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

Comparative Biochemistry and Physiology Part A 130 Ž2001. 551᎐564

Review

Molecular physiology of osmoregulation in eels and other teleosts: the role of transporter isoforms and gene duplication 夽 Christopher P. Cutler U , Gordon Cramb School 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 and the potential role of their products in osmoregulation in both freshwater and seawater environments. In particular details of isoforms of various ATPases, co-transporters, exchangers and ion channels in the eel as well as other teleost species are described. Many of the teleost transporter isoforms discovered so far, appear to occur as twin or duplicate copies compared to their homologous counterparts in higher vertebrates, although these duplicate isoforms often have distinct tissue-specific and developmental stage-dependent expression patterns. The possible meaning of this information will be examined in relation to the fish genome duplication debate. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Genome duplication; Ion transport; Na,K-ATPase; NarKr2Cl cotransporter; NarCl cotransporter; NarHCO3 cotransporter; CFTR chloride ion channel; ClrHCO3 exchanger; NarH exchanger; Aquaporins; V-type ATPase.

1. Introduction Studies concerning the number and evolution of homeobox Ž Hox . gene clusters in zebrafish Ž Danio rerio., have suggested the possibility that the genome of a teleost fish ancestor may have undergone a genome duplication or polyploidisation event in addition to those that have occurred 夽

This paper was originally presented at a symposium dedicated to the memory of Marcel Florkin, held within the Belgium, July ESCPB 21st International Congress, Liege, ` 24᎐28, 2000. U Corresponding author. Tel.: q44-1334-463531; fax: q441334-463600,. E-mail address: [email protected] ŽC.P. Cutler..

in other groups of higher vertebrates ŽAmores et al., 1998; Postlethwait et al., 1998; Wittbrodt et al., 1998; Meyer and Malaga-Trillo, 1999.. However, further studies on some teleost species such as pufferfish (Fugu rubripes., Japanese medaka (Oryzias latipes., and striped bass (Morone saxatilis. have so far failed to conclusively demonstrate additional hox gene clusters to the four found in mammals ŽMeyer and Malaga-Trillo, 1999; Stellwag, 1999.. It has been suggested that this may be due to the loss of chromosomal material Žincluding hox gene clusters: Stellwag, 1999. in these species and the smaller size of the pufferfish genome in particular, lends weight to this theory. However, the lack of additional hox gene clusters in these species has led to the

1095-6433r01r$ - see front matter 䊚 2001 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 Ž 0 1 . 0 0 4 3 5 - 4

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alternative hypothesis that the polyploidisation event may have occurred subsequent to the diversification of the ray-finned fish, possibly exclusively in zebrafish, but otherwise, in a much more limited but currently indeterminate range of fish species ŽStellwag, 1999.. Information coming from studies of fish chromosomal numbers or amounts of DNA in the genome does not readily yield an answer as to which hypothesis is correct. While there are teleost species which have obviously undergone relatively ancient tetraploidisation with subsequent assimilation of chromosomes into a diploid complement Že.g. cyprinids such as the common carp  Cyprinus carpio: 2 n s 1044 : Ohno, 1999., or that have remained polyploid including some other cyprinids and various salmoniforms ŽOhno, 1999; Stellwag, 1999., other cyprinid species including zebrafish Ž2 n s 50., show no signs of having unusually large chromosome complements compared to the majority of other teleosts or indeed mammals Že.g. homo sapiens: 2 n s 46.. The fact that the zebrafish does not have a significantly larger chromosome complement than the majority of other teleosts does not discount the possibility that these other species may also have a duplicated genome. One indication that widespread duplication has occurred would be the presence of larger genomes in teleosts compared to higher vertebrates, but in fact estimates suggest that Žwith 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 coding for ion and water transporters, in the eel and other teleost fish species, and to review the meaning of this information in relation to the fish genome duplication debate. In addition, this article will also act as a review of the latest published and unpublished data, produced by this laboratory and others, concerning molecular aspects of osmoregulation in teleost fish. From an osmoregulatory point of view, as well as from virtually any other perspective, the prospect of two complements of some or all of the genes connected with any particular physiological process, makes a fundamental difference to the investigation of the molecular basis of underlying mechanisms. As for example, the analysis 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 in osmoregulatory mechanisms, if a second ‘unknown’ regulatable isoform also exists. Furthermore, offsetting changes in the regulation of isoforms could be masked in studies concerned with the global action of a particular protein, for example in enzyme assays. Thus for fish molecular physiologists, it is of great importance to know whether the genome duplication event in zebrafish is species-specific or common to all teleosts, and in particular, whether the species used for experimentation in an investigators own laboratory has a duplicated genome. The European eel Ž Anguilla anguilla., like zebrafish, does not appear to have an enlarged chromosomal complement Ž2 n s 38: Salvadori et al., 1997. which gives no clue as to the likely presence of duplicate copies of osmoregulatory or other genes. The first evidence of duplicate copies of at least some genes in the eel came from work on the active ion transporting enzyme, the Na, K-ATPase.

2. Na,K-ATPase Na,K-ATPase actively transports intracellular sodium in exchange for extracellular potassium ions, consists of two essential components, namely the ␣ and ␤ subunits, and in mammals there are currently four known isoforms of each subunit. Full length sequences of the first ␣ subunit Ž ␣1. of Na,K-ATPase have now been published in a number of teleost fish species such as the white sucker Ž Catostomus commersoni: Schoenrock et al., 1991. and the eel ŽCutler et al., 1995a. and in addition, partial sequences have been reported for tilapia Ž Oreochromis mossambicus: Hwang et al., 1998. and rainbow trout Ž Oncorhynchus mykiss: Kisen et al. 1994; D’Cotta et al., 1996.. An ␣ 3 isoform has also has been shown to exist in the eel ŽCutler et al., 1996. and evidence from antibody studies suggests that an ␣ 3 subunit is also present in catfish Ž Ictalurus punctatus: Pressley, 1992. and tilapia Ž Oreochromis mossambicus: Lee et al., 1998.. Unlike the situation in mammals, where the ␣ 3 isoform is predominately expressed in the brain ŽYoung and Lingrel, 1987., both of the ␣ isoforms in the eel exhibit an ubiquitous tissue distribution ŽCutler et al., 1996.. Current information from the gene bank indicates that there are multiple Na,K-ATPase ␣ subunit iso-

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Fig. 1. The N-terminal amino acid sequences of Na,K-ATPase ␣ subunits from various fish species. Nucleotide sequences were obtained from the EMBL gene bank with accession numbers listed below, from which the putative amino acid sequences were determined: 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, AW281430: Tilapia ␣ 3, AF109409: Medaka ␣ , AF072312. Except for the Eel ␣ 3, Žunpublished data.. The isoform denomination follows mammalian nomenclature except for Zebrafish where the denomination is arbitrary. Symbols in the alignment are as follows: 䢇 conserved amino acid; N amino acid with conserved similarity; ᎐ amino acid deletion and ⭈ missing sequence data.

