effect of 1nitro2phenylethane in mice pancreatic acinar cells subjected to taurocholate : putative role of guanylyl cyclasederived 8nitrocyclicGMP

Cytoprotective

effect

of

1-nitro-2-phenylethane

in

mice

pancreatic

acinar

cells

subjected

to

taurocholate:

Putative

role

of

guanylyl

cyclase-derived

8-nitro-cyclic-GMP

Franc

¸

ois

Cosker

*

,

Francisco

J.B.

Lima,

Saad

Lahlou,

Pedro

J.C.

Magalha˜es

BiomedicalInstituteoftheBrazilianSemiarid(INCT-IBISAB-CNPq),DepartmentofPhysiologyandPharmacology,SchoolofMedicine,FederalUniversityof

Ceara´,Fortaleza,Ceara´ 60430-270,Brazil

1. Introduction

Acute pancreatitis (AP) is a common reason for hospital admission [1]. Its annual incidence ranges from 13 to 45 per 100,000personsworldwide[2–4].Initsnaturalhistory,theroleof reactiveoxygenspecies(ROS)productioninacinarcellshaslong

been debated and the efficacyof antioxidant regimen was not consistentlyestablished[5,6].Apartfromtheirdirectdetrimental oxidativeeffects,ROSandreactivenitrogenspecies(RNS)canalso function as second messengers in intracellular signaling [6]. Emergingevidencehasrevealedthatnitricoxide(NO)andROScan mediatesignaltransductionthatmaintainstheredoxstateofthe cell[7].Inmacrophages[8],astrocytes[9],heartmusclecells[10], and plant guard cells [11], NO and O2 react togethertoform

ONOO ,whichreactswithguanosinetriphosphate(GTP)toyield thenitrationproduct8-nitroguanosine-50_{-triphosphate} (8-nitro-GTP), a substrate for soluble guanylate cyclase (sGC) [12] that yields 8-nitroguanosine 30_,50_{-cyclic monophosphate} (8-nitro-cGMP).Thiscompoundisauniquesecondmessengerimplicated in the regulation of ROS signaling by reacting with particular proteins that possess a thiolmoiety toform cGMP adducts (S-guanylationreaction)[13].

In the pancreas, NO promotesthe exocytosis ofvesicles and secretory granules to prevent the intracellular accumulation of activated digestive enzymes during AP-inducing stimulus. By BiochemicalPharmacology91(2014)191–201

ARTICLE INFO

Articlehistory:

Received6June2014 Accepted28July2014 Availableonline12August2014

Keywords:

Acutepancreatitis Exocrinepancreas Bilesalts Guanylylcyclase 8-Nitro-cGMP

ABSTRACT

Thenitroderivative1-nitro-2-phenylethane(NPE)wasrecentlydescribedasacompoundpossessing heme-dependentsolubleguanylylcyclase(sGC)stimulatingpropertiesinvascularsmoothmusclecells. Inthisstudy,wetestedsuchpharmacologicalpropertyofNPEinmicepancreaticacinarcellssubjectedto thebilesalttaurocholate,atypeofpathologicalstimulusthatsimulatespancreatitis.Here,isolated acinarcellsweretreatedwithNPEinordertoassesstheroleofsGConthedetrimentaleffectsinducedby taurocholate.NPEreducedtaurocholate-elicitedCa2+_{overload,productionofreactiveoxygenspecies}

(ROS),apoptosis,necrosis,andexertedaprotectiveeffectagainstmitochondrialmembranepotential (DCm)dissipation.TheseNPE-inducedeffectswereabolishedbypretreatmentwithODQandKT5823, andaftertheblockadeofnitricoxide(NO)synthasewithL-NAME,inhibitorsofkeycomponentsofthe

sGC pathway. Contrarily to cGMP that alone increased DCm collapse and cell damage, the cytoprotectiveeffectofNPEonDCmandcellnecrosiswasalmostreproducedby8-nitro-cGMP,a secondmessengergeneratedbysGCunderoxidativestressconditions.Inconclusion,putativesGC stimulationwithNPErevealsitscytoprotectiveprofileonpancreaticcellssubjectedtotaurocholate. Moreover,ROSandNOconjunctlyappeartodrivesGCactivityinpancreaticacinarcellstoimplementan adaptivemechanisminresponsetooxidativeandCa2+_{stressthrough8-nitro-cGMPsynthesis.}

ß2014ElsevierInc.Allrightsreserved.

Abbreviations:DCm,mitochondrialmembranepotential;AP,acutepancreatitis;

8-Br-cGMP,8-bromoguanosine3’,5’-cyclicmonophosphate;CCK,cholecystokinin; CCCP,carbonylcyanidem-chlorophenylhydrazine;FAD,flavinadenine dinucleo-tide;GTP,guanosine-50_{-triphosphate;LawH}

2S,Lawesson’sreagent;L-NAME,N-v -nitro-L-argininemethylesterhydrochloride;mPTP,mitochondrialpermeability transitionpore;NADHandNAD+,nicotinamideadeninedinucleotide;NPE, 1-nitro-2-phenylethane;NO2-cGMP,8-nitroguanosine30_,50_{-cyclicmonophosphate;}

NO2-GTP,8-nitroguanosine-5’-triphosphate;ODQ,1H-[1,2,4]oxadiazolo[4,3-a ]quinox-alin-1-one;PI,propidiumiodide;PKG,proteinkinaseG;RNS,reactivenitrogen species;ROS,reactiveoxygenspecies;sGC,solubleguanylatecyclase;NO,nitric oxide;ATP,adenosine triphosphate;H2DCFDA,20,70-dichlorodihydrofluorescein

diacetate;DMSO,dimethylsulfoxide;NOS,nitricoxidesynthase. * Correspondingauthor.Tel.:+558533668334.

