Regulation of soluble guanylyl cyclase redox state by hydrogen sulfide

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ContentslistsavailableatScienceDirect

Pharmacological

Research

jo u r n al hom e p ag e :w w w . e l s e v i e r . c o m / lo c a t e / y p h r s

Regulation

of

soluble

guanylyl

cyclase

redox

state

by

hydrogen

sulﬁde

Zongmin

Zhou

a,1

_,

_Emil

_Martin

b,1

_,

_Iraida

_Sharina

b

_,

_Iolanda

_Esposito

c

_,

_Csaba

_Szabo

d

_,

Mariarosaria

Bucci

c

_,

_Giuseppe

_Cirino

c

_,

_Andreas

_{Papapetropoulos}

a,e,f,∗

a_1st_Department_of_Critical_Care_and_Pulmonary_Services,_Faculty_of_Medicine,_National_and_Kapodistrian_University_of_Athens,_Evangelismos_Hospital,

Greece

b_Division_of_Cardiology,_Department_of_Internal_Medicine,_University_of_Texas_Medical_School_at_Houston,_TX,_USA c_Department_of_Experimental_{Pharmacology,}_Faculty_of_Pharmacy,_University_of_{Naples—Federico}_II,_Italy d_Department_of_{Anesthesiology,}_University_of_Texas_Medical_Branch,_Galveston,_TX,_USA

e_Laboratory_of_{Pharmacology,}_Faculty_of_Pharmacy,_National_and_Kapodistrian_University_of_Athens,_Greece

f_Center_of_Clinical,_Experimental_Surgery_&_{Translational}_Research,_Biomedical_Research_Foundation_of_the_Academy_of_Athens,_Greece

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received28April2016

Receivedinrevisedform7June2016 Accepted30June2016

Availableonline1July2016

Keywords: H2S Nitricoxide cGMP ROS sGCactivators

a

b

s

t

r

a

c

t

Solubleguanylatecyclase(sGC)isareceptorfornitricoxide(NO).BindingofNO toferrous(Fe2+₎ hemeincreasesitscatalyticactivity,leadingtotheproductionofcGMPfromGTP.Hydrogensulfide (H2S)isasignalingmoleculethatexertsbothdirectandindirectanti-oxidanteffects.Inthepresent, studyweaimedtodeterminewhetherH2ScouldregulatesGCredoxstateandaffectits responsive-nesstoNO-releasingagentsandsGCactivators.Usingcultured rataorticsmoothmusclecells,we observedthattreatmentwithH2SaugmentedtheresponsetotheNOdonorDEA/NO,while attenu-atingtheresponsetotheheme-independentactivatorBAY58-2667thattargetsoxidizedsGC.Similarly, overexpressionofH2S-synthesizingenzymecystathionine-␥lyasereducedtheabilityofBAY58-2667 topromotecGMPaccumulation.Inexperimentswithphenylephrine-constrictedmouseaorticrings, treatmentwithrotenone(acompoundthatincreasesROSproduction),causedarightwardshiftofthe DEA/NOconcentration-responsecurve,aneffectpartiallyrestoredbyH2S.Whenringswerepre-treated withH2S,theconcentration-responsecurvetoBAY58-2667shiftedtotheright.Usingpurified recom-binanthumansGC,weobservedthattreatmentwithH2SconvertedferrictoferroussGCenhancing NO-donor-stimulatedsGCactivityandreducingBAY58-2667-triggeredcGMPformation.Thepresent studyidentifiedanadditionalmechanismofcross-talkbetweentheNOandH2Spathwaysatthelevel ofredoxregulationofsGC.OurresultsprovideevidencethatH2SreducessGChemeFe,thus,facilitating NO-mediatedcellularsignalingevents.

1. Introduction

Nitricoxide(NO)isa signalingmoleculethat affectsdiverse physiologicaland pathophysiologicalprocessesin practicallyall systems and organs [1,2]. In the cardiovascular system, NO regulatesvascular tone,inhibits platelet aggregation,promotes angiogenesis,modulatesinﬂammatoryresponsesandexerts car-dioprotectiveeffects[3].AlthoughNOiscapableofreactingwith aplethoraofmoleculartargets,theresponsetolowphysiological levelsofNOispredominantlymediatedbytheheterodimeric sol-ubleguanylylcyclase(sGC)[4].sGCisahemeproteinwithhigh

∗ Correspondingauthorat:LaboratoryofPharmacology,FacultyofPharmacy, Panepistimiopolis,Zografou,Athens15771,Greece.

E-mailaddress:apapapet@pharm.uoa.gr(A.Papapetropoulos).

1 _Zongmin_Zhou_and_Emil_Martin_contributed_equally.

afﬁnityandspeciﬁcitytowardsNO[5–7]thatconvertsGTPtothe secondmessengercGMP;sGCactivityisincreasedseveralhundred folduponbindingofNO[6].

MaintenanceofsGChememoietyintheferrousstateis essen-tialforthesGCNOsensingfunction[8].OxidationofsGChemeby speciﬁcagents[9]oroxidativestressinducedbyvarious patholog-icalconditions[10]renderssGCinsensitivetonormallevelsofNO andattenuatesNO/cGMPsignaling.Moreover,ithasbeen demon-stratedthatsGCwithoxidizedhemehasapropensitytolosethe hememoiety[11,12].Thepoolofferricorheme-freesGCismuch moresusceptibletodegradation[12,13].Aboutﬁfteenyearsago, NO-independentsGCactivatorsandstimulatorswerediscovered

[14,15];theseagentsenhancesGCactivityinaheme-dependent or−independentmanner.sGCactivatorsareuniquefortheir abil-itytoincreasethecatalyticactivityofheme-free/oxidizedsGC.sGC

http://dx.doi.org/10.1016/j.phrs.2016.06.029

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stimulatorshavealreadybeengrantedapprovalforhumanusefor certaintypesofpulmonaryhypertension[16].

Formanydecades,H2Swasconsideredatoxicgasthat pene-tratescellsbysimplediffusion[17].Followingthediscoverythat mammalian cells are capable of producing H2S, this molecule underwentadramaticmetamorphosisfromadangerouspollutant toabiologicallyrelevantmolecule,reminiscentofthe transforma-tionofNO[18,19].H2Sisnowacceptedasasignalingmoleculewith importantrolesinphysiology anddisease[20–22].H2Striggers manyofthesameresponsesasNOinthecardiovascularsystem;it reducesbloodpressure,promotesangiogenesisandlimitsinfarct sizefollowingischemia/reperfusioninjury[21,23].Although dis-tincteffectorshavebeenidentiﬁedforH2SandNO,bothagentsare capableofenhancingcGMPlevels.MuchlikeNOdonors,exposure ofcellsortissuestoH2SdonorsleadstointracellularcGMP accumu-lation[24,25].However,unlikeNOwhichstimulatessGC,H2Sraises cGMPbypreventingitsbreakdown[26],byenhancingendothelial NOsynthase(eNOS)activity[25,27]aswellasbyliberatingNOfrom stablebiologicalstoresofNO[28].TheinterdependenceofNOand H2Ssignalingisanareaofintenseinvestigation,withseveralgroups aimingtounravelitsimportancefornormalcellfunction,aswell asvariouspathophysiologicalstates.

