Molecular physiology of osmoregulation in eels and other teleosts: the role of transporter isoforms...

Ž .Comparative Biochemistry and Physiology Part A 130 2001 551�564

Review

Molecular physiology of osmoregulation in eels andother teleosts: the role of transporter isoforms and gene

duplication�

Christopher P. Cutler�, Gordon CrambSchool of Biology, Bute Medical Buildings, Uni�ersity of St Andrews, St Andrews, Fife, KY16 9TS, UK

Received 21 November 2000; received in revised form 16 March 2001; accepted 19 March 2001

Abstract

This review focuses on recent developments in the molecular biology of ion and water transporter genes in fish andthe potential role of their products in osmoregulation in both freshwater and seawater environments. In particulardetails of isoforms of various ATPases, co-transporters, exchangers and ion channels in the eel as well as other teleostspecies are described. Many of the teleost transporter isoforms discovered so far, appear to occur as twin or duplicatecopies compared to their homologous counterparts in higher vertebrates, although these duplicate isoforms often havedistinct tissue-specific and developmental stage-dependent expression patterns. The possible meaning of this informa-tion will be examined in relation to the fish genome duplication debate. � 2001 Elsevier Science Inc. All rights reserved.

Keywords: Genome duplication; Ion transport; Na,K-ATPase; Na�K�2Cl cotransporter; Na�Cl cotransporter; Na�HCO co-3transporter; CFTR chloride ion channel; Cl�HCO exchanger; Na�H exchanger; Aquaporins; V-type ATPase.3

1. Introduction

Studies concerning the number and evolutionŽ .of homeobox Hox gene clusters in zebrafish

Ž .Danio rerio , have suggested the possibility thatthe genome of a teleost fish ancestor may haveundergone a genome duplication or polyploidisa-tion event in addition to those that have occurred

� This paper was originally presented at a symposium dedi-cated to the memory of Marcel Florkin, held within theESCPB 21st International Congress, Liege, Belgium, July`24�28, 2000.

� Corresponding author. Tel.: �44-1334-463531; fax: �44-1334-463600,.

Ž .E-mail address: [email protected] C.P. Cutler .

Žin other groups of higher vertebrates Amores etal., 1998; Postlethwait et al., 1998; Wittbrodt et

.al., 1998; Meyer and Malaga-Trillo, 1999 . How-ever, further studies on some teleost species such

( .as pufferfish Fugu rubripes , Japanese medaka( . (Oryzias latipes , and striped bass Morone sax-

.atilis have so far failed to conclusively demon-strate additional hox gene clusters to the four

Žfound in mammals Meyer and Malaga-Trillo,.1999; Stellwag, 1999 . It has been suggested that

this may be due to the loss of chromosomalŽmaterial including hox gene clusters: Stellwag,

.1999 in these species and the smaller size of thepufferfish genome in particular, lends weight tothis theory. However, the lack of additional hoxgene clusters in these species has led to the

1095-6433�01�$ - see front matter � 2001 Elsevier Science Inc. All rights reserved.Ž .PII: S 1 0 9 5 - 6 4 3 3 0 1 0 0 4 3 5 - 4

( )C.P. Cutler, G. Cramb � Comparati�e Biochemistry and Physiology Part A 130 2001 551�564552

alternative hypothesis that the polyploidisationevent may have occurred subsequent to the diver-sification of the ray-finned fish, possibly exclu-sively in zebrafish, but otherwise, in a much morelimited but currently indeterminate range of fish

Ž .species Stellwag, 1999 . Information coming fromstudies of fish chromosomal numbers or amountsof DNA in the genome does not readily yield ananswer as to which hypothesis is correct. Whilethere are teleost species which have obviouslyundergone relatively ancient tetraploidisation withsubsequent assimilation of chromosomes into a

Ždiploid complement e.g. cyprinids such as the� 4common carp Cyprinus carpio: 2n �104 : Ohno,

.1999 , or that have remained polyploid includingsome other cyprinids and various salmoniformsŽ .Ohno, 1999; Stellwag, 1999 , other cyprinid

Ž .species including zebrafish 2n�50 , show nosigns of having unusually large chromosome com-plements compared to the majority of other

Žteleosts or indeed mammals e.g. homo sapiens:.2n�46 . The fact that the zebrafish does not

have a significantly larger chromosome comple-ment than the majority of other teleosts does notdiscount the possibility that these other speciesmay also have a duplicated genome. One indica-tion that widespread duplication has occurredwould be the presence of larger genomes inteleosts compared to higher vertebrates, but in

Žfact estimates suggest that with some notable.exceptions the haploid genomes of bony fish are

significantly smaller than those of mammals.One of the objects of this paper is to discuss

the data derived from osmoregulatory genes cod-ing for ion and water transporters, in the eel andother teleost fish species, and to review the mean-ing of this information in relation to the fishgenome duplication debate. In addition, this arti-cle will also act as a review of the latest publishedand unpublished data, produced by this labora-tory and others, concerning molecular aspects ofosmoregulation in teleost fish.

