mesenteric artery can be attributed to H2S. • EDRF activity attributable to NO is most prominent in large vessels such as the aorta, while in resistance vessels that regulate blood pressure more directly, NO’s effects are less evident. ON INFLAMMATION : • H2S donors display anti-inflammatory effects, inhibiting leukocyte-endothelium bonding and reducing carrageenan-induced paw edema. • H2S induces the formation of pro-inflammatory cytokines. ON NERVOUS SYSTEM : • H2S might acts as a Non-Adrenergic & Non-Cholinergic (NANC) neurotransmitter. • H2S donors elicit calcium waves in astrocytes and increase intracellular levels of calcium.
- 24. How does H2Srelax blood vessels? • A major component of EDRF activity involves hyperpolarization, a phenomenon that is not elicited by sGC; an endothelial-derived hyperpolarizing factor (EDHF). • Compounds postulated to mediate EDHF activity include a. prostacyclin generated from arachidonic acid by cyclooxygenase, b. epoxy eicosatrenoic acids generated from arachidonic acid by cytochrome P450 . c. hydrogen peroxide, potassium ions, C-type natriuretic peptide, electrical coupling through myoendothelial junctions mediated by connexins, and NO itself. • EDHF activity reflects opening of potassium channels. • The vasorelaxant effects of H2S are blocked by inhibitors of the ATP-sensitive potassium channel (KATP).
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- 26. How does H2Sstimulate KATP? KATP has 9 cysteines with C43, that lies close to the surface,influenced by oxidative insults. KATP is sulfhydrated with the sulfhydration abolished by mutations of C43 . Thus, H2S vasorelaxation reflects hyperpolarization mediated by the opening of KATP channels via their sulfhydration at C43. KATP is activated by binding of the phospholipid phosphatidylinositol (4,5)- bisphosphate (PIP2) . H2S donors markedly stimulate PIP2- KATP binding, as binding is markedly reduced in KATP- C43S mutants. As physiologic vasodilation is thought to be determined largely by EDHF, the evidence that EDHF activity is predominantly determined by H2S fits with a major role for H2S as an EDRF/EDHF.
- 27. ON INFLAMMATION : •Endogenous H2S is anti-inflammatory. • H2S donors display anti-inflammatory effects, inhibiting leukocyte-endothelium bonding and reducing carrageenan- induced paw edema. • H2S donors reduce visceral pain in a colorectal distension model & diminish colitis in rats. • H2S induces the formation of pro-inflammatory cytokines and chemokines by upregulating nuclear factor kappa-light-chain- enhancer of activated B cells (NF-κB) .
- 28. For instance, diclofenac derivatives that release H2S have been developed foruse as anti- inflammato ry drugs ( Wallace 2007). An H2S- releasing mesalamine derivative, ATB-429, displays analgesic and anti- inflammator y effects (Distrutti et al. 2006a,b). Abundant production from bacteria in the colon, bacterially generated H2S mediates the pathophysiol ogy of ulcerative colitis Butyrate, are thought to be important in maintainin g normal colonic mucosal function . Butyrate oxidation provides about 70% of colonic energy whereas the small intestine preferentiall y utilizes glucose and glutamine. H2S donors interfere with colonic butyrate metabolis m. the therapeutic effects of 5- aminosalicyl ate in ulcerative colitis reflect influences upon H2S, as patients treated with the drug. Pro-inflammatory role of H2S in colitis.
- 29. • CSE, neuronallocalizations were evident in the myenteric plexus of neurons in the small intestine suggesting that like NO and CO, H2S might be a Non- Adrenergic&Non-Cholinergic (NANC) neurotransmitter. • physiologic concentrations of H2S enhance long-term potentiation (LTP). • Sodium hydrogen sulfide (NaHS) applications and weak tetanic stimulation of rat hippocampal slices alone did not elicit LTP, while the simultaneous application of both led to robust LTP (Abe and Kimura 1996). • The effect of H2S on LTP was abolished by NMDA antagonists. Interestingly, NO and CO also induce LTP, but do so even when NMDA receptors are blocked (Zhuo et al. 1993). • NMDA receptors possess reactive cysteines and are known to be nitrosylated with resulting channel blockade (Lei et al. 1992; Choi et al. 2000). • Conceivably H2S regulates NMDA transmission by sulfhydrating NMDA receptors
- 30. H2S on astrocytes •H2S donors elicit calcium waves in astrocytes and increase intracellular levels of calcium. • The increased [Ca]ᵢ occurs rapidly following H2S exposure and decays slowly, whereas the oscillations of calcium decay rapidly. • The [Ca]ᵢ in astrocytes after H2S administration reflects calcium entry, reduced in Ca-free media and is associated with a direct influx of calcium similar to that elicited by Ca ionophores. • The type of calcium channel involved has not yet been established.
