cytoprotection by prostaglandins in rats

GASTROENTEROLOGY77:433-443,1979 ALIMENTARYTRACT CytoprotectionbyProstaglandinsinRats PreventionofGastricNecrosisProducedby Alcohol,HCl,NaOH,HypertonicNaCl,and ThermalInjury ANDRkROBERT,JAMESE.NEZAMIS,CLEOLANCASTER, andALEXANDERJ.HANCHAR DepartmentofExperimentalBiology,TheUpjohnCompany,Kalamazoo,Michigan Oraladministrationtofastedratsofeitherabsolute ethanol,0.6 Nhydrochloricacid,0.2 Nsodiumhy- droxide,25% sodiumchloride,orboilingwaterpro- ducedextensivenecrosisofthegastricmucosa.Pre- treatmentwithseveralprostaglandinsoftheA,E,or Ftype,eitherorallyorsubcutaneously,prevented suchnecrosis,andtheeffectwasdose-dependent. Thispropertyofprostaglandinsiscalledcyto- protection.Theprotectiveeffectagainstoralad- ministrationofabsoluteethanolwasalreadymaxi- mal1minafterPGE,givenorally,and15-30min afterPGE,givensubcutaneously.Cytoprotectionby prostaglandinsisunrelatedtotheinhibitionofgas- tricacidsecretionsince,(a)itismaximalatdoses thathavenoeffectongastricsecretion,and(b)anti- secretorycompounds(cimetidine,methscopolamine bromide)andantacidsarenotcytoprotective.AI- thoughthemechanismofgastriccytoprotectionis unknown,prostaglandinsappeartoincreasethere- sistanceofgastricmucosalcellstothenecrotizing effectofstrongirritants.Theseresultssuggestthat certainprostaglandins,byamechanismotherthan theinhibitionofgastricacidsecretion,maintainthe cellularintegrityofthegastricmucosa,andmightbe beneficialinthetreatmentofavarietyofdiseasesin whichgastricmucosalinjuryispresent. Severalprostaglandins(PG)havebeenshowntoin- hibitgastricsecretioninanimals-andhumans.4-6 ReceivedOctober13,1978.AcceptedFebruary27.1979. Addressrequestsforreprintsto:AndreRobert,M.D.,Ph.D,De- partmentofExperimentalBiology,TheUpjohnCompany,Kala- mazoo,Michigan49001. TheauthorsthankDr.EugeneD.Jacobson,UniversityofCin- cinnatiCollegeofMedicine,forhisvaluablesuggestions,andfor proposingthetermcytoprotectionafterexaminingourdata. 01979bytheAmericanGastroenterologicalAssociation. 0016-5085/79/090433-11/$02.00 Theyalsopreventulcerformationinanimals,,and accelerateulcerhealinginhumans.*.Itwasusually assumedthattheantiulcereffectwasduetothein- hibitionofgastricacidsecretion.Wenowpresent evidencethatPGcanpreventtheformationofgas- triclesions,evenmucosalnecrosis,producedinrats byavarietyofnecrotizingagents,withoutreducing gastricacidsecretion.Thispropertyofprostaglan- dinsiscalledcytoprotection.Anabstracthasbeen publishedearlier. Methods Inallthestudiesreportedhere,femalerats(Up- johnratsderivedfromtheSprague-Dawleystrain)ofa bodyweightof205-215gwereused.Food,butnotwater, wasremovedinthemorning.At~:OO pm,waterwasalso withheld,andtheanimalswereplacedinindividualcy- lindricalstainless-steelcageswithflatbottomsandwith perforationstoallowventilation.Thesecageslimitedtheir movementsandthuspreventedtheingestionofhairand feces.Thisprocedure,asdescribedearlier,didnotappear