From an osmoregulatory point of view, as wellas from virtually any other perspective, theprospect of two complements of some or all ofthe genes connected with any particular physio-logical process, makes a fundamental differenceto the investigation of the molecular basis ofunderlying mechanisms. As for example, the anal-ysis of results obtained from the study of a ‘known’housekeeping isoform may give a completely er-

roneous picture of the overall role of a protein inosmoregulatory mechanisms, if a second ‘un-known’ regulatable isoform also exists. Further-more, offsetting changes in the regulation of iso-forms could be masked in studies concerned withthe global action of a particular protein, for ex-ample in enzyme assays. Thus for fish molecularphysiologists, it is of great importance to knowwhether the genome duplication event in ze-brafish is species-specific or common to allteleosts, and in particular, whether the speciesused for experimentation in an investigators ownlaboratory has a duplicated genome.

Ž .The European eel Anguilla anguilla , like ze-brafish, does not appear to have an enlarged

Žchromosomal complement 2n�38: Salvadori et.al., 1997 which gives no clue as to the likely

presence of duplicate copies of osmoregulatory orother genes. The first evidence of duplicate copiesof at least some genes in the eel came from workon the active ion transporting enzyme, the Na,K-ATPase.

2. Na,K-ATPase

Na,K-ATPase actively transports intracellularsodium in exchange for extracellular potassiumions, consists of two essential components, namelythe � and � subunits, and in mammals there arecurrently four known isoforms of each subunit.

Ž .Full length sequences of the first � subunit �1of Na,K-ATPase have now been published in anumber of teleost fish species such as the white

Žsucker Catostomus commersoni: Schoenrock et. Ž .al., 1991 and the eel Cutler et al., 1995a and in

addition, partial sequences have been reportedŽfor tilapia Oreochromis mossambicus: Hwang et

. Žal., 1998 and rainbow trout Oncorhynchus mykiss:.Kisen et al. 1994; D’Cotta et al., 1996 . An �3

isoform has also has been shown to exist in theŽ .eel Cutler et al., 1996 and evidence from anti-

body studies suggests that an �3 subunit is alsoŽpresent in catfish Ictalurus punctatus: Pressley,

. Ž1992 and tilapia Oreochromis mossambicus: Lee.et al., 1998 . Unlike the situation in mammals,

where the �3 isoform is predominately expressedŽ .in the brain Young and Lingrel, 1987 , both of

the � isoforms in the eel exhibit an ubiquitousŽ .tissue distribution Cutler et al., 1996 . Current

information from the gene bank indicates thatthere are multiple Na,K-ATPase � subunit iso-

( )C.P. Cutler, G. Cramb � Comparati�e Biochemistry and Physiology Part A 130 2001 551�564 553

Fig. 1. The N-terminal amino acid sequences of Na,K-ATPase � subunits from various fish species. Nucleotide sequences wereobtained from the EMBL gene bank with accession numbers listed below, from which the putative amino acid sequences weredetermined: Eel �1, X76108: Tilapia �1, U8254: White Sucker � , X58629: Catfish � , BE212661: Zebrafish �1, AF286372: Zebrafish�2, AF286373: Zebrafish �3, AF286374: Zebrafish �4, AW280931: Zebrafish �5, AW422519: Zebrafish �6, AW280277: Zebrafish �7,

Ž .AW281430: Tilapia �3, AF109409: Medaka � , AF072312. Except for the Eel �3, unpublished data . The isoform denominationfollows mammalian nomenclature except for Zebrafish where the denomination is arbitrary. Symbols in the alignment are as follows: �

conserved amino acid; � amino acid with conserved similarity; � amino acid deletion and � missing sequence data.

Ž .forms expressed in zebrafish Fig. 1 , which isanalogous to the situation with homeobox geneclusters in this species.

Although the quality of expressed sequence tagŽ . ŽEST sequences is not 100% reliable as oftenindicated in the comments attached to EST

.genebank files , there do appear to be sevendistinct Na,K-ATPase � isoform sequences ex-pressed in zebrafish, which fall into three broad

Ž .categories, being either �1- zebrafish �1, 3�5 ,Ž . Ž�2- zebrafish �2 �3- zebrafish �6�7: 88% simi-

. Žlar to each other like following mammalian no-.menclature . However, on closer inspection the

�1-like sequences can be arranged into twogroups, based on the structure and sequence ho-

Žmologies of the N-terminal sequence this regionin mammalian isoforms is not very well con-

.served, but isoform specific ; these groups are theŽ .�1 and �4 82% similar to each other , and theŽ .�3 and �5 78% similar to each other , respec-

tively. The eel �1, tilapia �1 and white sucker �

isoforms are all somewhat more similar to thefirst of these groups. The zebrafish �2 isoform ismarginally more homologous to the �2 ratherthan to other mammalian and avian � isoformsŽ .when considering the whole sequence , althoughthis is not clear from the amino terminal se-quence presented in Fig. 1, as isoform-specificamino acid motifs, conserved between mam-malian and avian �2 isoforms, are not particu-larly well conserved in the sequence of the ze-brafish �2 isoform. Regarding the �3 isoforms,the tilapia �3 and medaka � sequences are simi-lar in structure to mammalian �3-isoforms. The

Ž .eel and zebrafish �3-like zebrafish �6�7 se-quences have an additional 13 conserved amino

Žacids near the N-terminus amino acids 3�16 in.Fig. 1 . There are some indications that other

species also have a number of Na,K-ATPase �-isoforms; collaborative work in this laboratorysuggests that there are at least five iso�splico-

Ž .forms expressed in Atlantic salmon Salmo salar ,


where at least two of these are �1-like isoforms.Currently there is nothing known about the func-tion, regulation or tissue-specific localisation ofthe additional zebrafish or salmon isoforms.