forms expressed in zebrafish ŽFig. 1., which is analogous to the situation with homeobox gene clusters in this species. Although the quality of expressed sequence tag ŽEST. sequences is not 100% reliable Žas often indicated in the comments attached to EST genebank files., there do appear to be seven distinct Na,K-ATPase ␣ isoform sequences expressed in zebrafish, which fall into three broad categories, being either ␣1- Žzebrafish ␣1, 3᎐5., ␣ 2-Žzebrafish ␣ 2. ␣ 3-Žzebrafish ␣6᎐7: 88% similar to each other. like Žfollowing mammalian nomenclature .. However, on closer inspection the ␣1-like sequences can be arranged into two groups, based on the structure and sequence homologies of the N-terminal sequence Žthis region in mammalian isoforms is not very well conserved, but isoform specific.; these groups are the ␣1 and ␣4 Ž82% similar to each other., and the ␣ 3 and ␣ 5 Ž78% similar to each other., respectively. The eel ␣1, tilapia ␣1 and white sucker ␣

isoforms are all somewhat more similar to the first of these groups. The zebrafish ␣ 2 isoform is marginally more homologous to the ␣ 2 rather than to other mammalian and avian ␣ isoforms Žwhen considering the whole sequence., although this is not clear from the amino terminal sequence presented in Fig. 1, as isoform-specific amino acid motifs, conserved between mammalian and avian ␣ 2 isoforms, are not particularly well conserved in the sequence of the zebrafish ␣ 2 isoform. Regarding the ␣ 3 isoforms, the tilapia ␣ 3 and medaka ␣ sequences are similar in structure to mammalian ␣ 3-isoforms. The eel and zebrafish ␣ 3-like Žzebrafish ␣6᎐7. sequences have an additional 13 conserved amino acids near the N-terminus Žamino 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 laboratory suggests that there are at least five isorsplicoforms expressed in Atlantic salmon Ž Salmo salar .,

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where at least two of these are ␣1-like isoforms. Currently there is nothing known about the function, regulation or tissue-specific localisation of the additional zebrafish or salmon isoforms. There are also published sequences of teleost fish Na,K-ATPase ␤ subunit isoforms. A homologue of the mammalian ␤1 subunit has been identified in the eel ŽCutler et al., 1995b. and a duplicate copy of this isoform has also recently been cloned and is called ␤233 ŽCutler et al., 2000.. The ␤233 has a much more limited tissue distribution of mRNA expression than the ␤1 isoform, however, it appears that the ␤233 isform is expressed at higher levels than the ␤1 isoform in osmoregulatory tissues such as the gill and intestine. There are also developmental differences between the two duplicate isoforms, while the ␤1 isoform has increased mRNA levels following seawater acclimation in yellow Žadult. or silver Ža later seawater migratory adult developmental stage. eels, the level of ␤233 expression is only upregulated in silver eels following seawater acclimation. In addition to ␤1 isoforms, a homologue of the mammalian Na,K-ATPase ␤3 was identified in zebrafish and a partial sequence is also available for the eel; in both of these species the ␤3 isoforms are predominately expressed in the brain ŽAppel et al., 1996, Cutler et al., 1997a.. Current information from the gene bank ŽEMBL or Genbank which share gene sequence data., indicates that there are multiple Na,K-ATPase ␤ subunit isoforms expressed in zebrafish ŽFig. 2.. The current sequences in the gene bank suggest there may be as many as four copies of the Na,K-ATPase ␤1 isoform in zebrafish, although two of these partial copies do not code for overlapping amino acid regions, and so may represent sequences from different parts of the same isoform Ž␤1c and ␤1d.. Furthermore, the quality of sequence information associated with ESTs is not always entirely accurate, leaving open the possibility that sequence differences between apparent isoforms may be created by error. There are some indications that other species may have duplicate Na,K-ATPase ␤1 isoforms, collaborative work Žsee acknowledgements. in this laboratory suggests that there are also three isoforms expressed in Atlantic salmon. As well as multiple isoforms of the ␤1 there is also evidence in the gene bank that there is a duplicate copy of the ␤3 in zebrafish Ž␤3b., although whether or how this

isoform differs in function, regulation, or tissuespecific or developmental expression in comparison to the original, is not known. There is also a homologue of the mammalian Na,K-ATPase ␤2 isoform in zebrafish which shares 67% amino acid homology with an eel ␤185b cDNA fragment and 57% amino acid homology with its possible duplicate, the eel ␤185 ŽCutler et al., 1996, 1997b.. The ␤185 mRNA is expressed solely in the brain and eye, in a similar fashion to mammalian ␤2 isoforms which are predominately found in nervous tissue ŽAppel et al., 1996..

3. Chloride–cation-cotransporters This gene family is comprised of a group of proteins whose activity is sensitive to various diuretic drugs and which have three identified ion transport specificities, cotransporting chloride ions together with sodium ŽNa᎐Cl., potassium ŽK᎐Cl. or sodium and potassium ŽNa᎐K᎐2Cl. ions. In addition, two isoforms of the Na᎐K᎐2Cl cotransporter have been identified and these are involved in ion transport mechanisms principally associated with either absorption or secretion respectively, although they may also play a role in cell volume homeostasis ŽRussell, 2000.. The first member of the diuretic-sensitive chloride cation co-transporter gene family to be cloned was the flounder thiazide-sensitive Na᎐Cl cotransporter ŽNCC: Gamba et al., 1993.. Since then, mammalian counterparts have been cloned both for NCC and for the secretory ŽNKCC1. and absorptive ŽNKCC2. isoforms of the Na᎐K᎐2Cl cotransporter as well as for four K᎐Cl cotransporter ŽKCC. isoforms ŽGamba et al., 1994; Delpire et al., 1994; Payne and Forbush, 1994; Mount et al., 1999.. In the eel, cDNA fragments coding for homologues of NKCC1, NKCC2 and NCC were cloned from the gill and intestine and have been designated cot 1, 2 and 4, respectively, ŽCutler et al., 1996.. Like its mammalian counterpart, mRNA coding for cot 1 is ubiquitously expressed in nearly all eel tissues. In yellow eels expression of cot 1 mRNA in the gill increases 6-fold during salinity acclimation but this response is absent in silver eels. This suggests that this protein may well play a role in ion extrusion in branchial chloride cells, at least in yellow eels, although a different mechanism may operate in silver eels.

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Fig. 2. Partial and complete teleost Na,K-ATPase ␤ subunit acid sequences Žderived from nucleotide sequences . reported to the gene bank. 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:- 䢇 conserved amino acid; N amino acid with conserved similarity; ᎐ amino acid deletion and spaces imply missing sequence data.

