CO cAMP pathwayisanevolutionarilyconservedmechanismforsensing regulated adenylylcyclasesfromcyanobacteria,suggestingthe sAC sensesbloodalkalosisandtriggerscompensatoryH animals arestilllimitedbutrapidlyaccumulating.Insharkgills, © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,663-672doi:10.1242/jeb.086157 *Author ([email protected]) for correspondence San Diego, California 9500 GilmanDrive, LaJolla, CA92093,USA. of Oceanography,Marine BiologyResearch Division,ScrippsInstitution of University (ADCY10) wasrecentlyidentified byLevinandBuck(Bucketal., Defer, 2001). A geneticallyunrelated,novel adenylylcyclase in someoftheirregulatoryproperties (Cooper, 2003;Hanouneand which differ intheirtissueand developmentalexpressionaswell regulated tmACs.MammalshaveninetmACgenes(ADCY1–9), type ofadenylylcyclase,afamilyhormoneandG-protein- Until recently, vertebrateanimalswerebelievedtohaveonlyone animals, belongtoClassIII. cyclase (sAC)andtransmembraneadenylyl(tmAC) from known eukaryoticadenylylcyclases,includingsoluble result ofconvergent evolution (LinderandSchultz,2008).All are unrelatedinsequenceandstructurebutallproducecAMP asa distributed throughoutBacteria,ArchaeaandEukarya;theseclasses ATP assubstrate.Therearesixdifferent classesofadenylylcyclases pathways. cAMP isproducedbyadenylylcyclaseenzymesthatuse of onethemostversatileandevolutionarilyconservedsignaling Cyclic adenosinemonophosphate(cAMP)isthesignalingmolecule pH sensing,Protonpump KEY WORDS:V-ATPase, Acid/base,cAMP, Carbonicanhydrase, suggests additionalfunctionsinwhichsACmaybeinvolved. knowledge onthephysiologicalrolesofsACinaquaticanimalsand present inmostanimalphyla.Thisreviewsummarizesthecurrent Bioinformatics andRT-PCR resultsrevealthatsACorthologsare the initiationofflagellarmovementandinacrosomereaction. water absorption.Andinseaurchinsperm,sACmayparticipate absorption. Intheintestineofbonyfishes,sACmodulatesNaCland by HCO nucleus andinsidemitochondria.sACactivityisdirectlystimulated distributed throughoutthecytoplasmanditmaybepresentin transmembrane adenylylcyclases(tmACs).MammaliansACis biochemically distinctfromtheclassicG-protein-regulated signaling moleculecyclicAMP (cAMP)thatisgeneticallyand Soluble adenylylcyclase(sAC)isarecentlyrecognizedsourceofthe Martin Tresguerres*, KatieL.Barott,Megan E.BarronandJinaeN.Roa sensing solubleadenylylcyclase(sAC)inaquaticanimals Established andpotentialphysiologicalrolesofbicarbonate- REVIEW and CO associate withcarbonicanhydrasestoactasa variety ofmammaliancelltypes.Inaddition,sACcanfunctionally Discovery of mammaliansAC Discovery of Introduction ABSTRACT 2 levels and/oracid/baseconditions.ReportsofsACinaquatic 2 3 . ThetwocatalyticdomainsofsACarerelatedtoHCO – , andsAChasbeenconfirmedtobeaHCO de facto 3 – sensor ofpH sensor ina 3 – + - in Kamenetskyetal.,2006;Tresguerres etal.,2011). Briefly, HCO mechanism isbelievedtoalsoapplymammaliansAC(reviewed adenylyl cyclaserelatedtosAC(Steegbornetal.,2005b);this are stimulatedbylowmicromolar [Ca heterotrimeric G-proteins,sACisunresponsive.MosttmACisoforms mammalian sACandtmACs.WhiletmACactivityismodulated by aspartate (Linder, 2006;Steegbornetal.,2005b). 2003; Litvinetal., 2003);mammaliansACisadditionally insensitive sAC withoutchangingits 1999; Chenetal.,2000),followingonfromearlierreportsofMn induces anallostericchangethatresultsinincreasethe effect ofHCO residue intmACs,whicharenotstimulatedbyHCO a serineinbacteriaandmollusks.Incontrast,thecorresponding on aspecificaminoacidresiduethatisthreonineinmammals and millimolar [Ca Storm, 2003;Willoughby andCooper, 2007),but inhibitedby pathways. causing researcherstorevisitpreviousstudiesonsignaltransduction microdomain modelhaschangedthecAMP-signalingparadigmand (reviewed inWong andScott,2004).ThecAMP-signaling allowing thespatio-temporaldynamicsofcAMP signaling (AKAPs), whichalsocoordinatePKA andPDEactivities,thus intracellular compartmentsbyA-kinaseanchoringproteins by phosphorylation.PKA and PDE aretetheredtospecific PKA, whichmodulatestheactivityofmultipledownstreamproteins Zaccolo etal.,2006).ThemostwidelystudiedcAMP effector is (reviewed inCooper, 2003;Tresguerres etal.,2011; Zaccolo,2009; gated channelsandexchangeproteinactivatedbycAMP (EPAC) cAMP-activated targets proteinkinaseA (PKA),cyclicnucleotide- phosphodiesterases (PDEs)asbarriersforcAMP diffusion, andthe 1),whichentailstmACsandsACassourcesofcAMP, (Fig. within cellssupportsthemodelofcAMP-signalingmicrodomains membrane bytmACs.ProductionofcAMP invariousfocalpoints cAMP isproducedexclusivelyintheproximityofcell The intracellularlocalizationofsACchangedtheassumptionthat cytoplasm andinorganelles (Zippinetal.,2003;Zippin2004). immunolocalization andbiochemicalstudiesfoundsACinthecell some activityisalsofoundassociatedwithmembranes.Subsequent its activityispreferentiallyfoundinthecytosolicfraction,although This enzymewastermed‘solubleadenylylcyclase’ (sAC)because Braun, 1975;1990;1991;BraunandDods,1975). stimulated cAMP activityinrattestishomogenates(Braun,1974; HCO (A/B) sensor(Chenetal.,2000).Themolecularmechanismof produce cAMP, turningsACintoaputativephysiologicalacid/base millimolar [Ca necessarily sACsfromotherorganisms, seebelow)isstimulated by site (Guillouetal.,1999).In contrast, mammaliansAC(butnot There areseveralotherbiochemicaldifferences between The paramountfeatureofsACisdirectstimulationbyHCO 3 – stimulation hasbeenelucidatedforCyaC,acyanobacterial 3 2+ 2+ – ], whichlowerssACK ], whichdisplacesthecofactorMg on sACandsAC-likeenzymesisstrictlydependent K m for substrateATP. Thestimulatory 2+ m ] viacalmodulin(Wang and for ATP (JaiswalandConti, 2+ from theactive 3 – , isan V max 3 663 – 2+ of to 3 – -