OurpreviousstudiesdemonstratedthatH2Sdoesnotdirectly affecttheactivityofferroussGC[25].Inthecurrentstudy,we inves-tigatedwhetherH2SaffectsthefunctionofsGCcarryingoxidized ferricheme.WereportthatH2SreducesferricsGChemeintoa ferrousstate,whichisaccompaniedbytherestorationofNO activa-tionofpuriﬁedandcellularsGC,andtherecoveryofNO-dependent vasodilation.Conversely,H2S-dependentreductionofsGCheme diminishestheresponsetoferric-sGCbyBAY58-2667;thislater observationhassigniﬁcantimplicationsforthepharmacologyof sGCactivators,especiallyinlightofthetranslationaleffortsofthis classofagents.

2. Materialsandmethods

2.1. Reagents

Cellculturemediaand serumwereobtainedfromLife Tech-nologiesGIBCO-BRL(Paisley,UK).Allcellcultureplasticwarewas purchasedfromCorning-CostarInc.(Corning,NY).DCProteinassay kit;penicillinandstreptomycinwerepurchasedfromApplichem (Darmstadt,Germany).BAY58-2667wasobtainedfromAdipogen AG(Switzerland).HiTrapdesaltingcolumnswerepurchasedfrom GEHealthcareBio-Sciences(Pittsburgh,PA).ThecGMPEIAkitwas obtainedfromEnzoLifeSciences(Farmingdale,NY). Guanosine-5-[(␣,␤)-methyleno]triphosphate (GpCpp) was purchased from JenaBioscience(Jena,Germany).Sodiumhydrosulﬁde(NaHS)and sodiumsulﬁde(Na2S),DEA/NO,sodiumnitroprusside(SNP),ODQ, rotenone,phenylephrine,protease/phosphataseinhibitorsandall otherchemicalsusedinsolutionsandbufferswerepurchasedfrom Sigma-AldrichCo.LLC(StLouis,MO).

2.2. Exvivostudies

AllanimalprocedureswereincompliancewiththeEuropean Communityguidelinesfor theuseof experimentalanimalsand approvedbytheCommitteeCentroServiziVeterinariofthe Univer-sityofNaples“FedericoII”;institutionalregulationsdonotrequire theuseofanimalprotocolnumbersforapprovedprotocols. Ani-mals(C57Blmice)weresacriﬁcedwithCO2 and thoracicaortas wererapidlyharvested,dissected,andcleanedofadherent con-nectiveandfattissue.Ringsofabout1mmlengthweredenuded oftheendothelium,cutandplacedinorganbaths(2.5ml)ﬁlled withoxygenated(95%O2-5%CO2)Krebssolutionmaintainedat

37◦C.Theringswereconnectedtoanisometrictransducer(type 7006, Ugo Basile, Comerio, Italy) and changes in tension were recorded continuouslywith a computerized system(Data Cap-sule 17400,UgoBasile, Comerio, Italy). Thecomposition of the Krebssolutionwasasfollow(mM):NaCl118,KCl4.7,MgCl21.2, KH2PO41.2,CaCl2 2.5, NaHCO3 25, andglucose 10.1.The rings werestretcheduntila restingtensionof1.5gwasreachedand allowedtoequilibrateforatleast45min,duringwhichtime ten-sionwasadjusted,asnecessary,to1.5gandbathingsolutionwas periodicallychanged.In each experiment,ringswereﬁrst chal-lenged with PE (1␮M) until the responses were reproducible. TheringswerethenwashedandcontractedwithPE(1␮M)and, onceaplateauwasreached,acumulativeconcentration-response curveoftheDEA/NOorBAY58-2667wereperformed.Somerings werepretreatedwithrotenone(10␮M)for15minwithorwithout Na2S(50␮M).Afterthe15minpre-incubationtime,cumulative concentration-responsecurveswereperformed.

2.3. Cellculture

Rataorticsmoothmusclecells(RASMC)wereisolatedfrom 12-to14-wk-oldmaleWistarrats,ﬁveratsperisolation,aspreviously described.Animalswereanesthetizedwithpentobarbitalsodium (40mg/kgip).Oncefullyanesthetizedasjudgedbythelackof reac-tiontoanoxiousstimulus,animalswereexsanguinated;thoracic aortaswerethenremoved.Morethan95%ofcellsisolatedstained positiveforsmoothmuscle␣-actin.Cellsbetweenpassages2and 5wereusedforallexperiments.RASMCwereroutinelyculturedin DMEMcontaining4.5g/lglucoseandsupplementedwith10%fetal bovineserumandantibiotics.

2.4. cGMPenzymeimmunoassay

Cellsweregrowntoconﬂuenceandwerewashedtwicewith phosphatebufferedsalineandthenincubatedinHBSSinthe pres-enceofisobutylmethylxanthine(IBMX;1mM)for5min.Cellswere thentreatedwithvehicle,rotenone(10␮M),Na2S(50␮M)ora Na2S/rotenonecombinationfor30min.Fifteenminutesafterthe exposuretoDEA/NO(1␮M)orBAY58-2667(1␮M),mediawere aspiratedand 200␮l of0.1N HClwereaddedintoeach wellto extractcGMP.After30min,HClextractswerecollectedand cen-trifugedat600gfor10mintoremovedebris.Thesupernatantswere directlyanalyzedforcGMPbyenzymeimmunoassay.

2.5. sGCexpressionandpuriﬁcation

HumanferroussGCwaspuriﬁedfromSf9cellsinfectedwith baculovirusesexpressing␣1and␤1sGCsubunitsfollowing the procedures described previously [29].Puriﬁed ferrous sGC was storedat−80◦_C_in₅₀_mM_TEA_pH7.4_and₁_mM_DTT,₁_mM_EDTA and1mMEGTAuntilfurtheruse.ToprepareferricsGC,the sam-pleofferroussGCwaspassedthroughaHiTrapdesaltingcolumnto removeDTT.sGCinthiol-freebufferwasthentitratedwith increas-ingconcentrationsofODQ(0.1–1␮M)untilsGChememoietywas fullyoxidized,asdemonstratedbytheconversionoftheSoretband from432nmto393nm.Thepreparationwasthenpassedthrough thedesaltingcolumnagaintoremoveODQ.Thispreparationof ferricsGCwasusedforspectroscopicstudiesandactivity measure-ments.

