research article hydrogen sulfide increases nitric oxide … · 2019. 7. 31. · research article...

Research ArticleHydrogen Sulfide Increases Nitric Oxide Productionand Subsequent S-Nitrosylation in Endothelial Cells

Ping-Ho Chen1 Yaw-Syan Fu2 Yun-Ming Wang3 Kun-Han Yang4

Danny Ling Wang5 and Bin Huang26

1 School of Dentistry College of Dental Medicine Kaohsiung Medical University Kaohsiung 80708 Taiwan2Department of Biomedical Science and Environmental Biology College of Life Science Kaohsiung Medical UniversityNo 100 Shihchuan 1st Road San Ming District Kaohsiung 80708 Taiwan

3Department of Biological Science and Technology Institute of Molecular Medicine and BioengineeringNational Chiao Tung University Hsinchu 30068 Taiwan

4 Institute of Biotechnology National Cheng Kung University Tainan 70101 Taiwan5 Institute of Medical Science College of Medicine Tzu Chi University Hualien County 97004 Taiwan6Department of Biological Sciences National Sun Yat-Sen University Kaohsiung 80424 Taiwan

Correspondence should be addressed to Bin Huang huangpin2yahoocomtw

Received 21 April 2014 Accepted 5 May 2014 Published 21 May 2014

Academic Editor Li-Yeh Chuang

Copyright copy 2014 Ping-Ho Chen et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Hydrogen sulfide (H2S) and nitric oxide (NO) two endogenous gaseous molecules in endothelial cells got increased attention with

respect to their protective roles in the cardiovascular system However the details of the signaling pathways between H2S and NO

in endothelia cells remain unclear In this study a treatment with NaHS profoundly increased the expression and the activity ofendothelial nitric oxide synthase Elevated gaseousNO levels were observed by a novel and specific fluorescent probe 5-amino-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid methyl ester (FA-OMe) and quantified by flow cytometry Further study indicatedan increase of upstream regulator for eNOS activation AMP-activated protein kinase (AMPK) and protein kinase B (Akt) By usinga biotin switch the level of NO-mediated protein S-nitrosylation was also enhanced However with the addition of the NO donorNOC-18 the expressions of cystathionine-120574-lyase cystathionine-120573-synthase and 3-mercaptopyruvate sulfurtransferase were notchanged The level of H

2S was also monitored by a new designed fluorescent probe 4-nitro-7-thiocyanatobenz-2-oxa-13-diazole

(NBD-SCN) with high specificity Therefore NO did not reciprocally increase the expression of H2S-generating enzymes and the

H2S level The present study provides an integrated insight of cellular responses to H

2S and NO from protein expression to gaseous

molecule generation which indicates the upstream role of H2S in modulating NO production and protein S-nitrosylation

1 Introduction

Gasmolecules that are produced by cells have been discussedfor several decades regarding their protective role in thevascular system Recently the diverse physiologic actions ofcarbon monoxide (CO) nitric oxide (NO) and hydrogensulfide (H

2S) and their role in preventing diseases through

the mediation of gas-regulating and -sensing mechanismshave attracted a great deal of interest [1] For exampleNO plays an important role in the regulation of the car-diovascular function through a posttranslational protein S-nitrosylation on the cysteine residue [2] In our previous

study a mechanical shear flow is regarded as protective forendothelial cells (ECs) leading to a series S-nitrosylation ofproteins [3] Investigating the reported mechanisms of NOon EC protection the NO-mediated S-nitrosylated proteinssuch as F1F0-ATPase reduced the generation of Ca2+ andROS inmitochondria during ischemiareperfusion injury [4]NO was also reported to be essential in the prevention ofirreversible oxidative stress and finally provided protectionfrom several diseases including cancer diabetes and neurondegeneration [5ndash7]

The toxic effects of hydrogen sulfide (H2S) on liv-

ing organisms have been recognized for nearly 300 years

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 480387 8 pageshttpdxdoiorg1011552014480387