The Journal of Experimental Biology modulation byG-protein-coupled receptorsandG-protein. Ca 664 A (PKA) orexchangeproteinactivatedbycAMP (EPAC) (notdepicted). ProductionofcAMP bysACisstimulatedincreased[HCO adenylyl cyclase(tmAC)];phosphodiesterases (PDEs)thatdegradecAMP, thusactingasbarriersforcAMP diffusion; andcAMP suchasproteinkinase targets movement ofsolubleadenylylcyclase (sAC)betweencompartments(notshown).Eachmicrodomaincontains asourceofcAMP transmembrane [sAC or mightinvolvethe the cytoplasm,(2)nucleus,(3)mitochondria, (4)thecellmembranevicinityand(5)internalizedendosomes. Additionalregulation Fig. sAC), and‘p-site’ inhibitorssuchas2 antagonists KH7andderivativesofcatecholestrogen(selectivefor tmACs alsodisplaydifferential sensitivitytothepharmacological is insensitivetoforskolin(Bucketal.,1999).MammaliansACand in vivo plant dipertenethathasbeenclassicallyusedtostudycAMP function to calmodulin.MosttmACsarepotentlyactivatedbyforskolin,a REVIEW 2008). Thebetter-characterized humansACsplicevariant,termed 2008; Gengetal.,2005;JaiswalandConti,2001;Moore sAC mRNA undergoes extensivealternativesplicing(Farrelletal., while humanshaveasinglesACgeneandonepseudogene.Human 2000). SomemammalshavemultiplesACgenes(e.g.dog,bonobo), regulatory functions(~140 terminus (~50 for tmAC)(Tresguerres etal.,2011). Mammalian sACiscomposedoftwocatalyticdomainsintheN- G wholegenomeshotgun transcriptomeshotgunassemblies transmembraneAC WGS TSA tmAC sAC H Na sodium/bicarbonatecotransporter G-protein-coupledreceptor expressedsequencetag NHE NBC exchangeproteinactivatedbycAMP GPCR cyanobacterialAC EST cysticfibrosistransmembraneconductanceregulator EPAC cyclicAMP carbonicanhydrase CyaC CFTR adenylylcyclase A-kinaseanchoringprotein cAMP acid/base CA AKAP AC A/B A solubleadenylylcyclase proteinkinaseA phosphodiesterase sAC sAC PKA PDE KCNa NKCC List of abbreviations List of 2+

.Intracellular cAMP-signalingmicrodomains. 1. , see‘Discoveryof mammaliansAC’ andFig. T FL (Seamon andDaly, 1981;Seamonetal.,1981);however, sAC kDa), andaC-terminusdomainwithmostlyunknown truncated sAC full-length sAC + + /K /H + + /2Cl exchanger – cotransporter

kDa) (Bucketal.,1999;Chen ′ ,5′ -dideoxyadenosine (selective

2 fordetails).Production ofcAMP bytmAC occurs inresponsetovariousextracellular ligandsanditrequires cAMP productionmayoccurindiscreteintracellularcompartments suchas(1)focalpointsthroughout and notbypHorCO (sAC catalytic domainsandis~10-foldmoreactivethanfull-lengthsAC assay conditionsthatsustainHCO than onefreeze/thawcycle(Barottetal.,2013),andthattheactivity methodological issueisthatsACrapidlylosesitsactivityaftermore genome (e.g.Tresguerres etal.,2010b). Oneimportant degenerate PCRtoclonesACfromspecieslackingasequenced detection ofsACbyRT-PCR andmakesitalmostimpossibletouse it doesnotprecluderobustexpression.However, itcomplicates sAC mRNA abundanceonlyimpliesalowproteinturnoverratebut all vertebrateanimalsbutnotnecessarilytoinvertebrates.Thelow Farrell etal.,2008;Geng2005).Thistrendseemstoapply exception oftestesbearingmaturingsperm(Bucketal.,1999; is thatsACmRNA abundanceistypicallyverylowincells,withthe complexity ofsACandmethodologicalissues.Amongtheformer catalytic domains. these parametersareexclusivelydeterminedbyresiduesinthetwo other (Chaloupkaetal.,2006;Geng2005),indicatingthat Pastor-Soler etal.,2003). shark (Tresguerres etal.,2010c)andmammals (Hessetal.,2005; 2005a), coral(Barottetal.,2013),seaurchin(Beltrán2007), to beeffective againstsACsfromcyanobacteria(Steegbornetal., However, KH7andderivativesofcatecholestrogenarenowproving impaired bythelackofpharmacologicalinhibitorsspecificforsAC. ‘truncated sAC’ (sAC The metals (Mg among species,especiallywhenitcomestotherequiredcatalytic (EC (Chaloupka etal.,2006).However, thehalf-maximumstimulation inhibitory domainlocatedC-terminaltothesecondcatalytic difference inspecificactivityisduetoanineaminoacidauto- sAC Mechanisms of sAC activation Mechanisms of Studies onsAChavebeenchallengingbecauseoftheintrinsic 50 FL in vitro FL ) forHCO ) (reflectedasahigher and othersplicevariantsareindistinguishablefromeach The JournalofExperimentalBiology(2014)doi:10.1242/jeb.086157 2+ activity ofsACisspecificallymodulatedby[HCO , Mn 3 – 2+ and the , Ca 2 (Chen etal.,2000;Tresguerres etal.,2010c). T ), isessentiallycomposedofjustthetwo 2+ ). Also,theinitialstudiesonsACwere K m V for Mg max in vivo in ) (Bucketal.,1999).Mostofthe 3 – stimulation arenotuniversal 2+ , Mn 3 – 2+ ] (andinsomecases and Ca 2+ of sAC 3 – T ] ,