Preliminary immunohistochemical localisation experiments show that chloride cells may be the principal site of expression of cot 1 in the gill, but a low level of expression was seen in all cell types. Initial experiments indicated that cot 1 mRNA is also expressed in the intestine and, subsequently

the localisation of this expression has been narrowed down solely to the mid gut region. The likely physiological role of a secretory NarKr2Cl co-transporter, such as cot 1, in the intestine, is probably associated with luminal fluid secretion for digestive purposes. However, a possible role

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for this isoform in volume regulation of cells within the mid gut region cannot be ruled out. During the cloning of cot 1, a further duplicate isoform was also identified in the eel, which was called cot 3. Expression of this isoform was mainly found in the brain, although in Northern blots low levels of mRNA expression could also be detected in the gills of yellow eels ŽCutler et al., 1996.. Currently the cellular location of branchial cot 3 expression or its role in salinity acclimation is unknown. A recent study in the mudskipper Ž Periophalmodon schlosseri: Wilson et al., 2000b. has suggested that a NarKr2Cl cotransporter is expressed in branchial chloride cells. However, while this information agrees with the data from the eel cot 1 isoform, it should be viewed with caution as many of the normal control experiments were not possible. How these data relate to the potential presence of secretory NarKr2Cl cotransporter isoforms in the mudskipper is also unclear. The absorptive type of NarKr2Cl co-transporter, cot 2, is the major isoform expressed in the intestine, although it is also expressed at reasonably high levels in the urinary bladder ŽCutler et al., 1996.. Expression predominates in the anterior and mid regions of the gut with reduced levels in the posterior intestine andror rectum. Expression in all regions of the intestine increase following salinity acclimation in both yellow and silver eels, although the levels vary considerably both between experiments and individuals. Mammalian NKCC 2 isoforms are almost exclusively expressed in the kidney ŽPayne and Forbush, 1994., and the completely different pattern of tissue expression of cot 2 in the eel suggests that this isoform might not be a direct counterpart of NKCC2. Not surprisingly then, recent cloning experiments in eel kidney led to the cloning of a fourth NKCC isoform which appears to be a duplicate of cot 2, designated as cot 6. Preliminary Northern blots have demonstrated its presence in the kidney but the level of mRNA expression is not significantly changed by salinity acclimation. This may mean that the expected decrease in NKCC 2 activity in SW kidney is produced 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 the posterior intestine andror rectum. Here the levels of expression are not changed following salin-

ity acclimation. The tissue-specifc expression of cot 4 in the eel is somewhat different to the equivalent gene found in the winter flounder, where it was additionally found to be expressed in the bladder, gonads, skeletal muscle, eye and brain, but not kidney or gill ŽGamba et al., 1993.. The presence of cot 4 in the posterior intestine andror rectum may explain why the level of cot 2 expression is decreased in this region of the gut. As the seawater imbibed by marine teleosts passes down the intestine, the potassium concentration decreases due to absorption via NarKrCl cotransport, suggesting further absorption of NaCl may be more efficiently achieved in the posterior gut andror rectum by a NarCl rather than a potassium-dependent cotransporter. Despite the lack of mRNA expression on Northern blots, a truncated form of cot 4 can be amplified from eel kidney using RT᎐PCR. However notwithstanding this, the low level of cot 4 in this tissue, Žwhich represents the major site of NCC expression in mammals; Gamba et al., 1994., suggested that cot 4 Žor flounder NCC. may not be the only or the most abundant homologue of mammalian NCC expressed in teleost fish kidney. Further cloning experiments showed this to be the case, as a cDNA fragment which was also homologous to mammalian NCC was recently cloned from eel kidney and was designated cot 5. Northern blots have demonstrated that, in the same fashion as mammalian NCC, cot 5 expression is almost exclusively located in the kidney. However, preliminary blots also suggest that cot 5 mRNA levels in the kidney are not greatly altered by salinity acclimation.

4. Ion channels A number of different ion channels are known to play roles in osmoregulatory processes. However, due to its central role in ion secretory processes in other species, a key member amongst these ion channels is a CFTR-like chloride ion channel. A gene fragment encoding a portion of this protein was originally identified in the eel ŽCutler et al., 1996. in addition however, the full length sequence from the killifish Ž Fundulus heteroclitus. has also now been determined ŽSinger et al., 1998.. The CFTR protein represents both a chloride ion channel as well as a regulator of other ion and water transporters in higher verte-

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brates ŽSchwiebert et al., 1999; Schreiber et al., 2000.. However, the amino acid and nucleotide sequences of teleost CFTR cDNAs have much lower homology to mammalian sequences than does the CFTR gene of the evolutionarily more ancient elasmobranch fish, the dogfish Ž Squalus acanthias: Marshall et al., 1991.. This suggests that teleost CFTR has a somewhat altered structure and function compared to CFTR in mammalian or elasmobranch species. However, so far killifish CFTR has been shown to represent a cAMP-regulated chloride ion channel with similar characteristics to mammalian counterparts ŽSinger et al., 1998.. Killifish CFTR mRNA expression has been demonstrated in the gills and opercular membrane as well as the intestine and brain, and has also been shown to increase 9-fold in the gill, 24 h after the onset of seawater acclimation ŽSinger et al., 1998; Marshall et al., 1999.. These results are in direct contrast to results in the eel where CFTR mRNA is only expressed at low levels in the gill and this does not alter following seawater acclimation. These apparent anomalies may reflect differences in the habitats occupied by the two species, as the killifish is a coastalrestuarine species, having to adapt to constantly changing salinities, whereas the eel would normally only expect to undergo freshwaterrseawater migrations twice during its entire lifetime. However, the differences in the patterns of CFTR expression between these two teleosts could be purely due to the evolutionary divergence of the functional roles of these proteins. Alternatively, the differences could also be due to the presence of two different CFTR-isoforms in each species, although there is no direct evidence currently to demonstrate that this is the case. However, two duplicate CFTR isoforms have been isolated from the Atlantic salmon Ždeposited in the EMBL gene bank under accession numbers AF155237 and AF161070., although the amino acid homology between these is so high Ž) 95%., that they probably result from a relatively recent duplication event in this species. More recently, as many as five isoforms are reported to be present in this species ŽChen et al., 1999.. A recent study in the mudskipper Ž Periophalmodon schlosseri: Wilson et al., 2000b. has suggested that a CFTR-protein is expressed in the apical region of branchial chloride cells. However, this infor-