tobestressful;actually,theanimalswereoftenobserved tobeasleepinsuchcages.Onthefollowingmorning,vari- ousnecrotizingagents(listedbelow)weregivenorally, andtheanimalswerekilled1 hrlater.Theirstomachs weredissectedout,openedalongthegreatercurvature, andthemucosawasexaminedbyanobserver,whowas unawareofthetreatmentgiven,witha2~binocularmag- nifierforthepresenceofnecroticlesionswhichwere counted. Theprostaglandinswereadministeredatmultipledose levels.Theresultswereplottedonsemilogpaper.Theor- dinateshowseithertheaveragenumberoflesionsper stomachassuchorasapercentofthecontrolvalues.The abscissashowsthedosesonalogscale.TheED,,,isthe doseofPGreducingtheaveragenumberoflesionsper stomachbyhalfincomparisonwithcontrolvalues,asde- terminedbygraphicinterpolationonaplotofthedoseon alogscale.Thenumberofanimalspergroupisindicated inthetablesandlegendsforfigures.Inallstudies,theStu- 434ROBERTETAL. GASTROENTEROLOGYVol. 77. No. 3 dentst-testwasusedtodeterminestatisticalsignificance. GastricLesionsProducedbyVarious NecrotizingAgents Oneofthefollowingagentswasgivenorallyina volumeof1ml:absoluteethanol,0.6NHCl,0.2NNaOH, 25%NaCl,andboilingwater.Inthecaseofboilingwater, theanimalswerefirstanesthetizedwithsodiummethoxi- tal(BrevitalSodium,EliLillyandCo.),12mgin0.6mlin- traperitoneally,topreventpain.Anesthesiawasfoundnot tointerferewiththeproductionofa gastricburn. EffectofPGonGastricLesionsProducedby SeveralNecrotizingAgents SeveralPGwereadministeredatvariousdoses,ei- therorallyorsubcutaneously,30minbeforeadministra- tionofeachofthenecrotizingagentsmentionedabove. ThesePGwere:PGE,,16,16-dimethylPGE,,15(S)-15- methylPGF,,,15(R)-15-methylPGF,,,and16,18dimethyl PGA,.ThePGwerefirstdilutedwithafewdropsof95% ethanolandthenmadeuptoavolumeof1 mlwitheither water(oral)orsaline(subcutaneous).Theanimalswere killed1 hrafterreceivingthenecrotizingagents. ComparisonofOralandSubcutaneous Routes BothPGE,and16,16-dimethylPGE,weregivenat variousdoselevels,eitherorallyorsubcutaneously,30 minbeforeoraladministrationof1 mlabsoluteethanol. Theanimalswerekilled1 hrafterreceivingethanol. OnsetofActionandDurationofAction PGE,(500pg/kg)wasgivenorallyorsubcutane- ouslyatvarioustimesupto5hrbeforeoraladministra- tionof1 mlofabsoluteethanol.Controlanimalsreceived 1mlofvehicleatthesametimeintervalsbeforeadminis- trationofethanol.Theanimalswerekilled1 hrafterre- ceivingethanol. EffectoftheVolumeofDiluent Tofindoutwhether,whenthePGweregiven orally,thecriticalfactorwasthetotalamountofPGorthe concentrationofPG,threedosesofPGE,wereadminis- teredinvariousvolumesofdiluent,namely0.5,1.0,and 2.0ml.ThedosesofPGE,were10,25,and100pg/kg.Ab- soluteethanol(1ml)wasgiven30minlater,andtheani- malswerekilled1 hrafterreceivingethanol. 