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1LactateprovidesastrongpH-independentventilatorysignalinthefacultativeair-breathingteleostPangasianodonhypophthalmusMikkelT.
Thomsen1,TobiasWang1,WilliamK.
Milsom2&MarkBayley1FishregulateventilationprimarilybysensingO2-levelsinthewaterandarterialblood.
Itiswellestablishedthatthissensoryprocessinvolvesseveralsteps,buttheunderlyingmechanismsremainfrustratinglyelusive.
HereweexaminetheeffectofincreasinglactateionsatconstantpHonventilationinateleost;specificallythefacultativeair-breathingcatfishPangasianodonhypophthalmus.
AtlactatelevelswithinthephysiologicalrangeobtainedbyNa-Lactateinjections(3.
5±0.
8to10.
9±0.
7mmolL1),gillventilationincreasedinadose-dependentmannertolevelscomparabletothoseelicitedbyNaCNinjections(2.
0molkg1),whichinducesahypoxicresponseandhigherthanthoseobservedinanylevelofambienthypoxia(lowestPO2=20mmHg).
Highlactateconcentrationsalsostimulatedair-breathing.
Denervationofthefirstgillarchreducedtheventilatoryresponsetolactatesuggestingthatpartofthesensorymechanismforlactateislocatedatthefirstgillarch.
However,sincearesidualresponseremainedafterthisdenervation,theothergillarchesorextrabranchiallocationsmustalsobeimportantforlactatesensing.
Weproposethatlactateplaysaroleasasignallingmoleculeinthehypoxicventilatoryresponseinfish.
AnimalsrelyonasuiteofphysiologicaladaptationstomaintainsufficientsupplyofO2totherespiringtissueswhenexposedtohypoxia.
Theseadaptationsvaryamongstvertebrateclasses,butnormallyincludeincreasedventilationandcardiovascularadjustments,withtheexacttypeandmagnitudeoftheresponsebeingaffectedbytheseverityanddurationofthehypoxia1–3.
ThefirststeprequiredinahypoxicresponseisobviouslyasensingmechanismrespondingtochangesinO2levels.
Yet,despitemucheffort,themechanismsunderlyingO2sensingremainfrustratinglyelusive,evenintheextensivelystudiedmammaliancarotidbody4.
DeterminingthemodalityofO2sensingisparamountinunderstandingventilatoryregulationandhypoxicresponses,whicharetypicallyassessedbyhypoxiaexposureperseorbyinjectionofvariouschemicalssuchasNaCN,whichstimulatesoxygensensitivereceptors5,6.
ThecapacitanceofO2isaround25–30timeslowerinwaterthaninair.
Hence,water-breathinganimalsmustventilatemorethanair-breatherstomeettheirmetabolicO2demand7,8.
ThesehighventilationratesresultinalowpartialpressureofCO2inarterialblood,andaventilatoryregulationofthearterialCO2-levelwouldunavoid-ablycompromiseO2uptake.
Hence,pH-regulationismainlyachievedbytransepithelialH+/HCO3–exchangeinfish9,10.
Inmammalsandotherair-breathingtetrapods,theventilatoryrequirementforO2uptakeislower,leadingtohigherarterialPCO2levels7,8.
ThisincreaseincombinationwiththehighO2availabilityhaschangedthemodeofventilatoryregulationinairbreathersintobeingprimarilycontrolledbyH+/CO2changesundernormoxicconditions11.
Thustheoxygenventilatorysignalinmostmammalshasutilityonlyinsituationsoutsidethenor-maladaptiverangesuchasathighaltitude,diving,burrowingorduringpulmonaryillness,butisthedominantregulatorymechanismofventilationinfish.
Hardarsonetal.
12showedforthefirsttimeinavertebratethatthelactateionpersegivesastimulationofven-tilationinratsthatisseparatefromtheeffectofthenormallyaccompanyingacidosis12,13Changetal.
14recentlydocumentedtheolfactoryreceptor78tobeinvolvedinthisresponsebydescribingahighlactateaffinityofthisreceptorinthecarotidbody.
Byknockingoutthecodinggene(Olfr78,alsoknownasOr51e2)inmicethey1DepartmentofBioscience,Zoophysiology,AarhusUniversity,Aarhus,Denmark.
2DepartmentofZoology,UniversityofBritishColumbia,Vancouver,BC,Canada.
CorrespondenceandrequestsformaterialsshouldbeaddressedtoM.
T.
T.