There are also published sequences of teleostfish Na,K-ATPase � subunit isoforms. A homo-logue of the mammalian �1 subunit has been

Ž .identified in the eel Cutler et al., 1995b and aduplicate copy of this isoform has also recently

Žbeen cloned and is called �233 Cutler et al.,.2000 . The �233 has a much more limited tissue

distribution of mRNA expression than the �1isoform, however, it appears that the �233 isformis expressed at higher levels than the �1 isoformin osmoregulatory tissues such as the gill andintestine. There are also developmental differ-ences between the two duplicate isoforms, whilethe �1 isoform has increased mRNA levels fol-

Ž .lowing seawater acclimation in yellow adult orŽsilver a later seawater migratory adult develop-

.mental stage eels, the level of �233 expression isonly upregulated in silver eels following seawateracclimation. In addition to �1 isoforms, a homo-logue of the mammalian Na,K-ATPase �3 wasidentified in zebrafish and a partial sequence isalso available for the eel; in both of these speciesthe �3 isoforms are predominately expressed in

Ž .the brain Appel et al., 1996, Cutler et al., 1997a .ŽCurrent information from the gene bank EMBL

.or Genbank which share gene sequence data ,indicates that there are multiple Na,K-ATPase �

Ž .subunit isoforms expressed in zebrafish Fig. 2 .The current sequences in the gene bank sug-

gest there may be as many as four copies of theNa,K-ATPase �1 isoform in zebrafish, althoughtwo of these partial copies do not code for over-lapping amino acid regions, and so may representsequences from different parts of the same iso-

Ž .form �1c and �1d . Furthermore, the quality ofsequence information associated with ESTs isnot always entirely accurate, leaving open thepossibility that sequence differences between ap-parent isoforms may be created by error. Thereare some indications that other species may haveduplicate Na,K-ATPase �1 isoforms, collabora-

Ž .tive work see acknowledgements in this labora-tory suggests that there are also three isoformsexpressed in Atlantic salmon. As well as multipleisoforms of the �1 there is also evidence in thegene bank that there is a duplicate copy of the �3

Ž .in zebrafish �3b , although whether or how this

isoform differs in function, regulation, or tissue-specific or developmental expression in compar-ison to the original, is not known. There is also ahomologue of the mammalian Na,K-ATPase �2isoform in zebrafish which shares 67% amino acidhomology with an eel �185b cDNA fragment and57% amino acid homology with its possible dupli-

Ž .cate, the eel �185 Cutler et al., 1996, 1997b . The�185 mRNA is expressed solely in the brain andeye, in a similar fashion to mammalian �2 iso-forms which are predominately found in nervous

Ž .tissue Appel et al., 1996 .

3. Chloride–cation-cotransporters

This gene family is comprised of a group ofproteins whose activity is sensitive to various di-uretic drugs and which have three identified iontransport specificities, cotransporting chloride ions

Ž . Ž .together with sodium Na�Cl , potassium K�ClŽ .or sodium and potassium Na�K�2Cl ions. In

addition, two isoforms of the Na�K�2Cl co-transporter have been identified and these areinvolved in ion transport mechanisms principallyassociated with either absorption or secretion re-spectively, although they may also play a role in

Ž .cell volume homeostasis Russell, 2000 . The firstmember of the diuretic-sensitive chloride cationco-transporter gene family to be cloned was theflounder thiazide-sensitive Na�Cl cotransporterŽ .NCC: Gamba et al., 1993 . Since then, mam-malian counterparts have been cloned both for

Ž .NCC and for the secretory NKCC1 and absorp-Ž .tive NKCC2 isoforms of the Na�K�2Cl co-

transporter as well as for four K�Cl cotransporterŽ . ŽKCC isoforms Gamba et al., 1994; Delpire etal., 1994; Payne and Forbush, 1994; Mount et al.,

.1999 . In the eel, cDNA fragments coding forhomologues of NKCC1, NKCC2 and NCC werecloned from the gill and intestine and have been

Ždesignated cot 1, 2 and 4, respectively, Cutler et.al., 1996 . Like its mammalian counterpart,

mRNA coding for cot 1 is ubiquitously expressedin nearly all eel tissues. In yellow eels expressionof cot 1 mRNA in the gill increases 6-fold duringsalinity acclimation but this response is absent insilver eels. This suggests that this protein maywell play a role in ion extrusion in branchialchloride cells, at least in yellow eels, although adifferent mechanism may operate in silver eels.


Ž .Fig. 2. Partial and complete teleost Na,K-ATPase � subunit acid sequences derived from nucleotide sequences reported to the genebank. Nucleotide sequences were obtained from the EMBL gene bank with accession numbers as follows: Eel �1, X76109: Eel �233,AJ239317: Zebrafish �1, AF286375: Zebrafish �1b, AW280780: Zebrafish �1c, AI617515: Zebrafish �1d, AW305551: Zebrafish �2,AF286376: Zebrafish �3, X89722: Zebrafish �3b, AF293369: Winter Flounder �1, AW013599: Catfish �1, BE212878: Medaka �1,AV668732. The isoform denomination follows mammalian nomenclature. Symbols in the alignment are as follows:- � conserved aminoacid; � amino acid with conserved similarity; � amino acid deletion and spaces imply missing sequence data.