2.6. ReductionofferricsGCbyhydrogensulﬁde

The reduction of ferric sGC by H2S was monitored using theUV–visspectrophotometer.FortitrationofNaHS3␮Mferric sGCwassupplemented withincreasingconcentrationsof NaHS

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(0.01–1000␮M)andthespecrumwasrecorded30safterthe addi-tionofNaHS.TomonitorthedynamicofsGCreductionbyNaHS, ∼1.5␮MpreparationofferricsGCwassupplementedwith200␮M NaHSandthespectralchangeswererecordedinakineticmode oftheinstrumentevery60s.Theapparentrateofreductionwas calculatedbasedonthedataﬁttingusingtheﬁrstorderreaction algorithm(UV–visChemStationsoftware,AgilentTechnologies). 2.7. AssayofsGCactivityinvitro

Enzymaticactivitywasassayedbytheformationof[32_P]_cGMP from␣[32_P]GTP_at₃₇◦_C_as_described_previously_[30]_._In_brief,_the reactionwasinitiatedadding1mMGTP/␣[32_P]GTP_to_0.1_␮g_sGC (ferricorferrous)in25mMTEA,pH7.5,1mg/mlBSA,1mMcGMP, 3mMMgCl2,0.05mg/mlcreatinephosphokinaseand5mM crea-tinephosphate.TomeasuretheresponseofferricorferroussGC tosodiumnitroprusside(SNP)orBAY58-2667,theactivatorswere addedtogetherwithGTP.After30minthereactionwasstoppedby zincacetatefollowedbyprecipitationofunreactedGTPbyZn car-bonate.cGMPwasseparatedfromtheremainingGTPbyalumina chromatographyandthelevelofsynthesizedcGMPwasquantiﬁed basedonCherenkovradiation.

2.8. Dataanalysis

Data are expressedas means±SEM. Statistical comparisons between groups were performed using ANOVA followed by a posthoctestorStudent’st-test,asappropriate.Differenceswere considered signiﬁcant when P<0.05. GraphPad Prism software (version4.02,GraphPadSoftware,SanDiego,CA)wasusedforall thestatisticalanalysis.

3. Results

3.1. H2SreversestheeffectsofsGChemeoxidationincells

Reactiveoxygenandnitrogenspecieshaveapotentialtooxidize thehememoietyofsGCandnegativelyaffectNO/cGMPsignaling

[14].WeinvestigatedtheabilityofhydrogensulfidetorestoresGC alteredresponsescausedbyoxidativestress.Togeneratea persis-tent,low-to-intermediateleveloxidativestressinrataorticsmooth musclecells we usedrotenone (10_␮M) toinhibit the electron flowofthemitochondrialrespiratorychain.Exposuretorotenone enhancedBAY58-2667-dependentcGMPaccumulationinRASMC (Fig.1A).Toinvestigateifhydrogensulfidecouldreversethe oxida-tionofsGChemecausedbyrotenone,cellswereco-treatedwithan H2S-yieldingsalt.AsshowninFig.1A,suchtreatmentsuppressed theenhancedabilityofBAY58-2667toincreasecGMPlevelsinthe presenceofrotenone.ToprobethereactivityofsGCtowardsNO, aconcentrationoftheNOdonorDEA/NOwaschosenthatdidnot increasecGMPlevels.ExposureofcellstoH2S,unmaskedthe cGMP-stimulatingcapacityofDEA/NO.Thisobservationisconsistentwith theabilityofH2Stodecreasethecontentofoxidizedcellularferric sGCandincreasetheamountofsGCthatcouldbeactivatedbyNO (ferroussGC).Inadifferentexperimentalsetting,oxidationofsGC hemewasachievedbyadministrationofODQ,aspecificsGCheme oxidant.EvenwiththisstrongsGCoxidizingagent,H2Sdiminished thefractionofferricsGC,asattestedbydiminishedcGMPsynthesis inresponsetoBAY58-2667(Fig.1B).

3.2. H2S-generatingcystathionine-lyasediminishesthecontent offerrichemesGC

Cystathionine_␥-lyase(CSE)isanenzymesofthe transulfura-tionpathwaythatgenerateshydrogensulﬁde[19].Totestwhether elevatedendogenousH2SaffectssGCfunctionweusedRASMCand

infectedthemwithanadenovirusexpressingCSEorgreen ﬂuo-rescentprotein(GFP)ascontrol.CSEoverexpressiondidnotalter sGC␣1or␤1levels(Fig.2A).WeobservedthatCSEover expres-sionresultedin reducedcGMP synthesisin RASMCin response to BAY58-2667 treated with ODQ (Fig. 2B). The ﬁndings with endogenouslygeneratedH2Sreplicatetheobservationsmadewith exogenouslyaddedH2Sandareconsistentwithdecreasedcellular contentofsGCwithferricheme.

3.3EffectsofH2SonNOandBAY58-2667-induced

vasorelax-ation.sGCplaysakeyroleinmediatingNO-dependentvasodilation

[6].Previousstudiesdemonstratedthatoxidativestressand var-iouspathological conditionsmayincreasethepoolofferricsGC or heme-deficient sGC in blood vessels and lead to impaired NOresponse invasculature.Therefore, wetestedwhetherH2 S-dependentreductionofsGChemetranslatesintoimprovedNO/sGC signalinginconditionsofoxidativestress.Wefirstevaluatedthe vasoreactivity of control and rotenone-treated mouse thoracic aorta.AsshowninFig.3A,rotenone-pretreatedaorticsegments exhibitedasignificantreductionin thesensitivitytoNOwitha rightwardshiftintheconcentration-responsecurvebeenobserved. TheapparentEC50forDEA/NOincreasedfrom149.9±1.30nMto 2.00_±1.29_␮M,forcontrolandrotenone-treatedvessels, respec-tively.However,whenH2Swasadministeredduringthelast10min ofrotenonetreatment,asubstantialrestorationofNO-dependent vasodilationwasobserved.Itshouldbenotedthatrotenone,H2Sor theirsimultaneousadditiondidnotaffecttheabilityofthetissue tocontractinresponsetophenylephrine.Additionalexperiments wereperformedtoconfirmthatH2SaffectsthefractionofferricsGC intheisolatedaorta.VesselswereexposedtoH2Sandthen relax-ationwastriggeredbyBAY58-2667.AsdemonstratedinFig.3B, vesselstreatedwithH2SwerelessresponsivetoBAY58-2667than controlvessels,suggestingthatinthepresenceofH2Slowerlevel ofsGCcarryingferrichemeexist.

3.3. H2SpromotesrestorationofferroussGC

TostudythemechanismofalteredresponsivenesstoNOand sGCactivatorsinthepresenceofH2S,weevaluatedifH2Sdirectly affectsthehemestatusand/oractivityofpuriﬁedferricsGC. There-fore,we testedifadditionofH2Scanchangetheredoxstateof sGChemebymonitoringtheUV–visabsorbanceofsGCheme.We observedthatH2Sinducedaconcentration-andtime-dependent conversionof 393nm Soretbandof ferricsGCintothe 432nm SoretbandofferroussGC(Fig.4),directlydemonstratingthatH2S reducessGChememoiety.Previousstudiesdemonstratedthatthe dynamicsofsGCresponsetoNOandCOgaseousligandsis differ-entinthepresenceofGTPsubstrate.Therefore,weevaluatedifGTP affectsthedynamicsofsGCreductionbyH2S.Wedeterminedthat theadditionofGpCpp(anon-hydrolyzableGTPanalog)doesnot altertheoverallprocessofsGChemereduction(Fig.4C).However, aslightdecreaseintheapparentreductionratefrom0.16±0.03to 0.11±0.04␮M/secwasobserved(Fig.4D).