- 31. H2S SERVE ASA NEUROPROTECTANT ! Glutamate neurotoxicit y in brain cultures involves, at least in part, inhibition of cystine uptake (Tan et al. 2001). The cystine/glut amate antiporter couples influx of cystine with efflux of glutamate. This process is blocked by high concentratio ns of exogenous glutamate which are cytotoxic via a process designated oxytosis (Tan et al. 2001). How does H2S act in this model? Glutamate reduces levels of intracellular glutathione, and H2S increases them both in untreated and in glutamate- exposed preparations (Kimura and Kimura 2004). Buthionine sulfoximine (Griffith 1982), which inhibits γ- glutmaylcysteine synthase, a rate limiting enzyme in glutathioine biosynthesis, prevents the H2S- elicited stimulation of glutathione levels and cell survival. H2S elicits augmented glutathione by stimulating cystine entry into cells, reversing the inhibition of cystine transport by glutamate (Kimura et. Al 2010).
- 32. Effects on VisceralNociceptors • H2S acts as a pro-nociceptor via CaV3 channels in mouse, whereas it acts as an anti-nociceptive molecule, mediated by KATP channels in the rat. • NaHS increases the frequency of action potentials in gut afferent neurons and in dorsal root ganglion neurons. • The effect is reduced by capsazepine, so TRPV1 has been implicated. • NaHS and H2S donor molecules reduce pain-related behaviors in healthy rats and rats with colitis, mediated by the opening of KATP channels. In mice, luminal release of H2S is nociceptive, involving CaV3 calcium channels.99
- 33. Effects on ColonicSecretion • In the rat colon, NaHS increases secretion of Cl¯ from the apical membrane of epithelial cells and secretion of K† from the basolateral membrane. • These secretory effects are mediated by nerves and via direct actions on calcium storage organelles in epithelial cells through Ryanodine receptors. • H2S activates KATP channels, inhibits and activates CaV1.2 calcium channels in different tissues, and activates CaV3calcium channels, TRPV1 and TRPA1 channels, and NaV1.5
- 34. Effects as a cardioprotective Recentstudy demonstrated that CSE expressed in rat aorta, tail artery, mesenteric artery, and pulmonary artery whereas the expression of CBS was not detectable. (“The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener. 2001
- 35. How H2S cancontrasts hypertension? It modulates the renin-angiotensin system , a hormone system that regulates blood pressure and water (fluid) balance. (“Hydrogen sulfide inhibits plasma renin activity. 2010”) may close voltage-gated calcium channels. Alternatively, H2S may directly inhibit voltage-gated calcium channels in vascular smooth muscle cells. THE OPENING OF THESE CHANNELS LEADS TO MEMBRANE HYPERPOLARIZATION H2S relaxes B.V and lowers B.P by opening ATP-sensitive K+channels in vascular smooth muscle (Gaso-transmitter hydrogen sulphide: potential new target in pharmacotherapy. 2010”)
- 36. Hydrogen sulfide downregulates cAMP production in some cell types by inhibiting adenylyl cyclase, suggesting the possibility that it may modulate renin release. It may also inhibits the up regulation of renin mRNA The release of renin is a process regulated by intracellular cAMP. This increases the volume of fluid in the body, also blood pressure. Angiotensin II —Aldosterone from adrenal cortex —reabsorption of H₂0 and Na⁺ into blood. Angiotensin II is a potent vaso-active peptide that causes blood vessels to constrict, blood pressure
- 37. ERK, extracellular signal-regulatedkinase; ARE, antioxidant response element; Keap1, Kelch-like ECH-associated protein 1; MPTP, mitochondrial permeability transition pore; PI3K, phosphatidylinositide 3-kinase; PKC, protein kinase C; RISK, reperfusion injury salvage kinase; ROS, reactive oxygen species; SERCA, sarco-endoplasmic reticulum Ca2+-ATPase. (Circ J 2014)Larry A. Barr et.al...
- 38. Macrophages produces H2Svia CSE Activity in the wake of inflammatory endotoxin lipopolysaccharide: an endogenous atherosclerotic treatment. Furthermore, administration of the H2S donor, sodium hydrosulfide (NaHS), can inhibit atherosclerotic precursors in macrophages, such as proatherogenic-oxidized low- density lipoprotein-induced foam cell formation. In another study,the H2S donor, sodium sulfide (Na2S), and NaHS were both able to inhibit leukocyte adherence. This is further evidence of the protective nature of H2S therapy during pathological stress states.
- 39. ANTI-OXIDANT ACTIVITY H₂S providesantioxidant effects in a number of tissues,in vascular smooth muscle cells,neurons,and cardiomyocytes. Reactive oxygen species (ROS) are produced from electron transfer reactions in the cell, they are scavenged by antioxidant molecules. The roaming ROS can cause a number of cellular maladaptations such as lipid peroxidation, protein oxidation to inactive states, and DNA strand breaks. H2S reduces this ROS derangement by upregulating antioxidant properties. It can scavenge the ROS superoxide and protect neuronal death by upregulating levels of glutathione, another antioxidant.
- 40. • These findingssuggest that H2S is an antioxidant itself, but can also upregulate antioxidant protective mechanisms. • One such mechanism is through the nuclear- factor-E2-related factor-2 (Nrf2) dependent signaling pathway. • Nrf2 is a member of the NF-E2 family of nuclear basic leucine zipper transcription factors, and activates enzymes that scavenge pro-oxidative stressors.