Preliminary immunohistochemical localisation ex-periments show that chloride cells may be theprincipal site of expression of cot 1 in the gill, buta low level of expression was seen in all cell types.Initial experiments indicated that cot 1 mRNA isalso expressed in the intestine and, subsequently

the localisation of this expression has been nar-rowed down solely to the mid gut region. Thelikely physiological role of a secretory Na�K�2Clco-transporter, such as cot 1, in the intestine, isprobably associated with luminal fluid secretionfor digestive purposes. However, a possible role


for this isoform in volume regulation of cellswithin the mid gut region cannot be ruled out.During the cloning of cot 1, a further duplicateisoform was also identified in the eel, which wascalled cot 3. Expression of this isoform was mainlyfound in the brain, although in Northern blotslow levels of mRNA expression could also be

Ždetected in the gills of yellow eels Cutler et al.,.1996 . Currently the cellular location of branchial

cot 3 expression or its role in salinity acclimationis unknown. A recent study in the mudskipperŽ .Periophalmodon schlosseri: Wilson et al., 2000bhas suggested that a Na�K�2Cl cotransporter isexpressed in branchial chloride cells. However,while this information agrees with the data fromthe eel cot 1 isoform, it should be viewed withcaution as many of the normal control experi-ments were not possible. How these data relate tothe potential presence of secretory Na�K�2Clcotransporter isoforms in the mudskipper is alsounclear.

The absorptive type of Na�K�2Cl co-trans-porter, cot 2, is the major isoform expressed inthe intestine, although it is also expressed at

Žreasonably high levels in the urinary bladder Cu-.tler et al., 1996 . Expression predominates in the

anterior and mid regions of the gut with reducedlevels in the posterior intestine and�or rectum.Expression in all regions of the intestine increasefollowing salinity acclimation in both yellow andsilver eels, although the levels vary considerablyboth between experiments and individuals. Mam-malian NKCC 2 isoforms are almost exclusively

Žexpressed in the kidney Payne and Forbush,.1994 , and the completely different pattern of

tissue expression of cot 2 in the eel suggests thatthis isoform might not be a direct counterpart ofNKCC2. Not surprisingly then, recent cloning ex-periments in eel kidney led to the cloning of afourth NKCC isoform which appears to be aduplicate of cot 2, designated as cot 6. Prelimi-nary Northern blots have demonstrated its pres-ence in the kidney but the level of mRNA expres-sion is not significantly changed by salinityacclimation. This may mean that the expecteddecrease in NKCC 2 activity in SW kidney isproduced by post-transcriptional regulation.

The eel homologue of the NCC cotransporter,cot 4, is only expressed at low levels in the eel,but it can be detected on Northern blots in theposterior intestine and�or rectum. Here the lev-els of expression are not changed following salin-

ity acclimation. The tissue-specifc expression ofcot 4 in the eel is somewhat different to theequivalent gene found in the winter flounder,where it was additionally found to be expressed inthe bladder, gonads, skeletal muscle, eye and

Ž .brain, but not kidney or gill Gamba et al., 1993 .The presence of cot 4 in the posterior intestineand�or rectum may explain why the level of cot 2expression is decreased in this region of the gut.As the seawater imbibed by marine teleosts passesdown the intestine, the potassium concentrationdecreases due to absorption via Na�K�Cl co-transport, suggesting further absorption of NaClmay be more efficiently achieved in the posteriorgut and�or rectum by a Na�Cl rather than apotassium-dependent cotransporter. Despite thelack of mRNA expression on Northern blots, atruncated form of cot 4 can be amplified from eelkidney using RT�PCR. However notwithstanding

Žthis, the low level of cot 4 in this tissue, whichrepresents the major site of NCC expression in

.mammals; Gamba et al., 1994 , suggested that cotŽ .4 or flounder NCC may not be the only or the

most abundant homologue of mammalian NCCexpressed in teleost fish kidney. Further cloningexperiments showed this to be the case, as acDNA fragment which was also homologous tomammalian NCC was recently cloned from eelkidney and was designated cot 5. Northern blotshave demonstrated that, in the same fashion asmammalian NCC, cot 5 expression is almost ex-clusively located in the kidney. However, prelimi-nary blots also suggest that cot 5 mRNA levels inthe kidney are not greatly altered by salinityacclimation.

4. Ion channels

A number of different ion channels are knownto play roles in osmoregulatory processes. How-ever, due to its central role in ion secretoryprocesses in other species, a key member amongstthese ion channels is a CFTR-like chloride ionchannel. A gene fragment encoding a portion ofthis protein was originally identified in the eelŽ .Cutler et al., 1996 in addition however, the full

Žlength sequence from the killifish Fundulus hete-. Žroclitus has also now been determined Singer et.al., 1998 . The CFTR protein represents both a

chloride ion channel as well as a regulator ofother ion and water transporters in higher verte-


Žbrates Schwiebert et al., 1999; Schreiber et al.,.2000 . However, the amino acid and nucleotide

sequences of teleost CFTR cDNAs have muchlower homology to mammalian sequences thandoes the CFTR gene of the evolutionarily more

Žancient elasmobranch fish, the dogfish Squalus.acanthias: Marshall et al., 1991 . This suggests

that teleost CFTR has a somewhat altered struc-ture and function compared to CFTR in mam-malian or elasmobranch species. However, so farkillifish CFTR has been shown to represent acAMP-regulated chloride ion channel with similar