Asexpected,ferricheme sGCwasnon-responsivetotheNO donorsodiumnitroprusside,butwasstronglyactivatedby100nM BAY58-2667(Fig.5).Onthecontrary,ferroussGCwasstrongly acti-vatedbysodiumnitroprussideandhad amoderateresponseto 100nMBAY58-2667.InthepresenceofH2S,ferricsGCexhibited adiminishedresponsetoBAY58-2667andarobustactivationtoa NOdonor,closelyresemblingthepropertiesofferroussGC.

4. Discussion

ThemainﬁndingofthepresentstudyisthatH2Siscapableof reducingtheprosthetichemegroupofsGCfromFe+3_to_Fe+2_, there-foreincreasingtheNO-activatablepoolofsGCincells.Theshiftin

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A

*

#

*

0

1

2

3

4

5 c

GMP

(pmole

/w

ell)

Basal DEA/NO BAY 58-2667

0

2

4

6

Vehicle H2S ODQ H2S + ODQ

c

GMP

(pmole

/w

ell)

B

*

Fig.1. H2SreversestheeffectsofrotenoneandODQoncGMPaccumulation.(A):Rataorticsmoothmusclecellswereincubatedwithvehicle,rotenone(10␮M),H2S(Na2S,

50␮M)oracombinationofH2Sandrotenonefor30min.CellswerethenincubatedwithDAE/NO(1␮M)orBAY58-2667(10␮M)for15mininthepresenceofIBMX(1mM).

CellularcGMPwasextractedwith0.1NHClandmeasuredbyEIA;n=4;*P<0.05vsrespectivevehiclegroup;#p<0.05vsrespectiverotenonegroup.(B):Rataorticsmooth musclecellswereincubatedwithvehicle,ODQ(0.1␮M),H2S(Na2S,50␮M)oracombinationofH2SandODQfor30min.CellswerethenincubatedwithBAY58-2667

(10␮M)for15mininthepresenceofIBMX(1mM).CellularcGMPwasextractedwith0.1NHClandmeasured*P<0.05vsODQ.

B

A

* Ad-GFP Ad-CSE ti GC 1 1 2 3 GFP CSE GFP+ODQ CSE+ODQ o le/ w ell) anti-sGC α1 anti-sGC β1 anti-CSE anti β actin α1 β1 1 cGM P (pm o anti-β-actin Basal BAY 58-2667 0

Fig.2.CSEoverexpressionattenuatessGCactivator(BAY58-2667)-inducedcGMPaccumulation.(A)Representativewesternblotfromsmoothmusclecellsinfectedwith CSEadenovirus.(B)CellswereinfectedwithaGFPoraCSEexpressingadenovirus(10m.o.i).FortyeighthourslatercellswashedandincubatedwithODQ(50nM)andthen exposedtoBAY58-2667(1␮M).CellularcGMPwasextractedwith0.1NHClandmeasuredbyEIA;n=4;*P<0.05*vsODQ.

Fig.3.H2Spartiallyrescuesrotenone-inducedreductionofDEA/NOvasorelaxation,whileitinhibitsBAY58-2667-triggeredvasorelaxation.Phenylephrine-constricted

aorticringswerepre-treatedwithrotenone(10␮M)orH2S+rotenonefor15min.ThesulﬁdesaltNa2S(50␮M)wasusedasasourceofH2S.Afterincubation,cumulative

concentration-responsecurvestoDEA/NO(A)P<0.05***rotenonevsvehicle;##H2S+rotenonevsvehicle;&H2S+rotenonevsrotenone)orBAY58-2667(B)**P<0.05H2S

vsvehicle)wereperformed.

sGCredoxstatecausedbyH2Shasimportantimplicationsnotonly foritsendogenousligand,butalsoimpactsonthepharmacological activityofsGCactivators.

Wehavepreviously shownthatincubationofcells with sul-ﬁdesaltsincreasescGMPaccumulationinrataorticsmoothmuscle

cells;thiseffectis attributedtoinhibition ofphosphodiesterase

[24,26].ToruleoutthatanychangesobservedincGMPlevelsin thecurrentseriesofexperimentsmightresultfromPDEinhibition, allofmeasurementswereperformedinthepresenceofthe non-selectivePDEinhibitorIBMX,ataconcentrationwhichinhibitsPDE

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A

B

0.25 0.3 0.35 Fe3+-sGC 1 μM 3 μM 10 μM 30 μM 0 08 0.1 0.12 0 sec 60 sec 120 sec 180 sec 200 μM H2S

A

0.1 0.15 0.2 OD, AU μ 100 μM 300 μM 1 mM 0.04 0.06 . OD, AU 300 sec 600 sec 900 sec 0 0.05 320 340 360 380 400 420 440 460 480 nm 0 0.02 320 340 360 380 400 420 440 460 480 nm 393 nm 432 nm 393 nm 432 nm 0.12 0.14 0 sec_{60 sec} 120 sec 180sec 0.08 0.09 0.10 K= 0.16 μM/sec 200 μM H2S +50 μM GPcPP

D

C

0 04 0.06 0.08 0.1 OD, AU 180 300 sec 600 sec 900 sec 0 03 0.04 0.05 0.06 0.07 ∆ OD 432, AU 200 M N HS K= 0.11 μM/sec 0 0.02 0.04 320 340 360 380 400 420 440 460 480 nm 0 250 500 750 1000 1250 0.00 0.01 0.02 0.03 Time sec ∆ 200 μM NaHS 200 μM NaHS + GPcPP 393 nm 432 nm Time, sec

Fig.4.H2S-dependentconversionofFe3toFe2+sGCisconcentration-andtime-dependent,andmildlyaffectedbyGTPsubstrate.(A):UV–visspectraoftheSoretregionfor

ferricsGC(3␮M)in50mMTEAbuffer(pH7.4)wererecorded1minaftertheadditionoftheindicatedamountofH2Sintheformofasulﬁdesalt(NaHS).(AU,absorbance

unit).(B):3␮MferricsGCwasmixedwithanequalvolumeofH2S(NaHS,200␮M)in50mMTEAbuffer(pH7.4)andspectralchangeswererecordedfor20min.(C):Spectral

changesforferricsGCsupplementedwith50␮MGpCppaftertheadditionofH2S.(D):Time-dependentaccumulationof432nmbandinsamplesofferricsGCaloneorinthe

presenceof50␮MGPcPPinresponsetoH2S.