16,26-DimethylPGE,andAbsoluteEthanol, bothGivenafterPylorusLigation Inafirststudy,boththenecrotizingagent(abso- luteethanol)andthecytoprotectiveagent(16,16-dimethyl PGE,)wereadministeredorallyafterligationofthepy- lorus,toseewhetherthePGcouldprotectbydirectcon- tactwiththegastricmucosa.Thepyloruswasfirstligated underetheranesthesia.16,16-DimethylPGE,wasthen givenorally2minlaterat1.5and5.0pg/kg.Tenminutes afterthePG,1mlofabsoluteethanolwasgivenorally. Theanimalswerekilled1 hrlater. Inanotherstudy,16,16-dimethylPGE,wasinjectedsub- cutaneouslyimmediatelyafterpylorusligation,at1.5and 5.0pg/kg.Thirtyminuteslater,inordertoallowenough timeforabsorptionofthePGfromtheinjectionsite,1 ml ofabsoluteethanolwasadministeredorally.Theanimals werekilled1 hrafterreceivingethanol. EffectofCytoprotectivePGonGastric Secretion TofindoutwhethercytoprotectivePGaffected gastricacidsecretion,severalPGweretestedintwodif- ferentpreparations:(a)Shayrats(pylorusligated),and(b) intact,unoperatedrats.TheadvantageoftheShayratis thatitpermitscollectionofallgastricjuicethataccumu- latesduringagiventimeinterval.Theadvantageofthein- tact,unoperatedratisthatthesecretionobtainedisinflu- encedonlybythetreatmentgiven,inthiscaseaPG,andis notalteredbypylorusligation,aprocedureknownto stimulategastricacidsecretion. ShayRats.Aftera24hrfast,theanimalsreceived orallyeither1mlofwater(controls)orofPG.ThePGs usedandtheirdosesareshowninTable2.Onehourlater, thepyloruswasligatedunderetheranesthesia.Theani- malswerekilled4hrafterpylorusligation,andgastric juicewascollected.Thevolumewasmeasured,andthe acidcontentwastitratedwithNaOH0.1NtopH7witha glasselectrode,usinganautomatictitrator(Copenhagen Radiometer).Theresultswereexpressedinmeq/liter (concentration)andmeq/4hr(output).ThedosesofPG werechosentobefrom40-500timesgreaterthanthecyto- protectivedosesinordertofindoutwhethertheseprosta- glandinsaffectedgastricsecretionevenwhengivenat dosesmuchinexcessofthecytoprotectivedose. Intact,UnoperatedRats.Theanimalswerefasted for24hrasaboveandthengiven16,16-dimethylPGE,,1 pg/kgorallyin1 ml.Thisdoseis20timeshigherthanthe cytoprotectiveED,,.Groupsof12ratswerekilledatthe followingintervalsaftertreatment:1,5,15,and30min. Controlsreceived1mlofwaterorallyandwerekilledat thesametimeintervals.Atautopsy,theesophagusandthe pyloruswereclamped,andgastricjuicewascollectedinto atesttube.Sincetherewastoolittlesecretionfordirect titration,thevolumewasfirstmeasured,andthen1mlof waterwasaddedtothetesttube.Theacidityofthedi- lutedjuicewasdeterminedbytitrating0.5mlwith0.1N NaOH.Theamountofacidinthestomachwascalculated asfollows: Vol.ofjuiceVol.ofjuicepeq/ literof beforedilutionxafterdilutionxdilutedjuice~2 Vol.ofjuiceafterdilutionminus1 mlX 1000 Acidin = stomach in peq September1979 GASTRICCYTOPROTECTIONBYPROSTAGLANDINS435 Example:0.4 x1.4X 6200 X 2 = 17.36peq/stomach [1.4- 1.01x 1000 Thisstudywasdonetofindoutwhethergastricacidse- cretionwasdecreasedatanytimeduringthe30minafter treatmentwithaPG,since,inotherexperimentsreported here,necrotizingagentswereadministered30 minaftera PG. EffectofAntisecretoryDrugsandofAntacids onGastricNecrosisProducedbyAbsolute Ethanol