2 The Scientific World Journal

101 50 100eN

OS

Actin

peN

OSS1

177

NaHS (2h)

(120583M)

(a)

eNO

SAc

tin

Ctrl 05 2 12 (h)

peN

OSS1

177

NaHS (50120583M)

(b)

Rela

tive f

old

101 50 100(120583M)

lowastlowastlowast

lowast

lowastlowast

35

3

25

2

15

1

05

0

4

(c)

Rela

tive f

old

Ctrl 05 2 12(h)

lowastlowast

lowastlowast

35

3

25

2

15

1

05

0

(d)

Figure 1 NaHS increases the expression and serine 1177 phosphorylation of eNOS (a) ECs pretreated by diluting concentrations of NaHS(1 120583M 10 120583M 50 120583M and 100120583M) for 2 h (b) ECs treated with NaHS (50120583M) for 05 2 and 12 h Blotted membranes were separatelyhybridized with eNOS and peNOSS1177 antibodies ((c) and (d)) Relative folds of protein levels shown as means plusmn SE compared to controlStatistical significance ( lowast119875 lt 005 lowastlowast119875 lt 001) analyzed using Fisherrsquos LSD

In recent years however interest has been directed towardsH2S as the third gaseous mediator which has been shown to

exhibit potent vasodilatory activity both in vitro and in vivoThis is assumed to be realized by opening vascular smoothmuscle KATP channels [8] Of the three enzymes cystathio-nine-120574-lyase (CSE) cystathionine-120573-synthase (CBS) and3-mercaptopyruvate sulfurtransferase (3-MST) can utilizeL-cysteine as a substrate to produce H

2S Deficiency of

H2S-producing enzymes results in some disorders such as

homocystinuria which is characterized by mental retarda-tion skeletal abnormalities increased urine homocysteineincreased risks of thromboembolism and early onset ofatherosclerosis [9ndash11] H

2S was also reported to protect

against vascular remodeling from endothelial damage [12]Recently a signaling molecule for H

2S was shown to regulate

vascular relaxation and angiogenesis via potassium channelS-sulfhydration [13ndash15]

With a similar physiological function it is interesting todiscuss the interactions between H

2S and NO in respond-

ing stimuli In the reports cited above H2S and NO in

synergy might regulate smooth muscle relaxation and alsomitochondrial integration [16 17] H

2S triggers late-phase

preconditioning in the postischemic small intestine by anNO- and p38 MAPK-dependent pathway [18] Despite H

2S

inhibiting NO production in lipopolysaccharide-stimulatedmacrophages the H

2S can also stimulate NO production

from other cells [19 20]Because of the technical difficulty in detecting gaseous

molecules in the current study not only monitoring the reg-ulations of theses enzymes but also quantifying themoleculesof H2S andNO specifically with the new designed fluorescent

probes Therefore we question here if H2S has any upstream

role in the regulation of endothelial NO production

2 Materials and Methods

21 Cell Culture and Drug Treatments The EAhy 926 cellline was kindly donated by Cora-Jean S Edgell University ofNorth Carolina Chapel Hill EAhy 926 cells were cultured inDMEM supplemented with fetal bovine serum (FBS 10)streptomycin (100 120583gmL) and penicillin (100UmL) ECswere replaced by the same medium containing 2 FBS andincubated overnight prior to the experimental NaHS andNOC-18 treatments

22 Cell Lysis and Protein Extraction ECs were washed withcord buffer after treatment [NaCl (014M) KCl (4mM)

The Scientific World Journal 3

Ctrl

Bright field FA-OMe2

h

(a)

Gate P1

Gate P1

V2-L

V2-L

496V2-R

V2-R

347

Cou

ntC

ount

FL1-H

FL1-H

0

200

400

600

800

0

200

400

600

800

103

104

105

10610

64

103

104

105

10610

64

455 664

(b)

FA-OMe DAPI Merged

Ctrl

2h

(c)

