The intestinal guanylin system and seawater adaptation in eels

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General and Comparative Endocrinology 152 (2007) 339–351

Review

The intestinal guanylin system and seawater adaptation in eels

Yoshio Takei *, Shinya Yuge

Laboratory of Physiology, Ocean Research Institute, The University of Tokyo, 1-15-1 Minamidai, Nakano, Tokyo 164-8639, Japan

Received 17 November 2006; revised 23 April 2007; accepted 7 May 2007Available online 13 May 2007

Abstract

Guanylin and uroguanylin are principal intestinal hormones secreted into the lumen to regulate ion and water absorption via a spe-cific receptor, guanylyl cyclase-C (GC-C). As the intestine is an essential organ for seawater (SW) adaptation in teleost fishes, the intes-tinal guanylin system may play a critical role in SW adaptation. Molecular biological studies identified multiple guanylins (guanylin,uroguanylin and renoguanylin) and their receptors (GC-C1 and GC-C2) in eels. The relative potency of the three ligands on cGMP pro-duction in transiently expressed receptors was uroguanylin > guanylin P renoguanylin for CG-C1 and guanylin P renoguany-lin > uroguanylin for GC-C2. Eel guanylin and GC-C genes are expressed exclusively in the intestine and kidney, and the level ofexpression is greater in SW eels than in freshwater (FW) eels except for renoguanylin. Physiological studies using Ussing chambersshowed that the middle and posterior intestine are major sites of action of guanylins, where they act on the mucosal side to decreaseshort circuit current (Isc) in a dose-dependent manner. The ID50 of guanylins for transport inhibition was 50-fold greater than that ofatrial natriuretic peptide that acts from the serosal side as an endocrine hormone. However, only guanylins reversed Isc to levels belowzero. Pharmacological analyses using various blockers showed that among transporters and channels localized on the intestinal cells ofSW teleost fish, the cystic fibrosis transmembrane conductance regulator Cl� channel (CFTR) on the apical membrane is the major tar-get of guanylins. Collectively, guanylins are synthesized locally in the intestine and secreted into the lumen to act on the GC-Cs in theapical membrane of eel intestinal cells. Then, intracellular cGMP production after ligand–receptor interaction activates CFTR and prob-ably induces Cl� and/or HCO3

� secretion into the lumen as suggested in mammals. The physiological significance of the anion secretioninduced by the luminal guanylin/GC-C system on SW adaptation may rival or exceed that of the serosally derived natriuretic peptides inthe euryhaline eel.� 2007 Elsevier Inc. All rights reserved.

Keywords: Guanylin; Guanylyl cyclase receptor; Intestinal absorption; CFTR

1. Role of intestine in seawater adaptation in teleost fish

It is well known that euryhaline teleosts such as eelmaintain their plasma osmolality at ca. 300 mOsmol/kgirrespective of environmental salinity over a broad range(Marshall and Grosell, 2005). In fresh water (FW), tele-osts gain water osmotically across the body surfaces, prin-cipally via the gills, and absorb monovalent ions (Na+

and Cl�) from the environment by ATP-driven mecha-nisms. The intestine of FW fish principally serves as thesite of nutrient absorption. In SW, however, teleost fisheslose water osmotically across the body surfaces. To com-

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doi:10.1016/j.ygcen.2007.05.005

* Corresponding author. Fax: +81 3 5351 6463.E-mail address: [email protected] (Y. Takei).

pensate for the loss, they drink environmental SW copi-ously. Since the intestinal lumen is technically outsidethe internal milieu, ingested SW must be absorbed bythe intestine to enter the organism. To this end, ingestedSW is diluted to become isotonic to body fluids duringits passage through the anterior part of alimentary tract(esophagus, stomach, etc.), reducing the work of waterabsorption in the posterior part. Accordingly, the intes-tine is a crucial organ for body fluid regulation in SW tel-eosts as are the gills and kidney for monovalent anddivalent ion excretion, respectively (Marshall and Grosell,2005). In fact, if the esophagus is tied to prevent ingestedSW from entering the intestine, fish eventually die becauseof severe hypovolemia and hypernatremia (Takei et al.,1998).