Twogastricantisecretorydrugs,cimetidine(ahis- tamineH,receptorantagonist)50mg/kgintraperitoneally, andmethscopolaminebromideorPamine@(ananti- cholinergic,UpjohnCo.)10mg/kgsubcutaneously,were giveneither30 min(cimetidine)or1hr(methscopolamine bromide)beforeoraladministrationof1mlofabsolute ethanol.Controlanimalsreceivedsaline.Twoantacids, sodiumbicarbonate0.15MandaTrisbufferatpH7(0.15 M),weregivenorallyin2mltoothergroups,1 minbefore oraladministrationof1mlofabsoluteethanol.Control animalsreceived2mlofwater.Theanimalswerekilled1 hrafterreceivingabsoluteethanol. Results GastricLesionsProducedbyVarious NecrotizingAgents Eachofthesefiveagentsproducedseveregas- tricdamagevisiblefromtheoutsideofthestomach asthickblackorredlines.Afteropeningthestom- ach,lesionswerefoundinthemucosaandconsisted ofelongatedbands,l-10mmlongbyl-3mmwide, usuallyparalleltothelongaxisofthestomach(Fig- ure1).Theircolorvariedwiththeagent:yellowor red(ethanol),red(25%NaCl,boilingwater),black (0.6NHCl,0.2NNaOH).Usually,15-20lesions couldbecounted.Theywerelocatedmostlyinthe corpus(theportionofthestomachsecretingacid andpepsin);theantrumwaslessaffected.Nogross lesiondevelopedintheforestomach(thenon- secretingpartcoveredwithasquamousepithelium). Histologically,thelesionsconsistedofnecrosis usuallyextendingdownthroughabouttwo-thirdsof themucosa(involvingthesurfaceepithelium,there- gionofmucusneckcells,andofparietalcells);occa- sionally,thenecrosisinvolvedthefullmucosal thickness.Necroticpatcheswerealsopresentinthe Figure1.Gastriclesionsproducedbyfivenecrotizingagents.Allagentsweregivenorallyin1ml,andtheanimalswerekilled1 hrlater. Stomachswereopenedalongthegreatercurvature.A.Control.B.Absoluteethanol.C.0.6NHCl.D.0.2NNaOH.E.25%NaCl. F.Boilingwater.Inallcases:extensivenecroticlesionsofthecorpus.Theantrumisalmostintactgrossly,althoughlesions werepresenthistologically.Theforestomach(upperwhiteportion)isintact. 436ROBERTETAL. GASTROENTEROLOGYVol.77,No.3 Figure2.Gastriccytoprotectionofprostaglandinsagainstabsoluteethanol.Onemlofabsoluteethanolwasgivenorallyandtheani- malswerekilled1hrlater.A.Controlvehiclegivenorally36minbeforeethanol.Multipleandseverenecroticlesionsofthe corpuscausedbyabsoluteethanol.InB,CandD,a prostaglandinwasadministered30minbeforeethanol.B.PGE,500pg/kg subcutaneously.C.PGE,150pg/kgorally.D.16,16DimethylPGA,50pgg/kgorally.Thesethreeprostaglandinspreventedthe formationofgastriclesionsbyabsoluteethanol. antralmucosa.Mucuscellsofthecorpuswerede- pletedofgranules(asshownbyPASstaining), sometimeseveninareasthatwerenotnecrotic. Largeglobsofmucuswerepftenfoundinthelumen andwereparticularlyabundantafteradministration ofboilingwater;sometimesthesemassesofmucus werestillattachedtothemucosaandcontainedmu- cosalcells,eitherisolatedorasdetachedfragments ofthemucosa.Themuscularismucosaewasintact. Thesubmucosaofthecorpus,theantrum,andthe forestomachwasmarkedlythickenedbyedema,but wasnotnecrotic.Theedematoussubmucosadidnot