Figure 2 Elevated NO levels monitored by specific fluorescent probes (a) ECs treated with NaHS (50 120583M) for 2 h stained by FA-OMe andobserved by fluorescent microscopy (b) FA-OMe signals calculated by flow cytometry (c) FA-OMe signals observed by confocal microscopyBar = 20 120583m

glucose (11mM) and HEPES (10mM pH 74)] and thenlysed with 100 120583L of lysis buffer [HEPES (250mM pH 77)EDTA (1mM) neocuproine (01mM) and CHAPS (04wv)] After centrifugation protein supernatant was collectedand protein concentrations were determined with BCAassay reagent (Thermo Fisher Scientific Inc Rockford ILUSA)

23 Western Blot Analysis Forty micrograms of cell lysateswith various treatments were mixed with an equal volume ofsample buffer [Tris-HCl (625mM pH 68) SDS (3 wv)2-mercaptoethanol (5 vv) and glycerol (10 vv)] andthen separated by SDS-PAGE The gel was transferred toPVDF membranes (Millipore MA USA) and immunoblot-tedwith antibodies eNOS (1 3000 Cell Signaling TechMA


AM

PKAc

tin

Ctrl 05 2 12 (h)

Akt

NaHS (50120583M)

(a)

Rela

tive f

old

AMPKAkt

Ctrl 05 2 12(h)

lowast

lowast

lowastlowast

lowastlowast

3

25

2

15

1

05

0

(b)

Figure 3The expression levels of AMPK and Akt in the presence ofNaHS (a) ECs treated with NaHS (50120583M) for 05 2 and 12 h Theblotted membranes hybridized with AMPK antibody (b) Relativefolds of protein levels shown as means plusmn SE compared to controlStatistical significance ( lowast119875 lt 005 lowastlowast119875 lt 001) analyzed usingFisherrsquos LSD

USA) peNOS1198781177 (1 2000 Cell Signaling Tech) AMPK(1 3000 Cell Signaling Tech) Akt (1 2000 Cell SignalingTech) cystathionine-120574-lyase (CSE 1 1000 Abnova TaipeiTaiwan) cystathionine-120573-synthase (CBS 1 1000 Abnova)and 3-mercaptopyruvate sulfurtransferase (3-MST 1 1000Abcam Cambridge UK) The membranes were visualizedwith the SuperSignal West Femto reagent (Thermo FisherScientific IL USA) on X-ray films The images from X-ray films were scanned using a digital scanner (MicrotekInternational Inc) and the density was calculated by theProgenesis Samespots v20 software (NonLinear DynamicsNewcastle UK)

24 Application of Fluorescent Probes and Imaging Con-ditions For NO detection 5-amino-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid methyl ester (FA-OMe) wasdesigned [21] ECs with NaHS treatment were coincubatedwith 10 120583M of FA-OMe for 4 h prior to imaging The ECswere washed three times with PBS buffer and then bathedin 2mL of PBS The images were obtained by the fluo-rescence microscope (120582ex 460 nm 120582em 524 nm Axiovert

40 CFL Zeiss) As for 4-nitro-7-thiocyanatobenz-2-oxa-13-diazole (NBD-SCN) that was used for detecting H

2S the

cells were incubated with 5120583M NBD-SCN for 30min andthen subjected to fluorescencemicroscope (120582ex 460 nm 120582em550 nm) [22] For confocal fluorescence images study ECswere seeded at a density of 2 times 105 cellswell on cover glasses(24times 24mm2) and grown for 24 hThe cells with 10 120583Mof FA-OMe incubation were fixed with 4 formaldehyde solutionfor 20min at room temperature Cell nuclei were stainedwith 406-diamidino-2-phenylindole (DAPI) Cover glassescontaining fixed ECs were mounted in a mixture of PBS andglycerol (1 1) on a microscopic slide The cells were observedusing a laser scanning confocal imaging system (OlympusFluoView 300) consisting of Olympus BX51 microscope anda 20mW output argon ion laser