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mation should be viewed with caution as many of the normal control experiments were not possible. How these data relate to the potential presence of CFTR-isoforms in the mudskipper is also unclear. In addition to CFTR, other ion channels may play a role in osmoregulatory processes. Recently two members of the CLC chloride ion channel were cloned from tilapia ŽCLC-3 and CLC-5: Miyazaki et al., 1999.. Both of these ion channels are expressed in osmoregulatory tissues such as the gill, intestine and kidney, however, the level of mRNA expression in these tissues is comparable in freshwater or seawater acclimated fish. Therefore, there is no indication that these ion channels are involved in chloride ion homeostasis during salinity acclimation. A further ion channel which may be involved in osmoregulatory processes is the inwardly rectifying potassium channel, eKir, which was recently cloned from eel gill ŽSuzuki et al., 1999.. This potassium channel shows increased mRNA expression in the gill, kidney and posterior intestine of the seawater eel in comparison to those acclimated to freshwater, and is located in the basolateral microtubular system of chloride cells of the branchial epithelium. These results taken together suggest that eKir may represent the channel on the basolateral surface of chloride cells, which recycles potassium ions entering through the NarKr2Cl cotransporter, back into interstitial body fluids during the process of NaCl excretion across the gills. Despite attempts in several laboratories, cloning of homologues of subunits of the epithelial Nachannel ŽeNaC. from teleost fish species has not so far proved possible. Together with V-type ATPase, this protein has long been postulated to play an important role in the absorption of Na across the apical surface of the gills in FW fish Žfor example see Evans et al., 1999.. Recent studies using mammalian antibodies raised against mammalian eNaC ␣ and ␤ subunits have suggested that the subunits of this protein may indeed be located on the apical surface of pavement Žrainbow trout and tilapia. and mitochondria-rich cells Žrainbow trout. of the branchial epithelium ŽWilson et al., 2000a.. However, again, this information should be viewed with caution as many of the normal control experiments were not possible.

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5. Bicarbonate transporters The band-3 anion exchanger ŽAE. was originally cloned from a rainbow trout spleen cDNA library. This protein functions as a chloride-bicarbonate exchanger, although it may also be able to transport taurine, sorbitol and urea ŽHubner et al., 1992; Fievet et al., 1998.. The mammalian anion exchanger gene family consists of 3-isoforms ŽAE1-3. and the structure of the trout AE is most similar to the original band 3 isoform ŽAE1. although the amino acid homology to mouse AE 1 is only 50% ŽHubner et al., 1992.. While chloride᎐bicarbonate exchangers are thought to have a role in acid᎐base regulation ŽPerry, 1997., its also possible that they also play a role in osmoregulatory processes, in tissues such as the gill, intestine and kidney. In the gill, anion exchangers have been postulated to be expressed on both the apical and basolateral membranes of epithelial cells and to be responsible for the uptake of chloride in freshwater fish ŽEvans et al., 1999.. The cell types associated with this uptake may be pavement cells ŽWood et al., 1998., however, chloride᎐bicarbonate exchanger mRNA has also been localised to cells on the primary lamella, and expression shared similar localisation to that characteristic of mitochondriarNa,K-ATPase-rich chloride cells ŽSullivan et al., 1996; Wilson et al., 2000a,b.. Chloride᎐bicarbonate exchange has also been suggested to play an indirect role in ion secretion on the basolateral surface of chloride cells in seawater acclimated teleosts ŽZadunaisky et al., 1995., and this activity may serve to regulate intracellular pH, where conditions favouring intracellular alkalosis augment chloride secretion ŽMarshall and Bryson, 1998.. In the intestine of seawater acclimated eels, a chloride᎐bicarbonate exchanger involved in bicarbonate extrusion has been suggested to be located on the brush border membranes of luminal epithelial cells ŽAndo and Subramanyam, 1990., although this finding was not confirmed by others ŽTrischitta et al., 1992a.. A chloride᎐bicarbonate exchanger has also been proposed to exist on the basolateral surface of intestinal enterocytes ŽTrischitta et al., 1992b., however, the results from these studies may also be explained by the presence of separate electrogenic sodium bicarbonate cotransporter and an as yet unidentified chloride efflux pathway. The brush border

membranes of eel kidney tubular cells ŽViella et al., 1997. may also contain a chloride᎐bicarbonate exchanger which may be involved in bicarbonate re-absorption. Complementary DNA fragments were recently isolated from the intestine of the eel and these encode three isoforms which are homologous to members of the chloride᎐bicarbonate exchanger family ŽEAE 1-3; unpublished data.. Preliminary studies indicate that EAE 1 is a direct counterpart of trout AE and its mRNA is expressed at high levels in gill, kidney, heart and brain, and with only just detectable levels of expression in the intestine, oesophagus and liver. Preliminary experiments show that expression of EAE 1 mRNA decreased in the gill and kidney following seawater acclimation. EAE 2 which appears to be a duplicate isoform of EAE 1, is expressed at high levels in kidney, oesophagus and stomach, with intermediate levels in heart and lower levels in brain, eye, intestine and gill. As with EAE 1, preliminary experiments show that EAE 2 mRNA expression was decreased in the gill and kidney following seawater acclimation. EAE 3 is most homologous to the mammalian AE 2 isoform, and has high levels of expression in the gill, with intermediate levels in kidney and lower levels in intestine, liver, skeletal muscle and heart. Again, preliminary experiments showed that EAE 3 mRNA expression decreased in the gill and kidney following seawater acclimation. This evidence suggests that these multiple isoforms may indeed play an osmoregulatory role in the gill and kidney of freshwater fish which is of less importance following seawater acclimation, however this preliminary information does not preclude an important role for any of these isoforms in transport mechanisms associated with acid-base balance. The sodium-bicarbonate co-transporter ŽNBC. was recently cloned from the kidneys of the salamander Ž Ambystoma tigrinum. and various isoforms have now been identified in mammals which show 30᎐35% amino acid homology with mammalian AEs ŽRomero et al., 1997; Romero and Boron, 1999.. In the eel, various complex ion flux and substitution experiments have indicated that a sodium bicarbonate cotransporter is present on the basolateral membrane of intestinal epithelial cells ŽAndo and Subramanyam, 1990.. Recent work from this laboratory has demonstrated the presence of a homologue of the NBC 1 sodium

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bicarbonate cotransporter isoform in the eel, and mRNA encoding this protein is expressed at highest levels in the intestine, with intermediate levels in kidney and lower levels in the gill. Seawater acclimation of eels leads to an increase in mRNA expression in the intestine, little change in the gill and decreases in expression in the kidney. This molecular evidence reinforces the physiological data suggesting that this protein may be involved in bicarbonate secretion into the lumen of the intestine and may also play a role in NaCl and water absorption in this tissue ŽAndo, 1990; Ando and Subramanyam, 1990.. The role played by the sodium bicarbonate cotransporter in intestinal NaCl and water absorption may be greater than first envisaged. Evidence from NBC 1 isoforms in other species suggests that the sodium bicarbonate transport produced is electrogenic with two or three bicarbonate ions transported per sodium ion ŽRomero and Boron, 1999.. If this was also the case for eel NBC 1, then the action of this transporter, would not only be to modulate intracellular pH, via bicarbonate transport Žwhich may in itself affect the level of NaCl absorption; Ando and Subramanyam, 1990., but also to produce hyperpolarisation Žof the cell membrane potential., which would increase the driving force for basolateral chloride efflux.