Fig.5.H2SreducesFe3+hemetoFe2+insGCleadingtochangesinenzyme

respon-sivenesstoNOandBAY58-2667.Puriﬁedsamplesofferric(Fe3+-sGC)orferrous (Fe2+-sGC)sGCwereincubatedwithorwithoutH2S(NaHS,25␮M)andstimulated

withsodiumnitroprusside(SNP;10␮M),orBAY58-2667(100nM).Theformationof [␣-32_P]_cGMP_from_[␣-32_P]_GTP_was_quantiﬁed;_n₌_6,_*_P_<_0.01_vs_ferric_sGC_without

H2S.

inratsmoothmusclecells[31,32].Indeed,exposureofcellstoNa2S alonedidnotcauseachangeincGMPlevelsinthepresenceofIBMX. Exposureofcellsto1␮MDEA/NOfailedtoincreasecGMP; how-ever,whencellswereincubatedwithH2S,1␮MDEA/NOpromoted anincreaseinintracellularcGMP,presumablyduetoconversionof existingcellularferrictoferroussGC.Ontheotherhand,whencells wereexposedto1␮MBAY58-2667asharpincreaseincGMP lev-elsweobserved,conﬁrmingpreviousobservations[33].Rotenone, anagentthatpromotesROSgenerationbyinhibitingcomplexIof therespiratorychain[34],increasedtheresponsetoBAY58-2667 thatselectivelytargetstheoxidized-heme/freepoolofsGC. Inter-estingly,thiseffectwasreversedbyincubatingcellswithasource ofH2S.Sulﬁdesalts(NaHSandNa2S)althoughwidelyusedinthe literaturetodeliverH2Stocellssufferamajordrawback[35];once dissolvedinaqueousbuffersinstantlydecomposeyieldingaburst

onH2S. TodeliverH2Sinaphysiologically-relevantmanner,we infectedcellswithanadenovirusthatoverexpressesCSE,amajor H2S-generatingenzymeinvasculartissue[17].CSE-overexpressing cellsdisplayedreducedresponsivenesstoBAY58-2667.Theabove datatakentogetherareinlinewiththenotionthatH2Sshiftsthe sGCredoxbalancetowardsferroussGC.

H2S is knownto have both direct and indirectanti-oxidant actions.H2SreactswithROSandreactivenitrogenspecies, includ-inghydrogenperoxide,andperoxynitrite,althoughwithrelatively modestrateconstants[36].Inaddition,H2Shasbeenshownto up regulate the expression/activity of antioxidant enzymes, to affecttheexpressionofROSgeneratingenzymesandtoincrease glutathionelevels[17,19,37].ThebeneficialeffectofH2SonNO signalinginourexperimentsthatemployedrotenonecouldresult fromneutralizationofROS,thuspreventingoxidationofsGCheme. Anotherpossiblemechanismofbeneficialeffectofsulfideson cel-lularsGCactivityunderconditionsofoxidativestressisthereaction of sulfide ion withoxidized cysteine residues. sGC hasa large numberofcysteineresiduesthataresusceptibletomodification by ROS.Proteomicanalysis of sGCfromcells thatwere chron-ically exposedtooxidative stressviaanaldosterone-dependent mechanismdemonstratedthattheCys122residueofthe␤1 sub-unitundergoesseveraloxidativemodifications[38].Thisspecific residuehasbeenimplicatedinmodulatingthepotencyofNO acti-vationofsGC[39].UnderphysiologicpH,cellularsulfidesaremore likelytoreactwithoxidizedproteinthiols,suchasproteinsulfenic acid,thanintracellularglutathione[40].Therefore,H2S-dependent restoration ofNO-inducible cGMP synthesis in cells exposedto rotenonecouldintheorybe,atleast,inpartexplainedbythe reduc-tionofsGCthiols.However,thisismostlikelynotthecase,asin thepurifiedenzymepreparationH2Srestoredresponsivenessof sGCexposedtotheselectivehemeoxidantODQthathasnoeffect

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onsGCthiols.ToevaluateifH2Scandirectlytargettheprosthetic hemegroupofsGCincells,weusedODQanagentthatselectively oxidizesthehememoietyofsGC[9].Inlinewithitsabilityto con-vertferroustoferricsGC,ODQ-treatedcellsexhibitedandincreased cGMPresponsetoBAY58-2667.ThiseffectofODQwasattenuated byH2S,suggestingthatH2ShasdirecteffectonsGCheme.

Tobetter characterize theeffect ofH2SonsGC redoxstate, weusedhumanrecombinantsGC.AfterpurificationsGCwas con-vertedtoitsferricstatebyareactionwithODQ;thesinglepeakat 393nmconfirmedthesuccessfulpreparationoffullyoxidizedsGC. Inagreementwiththecurrentliterature[15,41],theactivityofsGC withferrichemewasunaffectedbyNOreflecting thelow affin-ityofFe+3_sGC_for_NO._At_the_same_time,_Fe+3_sGC_responded_well toBAY58-2667,ansGCactivatorthathasahigheraffinitytosGC withoxidizedheme.TheoppositewastrueofferroussGC,which demonstratedonlyasmallchangeinactivityinresponsetoBAY 58-2667,butwasreadilyactivatedbyNO.IncubationofFe+3_sGC withH2S,causedaconcentration-dependentshiftintheabsorption maximumofhemefrom393nm,characteristicforferricsGCheme to432nm,whichisindicativeofferroushemesGC.Full conver-sionfromferrictoferroushemetook900s.TheactivityofFe+3_sGC towardsNOwasrestoredwhenincubatedwithH2S.Overall,the observationsmadewiththerecombinantsGCarewellinagreement withthosemadewiththeculturedsmoothmusclecells,reinforcing thenotionthatH2SkeepssGCinareduced,NO-responsivestate.

Ourobservationthathydrogensulfidereducesferricstateof sGChemeis inlinewithmanyobservations reportedforother heme-containingproteins[42].Sulfideevenatlowconcentrations canactaselectrondonortotheferricderivativeofcytochromeC oxidase[43].Similarheme-reducingactivityofhydrogensulfide wasreportedfortheferriccatalase[44]andseveralperoxidases

[45,46].HemoglobinandmyoglobininferricFe3+_state_bind_H

2S

ashemeligandandarerapidlyreducedtothedeoxyFe2+_and/or Fe2+_-O2_derivative_[47]_._It_should_be_noted_that_the_reaction_of_H2_S withoxygen-carryinghemoglobinand myoglobinresultsinthe formationofsulfheme,achlorine-typehemewithasulfuratom incorporatedintooneofthepyrrolerings.Generationofsulfheme wasalsodescribedfor catalaseandlactorperoxidase[48]. How-ever,inourspectralobservationsoftheinteractionbetweenferric orferroussGCwithH2,Sweobservednotracesofthe620nm opti-calbandcharacteristicforsulfheme(datanotshown).Therefore, weconcludethattheonlyeffectofH2SonsGCisthereductionof hemeironfromferrictoferrous.

SuperoxideaniongenerationleadstoNOscavengingandlimits NObiologicalactivity[2].Incubationofvesselswiththerotenone, causedashiftoftherelaxationcurvetoDEA/NOtotheright,due toproductionofROS.TheincreaseinEC50forNOdonorscanbe reversedbysuperoxidedismutaseandotherantioxidants. Incuba-tionofvesselswithH2SpartiallyrestoredtheDEA/NOresponse.As inthisvesselpreparationPDEwasnotinhibited,wecannotsafely concludethattheshiftoftheDEA/NOconcentrationcurveis exclu-sivelyduetoreductionofsGC.However,thisobservationiswellin accordancewithourcellcultureandpurifiedenzymeexperiments. Ina differentseriesofexperiments,vesselswerepre-incubated withH2SandthenexposedtoBAY58-2667.sGCactivatorresponses wereshiftedtotheright,asagreateramountofsGCisexpectedto beinitsreduced,BAY58-2667-unresponsiveform.Thesefindings confirmandextendourobservationsinvitroandprovidefunctional relevanceforthisH2S/NOinteractionatthesGClevel.