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340 Y. Takei, S. Yuge / General and Comparative Endocrinology 152 (2007) 339–351

When the eels are transferred from FW to SW, bothmorphological and functional changes occur profoundlyin the intestine over time (MacKay and Janicki, 1978);increased vascularization and wall thinning facilitate waterand ion absorption across the epithelia (Fig. 1). In fact,almost all ingested water and monovalent ions areabsorbed in parallel by the intestine of SW teleost (Skadha-uge, 1969; Loretz, 1995; Tsukada and Takei, 2006). By con-trast, only small amounts of divalent ions are absorbed bythe intestine, and these ions therefore accumulate in theposterior part (Parmelee and Renfro, 1983; Tsukadaet al., 2005). Eventually, the divalent cations precipitateas a complex of CaCO3 and MgCO3 (Walsh et al., 1991),which are visible through the thin intestinal wall as whiteprecipitates during the course of SW adaptation in eels(Fig. 1). The precipitation is considered to decrease theosmolality of luminal fluid and thus to accelerate waterabsorption (Wilson et al., 2002; Wilson and Grosell,2003). Thus, the eel intestine, which exhibits such obviousmorphological changes during the course of SW adapta-tion, should serve as an excellent model to explore the roleof the intestinal guanylin system in osmoregulation. In par-allel with the morphological changes, biochemical changesoccur in terms of transporters/channels and intercellularmatrices in the intestine during the course of SW adapta-tion in teleost fishes (Marshall and Grosell, 2005).

2. Mechanisms of intestinal absorption and its hormonal

regulation

Ion and water budgets differ greatly among animals indifferent habitats; terrestrial animals have developed

FW

3 h

24 h

1 wk

2 wk

anterior posterior

SW transfer

Fig. 1. Morphological changes in eel intestine after transfer from freshwater (FW) to seawater (SW). Blood vessels develop (red arrows) and thewall becomes thinner gradually after SW transfer. White precipitates(yellow arrows) are visible in the middle–posterior intestine.

mechanisms to retain both water and ions, while thesemechanisms are variable in aquatic fishes depending onthe environmental salinity. Consistently, the mechanismsof water and ion absorption by the intestine differ amonganimals in different habitats. In mammals, ingested wateris absorbed partly with Na+-coupled absorption of nutri-ents such as amino acids and glucose in the small intestine(Schultz, 1981). In the colon, a large capacity for waterabsorption exists (Geibel, 2005) and water follows Na+

and Cl� absorption via Na+/H+ exchanger (NHE) andCl�=HCO3

� exchanger in the anterior part and by epithe-lial Na+ channel (ENaC) and Cl� channels in the posteriorpart (Fig. 2). In addition, water and ion secretion can occurin the mammalian colon, where the cystic fibrosis trans-membrane conductance regulator Cl� channel (CFTR)on the apical membrane and secretory-type Na+–K+–2Cl� cotransporter (NKCC1) on the basolateral membraneplay major roles.

Compared with mammals, less is known about the mech-anism of ion and water absorption in fish intestine, exceptfor SW teleosts where the intestine plays an essential rolein body fluid regulation (Loretz, 1995; Ando et al., 2003;Marshall and Grosell, 2005). Initially, ingested SW isdiluted to isotonicity before transfer to the posterior intes-tine where it is absorbed copiously (Skadhauge, 1969; Hir-ano et al., 1976; Kirsch and Meister, 1982; Parmelee andRenfro, 1983; Loretz, 1995). In the eel, imbibed SW is firstdesalinated in the esophagus by dissipation of the concen-tration gradient of NaCl between ingested SW and plasma(Hirano and Mayer-Gostan, 1976). As a result, the NaClconcentration of ingested SW is decreased to half when itenters the stomach. The luminal fluid is further diluted inthe stomach and anterior intestine and becomes isotonicto plasma when it reaches the middle–posterior part ofthe intestine where active water absorption occurs (Hiranoet al., 1976; Kirsch and Meister, 1982; Ando et al., 2003;Tsukada et al., 2005). Thus, the posterior intestine is impor-tant for water absorption in SW teleost fish as is the case forthe colon in mammals (Loretz, 1995; Aoki et al., 2003; Mar-tinez et al., 2005; Marshall and Grosell, 2005). Althoughunabsorbed divalent ions (Mg2+, Ca2+, SO4