6. Sodium-hydrogen exchangers So far in mammals 6 different sodium᎐hydrogen exchanger isoforms have been identified, whereas in teleost fish, only a single exchanger, cloned from trout erythrocytes Ž␤-NHE: Borgese et al., 1992. has been published. Although the trout gene has a relatively low level of homology to mammalian isoforms, ␤-NHE is thought to represent a homologue of the mammalian NHE 1 isoform ŽNoel and Pouyssegur, 1995.. The sequences of homologous counterparts of ␤-NHE are also present in the EMBL gene bank for both the eel and carp ŽAccession numbers AJ006917 and AJ006916 respectively. as well as a partial sequence for the longhorned sculpin Ž M. octodecimspinosus: Accession number AF159880: Claiborne et al., 1999.. In addition to this, however, a partial sequence for a homologue of mammalian NHE-2 isoform has also been cloned from longhorned sculpin ŽAccession number AF159879: Claiborne et al., 1999.. As well as being important

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to the control of erythrocyte intracellular pH, sodium᎐hydrogen exchangers are also thought to play major roles in osmoregulatory tissues such as the gill, intestine and kidney. In the gill of freshwater fish, it has been suggested that a sodium᎐hydrogen exchanger is present on the basolateral surface of epithelial cells and is involved in the chloride uptake pathway ŽClaiborne, 1998., however in marine teleosts, sodium᎐hydrogen exchangers may be located either on the apical or basolateral surface of epithelial cells ŽClaiborne, 1998; Claiborne et al., 1999.. It has recently been speculated that sculpin ␤-NHE may be a housekeeping gene expressed on the basolateral membrane, whereas the NHE-2 isoform represents the exchanger on the apical membrane of branchial epithelial cells ŽClaiborne et al., 1999.. The intracellular alkalosis of epithelial cells produced by basolateral exchanger activity in marine teleosts may be used to augment the NaCl excretion mechanism ŽMarshall and Bryson, 1998., whereas apical activity would be counterproductive and is likely to be used to for acid-base balance at the expense of osmoregulatory requirements ŽClaiborne, 1998.. Recent studies using antibodies raised against mammalian NHE-2 and 3 isoforms have suggested that both of these isoforms may be expressed in various cell types within the branchial epithelium of both FW and marine teleosts ŽEdwards et al., 1999; Wilson et al., 2000a,b.. However, these data should be viewed with caution as many of the normal control experiments were not possible and multiple bands were obtained on Western blots. This is particularly true for the staining obtained with the NHE-3 antibody which has been suggested to be due to non-specific cross-reactivity ŽWilson et al., 2000a.. It has also been suggested that a sodium-hydrogen exchanger may be expressed in intestinal epithelial cells ŽViella et al., 1995., although this is in contrast to the results from other groups ŽSchettino et al., 1992; Trischitta et al., 1992a,b.. Sodium᎐hydrogen exchangers have also been localised to kidney brush border membranes of both freshwater and marine eels ŽViella et al., 1991; Zonno et al., 1994.. In the eel, cloning experiments in this laboratory have identified partial cDNA fragments of three sodium᎐hydrogen exchanger isoforms. The first of these is a direct copy of the eel ␤-NHE cDNA sequence in the EMBL gene bank, the second appears to be a

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duplicate copy of this isoform Žbased 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 the partial cDNA sequences encoding the eel and sculpin NHE-2 homologues are derived from different regions of the gene, it is not possible to tell how similar these two teleost proteins are to each other. The tissue-specific expression of the eel isoforms or other data concerning their role in osmoregulatory processes are currently unavailable.

7. V-type ATPase The V-type ATPase has been postulated to be a key component of the sodium uptake mechanism in the gills of freshwater teleosts ŽEvans et al., 1999., providing the driving force Ždue to hyperpolarisation of the membrane potential., for sodium entry into gill epithelial cells through an epithelial sodium channel. Although the putative sodium channel remains to be identified, the teleost V-type ATPase B subunit has been cloned from rainbow trout ŽPerry et al., 2000. and the enzyme has been localised to gill pavement cells both by in situ hybridisation and immunohistochemistry, and to mitochondria-rich cells by immunohistochemistry ŽSullivan et al., 1995, 1996; Wilson et al., 2000a,b.. In the eel, two isoforms of the V-type ATPase B subunit have been identified ŽNiederstatter and Pelster, 2000.. Although there are two isoforms of this subunit present in mammals Žknown as the brain and kidney forms., analysis of the conserved amino acid differences between these two mammalian forms when compared to the two eel isoforms, shows that of 39 conserved differences, both eel isoforms had 35 amino acids in common with the brain form and only four with the kidney form. This analysis suggests that the two eel isoforms are both related to, and are duplicate isoforms of, the mammalian brain V-type ATPase B subunit.

8. Aquaporins To date, ten aquaporin water channel isoforms have been identified in mammals ŽVan Os et al., 2000.. In teleost fish, there are several potential

roles for water transporters in osmoregulatory processes. These include the absorption of water across the intestine following drinking in marine teleosts, absorption of water across the gills, and the concomitant re-absorption of water together with ions in the kidney of freshwater fish. Studies in eels have led to the identification of a homologue of the mammalian AQP 3 water channel and its mRNA is expressed at high levels in the gill with low levels in the eye, oesophagus and intestine. Quantitative experiments revealed that a major site of AQP 3 mRNA expression was in the gill of freshwater yellow or silver eels and that levels decreased significantly to 3% of freshwater values in silver eels following seawater acclimation. The data suggest that the AQP3 homologue may play a role in the higher levels of osmotic water permeability found in the gill of freshwater eels Žcompared to seawater acclimated eels; Isaia, 1984.. In this tissue it may serve to release water entering all Žor a subset of. surface epithelial cells from the external environment. This process would prevent cell swelling and bursting, and would presumably occur because cell volume regulation could not easily be achieved, in the face of continuous water uptake, by the transport of ions or other osmolytes. A further possible role would be in the bulk flow of fluids suggested to occur in the basolateral tubular network of chloride cells as part of their ion transport mechanism ŽIsaia, 1984.. Despite these possibilities, they do not answer the fundamental question as to why gill osmotic water permeability Žor aquaporin expression. should be so different between FW and SW acclimated eels. Once the actual role of AQP 3 in the gill is determined, the answer may well become apparent. Although the AQP 3 homologue was expressed in the intestine, the low mRNA levels suggested the presence of other AQP homologues in this tissue and subsequently a homologue of mammalian AQP 1 was identified. This homologue has a wider tissue-specific distribution of mRNA expression than AQP 3, and is found in brain, eye, heart, pancreas, oesophagus, stomach and intestine with much lower levels in skeletal muscle, gill and kidney. Initial quantitative studies revealed that the level of AQP 3 mRNA expression in the intestine increased 10᎐25 = following seawater transfer. Messenger RNA abundance was also significantly decreased to 28% of FW values in the kidneys of seawater-acclimated fish, although