Žcharacteristics to mammalian counterparts Singer.et al., 1998 . Killifish CFTR mRNA expression

has been demonstrated in the gills and opercularmembrane as well as the intestine and brain, andhas also been shown to increase 9-fold in the gill,24 h after the onset of seawater acclimationŽ .Singer et al., 1998; Marshall et al., 1999 . Theseresults are in direct contrast to results in the eelwhere CFTR mRNA is only expressed at lowlevels in the gill and this does not alter followingseawater acclimation. These apparent anomaliesmay reflect differences in the habitats occupiedby the two species, as the killifish is acoastal�estuarine species, having to adapt toconstantly changing salinities, whereas the eelwould normally only expect to undergo freshwa-ter�seawater migrations twice during its entirelifetime. However, the differences in the patternsof CFTR expression between these two teleostscould be purely due to the evolutionary diver-gence of the functional roles of these proteins.Alternatively, the differences could also be due tothe presence of two different CFTR-isoforms ineach species, although there is no direct evidencecurrently to demonstrate that this is the case.However, two duplicate CFTR isoforms have been

Žisolated from the Atlantic salmon deposited inthe EMBL gene bank under accession numbers

.AF155237 and AF161070 , although the aminoŽ .acid homology between these is so high �95% ,

that they probably result from a relatively recentduplication event in this species. More recently,as many as five isoforms are reported to be pres-

Ž .ent in this species Chen et al., 1999 . A recentŽstudy in the mudskipper Periophalmodon

.schlosseri: Wilson et al., 2000b has suggested thata CFTR-protein is expressed in the apical regionof branchial chloride cells. However, this infor-

mation should be viewed with caution as many ofthe normal control experiments were not possi-ble. How these data relate to the potential pres-ence of CFTR-isoforms in the mudskipper is alsounclear.

In addition to CFTR, other ion channels mayplay a role in osmoregulatory processes. Recentlytwo members of the CLC chloride ion channel

Žwere cloned from tilapia CLC-3 and CLC-5:.Miyazaki et al., 1999 . Both of these ion channels

are expressed in osmoregulatory tissues such asthe gill, intestine and kidney, however, the levelof mRNA expression in these tissues is compara-ble in freshwater or seawater acclimated fish.Therefore, there is no indication that these ionchannels are involved in chloride ion homeostasisduring salinity acclimation. A further ion channelwhich may be involved in osmoregulatoryprocesses is the inwardly rectifying potassiumchannel, eKir, which was recently cloned from eel

Ž .gill Suzuki et al., 1999 . This potassium channelshows increased mRNA expression in the gill,kidney and posterior intestine of the seawater eelin comparison to those acclimated to freshwater,and is located in the basolateral microtubularsystem of chloride cells of the branchial epithe-lium. These results taken together suggest thateKir may represent the channel on the basolat-eral surface of chloride cells, which recyclespotassium ions entering through the Na�K�2Clcotransporter, back into interstitial body fluidsduring the process of NaCl excretion across thegills.

Despite attempts in several laboratories, cloningof homologues of subunits of the epithelial Na-

Ž .channel eNaC from teleost fish species has notso far proved possible. Together with V-type AT-Pase, this protein has long been postulated toplay an important role in the absorption of Naacross the apical surface of the gills in FW fishŽ .for example see Evans et al., 1999 . Recent stud-ies using mammalian antibodies raised againstmammalian eNaC � and � subunits have sug-gested that the subunits of this protein may in-deed be located on the apical surface of pave-

Ž .ment rainbow trout and tilapia and mitochon-Ž .dria-rich cells rainbow trout of the branchial

Ž .epithelium Wilson et al., 2000a . However, again,this information should be viewed with caution asmany of the normal control experiments were notpossible.


5. Bicarbonate transporters

Ž .The band-3 anion exchanger AE was origi-nally cloned from a rainbow trout spleen cDNAlibrary. This protein functions as a chloride-bi-carbonate exchanger, although it may also be able

Žto transport taurine, sorbitol and urea Hubner et.al., 1992; Fievet et al., 1998 . The mammalian

anion exchanger gene family consists of 3-iso-Ž .forms AE1-3 and the structure of the trout AE

is most similar to the original band 3 isoformŽ .AE1 although the amino acid homology to

Ž .mouse AE 1 is only 50% Hubner et al., 1992 .While chloride�bicarbonate exchangers arethought to have a role in acid�base regulationŽ .Perry, 1997 , its also possible that they also playa role in osmoregulatory processes, in tissues suchas the gill, intestine and kidney.

In the gill, anion exchangers have been postu-lated to be expressed on both the apical andbasolateral membranes of epithelial cells and tobe responsible for the uptake of chloride in fresh-

Ž .water fish Evans et al., 1999 . The cell typesassociated with this uptake may be pavement cellsŽ .Wood et al., 1998 , however, chloride�bi-carbonate exchanger mRNA has also been lo-calised to cells on the primary lamella, and ex-pression shared similar localisation to that char-acteristic of mitochondria�Na,K-ATPase-rich

Žchloride cells Sullivan et al., 1996; Wilson et al.,.2000a,b . Chloride�bicarbonate exchange has also

been suggested to play an indirect role in ionsecretion on the basolateral surface of chloride

Žcells in seawater acclimated teleosts Zadunaisky.et al., 1995 , and this activity may serve to regu-

late intracellular pH, where conditions favouringintracellular alkalosis augment chloride secretionŽ .Marshall and Bryson, 1998 .