SeverallevelsofH2S-NOcross-talkand interactions,ranging fromdirectchemicalinteractionstoconvergenceontodownstream signalingpathwayshavebeenidentiﬁed[49–52].Thedirect reac-tionbetweenH2SandNOyieldsnitroxylthattriggersvasodilation

[51]thatcanactivatesGC[53].Ontheotherhand,the nitrosoper-sulﬁde(SSNO−)formedfollowingreactionofS-nitrosothiolswith sulﬁde,liberatesNOtoactivatecGMP[50].H2Spromotes

phos-phorylationofeNOSonSer1177,leadingtoincreasedNOoutput

[25,27].eNOSdimerizationisessentialforenzymaticactivity;H2S preservesdimericeNOSbypersulﬁdationandinhibitionofCys443 nitrosation[54].H2Shasalsobeenshowntoinhibit phosphodi-esterase(PDE)activity,witha 30-foldselectivityfor PDE5[55]. IncreasedNObioavailabilityissupportedbyH2Sactivationof xan-thineoxidasetoreducenitriteinischemictissues[28].Allofthese events culminatein activation of the cGMP/PKGcascade regu-latingcardiovascularfunction.Asa consequence,vasorelaxation concentration-responsecurvestoH2Sareshiftedtotherightin eNOSKOmice,whilenostimulationofangiogenesisand cardiopro-tectiononresponsetoH2Sdonorscanbeobservedinmicelacking eNOS[25,27,56].TheabilityofH2StoaffectsGCresponsivenessto NOdemonstratedinthecurrentreportprovidesanadditionallevel ofcross-talkbetweenNOandH2S.

Inconclusion, wehavedemonstratedthatsGCregulatessGC redoxstate,favoring theexistenceof ferroussGC.Maintenance ofphysiological H2Slevelswould notonlypreventNO destruc-tionbyenhancinganti-oxidantpathways,butwouldalsopreserve NO-responsivenessofsGCthroughtheactionofH2Sontheheme moiety. Enhanced expressionof H2Sproducing enzymesmight counteract the oxidation of sGC encountered in disease states, whileadeclineincellularH2ScouldmakesGCrefractoryto nor-mallevelsofNO.AlthoughdiscretepoolsofsGCareknowntoexist (reducedandoxidized),thecellularconstituentsresponsible regu-latingthebalancebetweenthesetwoformsofsGCremainelusive

[57,58].H2Sbeingafreelydiffusiblesmallmoleculewouldbewell suitedtoserveasanendogenoussGCreducing agent.Our ﬁnd-ingshaveimportantpharmacologicalimplicationsasH2Sdonors whicharecurrentlyinclinicaldevelopmentmightrepresentaway torestoresGCresponsivenesstoNO.Furtherstudiesvalidatingsuch interactionsinvivoarerequired.

Authordisclosurestatement

Theauthorsreportnoconﬂictsofinterest.

Acknowledgements

Thisworkhasbeenco-ﬁnancedbytheEuropeanUnion (Euro-pean Social Fund − ESF) and Greek national funds through the Operational Program “Education and Lifelong Learning” of the National Strategic Reference Framework (NSRF)—Research Funding Program Aristeia 2011 (1436) to AP, by EU FP7 REG-POTCT-2011-285950-“SEE-DRUG”, bytheCOSTAction BM1005 (ENOG:Europeannetworkongasotransmitters),byaGrant-in-Aid 15GRNT25700101fromtheAmericanHeartAssociationtoEMand bytheNationalInstitutesofHealth(R01GM107846)toCS.

References

[1]K.Davis,E.Martin,I.Turko,F.Murad,Noveleffectsofnitricoxide,Annu.Rev. Pharmacol.Toxicol.41(2001)203–236.

[2]S.Moncada,E.A.Higgs,Endogenousnitricoxide:physiology,pathologyand clinicalrelevance,Eur.J.Clin.Invest.21(1991)361–374.

[3]U.Förstermann,W.C.Sessa,Nitricoxidesynthases:regulationandfunction, Eur.HeartJ.33(2012)829–837.

[4]J.Garthwaite,Newinsightintothefunctioningofnitricoxide-receptive guanylylcyclase:physiologicalandpharmacologicalimplications,Mol.Cell. Biochem.334(2010)221–232.

[5]E.M.Boon,M.A.Marletta,Liganddiscriminationinsolubleguanylatecyclase andtheH-NOXfamilyofhemesensorproteins,Curr.Opin.Chem.Biol.9 (2005)441–446.

[6]D.Koesling,M.Russwurm,E.Mergia,F.Mullershausen,A.Friebe,Nitric oxide-sensitiveguanylylcyclase:structureandregulation,Neurochem.Int. 45(2004)813–819.

[7]E.Martin,V.Berka,E.Bogatenkova,F.Murad,A.-L.Tsai,Ligandselectivityof solubleguanylylcyclase:effectofthehydrogen-bondingtyrosineinthedistal hemepocketonbindingofoxygen,nitricodieandcarbonmonoxide,J.Biol. Chem.281(2006)27836–27845.

(7)

[8]E.R.Derbyshire,M.A.Marletta,Structureandregulationofsolubleguanylate cyclase,Annu.Rev.Biochem.81(2012)533–559.

[9]J.Garthwaite,E.Southam,C.L.Boulton,E.B.Nielsen,K.Schmidt,B.Mayer, Potentandselectiveinhibitionofnitricoxide-sensitiveguanylylcyclaseby 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one,Mol.Pharmacol.48(1995) 184–188.

[10]J.P.Stasch,P.M.Schmidt,P.I.Nedvetsky,T.Y.Nedvetskaya,H.S.ArunKumar,S. Meurer,M.Deile,A.Taye,A.Knorr,H.Lapp,H.Müller,Y.Turgay,C.Rothkegel, A.Tersteegen,B.Kemp-Harper,W.Müller-Esterl,H.H.Schmidt,Targetingthe heme-oxidizednitricoxidereceptorforselectivevasodilatationofdiseased bloodvessels,J.Clin.Invest.116(2006)2552–2561.

[11]B.G.Fritz,X.Hu,J.L.Brailey,R.E.Berry,F.A.Walker,W.R.Montfort,Oxidation andlossofhemeinsolubleguanylylcyclasefrommanducasexta, Biochemistry50(2011)5813–5815.

[12]S.Meurer,S.Pioch,T.Pabst,N.Opitz,P.M.Schmidt,T.Beckhaus,K.Wagner,S. Matt,K.Gegenbauer,S.Geschka,M.Karas,J.-P.Stasch,H.H.H.W.Schmidt,W. Müller-Esterl,Nitricoxide—independentvasodilatorrescuesheme-oxidized solubleguanylatecyclasefromproteasomaldegradation,Circ.Res.105(2009) 33–41.