- 41. Knockout of CSEleads to downregulation of the Akt pathway, preventing activation of pro-angiogenic factors. Furthermore, H₂S upregulates the mitogen- activated protein kinase (MAPK) pathway, primarily the ERK (extracellular signal-regulated kinase) and p38 proteins, leading to enhanced cell proliferation. In a model of hypertension-induced heart failure, H₂S ameliorated the LV remodeling process, while upregulating angiogenesis. Additionally, administration of NaHS in drinking water increased VEGF expression and inhibited angiostatin and endostatin, 2 antiangiogenic factors. Also, the upregulation of both Akt and VEGF may cause this evident angiogenesis.
- 42. • H2S atphysiologically @ concentrations induced apoptosis of Human Aorta Smooth Muscle Cells (HASMCs) via the activation of mitogen-activated protein kinases (MAPK) and caspase-3. • Hydrogen sulfide promotes the activation of RAF- MEK-ERK pathway that leads to its downstream enzyme cascades, eventually activating caspase-3. (Hydrogen sulfide-induced apoptosis of human aorta smooth muscle cells …2004)
- 43. NovelMethodsofSulfhydrationDetection Measuring H2Slevels accurately is paramount in properly characterizing this molecule. In one such process, gas chromatography- chemiluminescence, fresh tissue homogenate is incubated in buffer, allowing for sulfide gas to be released. The released gas is measured in the headspace and quantified, but the incubation period takes time, removing the possibility for real-time sulfide release analysis. H2S levels are also measured with the methylene blue assay, which indicates sulfide concentrations through colorimetry.
- 44. BIOTIN SWITCH ASSAY Sulfhydration mediates the diverse physiologic act ions of H2S and a major post-translational modification (Mustafa et al., 2009) Sulfhydration diversity has been recently studied by Mustafa et al, using a modification of the biotin switch assay that traditionally detects S-nitrosylation (SNO). The biotin switch assay is normally able to detect nitrosylated thiols by selectively exposing them via ascorbate treatment followed by labeling of the sulfhydryl groups with biotin-HPDP.
- 45. BIOTIN SWITCH ASSAY The sulfhydration study found that biotin labeling still occurs without the ascorbate-induced exposure of nitrosylated thiols. These newly discovered thiols may be S-sulfhydrated (cysteine –SH groups are converted to –SSH), providing potential evidence of direct H2S modification of a number of key proteins.
- 46. BIOTIN SWITCH ASSAY 1.This would be analogous to NOS binding to SNO targets and the labile gasotransmitter itself quickly travels to the target proteins. 2. This novel method of identifying S- sulfhydrated proteins shows that H2S is a key factor in posttranslational modification. 3. An innovative method of labeling SNO has been developed by Kohr et al, providing for the first time an in-depth look at SNO occupancy in the myocardium. 4. Whole heart homogenates were used with a cysteine-reactive tandem mass tag labeling and enrichment kit.
- 47. H2S AS ATHERAPEUTIC H2S donors are on the rise, providing insights into instituting the molecule as an efficacious therapeutic. The most frequently utilized H2S donors are NaHS and Na2S, salts that can quickly release H2S in vivo. One such stable donor, diallyl trisulfide, is a derivative of garlic that is able to decrease infarct size and improve mitochondrial coupling in mice after acute myocardial ischemia. Another H2S donor has recently been synthetically developed,termed SG-1002, and was orally administered in a murine model of TAC.
- 48. H2S AS ATHERAPEUTIC water-soluble H2S donor has recently been developed, called GYY4137,slowly releases H₂S, such as vasodilation and antihypertensive activity. There is a recently developed H₂Sreleasing derivative of naproxen (ATV-346) that reduces the gastrointestinal damage induced by the toxicity of standard naproxen. This novel drug compound was also found to augment the chemotherapeutic effects of standard naproxen in a murine model of colon cancer.
- 49. REFERERNCES : HydrogenSulfide as a Gasotransmitter:Moataz M. Gadalla1 and Solomon H. Snyder1,2,3JOURNAL OF NEUROCHEMISTRY | 2010 | 113 | 14–26 Losing heart: the role of apoptosis in heart disease—a novel therapeutic target? CATHERINE GILL,*,† RUBEN MESTRIL,‡ AFSHIN SAMALI*,†, Carbon Monoxide, Hydrogen Sulfide, and Nitric Oxide as Signaling Molecules in the Gastrointestinal Tract: Gianrico Farrugia ,Joseph H. Szurszewski Johansen D, Ytrehus K, Baxter GF et al. Exogenous hydrogen sulfide (H2S)protects against regional myocardial ischemia-reperfusion injury –evidence for a role of KATP channels. Basic Res Cardiol 2006. Discoveries of Hydrogen Sulfide as a Novel Cardiovascular Therapeutic Larry A. Barr, PhD; John W. Calvert, PhD H2S SIGNALLING VIA S- SULFHYDRATION By MUSTAFA ET. AL. Hydrogen sulfide: a new EDRF:Rui Wang1 Department of Biology, Lakehead University, Thunder Bay, Ontario, Canada The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener. 2001