25 Flow Cytometry Assay After fluorescence microscopeobservation the ECs were washed twice with PBS anddetached by tryptic reaction ECs were collected by centrifu-gation and then resuspended in PBS The fluorescence wasimmediately measured by the Accuri C6 flow cytometer (BDNJ USA) with excitation and emission settings of 488 and530 nm respectively The fluorescence strength was obtainedfrom 1 times 104 cells and statistically calculated from threerepeats

26 Evaluation of Protein S-Nitrosylation The cell lysates(200120583g) after NaHS treatment were blocked by methylmethanethiosulfonate (MMTS) reduced by ascorbate andlabelled by biotin according to reported guideline [23] Thebiotinylated lysates were then subjected to a reductant-free SDS-PAGE and western blotted with streptavidin-HRP(1 3000) following a previous study [24]

3 Results and Discussion

31 NaHS Increased the Protein Level of eNOS and thePhosphorylation on Serine 1177 Residue Endothelial nitricoxide synthase (eNOS) is responsible for endothelial nitricoxide (NO) production and the enzyme activity is reportedto be highly affected by posttranslational phosphorylationon serine 1177 residue (S1177) [25] In this study with thetreatment of different concentrations of NaHS (1sim100 120583M)we found that 50120583M of NaHS can significantly enhanceboth the eNOS expression and the phosphorylation of theserine 1177 residue (peNOS1198781177) (Figures 1(a) and 1(c)) Thisconcentration conforms well with several vascular researcharticles [26] At this concentration the highest expressionlevel of eNOS and peNOS1198781177 was observed at 2 hours(Figures 1(b) and 1(d))

32 Cellular NO Was Precisely Determined by Specific Fluo-rescent Probes In addition to the expression of eNOS thelevels of NOmolecules were further measured by the specificfluorescent probe FA-OMe This can distinguish NO andother reactive oxygen species (ROS) from reactive nitrogenspecies (RNS) [21] After NaHS treatment the NO levelwas increased from 347 plusmn 29 to 664 plusmn 38 at 2 hours


Free Cys-SH was blocked by MMTS

H NO

S SN

NO

S SN

S

Ascorbate reductionand biotin labeling

S SN

SBiotin

Reductant-free SDS-PAGE and western blot

S

COOminus

COOminus

COOminus

CH3

CH3

(a)

Ctrl 05 2 12MW

(kD

a)

10090

80

70

60

50

40

30

20

Actin

NaHS (50120583M)

(b)

Figure 4 Detection of protein S-nitrosylation (a) Scheme representing the procedures of modified biotin switch Biotin-labeled lysateswere subjected to SDS-PAGE without any reducing agents in the buffers (b) ECs lysate (100120583g) treated with NaHS (50 120583M) for 05 2 and12 h separated by SDS-PAGE and the blotted membranes were hybridized with streptavidin-HRP Triangle indicates proteins with increasedS-nitrosylation Arrow head indicates proteins with decreased S-nitrosylation

(Figures 2(a) and 2(b)) By using confocalmicroscopy despitethe fact that the basal fluorescence in the control treatmentwas difficult to see the broadly distributed NO was observedin the cytosol and also in the nuclei (Figure 2(c))

33The Expression Profiles of AMPKandAkt in the Presence ofH2S 51015840 AMP-activated protein kinase (AMPK) is an enzyme

that plays a role in cellular energy homeostasis Besidesprotein kinase B (Akt) AMPK is also reported to activateeNOS by phosphorylating Ser1177 in response to variousstimuli [27] In the current study H

2S increased the protein

level of AMPK at 2 h and returned to a basal level at 12 hHowever sustainable expressions of Akt were observed from05 to 12 h (Figures 3(a) and 3(b))This indicated that H

2S can

stimulate eNOS activity through AMPK and Akt pathwaysSimilar findings were also reported recently [28 29]

34 H2S-Increased Bioavailability of NO That Can Enhance

Protein S-Nitrosylation According to previous data we con-firmed that NO level got elevated by H