E-mailaddress:fffcosker@yahoo.fr(F.Cosker).

ContentslistsavailableatScienceDirect

Biochemical

Pharmacology

j our na l ho me p a ge : w ww . e l se v i e r . com / l oc a te / b i och e mph a rm

http://dx.doi.org/10.1016/j.bcp.2014.07.030

stimulatingsGCenzymaticactivity,NOalsoexertsacytoprotective effectsagainstotherinjuriousstimulitopancreaticacinarcells[14– 16]possibly by counteracting the intensity of pathological Ca2+

signalstowardmorenormalpatterns[17].Interestingly,cGMPitself

[18,19]doesnotreduceCa2+_{signalsinpancreaticacinarcells.Thus,}

itishypothesizedthatthebeneficialactionsmediatedbytheNO/ sGCpathwayinahighROSproduction statecouldpreferentially involvethesynthesisof8-nitro-cGMPinsteadofcGMPalone.

Inthepresentstudy,wetestedsuchhypothesisbyevaluating the effects of 1-nitro-2-phenylethane (NPE), a compound that inducesNO-independentsGCstimulationinaorticsmoothmuscle

[20,21],infreshlyisolatedpancreaticacinarcellssubjectedtoanex vivomodelofAP.Ourresultsstronglyarguefortheinvolvementof sGC stimulation induced by NPE, which decreases both the intensity of Ca2+ _{signaling and ROS production in pancreatic}

acinarcellsinresponsetothedetrimentalstimuluspromotedby thebilesalttaurocholate.Moreimportantly,NPEisrevealedasa potentcytoprotectiveagentthatmaintains

DC

manddecreases the rate of cell apoptosis and necrosis. Finally, appears to be revealedas animportant productof sGC in this cytoprotective effectofNPE.

2. Materialsandmethods

2.1. Preparationofmousepancreaticacinarcells

ThestudywasapprovedbyourEthicsCommittee(protocolno. 76/2013).Pancreaticacinarcellswerefreshlyisolatedfrommale Swiss mice (25–30g). The mouse was sacrificed by cervical dislocation,and cells were isolated by mechanical dissociation afterincubationwithcollagenaseIa(8min,75UI)aspreviously described[22].Pancreaticacinar cellswereusedwithin4h. To simulateAP,cellswereincubatedwith6mMtaurocholateat378C for40–60minundervariousconditionsdetailedbelow.

2.2. ROSmeasurements

CellswereincubatedwiththeROSindicator20_,70 -dichlorodi-hydrofluoresceindiacetate(H2DCFDA)(3

m

mol/l)for35minaway

from light at room temperature and then washed to be then disposedonaconfocalmicroscope(excitation/emission:488nm/ 500–550nm)at378C.Thelaserintensitywasloweredto0.8%,and thesamplingratewasreducedto1frame/20s.Aftertaurocholate, cellexposuretoROSwasestimatedbycalculatingtheareaunder thecurve(AUC)over15min.Theresultsareexpressedastheratio offluorescence(F)dividedbytheinitialfluorescence(F0)(F/F0).

2.3. MeasurementofCa2+_levels

CellswereincubatedwithFluo-4AM(5

m

mol/l)for30minat roomtemperature.After washing,cells wereseeded on poly-L -lysine-coated coverslips on the top of an inverted confocal microscope(Olympus,IX81)at 37₈C(excitation/emission:488/ 500–550nm;imagesamplingfrequency:1frame/5s).Theresults areexpressedastheratiooffluorescence(F)dividedbytheinitial fluorescence(F0)(F/F0).

2.4. Assessmentofthemitochondrialmembranepotential(

DC

m)

Cells were loaded with 10

m

mol/l tetramethylrhodamine methylester (TMRM), or with rhodamine 123 (10

m

M) for 10minat378C. Attheconcentrationsapplied, thefluorescence wasquenchedwhenthedyewasaccumulatedbymitochondria

TMRM-loadedcellswereseededonpoly-L-lysine-coated cover-slipsonthetopofaninvertedconfocalmicroscope(Olympus,IX81) at37₈Candmonitored15minafterseeding(excitation/emission:

543/550–650nm;imagesamplingfrequency:1frame/10s).Allthe perfusion solutions contained 300nM of the dye to avoid any leakage.TheresultsareexpressedastheratioF/F0.Anincreasein

DC

mmanifestsbyadecreaseintheTMRMratioandviceversa. Rhodamine123-loadedcells werepreincubated(8min)with NPE,LawH2S,KT5823,8-Br-cGMP,NO2-cGMP,ODQ,orL-NAME. Taurocholatewasthenaddedfor1hat378Candcellimageunder aconfocalmicroscope(488/500–550nm).Mitochondrial depolar-ization induces dye diffusion throughout the cytosol. After anonymization, cells were counted and the loss of contrast of mitochondriawasinterpretedasacollapseof

DC

m.