In the intestine of seawater acclimated eels, achloride�bicarbonate exchanger involved in bi-carbonate extrusion has been suggested to belocated on the brush border membranes of lumi-

Žnal epithelial cells Ando and Subramanyam,.1990 , although this finding was not confirmed by

Ž .others Trischitta et al., 1992a . A chloride�bi-carbonate exchanger has also been proposed toexist on the basolateral surface of intestinal ente-

Ž .rocytes Trischitta et al., 1992b , however, theresults from these studies may also be explainedby the presence of separate electrogenic sodiumbicarbonate cotransporter and an as yet unidenti-fied chloride efflux pathway. The brush border

Žmembranes of eel kidney tubular cells Viella et.al., 1997 may also contain a chloride�bi-

carbonate exchanger which may be involved inbicarbonate re-absorption.

Complementary DNA fragments were recentlyisolated from the intestine of the eel and theseencode three isoforms which are homologous tomembers of the chloride�bicarbonate exchanger

Ž .family EAE 1-3; unpublished data . Preliminarystudies indicate that EAE 1 is a direct counter-part of trout AE and its mRNA is expressed athigh levels in gill, kidney, heart and brain, andwith only just detectable levels of expression inthe intestine, oesophagus and liver. Preliminaryexperiments show that expression of EAE 1mRNA decreased in the gill and kidney followingseawater acclimation. EAE 2 which appears to bea duplicate isoform of EAE 1, is expressed at highlevels in kidney, oesophagus and stomach, withintermediate levels in heart and lower levels inbrain, eye, intestine and gill. As with EAE 1,preliminary experiments show that EAE 2 mRNAexpression was decreased in the gill and kidneyfollowing seawater acclimation. EAE 3 is mosthomologous to the mammalian AE 2 isoform, andhas high levels of expression in the gill, withintermediate levels in kidney and lower levels inintestine, liver, skeletal muscle and heart. Again,preliminary experiments showed that EAE 3mRNA expression decreased in the gill and kid-ney following seawater acclimation. This evidencesuggests that these multiple isoforms may indeedplay an osmoregulatory role in the gill and kidneyof freshwater fish which is of less importancefollowing seawater acclimation, however this pre-liminary information does not preclude an impor-tant role for any of these isoforms in transportmechanisms associated with acid-base balance.

Ž .The sodium-bicarbonate co-transporter NBCwas recently cloned from the kidneys of the sala-

Ž .mander Ambystoma tigrinum and various iso-forms have now been identified in mammals whichshow 30�35% amino acid homology with mam-

Žmalian AEs Romero et al., 1997; Romero and.Boron, 1999 . In the eel, various complex ion flux

and substitution experiments have indicated thata sodium bicarbonate cotransporter is present onthe basolateral membrane of intestinal epithelial

Ž .cells Ando and Subramanyam, 1990 . Recentwork from this laboratory has demonstrated thepresence of a homologue of the NBC 1 sodium


bicarbonate cotransporter isoform in the eel, andmRNA encoding this protein is expressed at high-est levels in the intestine, with intermediate levelsin kidney and lower levels in the gill. Seawateracclimation of eels leads to an increase in mRNAexpression in the intestine, little change in the gilland decreases in expression in the kidney. Thismolecular evidence reinforces the physiologicaldata suggesting that this protein may be involvedin bicarbonate secretion into the lumen of theintestine and may also play a role in NaCl and

Žwater absorption in this tissue Ando, 1990; Ando.and Subramanyam, 1990 . The role played by the

sodium bicarbonate cotransporter in intestinalNaCl and water absorption may be greater thanfirst envisaged. Evidence from NBC 1 isoforms inother species suggests that the sodium bicarbo-nate transport produced is electrogenic with twoor three bicarbonate ions transported per sodium

Ž .ion Romero and Boron, 1999 . If this was alsothe case for eel NBC 1, then the action of thistransporter, would not only be to modulate intra-

Žcellular pH, via bicarbonate transport which mayin itself affect the level of NaCl absorption; Ando

.and Subramanyam, 1990 , but also to produceŽhyperpolarisation of the cell membrane poten-

.tial , which would increase the driving force forbasolateral chloride efflux.

6. Sodium-hydrogen exchangers

So far in mammals 6 different sodium�hydro-gen exchanger isoforms have been identified,whereas in teleost fish, only a single exchanger,

Žcloned from trout erythrocytes �-NHE: Borgese.et al., 1992 has been published. Although the

trout gene has a relatively low level of homologyto mammalian isoforms, �-NHE is thought torepresent a homologue of the mammalian NHE 1

Ž .isoform Noel and Pouyssegur, 1995 . The se-quences of homologous counterparts of �-NHEare also present in the EMBL gene bank for both

Žthe eel and carp Accession numbers AJ006917.and AJ006916 respectively as well as a partial

Žsequence for the longhorned sculpin M. octode-cimspinosus: Accession number AF159880: Clai-

.borne et al., 1999 . In addition to this, however, apartial sequence for a homologue of mammalianNHE-2 isoform has also been cloned from

Žlonghorned sculpin Accession number AF159879:.Claiborne et al., 1999 . As well as being important

to the control of erythrocyte intracellular pH,sodium�hydrogen exchangers are also thought toplay major roles in osmoregulatory tissues such asthe gill, intestine and kidney. In the gill offreshwater fish, it has been suggested that asodium�hydrogen exchanger is present on thebasolateral surface of epithelial cells and is in-