2S Since protein

S-nitrosylationdenitrosylation is regarded as important incardioprotection its investigation of protein S-nitrosylationis hence important for applied medical purposes [2 3 30]By using a modified biotin switch we could identify at least8 groups of increased S-nitrosoproteins and 2 groups ofdecreased S-nitrosoproteins (Figure 4) With the excellentperformance in analyzing S-nitrosoproteins mass spectrom-etry will be introduced in further identification of theseproteins [24]

35 NO Did Not Reciprocally Increase the Expression of H2S-

Generating Enzymes and theH2S Level After confirming that

H2S can increase NO at the cellular level we also examined

whether NO can be synchronized at elevated H2S levels

As shown in Figure 5 three key enzymes are involved inthe cellular H

2S synthesis CSE CBS and 3-MST These

were not changed by NOC-18 treatment (Figures 5(a) and5(b)) Although several studies indicated thatH

2S-generating

enzymes can also be exerted by NO [31] we could not finddifferences in our microscopic and flow cytometric analysis(460 plusmn 31 and 433 plusmn 48 separately) using NBD-SCNfluorescence probe (Figures 5(c) and 5(d)) The reportedstudy demonstrated that H

2S promotes NO production in

ECs via the activation of a cascade of phosphorylation eventsstarting from p38 MAPK and Akt to eNOS and this canbe through NO-dependent or NO-independent mechanismscascade Thus H

2S may be a key regulator for angiogenic

signalling pathways whether they required NO or not [32]This might indicate that NO works as a downstream gaseoustransmitter in the endothelium

4 Conclusion

In the present study hydrogen sulfide increased nitric oxideproduction This was not only concluded by studying relatedenzymes but also confirmed directly by detecting the finalproducts where NO levels were observed by a novel andspecific fluorescent probe FA-OMe and quantified by flowcytometry The level of H

2S was also monitored by a new


Ctrl 05 2 12CS

ECB

S3-

MST

Actin

NOC-18 (250120583M)

(a)

05 2 12Ctrl

Rela

tive f

old

14

12

1

08

06

04

02

3-MST

CSECBS

ns

(b)

Bright field NBD-SCN

Ctrl

2h

(c)

Gate P1

Gate P1

V1-L

V1-L

V1-R

V1-R

Cou

ntC

ount

FL1-A

FL1-A

540

567 433

10110

210

310

410

510

610

72

10110

210

310

410

510

610

72

460

500

0

500

0

1000

1000

(d)

Figure 5 Investigation of H2S biosynthesis under the treatment of NO (a) ECs which treated NO donor NOC-18 (250 120583M) for 05 2 and

12 h were subjected to western blot analysis with CSE CBS and 3-MST antibodies (b)The statistic data showed that no significance (119875 = ns)was observed between treatments (c) NBD-SCN was applied to detect cellular H

2S level specifically (d) Fluorescent signals were calculated

by flow cytometry

designed fluorescent probe NBD-SCN with high specificityThe present study provides an integrated insight of cellularresponses to two gaseous molecules from protein expres-sion to gaseous molecule generation which indicates theupstream role of H

2S in modulating NO production and

protein S-nitrosylation

Conflict of Interests

The authors declare no conflict of interests

Authorsrsquo Contribution

Ping-Ho Chen and Yaw-Syan Fu contributed equally to thispaper

Acknowledgments

The authors thank Dr Hans-Uwe Dahms for critical readingof the paper This study was supported by a Grant fromthe National Science Council (NSC 101-2320-B-037-041)


They are grateful to the Center for Resources ResearchampDevelopment of KaohsiungMedical School for the supportof fluorescent imaging

References

[1] M Kajimura R Fukuda R M Bateman T Yamamoto and MSuematsu ldquoInteractions of multiple gas-transducing systemshallmarks and uncertainties of CO NO and H