2.5. Apoptosisandnecrosisquantification

Cell apoptosis activation was detected using a caspase substrate, the rhodamine 110/aspartic acid amide (10

m

mol/l), whichreleasestherhodaminedyewithinthecellduringapoptosis. Propidiumiodide(PI)(10

m

g/ml)wasusedtodetectcellnecrosis. The cells were treated for 1hwith taurocholate and NPE and loaded with rhodamine 110/aspartic acid amide and PI. After washing,cellswereloaded(5min)withthenuclearstainHoechst 33342(50

m

g/ml)forcellcounting.Fluorescenceevaluationwas performed sequentially under confocal microscopy (Olympus, IX81; 488/500–530nm for R110/aspartic acidamide, 488/560– 660nm for PI, and 405/425–475nm for Hoechst 33342). A minimumof200cellswascountedforeachcondition.

2.6. Chemicals

Fluo-4AM,rhodamine123,rhodamine110/asparticacidamide, andHoechst 33342werefromInvitrogen(CARLSBAD,CA,USA). Poly-L-lysine, PI, Law H2S, collagenase Ia, H2DCFDA, carbonyl

cyanidem-chlorophenylhydrazone(CCCP),sodiumnitroprusside, N-

v

-nitro-L-arginine methyl ester hydrochloride (L-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one(ODQ),8-Br-cGMP,and KT 5823 were from SIGMA1 _{(St. Louis, MO, USA). NPE was}

extracted and purified as previously described [23]. It was dissolved in DMSO (SIGMA1_{, finalconcentration<0.02%). The}

extracellularsolutioncontained140mMNaCl,4.7mMKCl,10mM HEPES,1.13mMMgCl2,1mMCaCl2,and10mMglucose,pH7.4

withHCl(SIGMA1_{).8-Nitro-cGMPwaskindlygiftedbyDr.Takaaki}

Akaikegroup(TohokuUniversity,Sendai,Japan).

2.7. Statisticalanalysis

Thestatisticalanalysisofresultswasperformedusingone-way analysisofvariance(ANOVA)followedbytheBonferroniposthoc testorStudent’st-testwhenappropriate.ValuesofP<0.05were

considered statisticallysignificant. Theresultsare expressed as meanSEM.

3. Results

3.1. Preventionofthe

DC

mcollapsebyNPE

Firstly, cells were incubated with rhodamine 123, which accumulated and remainedinside themitochondria (A; T=0s, upper-left image). The dissipation of

DC

m by theinhibitor of oxidative phosphorylation carbonyl cyanide m-chlorophenyl hydrazone(CCCP,10

m

mol/l)inducedthedyediffusionintothe cytosol and loss of mitochondrial contrast (A; T=10–180s). Similarly,pancreaticacinarcellsexposedtotaurocholate(6mM), exhibited

DC

m depolarization in 70.05.4% of the cells, a percentage significantly higher than in vehicle-treated cells (Fig. 1B; 21.26.4%; n=5; P<0.001).The number of cellsthat

significantlyreducedwhentheywerepreincubatedfor8minwitha given concentration of NPE (20–325

m

mol/l; n=5). Although nonsignificant at 20

m

mol/l, the effect of NPE was maximal at 65

m

mol/l(Fig.3E,34.03.2%,P<0.001)andremainedsignificantat

higher concentrations (46.015.6 and 49.011.0% for 162 and 325

m

mol/lNPE,respectively).

Incubation oftetramethylrhodamine(TMRM)-loaded pancre-aticacinarcellswithNPE(65

m

mol/l)resultedinaslightdecrease inF/F0,indicatingmitochondrialhyperpolarizationthat,at15min

recordingtime,didnotattainstatisticalsignificanceincomparison withcontrol(Fig.1CandD).Asapositivecontrol,CCCP(10

m

mol/l) alsoinducedunequivocaldissipationof

DC

m(Fig.1C)andalostof Fig.1.ProtectiveeffectofNPEagainstmitochondrialdepolarizationcausedbytaurocholate:involvementofthesGC/PKGpathway.In(A)and(B),cellswereincubatedwith rhodamine123.LikeTMRM,rhodamine123accumulatedandremainedinsidethemitochondria(A;T=0s,upper-leftimage).Asinternalcontrol,panel(A)showsimagesof pancreaticacinarcellsincubatedwiththeinhibitorofoxidativephosphorylationCCCP(10mmol/l),whichdissipatedDCm,asindicatedbythedyediffusionintothecytosol andlossofmitochondrialcontrast(A;T=10–180s).In(B),asimilarprotocolusedtaurocholate(6mM;1hincubation)insteadofCCCP.Notice(B)thattaurocholate significantlyincreasedthepercentageofcellswithdepolarizedmitochondria,aphenomenonsignificantlydecreasedinthepresenceofarangeofNPEconcentrations(65– 325mmol/l).In(C)–(E),cellswereincubatedwithTMRM.In(C),NPE(65mmol/l)stabilizedandhyperpolarizedDCm,whiletaurocholate(6mM)inducedaslow depolarization(E).Theresultsareexpressedasmean SEM.,#_P

<0.001vs.vehicle;*P<0.05,**P<0.01,***P<0.001vs.Tauro(one-wayANOVAfollowedbyBonferroniposthoc test).Additionalcomparisonsbetweentreatmentsareindicatedbythehorizontallinesabovebars.

mitochondrial contrast similar to what we observed with rhodamine123(Fig.3A).Cellstreatedwithtaurocholate(6mM) showedaslowincreaseinF/F0indicativeof

DC

mdepolarization,a

phenomenonnot observed when cells were preincubated with NPE (65

m

mol/l; Fig. 1E). This deleterious process of

DC

m depolarizationelicitedbytaurocholatewasslowerandobservable after40minuntil1hincubation(pleaseseefurther).