(email:Mikkel.
thomsen@bios.
au.
dk)Received:16February2017Accepted:16June2017Published:xxxxxxxxOPEN2abolishedthecarotidbodyresponsestobothlactateionsandhypoxia14.
Thus,itseemsthatlactateionsinduceaventilatoryresponsesimilartohypoxemia(lowbloodO2levels)andtheauthorsarguedthatlactateandlac-tatesensingmayhaveanimportantroleinenhancingthehypoxicventilatoryresponse.
Lactateisproducedbymostvertebratecellsunderhypoxicconditionsduetoabuild-upoftheglycolyticend-productpyruvate,whichisenzymaticallyconvertedtolactateandreleasedtotheblood.
Sincelactateisproducedbyallvertebrates,thislactate-inducedcomponentofthehypoxicresponsemaybeauniversaltraitamongstvertebrates,makingittimelytostudytheinfluenceofthelactateiononventilationinothervertebrateclasses.
InthisstudyweexplorethepresenceofapH-independentlactateventilatoryresponseinateleost,namelythefacultativeair-breathingfishPangasianodonhypophthalmus,whichusesgillsandamodifiedswimbladderforO2uptake.
Sincetheteleostfirstgillarchisontogeneticallyhomologoustothemammaliancarotidbody15,16,wealsoexaminedthelactateresponsesfollowingdenervationofthefirstgillarch.
Wehypothesisedthatthisdenervationshouldreduceorabolishthelactateventilatoryresponse.
Finally,weexaminedthecardiorespiratoryresponsestoprogressivehypoxiaand/orhypercarbiatosimulatenaturalconditions17–20forcomparisonoftheresponseelicitedbylactateions.
ResultsIntra-arterialinjections.
Intra-arterialinjectionsoflactateinthenon-denervatedfishcausedadose-de-pendentincreaseinbothgillventilationrate(fR,p=0.
0042,p=0.
0003,p=0.
0003forthelow,mediumandhighlactatedoses,respectively;mixedmodelANOVAwithpairwisecomparisonandBenjamini-Hochbergp-valueadjustment,n=14,Figs1and2a)andgillventilationamplitude(Vamp,p=0.
006,p=0.
0003,p=0.
0002forthelow,mediumandhighlactatedoses,respectively,mixedmodelANOVAwithpairwisecomparisonandBenjamini-Hochbergp-valueadjustment,n=14,Figs1and2b)independentofchangesinpH(pH=7.
82±0.
01,nodifferencebetweentreatments,p=0.
70,orbetweenG1-denervatedandnon-denervatedfish,p=0.
37,mixedmodelANOVA,n=14,Fig.
3).
Thisresponsealsoincludedair-breathingatthetwohigherdoses(25%and50%non-denervatedfishand0%and33%G1-denervatedfishperformedatleast1observableair-breathfollowingthemediumandhighlactatedoses,respectively),butthisresponsecouldnotbequantifiedfurtherinourexperi-mentalsetup.
Whiledenervationofthefirstgillarchfailedtocompletelyabolishthelactateventilatoryresponse,itdidcauseareduction.
TheresponseoftheG1-denervatedfishtoelevatedlactatewasstillsignificantatthetwohighestdoses(Fig.
2;fRp=0.
10,p=0.
003andp=0.
003atthelow,mediumandhighlactatedoses,respectively;Vampp=0.
27,p=0.
003andp=0.
002,atthelow,mediumandhighlactatedoses,respectively,mixedmodelANOVAwithpairwisecomparisonandBenjamini-Hochbergp-valueadjustment,n=14).
Noneofthecontrolinjections–isosmoticsaline,1MNaCl,orpyruvate–affectedventilation(noair-breathswereobservedandnoincreaseineitherfRorVamp)orpH(Figs2and3),confirmingthattheresponsestoothertreatmentswerenotaresultofstressfrominjections,increasedbloodpressure,alteredosmolality/[Na+],orametabolicresponsefromtheconversionoflactatetopyruvate.
NaCNinjections(2.
0molkg1)initiatedclearventilatoryresponsesinbothintactanddenervatedfish(Fig.
2c,d)inadditiontoair-breathing(50%ofthefishinbothgroupsperformedatleast1observableair-breath)andescapebehaviour(notquantified).
ThetimebetweeninjectionandavisibleventilatoryresponsewassimilarforlactateandNaCNinjections,withthepeakresponseoccurringafter54±4secforlactateinjectionsandafter46±10secforNaCNinjections.
Hypoxiaandhypercarbia.
Hypoxiainducedchangesingillventilation(fRp=0.