The Journal of Experimental Biology each organism (~20and~5 HCO kidney (Gongetal.,2010;Paunescu2008).A thirdsourceof possibly mammalianepididymis(Pastor-Soler etal.,2003)and shark gills(Tresguerres etal.,2010c;Tresguerres etal.,2007),and in sACactivity, astheEC extracellular HCO straightforward mechanismofsACstimulationinvolves example membranepotential-drivenelectrogenicNa enters thecellacrossvoltage-dependentCa Finally, sACinmammalianINS-1cellsisstimulatedbyCa that dependsontheinterplaybetweencAMP fromdifferent sources. This illustratesapotential,moreintricate,mechanismofsACaction HCO is notdirectlymodulatedbyhormones,themechanismsthat load Soler etal.,2003)viaanunknownmechanism.Andalthough sAC REVIEW The hydrated intoHCO into thecell(bydiffusion orpossiblyacrossaquaporins),whereitis cells (Schmidetal.,2010).AnothermechanismistheentryofCO mammalian epididymisandkidney, orCFTRchannelsinairway cotransporters (NBCs)inastrocytes(Choietal.,2012)andprobably extra-cellular CO transporting proteinsmayrendersACa However, thepresenceofcarbonicanhydrase(CA)andA/Bion- conjunction withchangesin[HCO extracellular pHfrom6.6to7.8inHCO epididymis respondstophysiologicallyrelevantvariationsin stimulation maybemorecomplex.Forexample,sACinmammalian (Tresguerres etal.,2010b).Additional mechanisms forsAC mitochondria (Acin-Perezetal.,2009)orinthecytoplasm protein isclosetothenormal[HCO CA-catalyzed hydrationintoHCO the dose–response curve.However, sACislocatedinside cells, HCO G-protein-coupled receptor(GPCR)/G-protein/tmAC]leading to cells, thefollicle-stimulatinghormoneactivatesCFTRchannels [via implies thatsmallchangesin[HCO In vitro vitro In in vitro 3 3 3 – – – into acellmaybe.Forexample,inmouseovariangranulosa is CO entry andsubsequentsACstimulation(Chenetal.,2012a). versus EC 2 from mitochondrialaerobicrespiration,which,after 50 in vivo in for HCO 3 – 2 3 , pHand[HCO entering thecellviaiontransporterproteins,for – and H EC 50 3 – 50 is themeanpointinsteepest partof

for purifiedmammalianandshark sAC mmol + for HCO by intracellularCA,asobservedin 3

– l − ] andATP (Ramosetal.,2008). 3 3 3 1 – – – epciey al 1).This , respectively;Table , maystimulatesACinside ] willresultinlarge changes ] intheextracellularfluidof 3 – de facto 3 – ] 3 – 2+ -free medium(Pastor- in vivo channels, possiblyin sensor ofintra-and Fg 2).A (Fig. + /HCO 2+ that 3 – 2 fluids. Forexample,normalintracellular[HCO 1999) and1–4 type H Nonetheless, variationsinplasma[HCO 2012; Wood etal.,1990),dependingonthespeciesandcelltype. where theaverage[HCO alkalinization andtriggerscompensatoryH In theclearcellsofepididymis,sACisactivatedbylumen (1) EC changes inintracellular[HCO pathologic activation ofproteasesinpancreatic acinarcells gene expressionandestradiol production(Chenetal.,2012a), ganglion cellsandaxongrowth (Corredoretal.,2012),aromatase 2012), intraocularpressure(Lee etal.,2011), survivalofretinal metabolic couplingbetweenneurons andastrocytes(Choietal., the timeofwriting,sAChasadditionally beenshowntoregulatethe (reviewed inTresguerres etal.,2010a;Tresguerres etal., 2011). At beating inairwaysandoxidativephosphorylationmitochondria gene expression,developmentandcelldifferentiation, ciliary include epithelialionandfluidtransport,spermflagellarmovement, immune cells,bone,embryosandseveralcelllines.Thesefunctions such assperm,eye,airways,pancreas,colon,brain,nervoussystem, functions inresponsetoA/Bstatusdiversemammaliansystems Paunescu etal.,2008).sACalsoregulatesotherphysiological proposed forsACinkidneyintercalatedcells(Gongetal.,2010; 2008). SimilarrolesinA/Bsensingandhomeostasishave been factors); (2)thereportedintracellular[HCO not necessarilymatchtheconditionsinsidecells(forexample,co- between sACEC al., 1984;Hō al., 2010a;Tresguerres etal.,2011). also expressabundantintracellularCA (reviewedinTresguerres et In supportofthispossibility, sACismoreoftenpresentincellsthat mitochondria ortotheactionofCAs(e.g.Tresguerres etal.,2006a). to be10–15 cells havehigherlocal[HCO average withinwholecells.Itisthuspossiblethatfocalpointsinside Physiological roles of mammaliansAC of Physiological roles There areatleasttwopossiblereasonsforthesmalldiscrepancy 50 + -ATPases (Pastor-Soler etal.,2003;Pastor-Soler etal., is estimatedusingpurifiedsACprotein The JournalofExperimentalBiology(2014)doi:10.1242/jeb.086157 the CO hormones. (2)sACinthenucleusmaybestimulatedbyHCO exchangers andchannelscanpotentiallybemodulatedby Na membrane-transporting proteinssuchaselectrogenic generated CO inside mitochondriamaybestimulatedbymetabolically endoplasmic reticulumormitochondria(notdepicted).(4)sAC Ca mammals), whichenterthecellthroughvoltage-dependent Na cytoplasm maybestimulatedbycatalyticmetals(e.g.Ca derived fromallthesourceslistedabove.(3)sACin cytoplasm canbestimulatedbyHCO Fig. extruding transporters(HE)suchasV-type H CO (a) Carbonicanhydrase(CA)-dependenthydrationofexternal channels. (f)TheentryofHCO fibrosis transmembraneconductanceregulator(CFTR) be etal.,1984;Strobel2012;Wood etal.,1990). mmol 2+ + + 2 /HCO /H

. (b)CA-dependenthydrationofmetabolicCO .MechanismsofsACactivation 2. mmol channels (VDCC)orpotentiallybyCa + 50 2 exchangers fromthecellmaypreventslowingdownof

hydration reaction.(d,e)HCO l 3 for HCO − – 1 -cotransporters (NBCs),anionexchangersorcystic

l − in mammals(Betticeetal.,1984;Lodish, 1 2 3 through CA. in fish(Hō – ] issomewhatlowerthaninextracellular 3 3 – – and reportedintracellular[HCO ] duringstressconditions(Betticeet 3 – ] duetotheproximity be etal.,1984;Strobel 3 – across transporters, 3 – + ] resultinproportional 3 3 – secretion viaapicalV- 3 – – from varioussources. ] valuesrepresentan 3 in vivo.(1)sACthe that entersthrough – in vitro ] hasbeenreported 2+ + released fromthe -ATPase or , whichdoes 2 . (c)H 2+ + - in 665 3 – 3 – ]:

The Journal of Experimental Biology 666 physiological roles ofsACmaybeeasierinaquatic organisms than in aquaticorganisms thaninmammals,and(2)studying the implications: (1)thephysiological rolesofsACmaybemorecritical and themagnitudeofchanges. Thishastwoimportant variable comparedwiththatof mammals,intermsofbothstability in theinternalfluidsofaquatic organisms isgenerallymuch more (Kolodecik etal.,2012)andendocytosisofNa REVIEW recently, soinformationisstillscarce.Theconcentrationof HCO fishes), studiesonsACinaquaticorganisms havestartedonly organisms (especiallyvertebrates, e.g.boneyandcartilaginous (Middelhaufe etal.,2012)ofstillundefinedfunction. muscle havebeenidentifiedtocontainaheme-bindingdomain certain sACsplicevariantspresentinmammaliantestisandskeletal (Appukuttan etal.,2012)(reviewedinChen2012b).Lastly, coronary endothelialcells(Kumaretal.,2009)andcardiomyocytes In theheart,sACcontrolsmitochondria-dependentapoptosisin both 2013; Magroetal.,2012a;2012b;Zippin2010). aggressiveness ofcertainskinandprostatecancers(Flackeet al., Moreover, nuclearsAChasbeensuggested todeterminethe cultured cellsexposedtohypercapnia(Lecuonaetal.,2013). Table 1.Summaryofcurrentlydescribed Mollusks Cartilaginous fishes WGS contigs; details; present inPoriferans( Identification ofsACorthologsis basedonBLASTsearchesusing al., 2007; A A Bony fishes Co-factors Mammals accession no.) Organism (protein protein. ND, notdetermined;TSA,transcriptomicshotgunassembly;WGS, Cnidarians *Supports HCO Sea urchin ( EMP34522.1, Saccoglossus kowalevskii Buck etal.,1999; ccession numbersarefromNCBIunlessotherwise noted. Oyster Squalus acanthias Opsanus beta Homo sapiens Acropora spp.,Pocillopora Strongylocentrotus While manyofthesefunctionslikelyalsoapplytoaquatic (EKC32844.1) (ACA52542.1) and pharmacology) heterologous antibodies aurata sp. (NP_067716.1) (NP_060887.2); (NP_001020380.1) purpuratus homogenates) pharmacology ontissue damicornis G Tresguerres etal.,2010c; Crassostrea gigas N (based on Bookbinder etal.,1990; Alligator Ciona intestinalis (based on 3 , Sparus – stimulation; B Litvin etal.,2003; Rattus Amphimedon queenslandica

mississippiensis

XP_002731233); Amphioxus(

D D D Mantle,gill,hemocytes ND ND ND Mn ND ND Intestine Mn Mg Mn Mg Mg Mg ‡ maximum activity. maximum XP_002121952.1);andreptiles( 2+ 2+ 2+ 2+ 2+ 2+ 2+ +Mn +Ca * H P ‡,L ‡ ‡,A O ,M

~10 this paper; Zhu etal.,2001;

C XP_006273015.1, Chaloupka etal.,2006; 2+ 2+ * * ,B ,G

properties ofsACinmarineorganisms (mmol HCO ~5 ~11–25 20 I Roa etal.,2012; K G XP_003384443.1);Placozoans( P 3 – P  EC l Barron etal.,2012;
B–D 1 ) Branchiostoma floridae + 50 No Python /K

+ D Notinsymbiotic ND Yes No estvt isedsrbto Notes sensitivity Tissue pH distribution -ATPase in D Chen etal.,2000; Chrysemys pictabellii Chrysemys G D J

L Gill Carvalho etal.,2012; morulus the two catalytic domains of dogfish sAC (amino acids 1–485) a the twocatalyticdomainsofdogfishsAC(aminoacids1–485) wholegenomeshotgundatabase; EST,expressedsequencetag. 3 6 WGS contigs). 6WGS Q – Sperm Testes, eye,airways, Barott etal.,2013; (reviewed in muscle, kidney,epididymis cells, bone,embryos, nervous system,immune pancreas, colon,brain, or mucus mesenchymal cells cells G , rectalgland,redblood (Nomura etal., 2005),workthatreliedon traditionalprotein sperm fromthepurpleseaurchin sensor (seeTresguerres etal.,2011). sensor, and many evenbelievedittobeamammaliansperm-specific substantiated thebeliefthat sAC wasamammal-specificA/B zebrafish genomethatcame outseveralyearslaterfurther Van Haastert,2002). ThelackofsAC-relatedgenesindraftsthe that thesACgenehadbeenlostinmultiplelineages(Roelofs and Drosophila melanogaster, in cyanobacteria,butnotthefullysequencedgenomes of 2000s revealedsACorthologsintheslimemold genomes becameavailable.Bioinformaticsanalysesinthe early 1999), afewyearsbeforethefirstdraftsofmouseand rat Mammalian sACwasdiscoveredinthelate1990s(Bucket al., to measure. pronounced A/Bstress,whichmayresultinresponsesthatareeasier in mammalsbecauseitispossibletoexperimentallyinducemore sAC innon-mammalian organisms thaliana XP_002610143.1;XP_002610144.1) L–N H E

This view, however, wasupendedwiththe discoveryofsACin J,K Trichoplax adherens Tresguerres etal.,2011; , embryonicprimary

XP_005308389.1,XP_005314924.1, Q K Tresguerres etal.,2010b; E or ) Symbiodinium The JournalofExperimentalBiology(2014)doi:10.1242/jeb.086157 Saccharomyces cerevisiae R http://marinegenomics.oist.jp/; O P Presentin XP_002117857.1);Hemichordates

Present in K Kinetics determinedfromsemi-purified native Multiple splicevariants Present in Present inmost(all?)mammals,including Present in F m see ‘DiscoveryofmammaliansAC’ for AKZP01091244.1) rosmarus manatus GAPS01010686.1) contigs), WGS AHAT01020068.1), contigs),Lepisosteusoculata WGS EZ789694.1, EZ814745.1), Callorhinchus milii Callorhinchus milii protein. Presentin aug_v2a.10515.t1) Orcinus orca truncatus (XP_001623318.1) FC684349.1, WGSAMQO01004501.1) JO105222.1) and intestine (heterologousantibodies) hallerii contigs), forATP=~1–3 Caenorhabditis elegans L Nomura etal.,2005; gill, rectal gland, testes, muscle, eye, gill, rectalgland,testes,muscle, S Ruditapes philippinarium Oncorhynchus mykiss Acropora Leucoraja erinacea Triakis semifasciata ; Tunicates( (XP_004329889), (XP_004384133), (XP_004408373.1) Strongylocentrotus purpuratus (XP_004269766.1), Chelonia . Thisledtothehypothesis Latimeria  mmol S most likelyasingle Lottia gigantea (aug_v2a.21187.t1,

(WGS AAVX02006506.1) R Patiria miniata s thequery.Also Nematostella vectensis ; Nematostella Latimeria chalumnae F 

Oikopleura dioica l
1 B-D

mydas

menadoensis Dictyostelium M Odobenus (15WGS Salmo salar Trichechus Beltran et and ,