3.2. InvolvementoftheNO/sGCpathwayintheeffectsinducedbyNPE

Fig.2showsthatthemitoprotectiveeffectofNPE(65

m

mol/l) was significantly lost in presence of ODQ, (25

m

mol/l; n=5;

Fig.2A)orKT5823(1

m

mol/l;n=5;Fig.2B),whichrespectively inhibit sGC and protein kinase G (PKG). Interestingly, in the presenceof Nv-nitro-L-arginine methyl esterhydrochloride (L -NAME;200

m

mol/l;Fig.2C),aninhibitoroftheNOsynthase,NPE nomoreprotectedpancreaticcellstosuffer

DC

mdepolarization inresponsetothebilesalt.Indeed,almostallofthepancreatic cells(95.52.9%)showed

DC

mdissipationafter6mM taurocho-lateinthepresenceofthemixtureNPEandL-NAME.Inaseparateset ofexperiments(Fig.2D),themitoprotectiveeffectofNPEwasalso not seen in pancreatic acinar cells treated with the H2S donor

Lawesson’s reagent (Law H2S; 12.5

m

mol/l), which revealed

DC

m dissipation in a percentage similar to control cells (

DC

m dissipationtauro/NPE/NaHS=68.08.9%,

DC

m

dissipation-tauro/NPE=44.83.4%;n=4;P<0.05).ItisnoteworthythatODQ,

KT5823,L-NAMEorLawesson’sreagentdidnotchange

DC

mwhen employedalone.Finally,weassessedthemitoprotectiveeffectofa NO-donor,sodiumnitroprusside(NPS),onpancreaticacinarcells challenged by taurocholate. As shown in Fig. 2E, NPS (1nmol/l to 1

m

mol/l) significantly prevented

DC

m dissipation at all concentration tested in about 50% of cells (

DC

m dis-sipationtauro=64.06.7%,

DC

m dissipationtauro/NPS 10–8 mol/

l=31.810.8%;n=4;P<0.01).

3.3. NPEdecreasesCa2+_{overloadandROSproductioninducedbybile} acidinjury

Both Ca2+ _{overloadand high ROS production trigger mPTP}

opening,whichinturnfacilitate

DC

mdissipation.Weassessed successivelytheeffectofNPEonthesetwoparametersusingthe lowest protecting NPE (65

m

mol/l) concentration which pre-vents

DC

mdissipation.Resemblingthephysiologicalpatternof cholecystokinin(CCK)[22],taurocholate(6mM)promotedCa2+

oscillations in virtually all of the Fluo-4 AM-loaded cells (Fig.3A),whichslowlyinducedcytoplasmicCa2+_accumulation

followedbyastrongCa2+_{spike(Fig.3A,arrow),plasmalemmal}

disruptionand consequent cell death. In the presence of NPE (65

m

mol/l; Fig. 3B), taurocholate-induced Ca2+ _{peaks showed}

decreased amplitude from 1.280.09 to 0.840.10 (Fig. 3B; n=4;P<0.01),butwithoutchangesintheirfrequency(datanot

shown). The areas under the curve of the Ca2+_{-elicited waves}

causedbytaurocholatewerelowerinthepresenceofNPEthanin itsabsence(Fig.3panelsCandD;AUCtauro=68.09.1,AUCtauro/ NPE=48.17.9; P<0.05). The addition of ODQ (25

m

mol/l) to

taurocholate+NPE-treated pancreatic cells abolished (P<0.05)

the reduction of the AUC caused by NPE (Fig. 3D; AUCtauro/ NPE=48.17.9,AUCtauro/NPE/ODQ=67.610.7).

TheindicatorofROSproductionH2DCFDArevealedaslowbut

continuousincreasein[ROS]i(n=5;Fig.3E)incellssubjectedto 6mM taurocholate. NPE (65

m

mol/l) and vehicle both slightly decreasedH2DCFDA-dependentbaselinefluorescenceby

respec-tively 7.25.3% and 7.94.7% (n=7, Fig. 2G). However, only preincubationofNPE(65

m

mol/l)decreasedsharplythe[ROS]Isignal

aftertaurocholatechallenge(Fig.3FandG;

D

F/F0tauro=0.40.13,

D

F/F0NPE/tauro=0.170.14).