2�) accumulatein the distal intestine and increase the tonicity of luminalfluid, this effect on tonicity is moderated by precipitationas sulfates or carbonates by HCO3

� secretion into thelumen (Wilson et al., 2002; Wilson and Grosell, 2003).

At the cellular/molecular level, various transporters andchannels are localized on the apical and basolateral mem-brane of intestinal epithelial cells (Fig. 2). The primary driv-ing force for absorption is generated by Na+, K+-ATPase,but the location and abundance of other transporters/chan-nels differ markedly between mammals and fish (Loretz,1995; Schultz, 1998; Geibel, 2005; Marshall and Grosell,2005). In the intestine of SW teleost, where copious absorp-tion of NaCl and water occurs, critical transporters thus farsuggested are the absorptive-type NKCC2 and K+ channelon the apical membrane, and Na+, K+-ATPase, Cl� chan-nels and K+ channel on the basolateral membrane (Fig. 2).

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Fig. 2. Absorptive intestinal epithelia of seawater teleost (a) and mammals (b). Mammalian (human) colon was divided into proximal and distal segmentas major transporters and channels on these segments are different. For clarity, only major transporters and channels are depicted. For details, see text.

Y. Takei, S. Yuge / General and Comparative Endocrinology 152 (2007) 339–351 341

Furthermore, Cl�=HCO3� exchangers are implicated in Cl�

absorption at the basolateral side and HCO3� secretion at

the apical side (Wilson et al., 2002; Grosell et al., 2005).The presence of a small population of CFTR is also sug-gested by immunohistochemical study (Marshall et al.,2002). The electrophysiological characteristics of intestinalepithelia mounted in Ussing chambers also differ betweenteleosts and mammals; serosa-negative (teleosts) vs. ser-osa-positive (mammals) transepithelial potentials exist,which are in accord with the different ion transport mecha-nisms in the two groups. The serosa-negativity is muchgreater in SW teleosts than in FW fish, suggesting the pres-ence of active ion transport via electrogenic mechanisms inthe intestine of SW teleosts (Halm et al., 1985).

Hormones play key roles in various aspects of fishosmoregulation including water and ion transport by theintestine (Ando et al., 2003; Takei and Loretz, 2005).Among various hormones that are implicated in the regu-lation of intestinal absorption in teleost fish, atrial natri-

uretic peptide (ANP) appears to be the most potent andefficacious hormone for inhibition of NaCl absorption(O’Grady et al., 1985; Ando et al., 1992; Loretz and Takei,1997). Because of its profound effect, plasma Na+ concen-tration decreases after ANP infusion in SW eels (Takei andKaiya, 1998; Tsukada et al., 2005). Furthermore, the ANPgene is expressed in the eel intestine (Loretz et al., 1997).However, since ANP is principally an endocrine hormoneacting from the blood, other intestinal hormones may havepredominating roles in the local regulation of ion andwater transport across the teleost intestine.