[13]L.S.Hoffmann,P.M.Schmidt,Y.Keim,S.Schaefer,H.Schmidt,J.P.Stasch, Distinctmolecularrequirementsforactivationorstabilizationofsoluble guanylylcyclaseuponhaemoxidation-induceddegradation,Br.J.Pharmacol. 157(2009)781–795.

[14]O.V.Evgenov,P.Pacher,P.M.Schmidt,G.Hasko,H.H.H.W.Schmidt,J.-P.Stasch, NO-independentstimulatorsandactivatorsofsolubleguanylatecyclase: discoveryandtherapeuticpotential,Nat.Rev.DrugDiscov.5(2006)755–768.

[15]A.Papapetropoulos,A.J.Hobbs,S.Topouzis,Extendingthetranslational potentialoftargetingNO/cGMP-regulatedpathwaysintheCVS,Br.J. Pharmacol.172(2015)1397–1414.

[16]H.-A.Ghofrani,N.Galiè,F.Grimminger,E.Grünig,M.Humbert,Z.-C.Jing,A.M. Keogh,D.Langleben,M.O.Kilama,A.Fritsch,D.Neuser,L.J.Rubin,Riociguat forthetreatmentofpulmonaryarterialhypertension,N.Engl.J.Med.369 (2013)330–340.

[17]R.Wang,Physiologicalimplicationsofhydrogensulﬁde:awhiffexploration thatblossomed,Physiol.Rev.92(2012)791–896.

[18]L.Li,P.Rose,P.K.Moore,Hydrogensulﬁdeandcellsignaling,Annu.Rev. Pharmacol.Toxicol.51(2011)169–187.

[19]C.Szabo,Hydrogensulphideanditstherapeuticpotential,Nat.Rev.Drug Discov.6(2007)917–935.

[20]B.D.Paul,S.H.Snyder,H(2)Ssignallingthroughproteinsulfhydrationand beyond,Nat.Rev.Mol.CellBiol.13(2012)499–507.

[21]D.J.Polhemus,D.J.Lefer,Emergenceofhydrogensulﬁdeasanendogenous gaseoussignalingmoleculeincardiovasculardisease,Circ.Res.114(2014) 730–737.

[22]M.Whiteman,S.LeTrionnaire,M.Chopra,B.Fox,J.Whatmore,Emergingrole ofhydrogensulﬁdeinhealthanddisease:criticalappraisalofbiomarkersand pharmacologicaltools,Clin.Sci.(Lond.)121(2011)459–488.

[23]R.Wang,C.Szabo,F.Ichinose,A.Ahmed,M.Whiteman,A.Papapetropoulos, TheroleofH2Sbioavailabilityinendothelialdysfunction,TrendsPharmacol. Sci.36(2015)568–578.

[24]M.Bucci,A.Papapetropoulos,V.Vellecco,Z.Zhou,A.Zaid,P.Giannogonas,A. Cantalupo,S.Dhayade,K.P.Karalis,R.Wang,R.Feil,G.Cirino,

cGMP-dependentproteinkinasecontributestohydrogensulﬁde-stimulated vasorelaxation,PLoSOne7(2012)e53319.

[25]C.Coletta,A.Papapetropoulos,K.Erdelyi,G.Olah,K.Módis,P.Panopoulos,A. Asimakopoulou,D.Gerö,I.Sharina,E.Martin,C.Szabo,Hydrogensulﬁdeand nitricoxidearemutuallydependentintheregulationofangiogenesisand endothelium-dependentvasorelaxation,Proc.Natl.Acad.Sci.U.S.A.109 (2012)9161–9166.

[26]M.Bucci,A.Papapetropoulos,V.Vellecco,Z.Zhou,A.Pyriochou,C.Roussos,F. Roviezzo,V.Brancaleone,G.Cirino,Hydrogensulﬁdeisanendogenous inhibitorofphosphodiesteraseactivity,Arterioscler.Thromb.Vasc.Biol.30 (2010)1998–2004.

[27]A.L.King,D.J.Polhemus,S.Bhushan,H.Otsuka,K.Kondo,C.K.Nicholson,J.M. Bradley,K.N.Islam,J.W.Calvert,Y.X.Tao,T.R.Dugas,E.E.Kelley,J.W.Elrod, P.L.Huang,R.Wang,D.J.Lefer,Hydrogensulﬁdecytoprotectivesignalingis endothelialnitricoxidesynthase-nitricoxidedependent,Proc.Natl.Acad.Sci. U.S.A.111(2014)3182–3187.

[28]S.C.Bir,G.K.Kolluru,P.McCarthy,X.Shen,S.Pardue,C.B.Pattillo,C.G.Kevil, Hydrogensulﬁdestimulatesischemicvascularremodelingthroughnitric oxidesynthaseandnitritereductionactivityregulatinghypoxia-inducible factor-1␣andvascularendothelialgrowthfactor—dependentangiogenesis,J. Am.HeartAssoc.1(2012)e004093.

[29]E.Martin,V.Berka,I.Sharina,A.-L.Tsai,MechanismofbindingofNOto solubleguanylylcyclase:implicationforthesecondNObindingtotheheme proximalsite,Biochemistry51(2012)2737–2746.

[30]G.Schultz,Generalprinciplesofassaysforadenylatecyclaseandguanylate cyclaseactivity,MethodsEnzymol.38(1974)115–125.

[31]X.Pan,E.Arauz,J.J.Krzanowski,D.F.Fitzpatrick,J.B.Polson,Synergistic interactionsbetweenselectivepharmacologicalinhibitorsof phosphodiesteraseisozymefamiliesPDEIIIandPDEIVtoattenuate proliferationofratvascularsmoothmusclecells,Biochem.Pharmacol.48 (1994)827–835.

[32]A.Papapetropoulos,N.Marczin,M.D.Snead,C.Cheng,A.Milici,J.D.Catravas, Smoothmusclecellresponsivenesstonitrovasodilatorsinhypertensiveand normotensiverats,Hypertension23(1994)476–484.

[33]M.v.W.Rekowski,V.Kumar,Z.Zhou,J.Moschner,A.Marazioti,M.Bantzi,G.A. Spyroulias,F.vandenAkker,A.Giannis,A.Papapetropoulos,Insightsinto solubleguanylylcyclaseactivationderivedfromimprovedheme-mimetics,J. Med.Chem.56(2013)8948–8952.

[34]N.Li,K.Ragheb,G.Lawler,J.Sturgis,B.Rajwa,J.A.Melendez,J.P.Robinson, MitochondrialcomplexIinhibitorrotenoneinducesapoptosisthrough enhancingmitochondrialreactiveoxygenspeciesproduction,J.Biol.Chem. 278(2003)8516–8525.

[35]A.Papapetropoulos,M.Whiteman,G.Cirino,Pharmacologicaltoolsfor hydrogensulphideresearch:abriefintroductoryguideforbeginners,Br.J. Pharmacol.172(2015)1633–1637.