3.4. NPEprotectagainstnecrosisandapoptosis

BecauseNPE decreasedboth theintensityof theCa2+_signal

inducedbytaurocholateand

DC

mdissipation,wetestedwhether italsopreventscellnecrosis.After1hexposuretotaurocholate (6mM), cells loaded with PI and Hoechst were counted (Fig.4A)and 48.05.0% (n=5) ofthe cellsrevealed necrosis,a value higher than in cells not treated with the bile salt (necrosisControl=13.43.6%;Fig.4B).Asignificantdecreaseinthe

amountofdeadcellsbynecrosis(24.64.7%;n=5;P<0.001)was

observedinthecellstreatedwithtaurocholateandNPE(65

m

mol/l),a remarkable cytoprotection if we take into account that NPE was added 5min after taurocholate. We also observed that NPE’s cytoprotectiveeffectdecreasedat325

m

mol/l(34.011.3%;n=5), whileitalmostinhibitedtaurocholate-inducedeffectat650

m

mol/l (Fig.4B)(17.011.7%;n=5).

Then, we assessed the cytoprotective effect of NPE in cells challenged40minwithtaurocholate(6mM).Asrevealedbythe fluorescent dye rhodamine 110/aspartic acid amide, a caspase substrate, NPE (65

m

mol/l) completely reversed taurocholate-inducedproapoptoticeffect(Fig.4AandB).Inadifferentsetof experiment,incubationwithLawH2S(12.5

m

mol/l),preventedthe

protective effect of NPE against taurocholate. The number of apoptoticcellsunderthesecircumstanceswas63.74.0%(n=3),a valuethatdidnotattaindifferenceincomparisonwithtaurocholate group(P>0.05).

3.5. NO2-cGMP,butnotcGMP,protectsagainsttaurocholate-induced collapseof

DC

m

Fig. 5A shows that whereascontrol cells had the indicator rhodamine 123 accumulated inside the mitochondria (left image), cells treated with taurocholate (6mM) showed dye diffusionintothecytosol(rightimage),aphenomenonindicative of

DC

mdepolarization.Thepercentageofcellswithdepolarized

DC

minresponsetotaurocholatewas61.22.6%(n=5,P<0.05)

andpreincubationwithagivenconcentrationof8-bromoguanosine 30_,50_{-cyclicmonophosphate(8-Br-cGMP;0.3–100}

_m

_mol/l)wasnot abletodecreasethisrate.Indeed,theamountofcellsshowing

DC

m depolarization was significantly higher in the presence of low concentrationof8-Br-cGMP(1

m

mol/l,74.45.2%;P<0.05)than

initsabsence(Fig.5B).Inaseparatesetofexperiments(Fig.5C), pancreaticacinarcellsshowedasignificantdecreaseinthenumber ofcellswithtaurocholate-induced

DC

mdissipationwhen incubat-edwitharangeof8-nitro-cGMPconcentrations(0.3–100

m

mol/l) (

DC

m dissipationTauro=76.22.5%,

DC

mdissipation

Tauro/8-nitro-cGMP100mmol/l=52.04.2,n=5,P<0.001).

3.6. NO2-cGMP,butnotcGMP,protectsagainsttaurocholate-induced necrosis

Finally, we determinedthe effect of different concentrations (1–100

m

mol/l)of8-Br-cGMP(Fig.6A)and8-nitro-cGMP(Fig.6B) on taurocholate-induced cell necrosis. Incubation of pancreatic acinar cell with 8-Br-cGMP (1–100

m

mol/l) only tended to increase taurocholate-induced cell necrosis (Fig. 6A; necrosisTauro=46.51.2%, necrosisTauro/8-Br-cGMP at 1 mmol/l=52.8

2.2%),butthiseffectfailedtobestatisticallysignificant(P=0.053, n=4). Contrarily, 8-nitro-cGMP clearly decreased the amount of deadcells.Amongtheconcentrationstested,1

m

mol/l8-nitro-cGMP revealed thehighest protecting effect against taurocholate-elicited necrosis(Fig.6B;necrosisTauro=47.83.1%,necrosisTauro/8-nitro-cGMP1

mmol/l=31.42.2%,n=5,P<0.001).Intriguingly,thisprotectiveeffect

of8-nitro-cGMPdecreasedwhenhigherconcentrationwereusedand disappearedat100

m

mol/lNO2-cGMP.Totestthehypothesisthata

Fig.2.ProtectiveeffectofNPEagainstmitochondrialdepolarizationcausedbytaurocholateinvolvestheNO/sGCpathway.Theeffectsoftaurocholatealoneorincombination withNPEwerecomparedinpancreaticcellstreatedwithODQ(25mmol/l)(A)orKT5823(1mmol/l)(B).NoticethateitherODQorKT-5823pretreatmentfullyreversedthe NPE(65mmol/l)-inducedmitoprotectiveeffect.PancreaticcellschallengedwithtaurocholateandNPEintheabsenceorpresenceofL-NAME(200mmol/l)(C)orLawH2S

(12.5mmol/l)(D)revealedthatbothtreatmentsreversedtheNPE-inducedmitoprotectiveeffect,especiallyinL-NAME-treatedcells.TheNO-donor,NPS(1nmol/lto1mmol/

l)(E),alsohasamitoprotectiveeffect.Theresultsin(A)–(E)areexpressedasmean SEM.#_{P<0.001vs.vehicle;*P<0.05,**P<0.01,***P<0.001vs.Tauro(one-wayANOVA}

followedbyBonferroniposthoctest).Additionalcomparisonsbetweentreatmentsareindicatedbythehorizontallinesabovebars.