2S Gas Biologyrdquo

Antioxidants amp Redox Signaling vol 13 no 2 pp 157ndash192 2010[2] J Sun and E Murphy ldquoProtein S-nitrosylation and cardiopro-

tectionrdquo Circulation Research vol 106 no 2 pp 285ndash296 2010[3] B Huang S C Chen and D L Wang ldquoShear flow increases

S-nitrosylation of proteins in endothelial cellsrdquo CardiovascularResearch vol 83 no 3 pp 536ndash546 2009

[4] S Shiva M N Sack J J Greer et al ldquoNitrite augmentstolerance to ischemiareperfusion injury via the modulation ofmitochondrial electron transferrdquo The Journal of ExperimentalMedicine vol 204 no 9 pp 2089ndash2102 2007

[5] Y-Y Chen H-M Chu K-T Pan et al ldquoCysteine S-nitrosylation protects protein-tyrosine phosphatase 1B againstoxidation-induced permanent inactivationrdquoThe Journal of Bio-logical Chemistry vol 283 no 50 pp 35265ndash35272 2008

[6] T Nakamura and S A Lipton ldquoEmerging roles of S-nitrosylation in protein misfolding and neurodegenerative dis-easesrdquo Antioxidants amp Redox Signaling vol 10 no 1 pp 87ndash1012008

[7] B Lima M T Forrester D T Hess and J S Stamler ldquoS-nitrosylation in cardiovascular signalingrdquo Circulation Researchvol 106 no 4 pp 633ndash646 2010

[8] S Mani A Untereiner L Wu and R Wang ldquoHydrogen sulfideand the pathogenesis of atherosclerosisrdquo Antioxidants amp RedoxSignaling vol 20 no 5 pp 805ndash817 2014

[9] C AWagner ldquoHydrogen sulfide a new gaseous signalmoleculeand blood pressure regulatorrdquo Journal of Nephrology vol 22 no2 pp 173ndash176 2009

[10] G Yang L Wu B Jiang et al ldquoH2S as a physiologic vasore-

laxant hypertension in mice with deletion of cystathionine 120574-lyaserdquo Science vol 322 no 5901 pp 587ndash590 2008

[11] Y Wang X Zhao H Jin et al ldquoRole of hydrogen sulfide inthe development of atherosclerotic lesions in apolipoproteine knockout micerdquo Arteriosclerosis Thrombosis and VascularBiology vol 29 no 2 pp 173ndash179 2009

[12] T P Vacek W Gillespie N Tyagi J C Vacek and S C TyagildquoHydrogen sulfide protects against vascular remodeling fromendothelial damagerdquo Amino Acids vol 39 no 5 pp 1161ndash11692010

[13] A Papapetropoulos A Pyriochou Z Altaany et al ldquoHydrogensulfide is an endogenous stimulator of angiogenesisrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 106 no 51 pp 21972ndash21977 2009

[14] A KMustafaMMGadalla N Sen et al ldquoH2S signals through

protein S-Sulfhydrationrdquo Science Signaling vol 2 no 96 ArticleID ra72 2009

[15] A K Mustafa G Sikka S K Gazi et al ldquoHydrogen sulfideas endothelium-derived hyperpolarizing factor sulfhydratespotassium channelsrdquo Circulation Research vol 109 no 11 pp1259ndash1268 2011

[16] R Hosoki N Matsuki and H Kimura ldquoThe possible role ofhydrogen sulfide as an endogenous smooth muscle relaxant in

synergy with nitric oxiderdquoBiochemical and Biophysical ResearchCommunications vol 237 no 3 pp 527ndash531 1997

[17] F Bouillaud and F Blachier ldquoMitochondria and sulfide a veryold story of poisoning feeding and signalingrdquo Antioxidants ampRedox Signaling vol 15 no 2 pp 379ndash391 2011

[18] M Yusof K Kamada T Kalogeris F Spencer Gaskin and R JKorthuis ldquoHydrogen sulfide triggers late-phase precondition-ing in postischemic small intestine by an NO- and p38 MAPK-dependentmechanismrdquoAmerican Journal of PhysiologymdashHeartand Circulatory Physiology vol 296 no 3 pp H868ndashH8762009