(TSA Arabidopsis (EST (WGS (TSA Tursiups Urbatis I

(WGS (WGS (TSA 7

(5 (17 and

The Journal of Experimental Biology (Richards etal., 2003),inresponsetohypercapnia (compensatory (Wood etal.,2005),byexhaustiveexercise(metabolic acidosis) between 2and15 Mn dogfish andseaurchinsACarestimulatedbyHCO et al.,2005;NomuraandVacquier, 2006).LikemammaliansAC, urchin (semi-purifiednativeprotein)(Beltránetal.,2007;Nomura recombinant protein)(Tresguerres etal.,2010c) andfrompurplesea mammalian sACsarethosefromthedogfishshark(purified techniques. these speciesisduetoaglitchofsequencingandannotation fundamentally distinctmechanisms,orthattheabsenceofsACin REVIEW HCO catalytic domainsestablishedthatdogfishsACisstimulated by Studies usingpurifiedrecombinantproteincomprisingthe two unclear whethersACsplicevariantsorisoformsexistindogfish. bream (Sparusaurata)(Carvalhoetal.,2012). recently confirmedbyanotherresearchgroupworkingwithsea ( and waterabsorptionintheintestinalepitheliumoftoadfish acanthias) gills(Tresguerres etal.,2010c),andasregulator ofNaCl sensor forbloodA/Bhomeostasisindogfishshark(Squalus The firsttworeportsofsACinfishdescribeditasanessential distribution suggeststhateitherflyandzebrafishsenseHCO salmon, gar, coelacanth,chimera,shark,skate).Thisphylogenetic insects (e.g.moth,bee,wasp,mosquito)andotherfishestrout, reveal anysACorthologs;however, sACgenesarefoundinother BLAST searchesofgenomesfromfruitflyandzebrafishstilldonot controversial andsothistopicisnotdiscussedfurtherinreview. et al.,2008),eventhemerepresenceofcAMP inplantsis presence ofsACinplants(Lomovatskayaetal.,2008;Romanenko (Matsuda etal.,2011). Althoughsomeevidencesupportsthe phylum (Table sAC-related genescannowbefoundinvirtuallyeveryanimal among animals,noritisexclusivetosperm. in sharkgills,demonstratingthatsACisnotuniquetomammals 2010c). Furthermore,sACwasfoundtobepresentandfunctional sAC enzymefromanon-mammalianvertebrate(Tresguerres etal., shark a sACorthologwasidentifiedfromanEST libraryfromthedogfish purification techniquesinsteadofbioinformatics.A fewyearslater, metals andtheirconcentrations matches thenormal[HCO other fish,mollusksandcoral. reports frommarinefishandsomeinformationaboutsAC about dogfishandseaurchinsACaredescribedbelow, aswell sAC ismaximallystimulatedbymillimolarconcentrationsofMn relevant changesinpH(Tresguerres etal.,2010c).Purifieddogfish mammalian sAC,dogfishsACisinsensitivetophysiologically in sharkplasma[HCO while HCO Ca stimulated bymillimolarconcentrationsofMn sAC areinhibitedbymillimolarCa estrogens. However, unlikemammaliansAC,dogfishandseaurchin micromolar concentrationsofKH7andderivativescatechol sAC in sharks andboneyfishes sAC insharks Opsanus beta)(Tresguerres etal.,2010b).Thislatterrole was To date,theonlypublishedbiochemicalreportsonnon- Dogfish sACis~110 With theexplosioningenomicandtranscriptomicinformation, The steepestpartofthesACdose–response curveforHCO 2+ 2+ 3 (unlike mammaliansAC).Theidentityoftheactualcatalytic , butneitherbyMg Squalus acanthias,whichledtothefirstcharacterizationofa – to producecAMP withanEC 3 – stimulation issupportedbyacombinationofMg

1), aswellinotherorganisms suchasdiatoms

mmol 3

– kDa (Tresguerres etal.,2010c),butitisstill ] inducedbyfeeding(metabolic alkalosis) 2+

3 l − – ] indogfishplasmaaswell changes alone noracombinationofMg 1 (Tresguerres et al.,2010c),which in vivo 2+ 50 concentrations. Moredetails of ~5 remain tobedetermined. mmol 2+ , andinhibitedby

l − 1 . Likepurified 3 – , potently 3 – 2+ 2+ using 3 and and – 2+ is , dependent translocationofV-type H which stimulatessAC,inturnstriggersthemicrotubule- CO subpopulation thatexpressesV-type H al., 2007).CO ATPases pumpintracellularH CO blood tocounteracttheoriginalalkalosis. (4)TheexcessHCO intracellular CA II(CA channel (CC). (AE). Chlorideisprobably absorbedintothebloodacross abasolateral to seawaterinexchangeforchloride viaaPendrin-likeanionexchanger i.3,andisasfollows:excessHCO Fig. for sensingandcounteractingbloodalkalosisissummarizedin al., 2010c;Tresguerres etal.,2007).ThesAC-dependentmechanism (Tresguerres etal.,2005;Tresguerres etal.,2006b;Tresguerres et (increased [HCO metabolic alkalosis)(Heisler, 1988),andbytemperaturechanges During increasedbloodHCO Fig. basolateral membrane.Basolateral V-type H apical membraneintoseawater, mostlikelyviaPendrin-likeanion H alkalosis viacontinuousintravenousinfusionofNaHCO confirmed throughaseriesofexperimentsthatinducedmetabolic situations. physiological responsesduringthesephysiologicallyrelevant 1988). Therefore,sACispoisedtosensetheA/Bstatusandtrigger Tresguerres etal.,2007).CO and sAC(Tresguerres etal.,2005;Tresguerres etal.,2010c; alkalosis. Atthesametime,excessHCO V-type H to generatecAMP, whichtriggersthemicrotubule-dependent translocationof not beenexperimentallyconfirmed.ThenowbasolateralV-type H phosphorylation ofmicrotubulemotorproteins;however, thishas al., 2007).ThismechanismmostlikelyoccursviaPKA-dependent Tresguerres etal.,2006b;Tresguerres etal.,2010c;Tresguerres et vesicles tothebasolateralmembrane(Tresguerres etal.,2005; + -ATPase-rich cells,whereitishydratedbackintoH The roleofsACasasensorA/Bstatusinsharkgillshasbeen 2

2 .sAC-dependentsensingandcompensationofbloodalkalosis. 3. by extracellularcarbonicanhydrase(CA by extracellularCA IV locatedingillpillarcells(Gilmouret + -ATPase (blueicon)containingcytoplasmicvesiclestothe The JournalofExperimentalBiology(2014)doi:10.1242/jeb.086157 2 then entersthegillepithelialcells,including 3 – ] athighertemperature,andviceversa)(Heisler, II ). (3)TheelevatedintracellularHCO 3 – and pH,(1)HCO 2 + is hydratedbackintoH into theblood,thuscounteracting 3 + – + -ATPases fromcytoplasmic IV -ATPase reabsorbsH in plasmaisdehydratedinto + ). (2)CO -ATPase, intracellularCA 3 – in bloodisdehydratedinto 3 – is secretedacrossthe + and HCO 2 enters theV-type 3 – stimulates sAC + 3 – and HCO is secreted 3 – by + into the 667 3 + 3 – – - ,