Fig.3.Taurocholate-inducedCa2+_{signalsandROSproductionaremodulatedbyNPE.(A–C)RepresentativetracesthatshowtherelativeFluo-4fluorescence(F/F} 0)ofcells

treatedwith6mMtaurocholatealone(Tauro,A),afterincubationwith65mmol/lNPE(B),orinthepresenceof65mmol/lNPEplus25mmol/lODQ(C).Thearrow(inA) indicatesplasmamembranedisruption.Theareaundercurve(AUC;D),representingtheoverallCa2+_{loadinpancreaticacinarcells,wasquantifiedunderdifferentconditions.}

its cytoprotective effect at high concentrations, acinar cells were incubated with DL-propargylglycine (PAG), a cystathionine

g

-lyase inhibitor, whichimpairs H2S synthesisand, consequently,

8-nitro-cGMPmetabolizationincGMP[10].AsshowninFig.6C,whilePAG (3mM)didnotsignificantlymodifythenecrosisrateofacinarcells

exposed to taurocholate (6mM) and 8-nitro-cGMP (1

m

mol/l) in comparisonwithcellsnotexposedtoPAG,thecytoprotectiveeffectof thehigher8-nitro-cGMPconcentration(50

m

mol/l)wassignificantly improved(necrosisTauro/8-nitro-cGMP1mmol/l=35.54.3%,necrosisTauro/

8-nitro-cGMP50mmol/l=29.22.5%,n=4).

florescence(F/F0)recordedinpancreaticcellsexposedto6mMtaurocholatealone(E)orinpresenceof65mmol/lNPE(F).ROSexposurecausedbytaurocholatewas

quantifiedastheDF/F0withorwithoutpreincubationwithNPE(G).Theresultspresentedareexpressedasmean SEM.*P<0.05,#P<0.001vs.vehicle;***P<0.001vs.Tauro (one-wayANOVAfollowedbyBonferroniposthoctest).Thecomparisonbetweentreatmentsisindicatedbythehorizontallinesabovebars.

Fig.4.NecrosisandapoptosisinpancreaticcellssubjectedtotaurocholatearereducedbytreatmentwithNPE.In(A),imagesshownucleistainedwithHoechst(bluestaining) andnecrosiswasevaluatedbystainingcellswithpropidiumiodide(PI;redstaining),after1htreatmentwithtaurocholate(Tauro,6mM).Noticethedramaticreductionof necrosis(rightimageandpanelB)incellstreatedwith65mMNPEadded5minaftertheinitiationofpancreatitisbytaurocholate(Tauro+NPE).In(C),imagesshow evaluationofapoptosisinpancreaticacinarcellsstainedwithanindicatorofcaspaseactivation(fluorescentgeneralcaspasesubstrate)observed40minaftersaline(vehicle) ortaurocholate(5mM)intheabsence(Tauro)orpresenceofNPE(65mmol/l;Tauro+NPE).In(D),LawH2S(12.5mmol/l)preventedtheprotectiveeffectofNPE(65mmol/l)

againsttaurocholate.Theresultsareexpressedasmean SEM.(#_{P<0.001vs.vehicle;*P<0.05,***P<0.001vs.Tauro)(One-wayANOVAfollowedbyBonferronitest).Scalebars}

20mmol/l.

4. Discussion

GallstonesaretheleadingcauseofAP[24],andtheinterruption ofsuchapathophysiologicalprocessisstillachallenge,especially whenconsideringthatAPcanbelife-threateningandcurrentlyhas nospecifictreatment.Invivo,ductalinfusionoftaurocholateisa widelyemployed model toinduce experimental AP because it reproducespancreaticedema,hemorrhageandnecrosisinseveral animals [25]. In the present study, taurocholate triggered Ca2+

oscillationsthatwereinitiallyverysimilartophysiological Ca2+

signals. Then, over time, we observed a progressive Ca2+

accumulationwhichprecededplasmamembranedisruptionand celldeath.Suchinvitrofindingsarecompatiblewiththeexpected cellphenomenathatareconsideredcrucialtotriggerAPdisease

[26,27].Taurocholateconcentrationsemployedhereinareclose,or evenwithin,thenaturallycriticalmicellarconcentrationrange(1– 5mM)inbileducts.Asamatteroffact,ithasbeenreportedthat taurocholate can reach concentration above 20mM in patients withgallstones[28].Effectsofbileacidswereattributedtotheir interactionswithG-protein-coupledbileacidreceptor-1(GPBAR1) inapicalsideofpancreaticacinarcells[29],butconsideringthe physiologicalroleofbileacidsindietaryfatabsorption,adetergent

action should be also considered. However, the present study showed that changes in the deleterious effects caused by taurocholatemaybeselectivelymodulatedbyNPE,thus preclud-ingtheoccurrenceofunspecificlossofthemembranepermeability inresponsetoputativedetergenteffectsoftaurocholate.