[19] G-S Oh H-O Pae B-S Lee et al ldquoHydrogen sulfideinhibits nitric oxide production and nuclear factor-120581B via hemeoxygenase-1 expression in RAW2647 macrophages stimulatedwith lipopolysacchariderdquo Free Radical Biology amp Medicine vol41 no 1 pp 106ndash119 2006

[20] L Grossi ldquoHydrogen sulfide induces nitric oxide release fromnitriterdquo Bioorganic and Medicinal Chemistry Letters vol 19 no21 pp 6092ndash6094 2009

[21] T-W Shiue Y-H Chen C-M Wu et al ldquoNitric oxide turn-onfluorescent probe based on deamination of aromatic primarymonoaminesrdquo Inorganic Chemistry vol 51 no 9 pp 5400ndash5408 2012

[22] Y-H Chen J-C Tsai T-H Cheng S-S Yuan and Y-M Wang ldquoSensitivity evaluation of NBD-SCN towards cys-teinehomocysteine and its bioimaging applicationsrdquoBiosensorsamp Bioelectronics vol 56 pp 117ndash123 2014

[23] S R Jaffrey and S H Snyder ldquoThe biotin switch method forthe detection of S-nitrosylated proteinsrdquo Sciencersquos STKE SignalTransduction Knowledge Environment vol 2001 no 86 articlePl1 2001

[24] B Huang C L Liao Y P Lin S C Chen and D L WangldquoS-nitrosoproteome in endothelial cells revealed by a modifiedbiotin switch approach coupled with western blot-based two-dimensional gel electrophoresisrdquo Journal of Proteome Researchvol 8 no 10 pp 4835ndash4843 2009

[25] P M Bauer D Fulton Y C Boo et al ldquoCompensatoryphosphorylation and protein-protein interactions revealed byloss of function and gain of function mutants of multiple serinephosphorylation sites in endothelial nitric-oxide synthaserdquoTheJournal of Biological Chemistry vol 278 no 17 pp 14841ndash148492003

[26] C Szabo and A Papapetropoulos ldquoHydrogen sulphide andangiogenesis mechanisms and applicationsrdquo British Journal ofPharmacology vol 164 no 3 pp 853ndash865 2011

[27] S Dimmeler I Fleming B Fisslthaler C Hermann R Busseand A M Zeiher ldquoActivation of nitric oxide synthase inendothelial cells by Akt-dependent phosphorylationrdquo Naturevol 399 no 6736 pp 601ndash605 1999

[28] B L Predmore D Julian and A J Cardounel ldquoHydrogensulfide increases nitric oxide production from endothelial cellsby an Akt-dependent mechanismrdquo Frontiers in Physiology vol2 article 104 2011

[29] P Manna and S K Jain ldquoL-cysteine and hydrogen sulfideincrease PIP3 and AMPKPPAR120574 expression and decrease ROSand vascular inflammation markers in high glucose treatedhuman U937 monocytesrdquo Journal of Cellular Biochemistry vol114 no 10 pp 2334ndash2345 2013

[30] B Huang F A Li C H Wu and D L Wang ldquoThe roleof nitric oxide on rosuvastatin-mediated S-nitrosylation andtranslational proteomes in human umbilical vein endothelialcellsrdquo Proteome Science vol 10 no 1 article 43 2012


[31] C Coletta A Papapetropoulos K Erdelyi et al ldquoHydrogen sul-fide and nitric oxide are mutually dependent in the regulationof angiogenesis and endothelium-dependent vasorelaxationrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 109 no 23 pp 9161ndash9166 2012

[32] Z Altaany G Yang and RWang ldquoCrosstalk between hydrogensulfide and nitric oxide in endothelial cellsrdquo Journal of Cellularand Molecular Medicine vol 17 no 7 pp 879ndash888 2013

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Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