containpolymorphonuclearleucocytes1hrafterad- ministrationofthenecrotizingagents.Inotherstud- iesinwhichtheanimalswerekilled24hrafterthe necrotizingagents,thesubmucosawasstill markedlyedematousand,inaddition,wasinfil- tratedwithpolymorphonuclearleucocytes.Atthat time(24hr),mostofthemucosahadbecomene- crotic.Inanimalspretreatedwithacytoprotective PGandinwhichnogrosslesionswerevisible,the histologyofthestomachshowedanormalmucosa (nonecrosis,intactmucosalcells)andanormalsub- mucosa(noedema,noinfiltrationwithpolymorpho- nuclearleucocytes).Thesehistologicalfindingswill bereportedindetailelsewhere. September1979 GASTRICCYTOPROTECTIONBYPROSTAGLANDINS 437 Figure3.Gastriccytoprotectionbyprostaglandinsagainstboilingwater.RatswhosestomachsareshowninB,C,andDreceived1mlof boilingwaterorallyandwerekilled1 hrlater.A.Untreatedcontrol.B.Controlvehiclegivenorally30minbeforeboilingwa- ter.Massivenecrosisofthecorpuscausedbyboilingwater.CandD.X,16-DimethylPGE,5pg/kgeitherorally(C)orsubcuta- neously(D),30minbeforeboilingwater.Completeprotection. EffectofPGonGastricNecrosisProducedbypg/ kg,whereasothersrequiredupto500pg/ kgtoin- SeveralAgents hibitthelesions.Figure4showsthecytoprotective effectoffivePG,givenorally,againstgastricmu- ThePGsusedpreventedtheformationofgas-cosalnecrosisproducedbyabsoluteethanol.The triclesions,regardlessofthenecrotizingagentusedprotectionwasdose-dependent.ThemostpotentPG (Figures2and3).TheonlydifferenceamongthePGswas16,16-dimethylPGE,.Figure5showstheeffect wastheirpotency:somewereactiveatlessthan1ofl6,16-dimethylPGE,,givenorally,ongastricmu- 438ROBERTETAL. GASTROENTEROLOGYVol.77,No.3 GASTRI CLESI ONS 100 5101525501 150( 500 0. 025 pgl hgORALLY 100300 Figure4.Gastriccytoprotectionbyfiveprostaglandinsagainst absoluteethanol.Theprostaglandinswereadminis- teredorally30minbeforegiving1mlofabsoluteeth- anolorally.Animalswerekilled1hrafterethanol.The verticalaxisshowstheaveragenumberoflesionsper stomachineachgroup,expressedaspercentofcon- trols(controlanimalsreceivedethanolbutnoprosta- glandin).Eachprostaglandinreducedthenumberofle- sionsperstomachinadose-dependentmanner.The mostpotentprostaglandinwas16,16-dimethylPGE,. Tenratsperpoint. cosallesionsproducedbyfivedifferentagents.Inall cases,thecytoprotectiveeffectwasdose-dependent. ThecytoprotectiveED,,of16,16-dimethylPGE, rangedfrom0.03to0.15pg/kg,dependingonthe necrotizingagent. ComparisonofOralandSubcutaneous Routes. PGE,and16,16-dimethylPGE,wereabout3-4 timesmorecytoprotectivewhengivenorallythan whengivensubcutaneously(Figure6).Theircyto- protectiveED,,wereasfollows:PGE,,25pg/kg orallyand100pg/kgsubcutaneously;16,16-dimethyl PGE,,0.05pg/kgorallyand0.15pg/kgsubcutane- ously.Byeitherroute,16,16-dimethylPGE,wasap- proximately500timesmorepotentthanPGE,. OnsetofActionandDurationofAction WhenPGE,wasgivenatthesametimeasab- soluteethanol,thenumberofgastriclesionswasre- ducedby34%(14.5lesionsincontrols,9.6lesionsin PGE,-treatedanimals,justbelowstatisticalsignifi- cance)(Figure7).WhenPGE,wasgivenfrom1 to30 minbeforeethanol,theinhibitionwasnearlycom- plete(85-97%).Theonsetofcytoprotectionafter subcutaneousadministrationofPGE,waslonger: ThePChadtobegiven2.5minbeforeethanoltore- ducethenumberofgastriclesionsbyhalf.Nearto- talinhibition(88-100%)wasachievedwhenitwas GASTRI CLESI ONS 56 B z4 2 . ; ' . . . .. ; . . . .. "0.. - s - i y/ I I \ 0. 