NPEsignificantlyprotectedpancreaticacinarcellsagainstthe deleteriouseffectsoftaurocholateandstimulationofsGCappears tobethemodebywhichitinducessuchactions.Itreducedthe intensityofCa2+_{signalsandintracellularROSaccumulationand}

stabilized

DC

m, resulting in decreases in both apoptosis and necrosis.ThemodulationofCa2+_{spikesappearstobeinvolvedin}

thecytoprotectiveeffectofNPEsinceCa2+overloadinpancreatic acinar cells has been implicated as a cardinal event in the establishmentofpancreatitis[30].Inapreviousstudy,ithasbeen demonstratedthattheactivationofsGCdecreasestheintensityof pathological Ca2+ _{signals induced by CCK in acinar cells and}

protectsagainstcaerulein-inducedpancreatitis[17].Similarly,we observedthatNPEdecreasedtheintensityofCa2+_{signalsinduced}

bysupraphysiologicalCCKconcentration(datanotshown). Likeinthevascularsmoothmuscle[20],theeffectsinducedby NPEwerereversedbyODQandKT5823,findingsthatsupportthe involvement of sGC and PKG on its actions. In this study, it Fig.5.ProtectiveeffectofNPEagainstmitochondrialdepolarizationcausedbytaurocholateinvolves8-nitro-cGMPratherthancGMPsynthesis.Rhodamine123accumulated andremainedinsidethemitochondria(A;leftimage,CTnegasnegativecontrol).Incubationwithtaurocholate(6mM)dissipatedDCm,allowingthedyetodiffuseintothe cytosol(A;rightimage).Taurocholate(6mM;1hincubation)significantlyincreasedthepercentageofcells(#_{P<0.001,vs.vehicle)thatexhibited mitochondrial}

appearedlogicalthatcGMP,themostknownproductofsGC,could bedirectlyresponsibleforthesebeneficialeffectsofNPE.However, the cGMP analog 8-Br-cGMP did not reproduce any of its cytoprotective effects. Contrarily, 8-Br-cGMP revealed rather a detrimentalprofileworseningthemaintenanceof

DC

mandcell survivalat amicromolarconcentration. Thus, itisunlikely that cGMPisinvolvedinthemodulationoftaurocholate-inducedCa2+ signalinginducedbyNPE.Inlinewiththishypothesis,ithasbeen reportedthatcGMPactivatesIP3synthesisbyphospholipaseCand

thecapacitiveCa2+_{entryinpancreaticacinarcells[18,31].These}

phenomenastronglyparticipatetotheCa2+_{overloadinvarious}

modelsofAP.Thus,thesynthesisofonlycGMPwouldfavorCa2+

overloadbutnotitsreductionperse.

During AP,the Ca2+_{overloadresponsiblefor cell necrosisis}

increased by mitochondria dysfunction [23]. Indeed, bile acids induce an excessive ROS production and a decrease of ATP synthesis[32],whichtogether,mayinduceamarkedinhibitionof

the plasma Ca2+_{-ATPase [33]. Under the present experimental}

conditions,NPE pretreatmentsignificantlyslowed taurocholate-inducedH2DCFDAfluorescenceincrease,reflectingadecreasein

ROS production or an increase in cell antioxidant activity.We hypothesize that NPE-dependent Ca2+_{modulationcouldnot be}

entirely explained by the inhibition of the intracellular ROS accumulation.Indeed,theuseofantioxidantsinAPisnotsufficient topreventcelldeath,anditcanevenincreasecelldeathbynecrosis

[5,6,34].Interestingly,weobservedthatNPE,andNO2-cGMP,also

prevented

DC

mcollapse and couldeven inducemitochondrial hyperpolarization.Noteworthy,8-nitro-cGMPhasbeenreported asanovelNOderivativeinvolvedonsignaltransductioninhealth and disease [34,35].Therefore, it appearsthat NPE, through 8-nitro-cGMP synthesis, maintains mitochondrial integrity and function.Theseresultssupportthehypothesisthat8-nitro-cGMP couldbeasecondmessengersynthesizedunderstressconditions toregulatecelloxidativestate.Nonetheless,itisnoteworthythat Fig.6.8-Nitro-cGMPbutnot8-Br-cGMPreducedcellnecrosisinducedbytaurocholate.Thenumberoftaurocholate-inducednecroticcellswasevaluatedinpresenceof 8-Br-cGMP(1–100mmol/l;panelA)or8-nitro-cGMP(1–100mmol/l;panelB),butonly8-nitro-cGMPdecreasedthenumberofcellsthatenteredinnecrosis.InC,theinhibitionof 8-nitro-cGMPmetabolismbyPAGrestored8-nitro-cGMPdose-dependentcytoprotectiveeffect.Theresultsareexpressedasmean SEM.#_{P<0.001,vs.vehicle,*P<0.05,}

**P<0.01,***P<0.001(one-wayANOVAfollowedbyBonferronitest).Additionalcomparisonsbetweentreatmentsareindicatedbythehorizontallinesabovebars.

8-nitro-cGMPonlyprevented

DC

mdissipationbytaurocholateat concentrationsequalorhigherthan10

m

mol/l,whileitprevented abouthalfofcellnecrosisat1

m

mol/l(pleaseseeFig.6).Atthe presentmoment,wedidnotfindanyclueforsuchdiscrepancybut wecanspeculatethatitcouldbeexplainedbyastrongglycolytic ATPsynthesis tofueltheATPases anda separatestudywillbe neededtoinvestigatewhetherNPEand8-nitro-cGMPrestoreATP productionduringAPstatus.