[36]S.Carballal,M.Trujillo,E.Cuevasanta,S.Bartesaghi,M.N.Möller,L.K.Folkes, M.A.García-Bereguiaín,C.Gutiérrez-Merino,P.Wardman,A.Denicola,R. Radi,B.Alvarez,Reactivityofhydrogensulﬁdewithperoxynitriteandother oxidantsofbiologicalinterest,FreeRadic.Biol.Med.50(2011)196–205.

[37]Y.Ju,W.Zhang,Y.Pei,G.Yang,H2Ssignalinginredoxregulationofcellular functions,Can.J.Physiol.Pharmacol.91(2013)8–14.

[38]B.A.Maron,Y.Y.Zhang,D.E.Handy,A.Beuve,S.S.Tang,J.Loscalzo,J.A. Leopold,Aldosteroneincreasesoxidantstresstoimpairguanylylcyclase activitybycysteinylthioloxidationinvascularsmoothmusclecells,J.Biol. Chem.284(2009)7665–7672.

[39]N.Sayed,P.Baskaran,X.Ma,F.vandenAkker,A.Beuve,Desensitizationof solubleguanylylcyclasetheNOreceptor,byS-nitrosylation,Proc.Natl.Acad. Sci.U.S.A.104(2007)12312–12317.

[40]P.Nagy,C.C.Winterbourn,Redoxchemistryofbiologicalthiols,Adv.Mol. Toxicol.4(2010)182–222.

[41]J.P.Stasch,A.J.Hobbs,NO-independent,haem-dependentsolubleguanylate cyclasestimulators,Handb.Exp.Pharmacol.(2009)277–308.

[42]P.Nagy,Mechanisticchemicalperspectiveofhydrogensulﬁdesignaling, MethodsEnzymol.554(2015)3–29.

[43]J.P.Collman,S.Ghosh,A.Dey,R.A.Decreau,Usingafunctionalenzymemodel tounderstandthechemistrybehindhydrogensulﬁdeinducedhibernation, Proc.Natl.Acad.Sci.U.S.A.106(2009)22090–22095.

[44]P.Nicholls,Theactionofanionsoncatalaseperoxidecompounds,Biochem.J. 81(1961)365–374.

[45]S.Nakamura,M.Nakamura,I.Yamazaki,M.Morrison,Reactionsofferryl lactoperoxidase(compoundII)withsulﬁdeandsulfhydrylcompounds,J.Biol. Chem.259(1984)7080–7085.

[46]Z.Palinkas,P.G.Furtmuller,A.Nagy,C.Jakopitsch,K.F.Pirker,M.Magierowski, K.Jasnos,J.L.Wallace,C.Obinger,P.Nagy,Interactionsofhydrogensulﬁde withmyeloperoxidase,Br.J.Pharmacol.172(2014)1516–1532.

[47]R.Pietri,E.Roman-Morales,J.Lopez-Garriga,Hydrogensulﬁdeand hemeproteins:knowledgeandmysteries,Antioxid.RedoxSignal.15(2011) 393–404.

[48]B.B.Rios-Gonzalez,E.M.Roman-Morales,R.Pietri,J.Lopez-Garriga,Hydrogen sulﬁdeactivationinhemeproteins:thesulfhemescenario,J.Inorg.Biochem. 133(2014)78–86.

[49]S.-I.Bibli,G.Yang,Z.Zhou,R.Wang,S.Topouzis,A.Papapetropoulos,Roleof cGMPinhydrogensulﬁdesignaling,NitricOxide46(2015)7–13.

[50]M.M.Cortese-Krott,G.G.C.Kuhnle,A.Dyson,B.O.Fernandez,M.Grman,J.F. DuMond,M.P.Barrow,G.McLeod,H.Nakagawa,K.Ondrias,P.Nagy,S.B.King, J.E.Saavedra,L.K.Keefer,M.Singer,M.Kelm,A.R.Butler,M.Feelisch,Key bioactivereactionproductsoftheNO/H(2)SinteractionareS/N-hybrid species,polysulﬁdes,andnitroxyl,Proc.Natl.Acad.Sci.U.S.A.112(2015) E4651–E4660.

[51]M.Eberhardt,M.Dux,B.Namer,J.Miljkovic,N.Cordasic,C.Will,T.I.Kichko,J. delaRoche,M.Fischer,S.A.Suárez,D.Bikiel,K.Dorsch,A.Lefﬂer,A.Babes,A. Lampert,J.K.Lennerz,J.Jacobi,M.A.Martí,F.Doctorovich,E.D.Högestätt,P.M. Zygmunt,I.Ivanovic-Burmazovic,K.Messlinger,P.Reeh,M.R.Filipovic,H(2)S andNOcooperativelyregulatevasculartonebyactivatinganeuroendocrine HNO–TRPA1–CGRPsignallingpathway,Nat.Commun.5(2014)4381.

[52]G.K.Kolluru,X.Shen,C.G.Kevil,Ataleoftwogases:NOandH(2)Sfoesor friendsforlife?RedoxBiol.1(2013)313–318.

[53]T.W.Miller,M.M.Cherney,A.J.Lee,N.E.Francoleon,P.J.Farmer,S.B.King,A.J. Hobbs,K.M.Miranda,J.N.Burstyn,J.M.Fukuto,Theeffectsofnitroxyl(HNO) onsolubleguanylatecyclaseactivity:interactionattheferoushemeand cysteinethiols,J.Biol.Chem.284(2009)21788–21796.

[54]Z.Altaany,Y.Ju,G.Yang,R.Wang,ThecoordinationofS-sulfhydration, S-nitrosylation,andphosphorylationofendothelialnitricoxidesynthaseby hydrogensulﬁde,Sci.Signal.7(2014)ra87.

[55]P.Panopoulos,G.Yang,A.Asimakopoulou,S.Topouzis,R.Wang,C.Szabo,A. Papapetropoulos,Selectivityofhydrogensulﬁdetowardscyclicnucleotide phosphodiesterases,NitricOxide47(2015)S39.

[56]S.-I.Bibli,I.Andreadou,A.Chatzianastasiou,C.Tzimas,D.Sanoudou,E. Kranias,P.Brouckaert,C.Coletta,C.Szabo,D.T.Kremastinos,E.K.Iliodromitis, A.Papapetropoulos,CardioprotectionbyH2SengagesacGMP-dependent proteinkinaseG/phospholambanpathway,Cardiovasc.Res.106(2015) 432–442.

[57]M.Follmann,N.Griebenow,M.G.Hahn,I.Hartung,F.-J.Mais,J.Mittendorf,M. Schäfer,H.Schirok,J.-P.Stasch,F.Stoll,A.Straub,Thechemistryandbiology ofsolubleguanylatecyclasestimulatorsandactivators,Angew.Chem.Int.Ed. Engl.52(2013)9442–9462.

[58]J.-P.Stasch,P.Pacher,O.V.Evgenov,Solubleguanylatecyclaseasanemerging therapeutictargetincardiopulmonarydisease,Circulation123(2011) 2263–2273.