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overall levels in the kidney were much lower than the intestine. The high level of AQP 1 mRNA expression in the intestine of marine eels suggests AQP 1 may play a role in the absorption of water by this tissue. As only limited expression of aquaporins was found in eel kidney Ža major site of mammalian aquaporin expression. a further search led to the discovery of a third aquaporin which appears to be a duplicate of the AQP 1 homologue ŽAQP 1 dup.. Initial experiments indicate that mRNA for this isoform is only detectable in the oesophagus and kidney, and that the high level of mRNA expression in the kidney is dramatically down-regulated when eels are transferred from FW to SW. The physiological role of this change in expression is, on the face of it, difficult to understand when considering that the role of the kidney in FW fish is to produce copious quantities of dilute urine, presumably with a minimal level of water re-absorption. However, the production of large amounts of dilute urine involves relatively high levels of glomerular filtration and urine flow rates Žin freshwater compared to seawater acclimated fish. necessitating the absorption of large quantities of salts, which consequently requires a certain amount of concomitant water absorption. As a consequence, the level of water transport is presumably much greater in freshwater than in seawater-acclimated fish. Both the AQP 1 and AQP 1 dup isoforms may therefore play a role in re-absorption of water associated with salt retention in the kidney of freshwater fish. However, despite this explanation, the exact role of these aquaporins remains to be determined. A major indication of the likely roleŽs. of these proteins will depend on the cellular and sub-cellular locations of their expression. As with AQP 3 in the gill, in the FW eel kidney, the most likely role for aquaporins would be to protect tubular epithelial cells from the effects of swelling due to water infiltration from hypo-osmotic tubular fluid and this would suggest a basolateral cellular localisation for aquaporin expression. While this may represent the most likely possibility, other roles in other cells types can by no means be excluded. The presence of expression of all three aquaporin homologues in the oesophagus is somewhat puzzling considering that this tissue is thought to represent a particularly impermeable epithelium ŽHirano and Mayer-Gostan, 1976.. Although reports suggest that there is a small net osmotic

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water movement across the oesophagus ŽNagashima and Ando, 1993., uni-direction fluxes were not measured, leaving open the possibility of larger Žnearly equivalent. uni-direction fluxes across this epithelium, Žas 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 and water, but a roughly equivalent amount of water is lost by osmosis into the hyper-osmotic lumen contents of the oesophagus.

9. Perspectives Any of the duplicate copies of ion and water transporter genes present in the eel and other teleosts may individually be the result of isolated tandem duplication events. However, taken together, the number of potential duplicate genes that have started to be characterised, both in terms of their existence and subsequently their function and regulation, lends support to Žalthough by no means proves. the hypothesis that an ancient teleost progenitor species underwent a genome duplication event which occurred some time after the divergence of the lineage leading to the evolution of the higher vertebrates such as mammals. This duplicated genome would then have been carried forward into most if not all teleost fish species, but if this was the case, a very significant loss of chromosomal material may have occurred, which would explain the generally smaller size of the haploid genome of teleost fish, in comparison to that of mammals. This loss would also explain why individual species or even whole super-orders, such as the acanthopterygians Žwhich includes fugu, medaka and striped bass., may have lost several hox gene clusters. It also suggests that while a significant number of genes involved in osmoregulation in extant species, may have duplicate copies, it can be predicted that a considerable number of duplicates must have been lost. From data on gene duplicates that are now accumulating, it is clear that most of the isoforms retained in current day teleost species have developed separate patterns of expression and regulation. This suggests that they now play distinct roles, which may have allowed teleosts to develop the physiological plasticity to adapt to the many different environmental niches that they now inhabit.

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Acknowledgements The authors would like to thank the Natural Environment Research Council and The Wellcome Trust for research funding. Studies involving the cloning of various Atlantic salmon transporters were conducted in collaboration with Michel Seidelin and Steffen Madsen, of the University of Southern Denmark, Odense, Denmark. References Amores, A., Force, A., Yan, Y.L. et al., 1998. Zebrafish hox clusters and vertebrate genome evolution. Science 282, 1711᎐1714. Ando, M., 1990. Effects of bicarbonate on salt and water transport across the intestine of the seawater eel. J. Exp. Biol. 150, 367᎐379. Ando, M., Subramanyam, M.V.V., 1990. Bicarbonate transport systems in the eel intestine of the seawater eel. J. Exp. Biol. 150, 381᎐394. Appel, C., Gloor, S., Schmalzing, G., Schachner, M., Bernhardt, R.R., 1996. Expression of a Na,K-ATPase beta3 subunit during development of the zebrafish central nervous system. J. Neurosci. Res. 46, 551᎐564. Borgese, F., Sardet, C., Cappadoro, M., Pouyssegur, J., Motais, R., 1992. Cloning and expression of a cAMP-activated NaqrHq exchanger-evidence that the cytoplasmic domain mediates hormonal-regulation. Proc. Natl. Acad. Sci. USA 89, 6765᎐6769. Chen, J.M., Jacques, C., Cutler, C., Mercier, B., Boeuf, G., Ferec, C., 1999. Cloning and sequence analysis of five distinct cDNAs encoding cystic fibrosis transmembrane conductance regulator from the Atlantic salmon, Salmo salar. Am. J. Human Genet. 65 Ž4., S1007. Claiborne, J.B., 1998. Acid-base regulation. In: Evans, D.H. ŽEd.., The Physiology of Fishes. Boca Raton, USA. CRC Press, pp. 177᎐198. Claiborne, J.B., Blackston, C.R., Choe, K.P. et al., 1999. A mechanism for branchial acid excretion in marine fish: Identification of multiple NaqrHq antiporter ŽNHE. isoforms in gills of two seawater teleosts. J. Exp. Biol. 202, 315᎐324. Cutler, C.P., Sanders, I.L., Hazon, N., Cramb, G., 1995a. Primary sequence tissue specificity and expression of the Naq,Kq-ATPase ␤1 subunit in the European eel Ž Anguilla anguilla.. Comp. Biochem. Physiol. 111B, 567᎐573. Cutler, C.P., Sanders, I.L., Hazon, N., Cramb, G., 1995b. Primary sequence, tissue specificity and expression of the Naq, Kq-ATPase ␤1 subunit in the European eel Ž Anguilla anguilla.. Fish Physiol. Biochem. 14, 423᎐429.