The dissipation of

DC

m is an earlymarker of the onset of apoptosis, whereas its complete collapse can be a marker of necrosis.Ourresultsalsoarguefortheinvolvementofthe sGC/8-nitro-cGMPpathwayinthepreventionof

DC

mcollapsebecause theinhibitionofthispathwayeitherbyODQorL-NAMEincreased cellulardamageinducedbythebilesalttaurocholate.Althoughthe relaxanteffectsofNPEoccurredinaNO-independentmannerin vascularsmoothmuscle[20],thepresentfindingsrevealthatNPE somehowrecruitsNOinpancreaticacinarcellstostrengthenits endogenousdefensemechanisms.Interestingly,arecentstudyby Buchwalowetal.reportedupregulationoftheentireNOsignaling machinery in pancreatitis, especially within the exocrine com-partment [35]. Therefore, under stressful NO-producing condi-tions, intracellular nitration reactions could occur to yield byproductssuchasNO2-GTP,whichisconvertedto8-nitro-cGMP

bythedualactionofsGC[7].OurexperimentswithL-NAMEand ODQreinforcethehypothesisthatthismechanismactuallypasses

throughtheactivationofsGCanditisnotadirectnitrosylation reactionofcGMP.Putatively,thehighintracellularavailabilityof theelectrophilic nucleotide NO2-GTPmay beimportant tothis

enzymaticconversionandtopreventionofcelldeathinpancreatic acinarcellsduringAP(Fig.7).

Usingapharmacologicalapproach,wefurtherelucidatedhow 8-nitro-cGMPtriggersitsadaptivemechanisminacinarcells.Like cGMP,8-nitro-cGMPactsthroughPKGactivation.Additionally,it actsthroughposttranslationalmodificationsofproteinthiolsvia cGMPadduction(proteinS-guanylation)[13].Indeed,8-Br-cGMP was unable toreproduce the cytoprotective effectof NPE. The experimentwithKT5823showedthatPKGwasnecessaryforthe protective effectof NPE. Such resultssoundimportant because theysupportthehypothesisthatthemitoprotectiveeffectofNPE cannotbeexplainedbycGMP,especiallywithinthephysiological range of concentrations used herein. At low concentration (1

m

mol/l),8-Br-cGMPincreased

DC

mdissipation.Ifthe activa-tion of PKG was necessary but insufficient by itself to confer complete protection for pancreatic cells, thus S-guanylation appearedtohave moreof aprotective influencein thepresent protective effects of NPE. Such reasoning is supported by the reversalofthesemitoprotectiveeffectsafterthesulfhydrationof 8-nitro-cGMPwithLawH2S,confirmingthatthismoleculeensures

theimplementationofanadaptiveresponsetooxidativestressin different cell types [13,36]. Moreover, we showed that the

L-Arg

NO

NOS

O

2

-ONOO

-GTP

8-nitro-cGMP

cGMP

sGC

PKG

Tauro

cholate

S-guanylatio

n ?

Adaptativ

e ce

ll res

ponse:

Δψm s

tabiliz

ation

8-nitro-GTP

ODQ NPE

ODQ NPE L-NAME

ROS

KT-5823 8-Br-cGMP

Lack of

adaptative ce

ll

response

and necros

is

LawH2S PAG

8-nitro-cGMP

&

Ca

2+

&

NPS NPS

progressivelossofcytoprotectiveeffectsusinghighconcentrations of 8-nitro-cGMP could be reverted be the inhibition of its metabolismincGMP.Theseresultsareinagreementwithprevious resultsincardiactissue[10].

ThepresentstudyrevealsthatthesGCstimulatingpropertiesof NPEispotentiallyofconsiderablepathophysiologicalimportance. NPE prevented necrosis in pancreatic acinar cells exposed to taurocholate,mostprobablythrough8-nitro-cGMPsynthesisby thesGC. Altogether, thesetwo biological products of thesame enzyme, namely sCG, appear to have opposite effects on cell survival in AP (Fig. 7). Supporting this possibility, the total dissipationof

DC

mandcompletelossoftheprotectiveeffectof NPEobservedaftertheblockadeofNOsynthesisbyL-NAMEare highlyrevealing.Indeed,intheabsenceofNO,8-nitro-cGMPcould notbefurther synthesized, andsubsequentsCG stimulationby NPEonly ledtocGMP production and celldeath. Thus, likean adaptationtostresspreviouslydescribedinplantguardcellsin whichcGMP and8-nitro-cGMPwereshowntoplay differential rolesinthesignalingpathwaysandcontrolstomataopeningand closing[11],thepresentstudydemonstratesthatoxidativestress andNOtogethermodulatealsoinvertebratestheeffectsofcGMP andinduceanadaptivemechanisminresponsetooxidativeand Ca2+_{stressinpancreaticacinarcells.}

Acknowledgements

TheauthorsthankDr.J.G.Maia,Dr.P.J.Sousa,Dr.M.H.Souza, andDr.L.V.Costa-Lotufoforkindlyprovidinguswith 1-nitro-2-phenylethane,LawH2S,Rhodamine123andPI.Theauthorsalso

expressmanythankstoDr.TakaakiAkaikeforthesynthesisand kind donation of NO2-cGMP. This work was supported by the

ConselhoNacional deDesenvolvimentoCientı´fico eTecnolo´gico (CNPq; grant no. 573928/2008-8 INCT) and Fundac¸a˜o Cearense paraoDesenvolvimentoCientı´ficoeTecnolo´gico(FUNCAP).

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