The Journal of Experimental Biology regulatory rolesinacid-secretingNa ATPase-rich cells(Tresguerres etal.,2010c),suggestingadditional correlates withsignificantV-type H HCO stomach (toaidwithfooddigestion),whichismatchedbyequimolar and H 668 regulatory: oncenormalblood[HCO independently ofanyhormonalcues,andintheoryisself- transporters (Piermarinietal.,2002).Thisprocessseemstowork REVIEW genome shotgun(WGS)assembliesofsalmon( al., 1991;Wilson etal.,2009;Wilson etal.,2002). for osmoregulationofmarineteleostfishes(Grosell,2011; Walsh et type H activities ofNa precipitation withNaClandwaterabsorptionbymodulatingthe in theteleostintestineistocoordinateintestinalcarbonate Tresguerres etal.,2010b).Theproposedphysiological roleofsAC bream intestineusingsACantagonists(Carvalhoetal.,2012; and inhibitionofNaClwaterabsorptionintoadfishsea intestine usingheterologousantibodies(Tresguerres etal.,2010b) fishes issofarlimitedtoimmunologicaldetectionintoadfish (Shuttleworth etal.,2006;Wood etal.,2007b). including thestimulationofNaClsecretionacrossrectalgland alkalotic post-feedingperiod(Walsh etal.,2006;Wood etal.,2008), changes thattakeplacethroughouttheshark’s bodyduringthe is agoodcandidatetomediatesomeoftheprofoundmetabolic 4)(seealsoRoaetal.,2012),it in multipleothersharktissues(Fig. pavement cellsandpillarcells.Furthermore,assACisalsopresent no longerstimulated,permittingtheretrievalofV-type H away fromthebasolateralmembrane. present inmostgillepithelialcellsandnotjustV-type H triggering theA/Bcompensatorymechanisms.Moreover, sACis sAC beingresponsibleforsensingpost-feedingalkalosisand cells (Tresguerres etal.,2007).Altogether, theevidencepointsto compensated forbyanupregulationofbranchialHCO (Wood etal.,2005;Wood etal.,2009).Thebloodalkalosisis sAC genesarepresentinthesequencedgenomesorwhole The publishedevidenceforsACpresenceandactivityinteleost In thepost-feedingperiod,dogfishgastriccellssecreteH 3 – + + -ATPase andanionexchangers.Theseprocessesareessential absorption intotheblood,resultinginpronouncedalkalosis absorption acrossthegills(Wood etal.,2007a),which + /K + -ATPase, Na + /K + + -ATPase translocationinthegill /Cl 3 – ] hasbeenrestored,sACis – ornpre (NKCC), V- cotransporter + /K + -ATPase-rich cells, Salmo salar), 3 – + -ATPases secretion + into the + - activity inresponsetoMn was notstimulatedbyforskolinorG-proteins,andshowedhigh which wasthefirstACtobeidentifiedfromanimalspermatozoa, values seemhighcomparedwith[HCO its maximumat50 not identifiedasaseaurchinsACanditssensitivitytoHCO not bediscovereduntilalmostadecadelater, theseaurchinACwas chromatography (Bookbinderetal.,1990).The190 identification andpurificationofanACenzymebyimmunoaffinity in urchinspermatozoa(GarbersandKopf,1980)ledtothe in thepurpleseaurchin, Among marineinvertebrates,sAChassofaronlybeencharacterized stimulated byHCO and Ishida,1987;Zillietal.,2008),atleastinsalmonidsitis sperm motilitydependsoncAMP inseveralfishspecies(Morisawa biology infishesasitismammals.Intriguingly, theinitiationof unpublished observations),suggestingsACisimportantforsperm testis ofdogfishsharkandrainbowtrout(M.T. andJ.N.R., (Roa etal.,2012).We havealsodetectedsACbyRT-PCR inthe hallerii) bywesternblottingusingantibodiesagainstdogfishsAC leopard shark( protein ingill,rectalgland,whitemuscle,intestineandeyeof the ubiquityofsACinelasmobranchsincludesdetection chimera ( stimulated byHCO sAC. UnlikethepH-insensitivemammalian(Chenetal.,2000)and raising doubtsaboutthephysiologicalsignificanceofseaurchin 2005). length) withseveralpotentialphosphorylationsites(Nomuraetal., contains multipleaminoacidinsertions(16–74acidsin et al.,2005).ComparedwithmammaliansAC,seaurchinsAC sequence indicateditwasahomologofmammaliansAC(Nomura not testedatthetime.Eventually, analysisoftheseaurchinAC sAC inseaurchin rainbow trout( oculatus), andintranscriptomeshotgunassemblies(TSA)of coelacanth ( In thepresenceofMg Callorhinchus milii The JournalofExperimentalBiology(2014)doi:10.1242/jeb.086157 Latimeria chalumnae Oncorhynchus mykiss Triakis semifasciata

3 mmol 3 – – (Morisawa andMorisawa,1988). with anEC S. purpuratus.HighlevelsofACactivity

l 2+ − 2+ 1 , partiallypurifiedseaurchinsACis . However, asmammaliansACwould (Beltrán etal.,2007).However, these ), littleskate( 50 al., 2010c). described previously(Tresguerres et green). Methodsfollowedthose nuclear localization(nucleistainedin showing cytoplasmicandpotentially sAC (brown)inrectalglandcells, gland. (D)Immunolocalizationof in gill(positivecontrol)andrectal (C) sACmRNA detectedbyRT-PCR predicted ~110 blood cells;thebandmatches immunoblot inrectalglandandred protein detectedbywestern dogfish sharktissues.(A,B)sAC Fig. ) andspottedgar( of ~20 ) (Table ) androundray(Urobatis

.ExpressionofsACin 4. 3 – ] inaquaticinvertebrates, mmol

1). Furtherevidencefor Leucoraja erinacea

kDa sACprotein.