Žvolved in the chloride uptake pathway Claiborne,.1998 , however in marine teleosts, sodium�hydro-

gen exchangers may be located either on theapical or basolateral surface of epithelial cellsŽ .Claiborne, 1998; Claiborne et al., 1999 . It hasrecently been speculated that sculpin �-NHE maybe a housekeeping gene expressed on the basolat-eral membrane, whereas the NHE-2 isoform rep-resents the exchanger on the apical membrane of

Ž .branchial epithelial cells Claiborne et al., 1999 .The intracellular alkalosis of epithelial cells pro-duced by basolateral exchanger activity in marineteleosts may be used to augment the NaCl excre-

Ž .tion mechanism Marshall and Bryson, 1998 ,whereas apical activity would be counterproduc-tive and is likely to be used to for acid-basebalance at the expense of osmoregulatory re-

Ž .quirements Claiborne, 1998 .Recent studies using antibodies raised against

mammalian NHE-2 and 3 isoforms have sug-gested that both of these isoforms may be ex-pressed in various cell types within the branchial

Žepithelium of both FW and marine teleosts Ed-.wards et al., 1999; Wilson et al., 2000a,b . How-

ever, these data should be viewed with caution asmany of the normal control experiments were notpossible and multiple bands were obtained onWestern blots. This is particularly true for thestaining obtained with the NHE-3 antibody whichhas been suggested to be due to non-specific

Ž .cross-reactivity Wilson et al., 2000a .It has also been suggested that a sodium-hydro-

gen exchanger may be expressed in intestinalŽ .epithelial cells Viella et al., 1995 , although this

is in contrast to the results from other groupsŽ .Schettino et al., 1992; Trischitta et al., 1992a,b .Sodium�hydrogen exchangers have also been lo-calised to kidney brush border membranes of

Žboth freshwater and marine eels Viella et al.,.1991; Zonno et al., 1994 . In the eel, cloning

experiments in this laboratory have identified par-tial cDNA fragments of three sodium�hydrogenexchanger isoforms. The first of these is a directcopy of the eel �-NHE cDNA sequence in theEMBL gene bank, the second appears to be a


Žduplicate copy of this isoform based on derived.amino acid homology and the third eel isoform

Žin a similar way to the longhorned sculpin NHE-2.isoform , shares greatest homology with the

NHE-2 isoform of mammals. However, as thepartial cDNA sequences encoding the eel andsculpin NHE-2 homologues are derived from dif-ferent regions of the gene, it is not possible to tellhow similar these two teleost proteins are to eachother. The tissue-specific expression of the eelisoforms or other data concerning their role inosmoregulatory processes are currently unavail-able.

7. V-type ATPase

The V-type ATPase has been postulated to bea key component of the sodium uptake mecha-

Žnism in the gills of freshwater teleosts Evans et. Žal., 1999 , providing the driving force due to

.hyperpolarisation of the membrane potential , forsodium entry into gill epithelial cells through anepithelial sodium channel. Although the putativesodium channel remains to be identified, theteleost V-type ATPase B subunit has been cloned

Ž .from rainbow trout Perry et al., 2000 and theenzyme has been localised to gill pavement cellsboth by in situ hybridisation and immunohis-tochemistry, and to mitochondria-rich cells by

Žimmunohistochemistry Sullivan et al., 1995, 1996;.Wilson et al., 2000a,b . In the eel, two isoforms of

the V-type ATPase B subunit have been identi-Ž .fied Niederstatter and Pelster, 2000 . Although

there are two isoforms of this subunit present inŽ .mammals known as the brain and kidney forms ,

analysis of the conserved amino acid differencesbetween these two mammalian forms when com-pared to the two eel isoforms, shows that of 39conserved differences, both eel isoforms had 35amino acids in common with the brain form andonly four with the kidney form. This analysissuggests that the two eel isoforms are both re-lated to, and are duplicate isoforms of, the mam-malian brain V-type ATPase B subunit.

8. Aquaporins

To date, ten aquaporin water channel isoformsŽhave been identified in mammals Van Os et al.,

.2000 . In teleost fish, there are several potential

roles for water transporters in osmoregulatoryprocesses. These include the absorption of wateracross the intestine following drinking in marineteleosts, absorption of water across the gills, andthe concomitant re-absorption of water togetherwith ions in the kidney of freshwater fish. Studiesin eels have led to the identification of a homo-logue of the mammalian AQP 3 water channeland its mRNA is expressed at high levels in thegill with low levels in the eye, oesophagus andintestine. Quantitative experiments revealed thata major site of AQP 3 mRNA expression was inthe gill of freshwater yellow or silver eels and thatlevels decreased significantly to 3% of freshwatervalues in silver eels following seawater acclima-tion. The data suggest that the AQP3 homologuemay play a role in the higher levels of osmoticwater permeability found in the gill of freshwater

Žeels compared to seawater acclimated eels; Isaia,.1984 . In this tissue it may serve to release water

Ž .entering all or a subset of surface epithelial cellsfrom the external environment. This processwould prevent cell swelling and bursting, andwould presumably occur because cell volume reg-ulation could not easily be achieved, in the faceof continuous water uptake, by the transport ofions or other osmolytes. A further possible rolewould be in the bulk flow of fluids suggested tooccur in the basolateral tubular network of chlo-ride cells as part of their ion transport mechanismŽ .Isaia, 1984 . Despite these possibilities, they donot answer the fundamental question as to why

Žgill osmotic water permeability or aquaporin ex-.pression should be so different between FW and

SW acclimated eels. Once the actual role of AQP3 in the gill is determined, the answer may wellbecome apparent.