101 50 100eN

OS

Actin

peN

OSS1

177

NaHS (2h)

(120583M)

(a)

eNO

SAc

tin

Ctrl 05 2 12 (h)

peN

OSS1

177

NaHS (50120583M)

(b)

Rela

tive f

old

101 50 100(120583M)

lowastlowastlowast

lowast

lowastlowast

35

3

25

2

15

1

05

0

4

(c)

Rela

tive f

old

Ctrl 05 2 12(h)

lowastlowast

lowastlowast

35

3

25

2

15

1

05

0

(d)

Figure 1 NaHS increases the expression and serine 1177 phosphorylation of eNOS (a) ECs pretreated by diluting concentrations of NaHS(1 120583M 10 120583M 50 120583M and 100120583M) for 2 h (b) ECs treated with NaHS (50120583M) for 05 2 and 12 h Blotted membranes were separatelyhybridized with eNOS and peNOSS1177 antibodies ((c) and (d)) Relative folds of protein levels shown as means plusmn SE compared to controlStatistical significance ( lowast119875 lt 005 lowastlowast119875 lt 001) analyzed using Fisherrsquos LSD

In recent years however interest has been directed towardsH2S as the third gaseous mediator which has been shown to

exhibit potent vasodilatory activity both in vitro and in vivoThis is assumed to be realized by opening vascular smoothmuscle KATP channels [8] Of the three enzymes cystathio-nine-120574-lyase (CSE) cystathionine-120573-synthase (CBS) and3-mercaptopyruvate sulfurtransferase (3-MST) can utilizeL-cysteine as a substrate to produce H

2S Deficiency of

H2S-producing enzymes results in some disorders such as

homocystinuria which is characterized by mental retarda-tion skeletal abnormalities increased urine homocysteineincreased risks of thromboembolism and early onset ofatherosclerosis [9ndash11] H

2S was also reported to protect

against vascular remodeling from endothelial damage [12]Recently a signaling molecule for H

2S was shown to regulate

vascular relaxation and angiogenesis via potassium channelS-sulfhydration [13ndash15]

With a similar physiological function it is interesting todiscuss the interactions between H

2S and NO in respond-

ing stimuli In the reports cited above H2S and NO in

synergy might regulate smooth muscle relaxation and alsomitochondrial integration [16 17] H

2S triggers late-phase

preconditioning in the postischemic small intestine by anNO- and p38 MAPK-dependent pathway [18] Despite H

2S

inhibiting NO production in lipopolysaccharide-stimulatedmacrophages the H

2S can also stimulate NO production

from other cells [19 20]Because of the technical difficulty in detecting gaseous

molecules in the current study not only monitoring the reg-ulations of theses enzymes but also quantifying themoleculesof H2S andNO specifically with the new designed fluorescent

probes Therefore we question here if H2S has any upstream

role in the regulation of endothelial NO production

2 Materials and Methods

21 Cell Culture and Drug Treatments The EAhy 926 cellline was kindly donated by Cora-Jean S Edgell University ofNorth Carolina Chapel Hill EAhy 926 cells were cultured inDMEM supplemented with fetal bovine serum (FBS 10)streptomycin (100 120583gmL) and penicillin (100UmL) ECswere replaced by the same medium containing 2 FBS andincubated overnight prior to the experimental NaHS andNOC-18 treatments

22 Cell Lysis and Protein Extraction ECs were washed withcord buffer after treatment [NaCl (014M) KCl (4mM)


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by flow cytometry








Acknowledgments




References


2S Gas Biologyrdquo









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


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by flow cytometry








Acknowledgments




References


2S Gas Biologyrdquo









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


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Acknowledgments




References


2S Gas Biologyrdquo









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



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Actin

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1000

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by flow cytometry








Acknowledgments




References


2S Gas Biologyrdquo









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Ctrl 05 2 12CS

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by flow cytometry








Acknowledgments




References


2S Gas Biologyrdquo









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



References


2S Gas Biologyrdquo









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of








Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

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