3. Guanylin peptides and their receptors

The discovery of guanylin arose from research on diar-rhea in mammals, which is caused by the heat-stableenterotoxin (STa) product of enterotoxigenic Escherichiacoli (Field, 2003; Forte, 2004). STa binds guanylylcyclase-C (GC-C) located on the apical membrane of


intestinal epithelial cells and causes diarrhea by cGMPaccumulation in the epithelial cells (Schulz et al., 1990;Forte, 2004). Subsequently, the endogenous ligand guany-lin (Currie et al., 1992) and uroguanylin (Hamra et al.,1993) were identified in the intestine and kidney, respec-tively. They are produced in the intestine and kidney, andinfluence body fluid regulation through binding to GC-C(Forte et al., 2000). The basic structure of mature guanylinsis 15–16 amino acid peptide with two disulfide bonds(Fig. 3a). These disulfide bonds are indispensable for opti-mal biological actions. Although two topological isomersexist and are easily interchangeable, only one of these isbioactive (Chino et al., 1998). This isomerization potentialmakes the quantitative assessment of guanylin bioactivitydifficult. Compared with guanylins, STa has an additionaldisulfide bond, which may stabilize the tertiary structureand enhance bioactivity of this exogenous peptide (Nakaz-ato, 2001). Guanylin is synthesized widely in the villus epi-thelial cells and Paneth cells of human intestine (Date et al.,1996) or in the goblet cells of the rat intestine (Li et al.,

Guanylin

Uroguanylin

C C

C

N

C C

C

C

C

Y/F

Fig. 3. (a) Secondary structure of guanylin and uroguanylin. (b) Amino acidfish. Accession numbers and the genome scaffold numbers (SCAF) are noted fshadowed. Ref. 1, Cramb et al. (2005); Ref. 2, Forte et al. (1999).

1995), and secreted mostly into the lumen, while uroguany-lin is produced and stored in the enterochromaffin cells(Perkins et al., 1997) and secreted both into the lumenand into the blood. In fact, Fujita et al. (1974) reportedmore than 30 years ago that granular content is releasedfrom enterochromaffin cells of rabbit intestinal epitheliain response to cholera toxin administration. It is possiblethat the released uroguanylin may competitively bind tothe GC-C to ameliorate diarrhea and to stimulate peristal-tic movement to purge luminal toxin.

The GC-C is one of the membrane guanylyl cyclasesthat catalyze the production of a second messenger, cGMP,which further activates phosphokinase G-II to modify tar-get protein (Vaandrager, 2002; Nakauchi and Suzuki,2005). The GC-C consists of four major domains, extracel-lular ligand-binding domain (ECD), membrane-spanningdomain (MSD), kinase-like domain (KLD) and guanylylcyclase domain (GCD) (Fig. 4). The receptor is located pre-dominantly on the apical surface of the intestinal epitheliaand renal proximal tubules (Vaandrager, 2002; Forte,

sequences of guanylin peptides thus far identified in mammals and teleostor reference. Amino acid residues conserved in more than half species are

Fig. 4. Localization and gross structure of guanylyl cyclase-C (GC-C)receptor and its signal transduction pathway for CFTR activationsuggested in the enterocyte of mammals. ECD, extracellular domain;GCD, guanylyl cyclase domain; GN, guanylin; KLD, kinase-like domain;MSD, membrane-spanning domain; PKG-II, protein kinase G-II; UGN,uroguanylin.


2004). Guanylin and uroguanylin secreted into the intesti-nal lumen act on the apical GC-C. Circulating guanylinsmay act on the kidney but not on the intestine, since guany-lin applied to the basolateral side of intestinal epitheliadoes not exhibit biological activities (Guba et al., 1996;Joo et al., 1998).

The major function of guanylin and uroguanylin is toinduce electrogenic Cl� secretion into the intestinal lumenthrough CFTR, and to activate the peristaltic movementof intestine, resulting in diarrhea (Nakazato, 2001; Forte,2004). In addition, guanylins stimulate HCO3

� secretionthrough CFTR into the intestinal lumen (Guba et al.,1996; Joo et al., 1998). In the kidney, guanylin and urogu-anylin induce natriuresis, kaliuresis and diuresis withuroguanylin much more potent than guanylin (Fonteleset al., 1998; Forte et al., 2000). This can be explained bythe higher resistance of uroguanylin against degradingenzymes located on the brush border membrane of proxi-mal tubular cells. Thus uroguanylin can act on the tubularcells from the luminal side.