Although the AQP 3 homologue was expressedin the intestine, the low mRNA levels suggestedthe presence of other AQP homologues in thistissue and subsequently a homologue of mam-malian AQP 1 was identified. This homologue hasa wider tissue-specific distribution of mRNA ex-pression than AQP 3, and is found in brain, eye,heart, pancreas, oesophagus, stomach and intes-tine with much lower levels in skeletal muscle, gilland kidney. Initial quantitative studies revealedthat the level of AQP 3 mRNA expression in theintestine increased 10�25� following seawatertransfer. Messenger RNA abundance was alsosignificantly decreased to 28% of FW values inthe kidneys of seawater-acclimated fish, although


overall levels in the kidney were much lower thanthe intestine. The high level of AQP 1 mRNAexpression in the intestine of marine eels suggestsAQP 1 may play a role in the absorption of waterby this tissue.

As only limited expression of aquaporins wasŽfound in eel kidney a major site of mammalian.aquaporin expression a further search led to the

discovery of a third aquaporin which appears toŽbe a duplicate of the AQP 1 homologue AQP 1

.dup . Initial experiments indicate that mRNA forthis isoform is only detectable in the oesophagusand kidney, and that the high level of mRNAexpression in the kidney is dramatically down-reg-ulated when eels are transferred from FW to SW.The physiological role of this change in expres-sion is, on the face of it, difficult to understandwhen considering that the role of the kidney inFW fish is to produce copious quantities of diluteurine, presumably with a minimal level of waterre-absorption. However, the production of largeamounts of dilute urine involves relatively highlevels of glomerular filtration and urine flow ratesŽin freshwater compared to seawater acclimated

.fish necessitating the absorption of large quanti-ties of salts, which consequently requires a cer-tain amount of concomitant water absorption. Asa consequence, the level of water transport ispresumably much greater in freshwater than inseawater-acclimated fish. Both the AQP 1 andAQP 1 dup isoforms may therefore play a role inre-absorption of water associated with salt reten-tion in the kidney of freshwater fish. However,despite this explanation, the exact role of theseaquaporins remains to be determined. A major

Ž .indication of the likely role s of these proteinswill depend on the cellular and sub-cellular loca-tions of their expression. As with AQP 3 in thegill, in the FW eel kidney, the most likely role foraquaporins would be to protect tubular epithelialcells from the effects of swelling due to waterinfiltration from hypo-osmotic tubular fluid andthis would suggest a basolateral cellular localisa-tion for aquaporin expression. While this mayrepresent the most likely possibility, other roles inother cells types can by no means be excluded.

The presence of expression of all three aqua-porin homologues in the oesophagus is somewhatpuzzling considering that this tissue is thought torepresent a particularly impermeable epitheliumŽ .Hirano and Mayer-Gostan, 1976 . Although re-ports suggest that there is a small net osmotic

water movement across the oesophagusŽ .Nagashima and Ando, 1993 , uni-direction fluxeswere not measured, leaving open the possibility of

Ž .larger nearly equivalent uni-direction fluxesŽacross this epithelium, as indeed is suggested by

the results in a study by Parmelee and Renfro,.1983 . This suggests that when marine fish drink,

the oesophagus attempts to absorb both ions andwater, but a roughly equivalent amount of wateris lost by osmosis into the hyper-osmotic lumencontents of the oesophagus.

9. Perspectives

Any of the duplicate copies of ion and watertransporter genes present in the eel and otherteleosts may individually be the result of isolatedtandem duplication events. However, takentogether, the number of potential duplicate genesthat have started to be characterised, both interms of their existence and subsequently their

Žfunction and regulation, lends support to al-.though by no means proves the hypothesis that

an ancient teleost progenitor species underwent agenome duplication event which occurred sometime after the divergence of the lineage leading tothe evolution of the higher vertebrates such asmammals. This duplicated genome would thenhave been carried forward into most if not allteleost fish species, but if this was the case, a verysignificant loss of chromosomal material may haveoccurred, which would explain the generallysmaller size of the haploid genome of teleost fish,in comparison to that of mammals. This losswould also explain why individual species or evenwhole super-orders, such as the acanthoptery-

Žgians which includes fugu, medaka and striped.bass , may have lost several hox gene clusters. It

also suggests that while a significant number ofgenes involved in osmoregulation in extantspecies, may have duplicate copies, it can bepredicted that a considerable number of dupli-cates must have been lost. From data on geneduplicates that are now accumulating, it is clearthat most of the isoforms retained in current dayteleost species have developed separate patternsof expression and regulation. This suggests thatthey now play distinct roles, which may haveallowed teleosts to develop the physiological plas-ticity to adapt to the many different environmen-tal niches that they now inhabit.


Acknowledgements

The authors would like to thank the NaturalEnvironment Research Council and The Well-come Trust for research funding. Studies involv-ing the cloning of various Atlantic salmontransporters were conducted in collaboration withMichel Seidelin and Steffen Madsen, of the Uni-versity of Southern Denmark, Odense, Denmark.

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