Cutler, C.P., Sanders, I.L., Luke, G., Hazon, N., Cramb, G., 1996. In: Ennion, S.J., Goldspink, G. ŽEds.., Ion transport in teleosts: identification and expression of ion transporting proteins in the branchial and intestinal epithelia of the European eel Ž Anguilla anguilla.. Cambridge University Press, Cambridge, pp. 43᎐74. Society of Experimental Biologists, Seminar Series, 58. Cutler, C.P., Sanders, I.L., Cramb, G., 1997a. Expression of Naq,Kq-ATPase ␤ subunit isoforms in the European eel Ž Anguilla anguilla.. Fish Physiol Biochem. 17, 371᎐376. Cutler, C.P., Sanders, I.L., Cramb, G., 1997b. Isolation of six putative P-type ATPase ␤ subunit PCR fragments from the brain of the European eel Ž Anguilla anguilla.. Annals. NY Acad. Sci. 834, 123᎐125. Cutler, C.P., Brezillon, S., Bekir, S., Sanders, I.L., Hazon, N., Cramb, G., 2000. Expression of a duplicate Naq,Kq-ATPase ␤1 isoform in the European eel Ž Anguilla anguilla.. Am. J. Physiol. 279, R222᎐R229. Delpire, E., Rauchman, M.I., Hebert, S.C., Gullans, S.R., 1994. Molecular cloning and chromosome localization of a putative basolateral Naq-Kq-2Cl- cotransporter from mouse inner medullary collecting duct ŽmIMCD-3. cells. J. Biol. Chem. 269, 25677᎐25683. D’Cotta, H.C., Gallais, C., Saulier, B., Prunet, P., 1996. Comparison between parr and smolt Atlantic salmon Ž Salmo salar . ␣ subunit gene expression of NaqrKq ATPase in gill tissue. Fish Physiol. Biochem. 15, 29᎐39. Edwards, S.L., Tse, C.M., Toop, T., 1999. Immunolocalization of the NHE3-like immunoreactivity in the gills of the rainbow trout Ž Oncorhynchus mykiss. and the blue-throated wrasse Ž Pseudolabrus tetrious.. J. Anat. 195, 465᎐469. Evans, D.H., Piermarini, P.M., Potts, W.T.W., 1999. Ionic transport in the fish gill epithelium. J. Exp. Zool. 283, 641᎐652. Fievet, B., Perset, F., Gabillat, N. et al., 1998. Transport of uncharged organic solutes in Xenopus oocytes expressing red cell anion exchangers ŽAE1s.. Proc. Natl. Acad. Sci. USA 95, 10996᎐11001. Gamba, G., Saltzberg, S.N., Lombardi, M. et al., 1993. Primary structure and functional expression of a cDNA encoding the thiazide-sensitive, electroneutral sodium-chloride cotransporter. Proc. Natl. Acad. Sci. USA 90, 2749᎐2753. Gamba, G., Miyanoshita, A., Lombardi, M. et al., 1994. Molecular cloning, primary structure, and characterisation of two members of the mammalian electroneutral sodium - Ž potassium . -chloride co transporter family expressed in kidney. J. Biol. Chem. 269, 17713᎐17722.

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

Hirano, T., Mayer-Gostan, N., 1976. Eel esophagus as an osmoregulatory organ. Proc. Natl. Acad. Sci. USA. 73, 1348᎐1350. Hubner, S., Michel, F., Rudloff, V., Appelhans, H., 1992. Amino acid sequence of band-3 protein from rainbow trout erythrocytes derived from cDNA. Biochem. J. 285, 17᎐23. Hwang, P.P., Fang, M.J., Tsai, J.C., Huang, C.J., Chen, S.T., 1998. Expression of mRNA and protein of Naq-Kq-ATPase alpha subunit in gills of tilapia Ž Oreochromis mossambicus.. Fish Physiol. Biochem. 18, 363᎐373. Isaia, J., 1984. Water and nonelectrolyte permeation. In: Hoar, W.S., Randall, D.J. ŽEds.., Fish Physiology. vol. X. Gills. Part B. Ion and water transfer, X. Academic Press, London, pp. 1᎐38. Kisen, G., Gallais, C., Auperin, B. et al., 1994. Northen blot analysis of the Naq,Kq-ATPase ␣-subunit in salmonids. Comp. Biochem. Physiol. 107B, 255᎐259. Lee, T-H., Tsai, J-C., Fang, M-J., Yu, M-J., Hwang, P-P., 1998. Isoform expression of Naq-Kq-ATPase ␣-subunit in gills of the teleost Oreochromis mossambicus. Am. J. Physiol. 275, R296᎐R932. Meyer, A., Malaga-Trillo, E., 1999. Vertebrate genomics: More fishy tales about Hox genes. Curr. Biol. 9, R210᎐R213. Marshall, J., Martin, K.A., Picciotto, M., Hockfield, S., Nairn, A.C., Kaczmarek, L.K., 1991. Identification and localisation of a dogfish homologue of Human Cystic Fibrosis Transmembrane conductance Regulator. J. Biol. Chem. 266, 22749᎐22754. Marshall, W.S., Bryson, S.E., 1998. Transport mechanisms of seawater teleost chloride cells: An inclusive model of a multifunctional cell. Comp. Biochem. Physiol. 119A, 97᎐106. Marshall, W.S., Emberley, T.R., Singer, T.D., Bryson, S.E., McCormick, S.D., 1999. Time course of salinity adaptation in a strongly euryhaline esturine teleost, heteroclitus: A multivariable approach. J. Exp. Biol. 202, 1535᎐1544. Miyazaki, H., Uchida, S., Takei, Y., Hirano, T., Marumo, F., Sasaki, S., 1999. Molecular cloning of CLC chloride channels in Oreochromis mossambicus and their functional complementation of yeast CLC gene mutant. Biochem. Biophys. Res. Commun. 255, 175᎐181. Mount, D.B., Mercado, A., Song, L., Xu, J., George, Jr. A.L., Delpire, E., Gamba, G., 1999. Cloning and characterization of KCC3 and KCC4, New members of the chloride-cation cotransporter gene family. J. Biol. Chem. 274, 16355᎐16362. Nagashima, K., Ando, M., 1993. Characterization of esophageal desalination in the seawater eel, Anguilla japonica. J. Comp Physiol. B 164, 47᎐54. Niederstatter, H., Pelster, B., 2000. Expression of two vacuolar-type ATPase B subunit isoforms in swim-