05I 0. 1 0. 150. 30. 51. 5,0. 025 0. 03 16. WOI METHYLPGE2: pg/ kg ORALLY,30 MI NBEFORENECROTI ZI NGAGENT Figure5.Cytoprotectionof16,16-dimethylPGE,againstfivedif- ferentnecrotizingagents.16,16-DimetbylPGE,was givenorally30minbeforethenecrotizingagents(1ml ofeitherabsoluteethanol,0.6NHCl,0.2NNaOH,25% NaCl,orboilingwater).Theverticalaxisshowstheav- eragenumberoflesionsperstomachineachgroup.For eachnecrotizingagent,16,16-dimethylPGE,reduced thenumberoflesionsandthedegreeofprotectionwas dosedependent.Tentosixteenratsperpoint. GASTRI CLESI ONS 100 16. 16. LI tMETHYL PGE2PGEz pdkg Figure6.GastriccytoprotectionofPGE,and16,16-dimethyl PGE,againstabsoluteethanol:comparisonoforaland subcutaneousadministration.Theprostaglandinswere given30minbeforeoraladministrationof1mlofabso- luteethanol.Theverticalaxisshowstheaveragenum- beroflesionsperstomachin eachgroup,expressedas percentofcontrols(controlanimalsreceivedethanol butnoprostaglandin).Eachprostaglandinwas3-4 timesmorecytoprotectiveafteroralthanaftersubcuta- neousadministration.Tenanimalsperpoint. September1979 GASTRICLESIONS P 100- 60- 6_ E 5 60 - ii_ I- t g40- i!i 20- 3,PGE2 \Subcutaneously , 12.5510153060120180300 Figure7. MINUTESBEFOREETHANOL 90150240 Timeofonsetanddurationofactionofcytoprotection byPGE,.PGE,,500&kg,wasgiveneitherorallyor subcutaneously,eitheratthesametime(Time0)asab- soluteethanol(Imlorally)oratvarioustimeintervals beforeabsoluteethanol;thesetimeintervalsareshown onthehorizontalaxis.Afteroraladministration,cyto- protectionwasalmostimmediate.Aftersubcutaneous administration,cytoprotectionwasapparentat-2.5 minandmaximalat-30min.Thedurationofaction wasabouttwiceaslongafteroralthanaftersubcuta- neousadministration.Tentotwenty-fiveratsperpoint. injected15-60minbeforeethanol(Figure7).Cyto- protectiondiminishedwhenPGE,wasgivenlonger than1 hrbeforeethanol.Gastriclesionshadre- turnedto50%ofcontrolvalues4hrafteroraladmin- istrationofPGE,,andabout2hraftersubcutaneous administration,asdeterminedbygraphicinter- polationonaplotofthetimeonalogscale, EffectoftheVolumeofDiluent PGE,givenatalowdose(10pg/kg)wasmore GASTRICCYTOPROTECTIONBYPROSTAGLANDINS439 GASTRICLESIONS 100- 80- 20- CONTROL0.5120.512 MLOFDILUENT DOSEOFPGE:,top9lk925p9/k9 ml 0.512 100p9lk9 Figure8Effectofthevolumeofdiluentonthecytoprotectiveac- tivityofPGE,.PGE,(atthreedoselevels)wasgiven orallyineither0.5,1.0,or2.0ml,30minbeforeabsolute ethanol.Thedilutionfactorplayedaroleonlyatthe lowestdose.NS=Notstatisticallysignificantwhen comparedtocontrolgroup.*=P