002;Vampp=0.
002,mixedmodelANOVA,n=22),withmoderatedecreasesinwaterPO2(PwO2=40–60mmHg)elicitingincreasedgillventilationwiththelargestelevationinVamp,whereasmoreseverehypoxia(PwO2140–20mmHg,stepwiseover7hours),B)Progressivehypercarbia(n=6,PwCO2:0–37.
5mmHg,stepwiseover6hours,C)Progressivehypoxiainconstanthypercarbia(n=6,PwCO2:22.
5mmHg,PwO2asinA),D)Progressivehypercarbiainconstanthypoxia(n=6,PwO2:40mmHg,PwCO2asinB),E)control(n=10,PwO2:>140mmHg,PwCO2:0mmHgfor7hours).
Eachexposuresteplasted1hourtoallowventilationtostabi-lise.
1hstepswerechosenaschangingthewaterPO2andPCO2tookseveralminutes,preventingtherecordingofreliableacutehypoxiaresponsesdirectlycomparabletothoseinducedbythechemicalinjections.
Datasamplingandanalysis.
Measurementsfromthepressuretransducersandthegasanalyserwerecol-lectedwithaMP100BIOPACsystem(BiopacSystemsInc.
,SantaBarbara,CA,USA)at200HzandstoredinAcqKnowledgev.
3.
9.
1,wheredatawassmoothedbyafactor25toreducethenoiseandallowforautomatizationoftheanalysisinmostdata.
Intheambientgasexperimentthereportedvaluesaretakenfromthelast15minof8eachhouroftheprogressivehypoxiaorhypercarbia(45minutestostabilise).
Spontaneoushighactivitylevelswereexcludedbothtoreducetherandomvariationandbecauseofthehighlevelofnoiseonthesignalsduringtheseperiods.
Intheintra-arterialinjectionsexperiment,thereportedpost-injectionvaluesarethepeakresponseaveragedover30secondswhereasthepre-injectionvaluesaretheaverageof30secondimmediatelypriortoinjection.
Periodswithnoiseduetomovementwerefewandwereavoidedintheanalysis.
Alldataisavailableathttps://pastebin.
com/44d7jRdG.
Statistics.
Alldataarepresentedasmean±s.
e.
m.
Atwo-waymixedmodelanalysisofvariancefollowedbyapairwisecomparisonandaBenjamini-Hochbergp-valuecorrectionwasusedtoanalysemostresults.
Fortheinjectionexperiment,subjectwasincludedasarandomfactorandtreatmentandgroupasfixedfactors.
Thep-valuesreportedforaneffectofaninjectionaretheadjustedp-valuesfromthepairwisecomparisonbetweenpre-andpost-injectionforagivensubstanceforagivenvariable.
Fortheambientgasexperiment,subjectwasincludedasarandomfactorandtimeandgroupincludedasfixedfactors.
Pairwisecomparisonwasmadeateachtime-point,comparingthespecifictreatmenttothecontrolgroup.
TheanalysiswasperformedusingInVivoStatsoftware(v.
3.
6.
0.
0).
Alldataweretestedforvariancehomoscedacitywithresidualplotsandnormalitywithanormalprobabilityplot.
Inthefewcasesofheteroscedacity(air-breathingdataandgillventilationamplitudedataforhypoxiaexposureinnormocarbia)thedatawasrank-transformedpriortoperformingthemixedmodelANOVA.
Themaximaleffectofhypoxiaongillventilationvariables(Fig.
2c,d.
)wereanalysedwithatwotailedpairedT-test.
Differenceswereconsideredsignificantwhenp<0.
05.
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AcknowledgementsTheauthorswouldliketothankHeidiMeldgaardandPerHenriksenforanimalcare.
ThisstudywasfundedbytheGraduateSchoolofScienceandTechnology,AarhusUniversity,Denmark,theDanishMinistryofForeignAffairs(DANIDA)[DFCno.
12-014AU]andbytheDanishNaturalScienceResearchCouncil.
AuthorContributionsM.
T.
T.
conductedtheexperiments,analyseddata,preparedfiguresanddraftedthemanuscript.
Allauthorsplannedanddesignedthestudy,interpretedtheresults,editedthemanuscriptandapproveditsfinalversion.
AdditionalInformationSupplementaryinformationaccompaniesthispaperatdoi:10.
1038/s41598-017-06745-4CompetingInterests:Theauthorsdeclarethattheyhavenocompetinginterests.
Publisher'snote:SpringerNatureremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations.
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