563

bladder gas gland cells of the European eel: nucleotide sequences and deduced amino acid sequences. Biochim. Biophys. ACTA 1491, 133᎐142. Noel, J., Pouyssegur, J., 1995. Hormonal regulation, pharmacology, and membrane sorting of vertebrate NaqrHq exchanger isoforms. Am. J. Physiol. 268, C283᎐C296. Ohno, S., 1999. Gene duplication and the uniqueness of vertebrate genomes circa 1970᎐1999. Cell Dev. Biol. 10, 517᎐522. Parmelee, J.T., Renfro, J.L., 1983. Esophageal desalination of seawater in flounder: role of active sodium transport. Am. J. Physiol. 245, R888᎐R893. Payne, J.A., Forbush, IIIB., 1994. Alternatively spliced isoforms of the putative renal Na-K-Cl cotransporter are differentially distributed within the rabbit kidney. Proc. Natl. Acad. Sci. USA 91, 4544᎐4548. Perry, S.F., 1997. The chloride cell: Structure and function in the gills of freshwater fishes. Annu. Rev. Physiol. 59, 325᎐347. Perry, S.F., Beyers, M.L, Johnson, D.A., 2000. Cloning and molecular characterisation of the trout Ž Oncorhynchus mykiss. vacuolar H q-ATPase B subunit. J. Exp. Biol. 203, 459᎐470. Postlethwait, J.H., Yan, Y.L., Gates, M.A. et al., 1998. Vertebrate genome evolution and the zebrafish gene map. Nat. Genet. 18, 345᎐349. Pressley, T.A., 1992. Phylogenetic conservation of isoform-specific regions within the ␣-subunit of NaqKq-ATPase. Am. J. Physiol. 262, C743᎐C751. Romero, M.F., Boron, W.F., 1999. Electrogenic NaqrHCO 3- co-transporters: Cloning and physiology. Annu. Rev. Physiol. 61, 699᎐723. Romero, M.F., Hediger, M.A., Boulpaep, E.L., Boron, W.F., 1997. Expression cloning and characterization of a renal electrogenic NaqrHCO 3- cotransporter. Nature 387, 409᎐413. Russell, J.M., 2000. Sodium-Potassium-Chloride Cotransport. Physiol. Rev. 80, 211᎐276. Salvadori, S., Deiana, A.M., Coluccia, E., Milia, A., Cau, A., 1997. The different banding patterns produced by restriction endonuclease digestion in mitotic chromosomes of the American and European eel. J. Fish Biol. 50, 668᎐671. Schettino, T., Trischitta, F., Denaro, M.G., Faggio, C., Fucile, I., 1992. Requirement of HCO 3 for Cl-absorption in seawater-adapted eel intestine. Pflugers Arch. 421, 146᎐154. Schoenrock, C., Morley, S.D., Okawara, Y., Lederis, K., Richter, D., 1991. Sodium and potassium ATPase of the teleost fish Catostomus commersoni: sequence, protein structure and evolutionary conservation of the alpha-subunit. Biol. Chem. Hoppe-Seyler 372, 16895᎐16903.

564

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

Schreiber, R., Pavenstadt, H., Greger, R., Kunzelmann, K., 2000. Aquaporin 3 cloned from Xenopus laevis is regulated by the cystic fibrosis transmembrane conductance regulator. FEBS Lett. 475, 291᎐295. Schwiebert, E.M., Benos, D.J., Egan, M.E., Jackson, Stutts, M., Guggino, W.B., 1999. CFTR is a conductance regulator as well as a chloride channel. Phys. Rev. 97, S145᎐S166. Singer, T.D., Tucker, S.J., Marshall, W.S., Higgins, C.F., 1998. A divergent CFTR homologue: highly regulated salt transport in the euryhaline teleost F. heteroclitus. Am. J. Physiol. 274, C715᎐C723. Stellwag, E.J., 1999. Hox gene duplication in fish. Cell Dev. Biol. 10, 531᎐540. Sullivan, G., Fryer, J., Perry, S.F., 1995. Immunolocalization of proton pumps ŽHq-ATPase. on pavement cells of rainbow trout gill. J. Exp. Biol. 198, 2129᎐2619. Sullivan, G., Fryer, J., Perry, S.F., 1996. Localization of mRNA for proton pump ŽHq-ATPase. and ClrHCO 3- exchanger in rainbow trout gill. Can. J. Zool. 74, 2095᎐2103. Suzuki, Y., Itakura, M., Kashiwagi, M. et al., 1999. Identification by differential display of a hypertonicity-inducible inward rectifier potassium channel highly expressed in chloride cells. J. Biol. Chem. 274, 11376᎐11382. Trischitta, F., Denaro, M.G., Faggio, C., Schettino, T., 1992a. Comparison of Cl-absorption in the seawaterand freshwater-adapted eel, Anguilla anguilla: evidence for the presence of an Na-K-Cl cotransport system on the luminal membrane of the enterocyte. J. Exp. Zool. 263, 245᎐253. Trischitta, F., Denaro, M.G., Faggio, C., Schettino, T., 1992b. An attempt to determine the mechanisms of Cl-exit across the basolateral membrane of eel intestine: Use of different Cl-transport pathway inhibitors. J. Exp. Zool. 264, 11᎐18. Van Os, C.H., Kamsteeg, E.-J., Marr, N., Deen, P.M.T., 2000. Physiological relevance of aquaporins: luxury or necessity. Pflug. Arch. 440, 513᎐520. Viella, S., Ingrosso, L., Zonno, V., Schettino, T., Storelli,

C., 1997. An electroneutral anion exchange mechanism is present in brush᎐border membranes isolated from eel kidney. Am. J. Physiol. 272, R1143᎐R1148. Viella, S., Zonno, V., Cassano, G., Maffia, M., Storelli, C., 1991. NaqrHq exchange in the kidneys of eels Ž Anguilla anguilla. adapted to seawater or to freshwater environments: studies with brush border membrane vesicles. Comp. Biochem. Physiol. 100A, 455᎐460. Viella, S., Zonno, V., Lapadula, M., Verri, T., Storelli, C., 1995. Characterization of plasma membrane Naq-Hq exchange in eel Ž Anguilla anguilla. intestinal epithelial cells. J. Exp. Zool. 271, 18᎐28. Wilson, J.M., Laurent, P., Tufts, B.L. et al., 2000a. NaCl uptake by the branchial epithelium in freshwater teleost fish: an immunological approach to iontransport protein localization. J. Exp. Biol. 203, 2279᎐2296. Wilson, J.M., Randall, D.J., Donowitz, M., Vogl, A.W., IP, A.K.-Y., 2000b. Immunolocalization of ion-transport proteins to branchial epithelium mitochodriarich cells in the mudskipper Ž Periophthalmodon schlosseri .. J. Exp. Biol. 203, 2297᎐2310. Wittbrodt, J., Meyer, A., Schartl., M., 1998. More genes in fish? Bioessays 20, 511᎐515. Wood, C.M., Gilmour, K.M., Part, P., 1998. Passive and active transport properties of a gill model, the cultured branchial epithelium of the freshwater rainbow trout Ž Oncorhynchus mykiss). Comp Biochem. Physiol. 119A, 87᎐96. Young, R.M., Lingrel, J.B., 1987. Tissue distribution of mRNAs encoding the ␣ isoforms and ␤ subunit of rat Naq,Kq-ATPase. Biochem. Biophys. Res. Commun. 145, 52᎐58. Zadunaisky, J.A., Cardona, S., Au., L. et al., 1995. Chloride transport activation by plasma osmolarity during adaptation to high salinity of Fundulus heteroclitus. J. Membr. Biol. 143, 207᎐217. Zonno, V., Viella, S., Storelli, C., 1994. Salinity dependence of NaqrHq exchange activity in the eel Ž Anguilla anguilla. renal brush border membrane vesicles. Comp. Biochem. Physiol. 107A, 133᎐140.