Intestinal and renal guanylin and uroguanylin seem tobe regulated by changes in luminal NaCl level. High saltintake promotes guanylin secretion from the intestine (Kitaet al., 1999) and uroguanylin gene expression in the kidney(Potthast et al., 2001). By contrast, low salt intake down-regulates guanylin expression in the intestine (Li et al.,1996). Lennane et al. (1975) showed that oral sodium loadis a stronger stimulus for natriuresis than the same loaddelivered directly to the blood stream. Thus, it is likely that

intestinal uroguanylin is secreted into blood in response tothe sodium load to the lumen, which then acts on the kid-ney to induce potent natriuresis (Forte et al., 2000). Thepresence of uroguanylin in the endocrine enterochromaffincells supports the endocrine nature of intestinal uroguany-lin (Perkins et al., 1997).

4. The guanylin–GC-C system in non-mammalian species

Guanylin-like peptide and GC-C-like protein have beendetected using antibodies raised against mammalianguanylin or radio-labeled STa in birds (Krause et al.,1995) and reptiles (Krause et al., 1997). Furthermore, acDNA coding for uroguanylin has been cloned in the eel,Anguilla anguilla (Comrie et al., 2001a). Concerning thereceptors, two isoforms of GC-C, named OlGC6 (Man-toku et al., 1999) and OlGC9 (Iio et al., 2005), have beenidentified in the medaka (Oryzias latipes). Therefore, it isstrongly evident that the guanylin–GC-C system exists inteleost fish and other non-mammalian species. In addition,partial cDNAs coding for GC-C-like sequences have beencloned in A. anguilla (Comrie et al., 2001b). Based on thesestudies, we have started molecular biological and physio-logical studies on the guanylin and GC-C system usingthe Japanese eel, A. japonica.

4.1. The guanylin family in eel

From the eel intestine, we cloned cDNAs encoding threedistinct guanylins, guanylin, uroguanylin and renoguanylin(Yuge et al., 2003). We named the third guanylin ‘reno’-guanylin as it is expressed most abundantly in the kidneyamong the three. The molecules were distinguished basedon a critical amino acid position; guanylin and uroguanylinhave an aromatic amino acid (Phe in eel and Tyr in mostmammals) and a non-aromatic amino acid (Asn) at theninth position, respectively (Fig. 3b). Further, eel urogu-anylin has an additional residue at the C-terminus asobserved in some mammalian uroguanylins. Renoguanylinis a hybrid type, with an aromatic residue (Phe) at the ninthposition as guanylin, and an additional Leu at the C-termi-nus as uroguanylin. The C-terminal addition is thought todecrease the conversion of active peptide to a non-activestereoisomer and thus to stabilize the biological activity(Schulz et al., 1998). More recently, cDNAs encoding tele-ost guanylin and uroguanylin have been cloned (Crambet al., 2005) or identified in the genome and EST databasesof pufferfish, medaka and zebrafish (Fig. 3b). Third guany-lins are also identified in the green pufferfish (Tetraodon

nigroviridis) and opossum (lymphoguanylin, Forte et al.,1999). The pufferfish peptide has non-aromatic Ser at theninth position. Thus it is more similar to uroguanylin asis the case with lymphoguanylin, and it may not be anortholog of eel renoguanylin. Two guanylins in salmonidspecies may be due to the tetraploidy of this species. Phy-logenetic analyses using teleost and mammalian guanylinsshow that guanylin and uroguanylin of each vertebrate


class are clustered into a separate groups and eel renogu-anylin is localized close to teleost guanylins (Fig. 5). Thisindicates that two guanylin peptides (guanylin and urogu-anylin) already existed when ray-finned fish and lobe-finned fish (tetrapods) diverged into different lineages,and that the third guanylin peptide was produced afterthe divergence. This event will be discussed in some detailbelow (Section 4.3).