l − 1 , anditreaches

kDa protein, Lepiosteus 3 – was ),

The Journal of Experimental Biology motility andacrosomereaction. 2001), suggestingvariousphysiologicalrolesinadditiontosperm is alsopresentinembryonicprimarymesenchymecells(Zhuetal., Searches ofEST andTSA databasesrevealseaurchinsACmRNA not essential,forthespermacrosomereaction(Beltránetal.,2007). area (Beltránetal.,2007).Indeed,seaurchinsACisimportant,but revealed seaurchinsACtoalsobepresentintheheadandacrosomal speract andpHchanges.Furtherresearchusingconfocalmicroscopy proposed thatthiscomplexmodulatesspermmotilityinresponseto α- andβ-tubulins(NomuraVacquier, 2006).Theauthors phosphodiesterase 5A,thereceptorforeggpeptidesperact,and kinase, membrane-boundguanylylcyclase,cGMP-specific nucleotide-gated ionchannel,sperm-specificcreatine HCO from allotheranimalsexaminedsofar, butmatchesthemotifsof REVIEW HCO serine inthepositionofresiduesuggestedtobeinvolved sAC aminoacidsequencesfromthesethreemolluskspecieshavea philippinarium) andtheowllimpet( identifiable sACorthologsincludetheVenus clam( unknown. Inadditiontooysters,othermolluskswithreadily al., 2012).However, thephysiologicalrolesofoystersACremain sensitive cAMP productioninmantletissuehomogenates(Barronet by RT-PCR inmantle,gillandhemocytes,haveobservedKH7- oyster (Crassostrea gigas).Moreover, wehavedetectedsACmRNA sAC hitsarepresentinthegenome,TSAsandESTs ofthePacific However, thehighEC steep sensitivitytopHbetween7.0and7.5(Beltránetal.,2007). dogfish sAC(Tresguerres etal.,2010c),seaurchinsACdisplaysa been explained byattributingactivitytoorthologs relatedtothe and Drummond,1985).Although theresultsofthesestudieshave by, forskolin(Manceboetal.,1991;Valbonesi etal.,2004;Weiss Aplysia californica galloprovincialis cAMP productioninsomebivalve speciessuchas responses predatesthediscovery ofsACinmammals.Forexample, tmACs. However, theliteratureoncAMP anditsphysiological in molluskshasbeenexclusivelyattributedtotheactivation of GPCRs (FabbriandCapuzzo,2010).Asaresult,cAMP production noradrenaline andadrenaline,whichareextracellularligands for relied heavilyontheuseofbiogenicamines5-HT, dopamine, in FabbriandCapuzzo,2010).ResearchoncAMP inbivalveshas relaxation afterthe‘catchresponse’,andstressresponse(reviewed reproduction, mantleandsiphonmovement,adductormuscle beating andactivation,spawninginduction,cardiaccontraction, implied toplayaroleinregulatingglycogenbreakdown, cilia unknown. Steegborn etal.,2005b).Theimplicationsofthesedifferences are chains 7and9,sperm-specificNa of theplasmamembraneandaxoneme,includingdyneinheavy al., 1998).SeaurchinsACistightlycomplexedwithseveralproteins dependent phosphorylationofflagella-associatedproteins(Brachoet triggers theinitiationofspermmotility, whichdependsoncAMP- midpiece (Bookbinderetal.,1990);thissuggestedseaurchinsAC proximal halfofthespermflagellumnearmitochondrial catalytic metals,salts,etc.). or ofnotutilizingphysiologicallyrelevantassayconditions(e.g. artifacts ofworkingwithsemi-purifiedseaurchinsACpreparations sAC inmollusks In bivalves,pathwaysinvolvingcAMP havebeenfoundor Initial experimentsfoundseaurchinsACtobeconcentratedinthe 3 3 – – -sensing bacterialsAC-likeenzymes(Linder, 2006; stimulation, whichisdifferent fromthethreonineinsACs and , areunresponsiveto,oronlyslightly stimulated Tapes philippinarum , aswelltheseahare, 50 value andpHsensitivitymayreflect + Lottia gigantea /H + exchanger (NHE),cyclic ) (Table Ruditapes

Mytilus 1). The homogenates issignificantlystimulatedbyHCO organism. IndicativeofsACactivity, cAMP production in symbiotic zooxanthellaeisamongthehighestobservedfromany vectensis. Total ACactivityincoraltissuehomogenatesdevoidof well asinthegenomeofstarletseaanemone pistillata freshwater clam stimulation ofsAC.Forexample,[HCO [HCO A/B homeostasis,photosynthesisandcalcification.Because question, butmultiplerolesarepossible,includingtheregulationof kinetic studiesusingpurifiedprotein. KH7 dose–responseofcoralsACneedtobedeterminedfrom significant elevationsinhemolymph[HCO hypercapnia (Gutowskaetal.,2010);bivalvesmayalsoexperience in atleasttwoothercoralspecies( the coral We haverecentlyidentifiedtwoisoformsofsACinthegenome establishing thesensitivityofmollusksACtoHCO plays anyphysiologicalroleinthesesituationsmuststartby still doubleorinsomecasesquadruple.ExploringwhethersAC much moremodestcomparedwiththatinactivecephalopods,itcan accumulation inbivalvesresponsetohypercapniaistypically increases inthe[HCO supply analternative,equallyplausible,explanation. forskolin-insensitive tmACIXisoform,orthologsofsACcould ( adherens), sponge(Amphimedonqueenslandica aquatic animals,includingthosefromplacozoa( Table dioica identifiable inWGSdatabasesofappendicularia( and seasquirt( is duetosACactivityremainsbedetermined. in vivo most activeinthelight.Indeed,endogenouscAMP levelsincorals be sensitivetophysiologicallyrelevantvariationsin[HCO by 50 μmol 100 cuttlefish ( from freshwatertobrackishwater(ByrneandDietz,2006).The 6.5 seasonal basis(Lopes-Limaetal.,2009),anddoublesfrom3to inhibitors, andbyidentifyingthetissueswhereitisexpressed. exposure toair(Michaelidisetal.,2005a).AlthoughHCO acidification (Lannigetal.,2010;Michaelidis2005b)and EC ~2 ( ( orthologs frommarinemammals includethosefromorcawhale (reviewed inWang etal.,2001)].Available sequencesforsAC undergoes averypronouncedpost-feedingbloodalkalosis not anaquaticanimalbut,like mostreptilesandamphibians, mississippiensis), aswellinsnake( Python morulus)[whichis ( sAC incorals sAC inotheraquatic animals Saccoglossus kowalevskii Trichechus manatus ) andwalrus(Odobenusrosmarus Orcinus orca), bottlenosedolphin( Chrysemys picta)andAmericanalligator( The biologicalroleofsACincoralsremainsanoutstanding Mollusks typicallyexperiencemanysituationsassociatedwith mmol 50 mmol mmol of ~10 1 containsanon-exhaustivelistofsACorthologsfromother 3 ), green seaturtle( ), – are highestinthelight(Barottetal.,2013),butwhetherthis ] incoraltissuesrangesfrom~4 ; M.T., K.L.B. andM.E.B.,unpublishedobservations),as Acropora digitifera

l l

− l − Sepia officinalis 1 − 1 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.086157 l 1 mmol − to over10 in zebramussel( 1 in thelight(Furlaetal.,2000),coralsACislikelyto KH7 (Barottetal.,2013).TheHCO Ciona intestinalis Anodonta cygnea l − 1 , andtheHCO 3 – ] intheirinternalfluids,whichcouldleadto mmol ) canaccumulateHCO ), amphioxus(Branchiostomafloridae (Barott etal.,2013),andsACispresent Dreissena polymorpha l hlnamydas),turtle painted Chelonia − 1 to compensateforenvironmental ). PartialsACsequencesarealso rises from5to12 3 – -stimulated activityisinhibited Tursiups truncatus

A. yongei 3 mmol – ] inthehemolymphof 3 – ] inresponsetoocean l − 1 3 in thedarktoover – 3 – with anapparent and ), acornworm 3 in plasmafrom ) upontransfer – mmol Nematostella EC Oikopleura 3 Trichoplax ) (Table Stylophora ), manatee – 50 , pHand Alligator

l and the 3 − – 1 ] and on a 669

1). 3 – )

The Journal of Experimental Biology from HCO initiate? Onepotentialmechanisminvolvestheintegrationofinputs express sAC,buthowdoessAC‘know’ whichresponseitshould bicarbonate-secreting cellsofsharkgillsandmammaliankidney difference betweenthedifferent typesofstress.Bothacid-and A/B disturbances,cellsandorganisms mustbeabletotellthe is exactlytheoppositeofresponsetoalkalosis.To correctfor HCO tmACs mayactuallydependonsAC. existing literature,asmanyofthefunctionscurrentlyascribedto cAMP indifferent intracellularlocations warrantsarevisitingofthe roles asasensorofA/Bstress.Theexistencemultiplesources systems, sACispoisedtoplaymultipleimportantphysiological 670 months. Program inMarineBiotechnologygrant.DepositedPMCforreleaseafter12 J.N.R., whoisalsopartiallysupportedbyaNationalInstitutesofHealthTraining Science FoundationgraduatefellowshipstoM.E.B.,andSanDiegoFellowship 1226396 toK.L.B.,ScrippsInstitutionofOceanographyRegentsandNational EF-1220641 toM.T., NationalScienceFoundationOCEpostdoctoralfellowship This workwassupportedbySIOfundstoM.T., NationalScienceFoundationgrant presented inFig. All authorswrotethearticle.M.T. producedthefigures,includingdata The authorsdeclarenocompetingfinancialinterests. Buck andDrLonnyR.Levin(Weill CornellMedicalCollege). We appreciatetheuseful comments byDrVic Vacquier (SIO-UCSD),DrJochen Because oftheubiquitybothHCO combination withdifferential cellmembranepermeabilitytoCO Brown andWagner, 2012;Tresguerres etal.,2010a),possiblyin alkalosis arecharacterizedbyelevated complex. Forexample,bothrespiratoryacidosisandmetabolic its potentialfunctionduringotherA/Bstressconditionsismore While thesensoryroleofsACduringalkalosisisstraightforward, REVIEW cnPrz . aaa,E,Kmntk,M,Bc,J,Lvn .R n Manfredi, and L.R. Levin, J., Buck, M., Kamenetsky, E., Salazar, R., Acin-Perez, References Funding Author contributions Competing interests Acknowledgements Conclusions acidsensor(s) Need for complementary to acidosisrequiressecretionofH result insACstimulation.However, thehomeostaticA/Bresponse puutn . ascet .A,Mcoulr,M,Fak,J-. ua,S., Kumar, J.-P., Flacke, M., Micoogullari, S.A., Kasseckert, A., Appukuttan, aot .L,Hla,Y,Hrmt,L,Bro,M . es .C,Bc,J,Levin, J., Buck, K.C., Hess, M.E., Barron, L., Haramaty, Y., Helman, K.L., Barott, etá,C,Vcue,V . o,G,Ce,Y,Bc,J,Lvn .R n Darszon, and L.R. Levin, J., Buck, Y., Chen, G., Moy, V. D., Vacquier, C., Beltrán, M. Tresguerres, and J.N.B. Roa, M.E., Barron, rco .E,Fic,J .adTs,J.S. Tash, and J. Fritch, G.E., Bracho, okidr .H,My .W n aqir V. D. S. Vacquier, and Riley, G.W. Moy, L.H., and Bookbinder, D. Owens, J.A., Bettice, ru,T. Braun, phosphorylation. G. ot,A,Adla,Y,Pt,L,Ruc,H .adLdlv Y. Ladilov, and H.P. cardiomyocytes undersimulatedischaemia/reperfusion. Reusch, L., Pott, adenylyl cyclasemediatesmitochondrialBaxtranslocationandapoptosisofadult Y., rat Abdallah, A., Woste, fluctuations incoralssuggestcentralphysiologicalrole. M. Tresguerres, and L. R. 349. acrosome reaction. A. FASEB J. and hemocytesexpressthebicarbonate-sensingenzymesolubleadenylylcyclase. 242 that initiatetheactivationofspermmotility invivo. intracellular pHandbicarbonate. sperm adenylatecyclase. systems inrattestis. Curr. Top. Mol. Endocrinol. , 231-237. (2007). Particulateandsolubleadenylylcyclasesparticipateinthesperm 3 (2009). CyclicAMP producedinsidemitochondriaregulatesoxidative – . (1974). Evidencefor multiple,cellspecific,distinctiveadenylate cyclase 26, 1070.2. 3 – -responsive sACandoneorseveralH

4. Cell Metab. Biochem. Biophys.Res.Commun. J. CellBiol. (2013). HighadenylylcyclaseactivityandinvivocAMP 9 , 265-276. Respir. Physiol. 111 + , 1859-1866. and absorptionofHCO (1998). Identificationofflagellarproteins (1984). Theeffects ofhypocapniaon P 1 3 55, 121-130. , 243-264. CO – Biochem. Biophys.Res.Commun. (1990). Identificationofseaurchin (2012). Pacificoystermantle,gill and cAMP inbiological 2 and [HCO 358 Sci Rep Cardiovasc. Res. , 1128-1135. 3 , 1379. + 3 (2012). Type 10 sensors (see – ] andcould 3 – , which 93, 340- 2 or es .C,Jns .H,Mruz . hn . r,T . aeesy M., Kamenetsky, T. S., Ord, Y., Chen, B., Marquez, B.H., Jones, K.C., Hess, ese,N. Heisler, ru,T. Braun, T. Braun, ru,T n os R.F. Dods, and T. Braun, ulo,J . aaa .adCoe,D.M. Cooper, and H. Nakata, J.L., Guillou, M. Grosell, H and G. Claireaux, C., Bock, M., Langenbuch, F., Melzner, M.A., Gutowska, aon,J n ee,N. Defer, and J. Hanoune, ru,T. Braun, og . laoa . mlk . i . aed . emn,D,Hlos K. 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