Immunohistochemical studies using a specific antiserumraised against eel guanylin showed that guanylin is found inthe goblet cells of eel intestine as it is found in the rat (Liet al., 1995), but not in enterochromaffin cells that produceuroguanylin in mammals (Yuge et al., 2003). Therefore,guanylin may be secreted into the intestinal lumen withmucus in teleost fish as is the case suggested in the rat.The cellular localization of uroguanylin and renoguanylinin the eel intestine and kidney has not been examinedbecause of the lack of specific antiserum to these peptides.

After transfer of eels from FW to SW, mRNA expres-sion in intestine of guanylin and uroguanylin did notchange for 3 h, but it increased gradually and the increasebecame significant after 24 h (Yuge et al., 2003). Theincreases for guanylin and uroguanylin were profoundafter adaptation to SW for more than a week, but thiswas not so for renoguanylin (Fig. 6). Uroguanylin tran-scripts were also up-regulated in the kidney of SW-adaptedeels. Similar results are reported in the FW-type yellow eelsand silver eels that are prepared to migrate into the sea(Comrie et al., 2001a). To our knowledge, guanylin anduroguanylin are the only osmoregulatory hormones thatexhibit such profound increases in the gene expression afterSW adaptation in teleost fish. Thus, the eel guanylin familymay have important functions for SW adaptation. As inmammals, it is likely that both guanylin and uroguanylinare secreted in response to an increase in luminal fluid

humanUGN

ratUGN

medakaUGN trout

UGN

eelUGN

ratGN

humanGN eel

RGNeelGN

troutGN

medakaGN

10036

39

87

4036

3295

Guanylin family

Fig. 5. Phylogenetic tree of (a) guanylins and (b) their receptor GC-Cs in telmethod based on the precursor sequence. Analyses by the Bayesian method i(2003, 2006).

osmolality after transfer of eels from FW to SW, as sup-ported by the enhanced gene expression after more than3 h.

4.2. Guanylin receptors in eels

We cloned full-length cDNAs encoding two isoforms ofGC-C, named GC-C1 and GC-C2, from the intestine of A.

japonica (Yuge et al., 2006). These receptors correspondedto the two GC-C-like partial cDNAs identified in A. angu-

illa, as sequence similarities were more than 98% at corre-sponding regions of 529 bp for GC-C1 (3802 bp in total)and 2765 bp for GC-C2 (5065 bp in total) (Comrie et al.,2001b). A third GC-C was extensively sought in the eelintestinal cDNA library using sequences for the intracellu-lar domains that are conserved even between eel and mam-mals. However, no more GC-Cs could be identified in theeel. The cloned GC-C proteins consisted of four domains,ECD, MSD, KLD, and GCD as in mammals; of these,the intracellular KLD and GCD were more conserved thanthe ligand-binding ECD (Fig. 4). Other GC-C-specificsequences detected in mammals were largely conserved ineels except for some amino acid residues important forthe ligand-binding in the ECD, indicating different ligandselectivity between fish and mammals (Yuge et al., 2006).Phylogenetic analyses showed that cloned GC-Cs aregrouped with other GC-Cs but not with GC-As and GC-Bs (Yuge et al., 2006) that are receptors for natriuretic pep-tides (Takei and Hirose, 2002). However, two eel GC-Csappear to have undergone unique structural evolutioncompared with other teleost GC-Cs, as they are not clus-tered separately into two different groups of GC-C of otherteleost species (Fig. 5).

To test the ligand specificity of the cloned eel GC-Cs,each receptor protein was transiently expressed in the

TakifuguGC-CII

humanGC-C

ratGC-C

medakaOlGC9

medakaOlGC6

TakifuguGC-CI

frogGC-C

100

100 100

52

46

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eel GC-C2

Guanylyl cyclase C

eost fishes and mammals. Tree was depicted by the maximum likelihoodn MrBayes program produced similar results. Modified from Yuge et al.

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esophagus stomach a. intestine p. intestine kidneyFW SWFW SW FW SW FW SWFW SW

eel GC-C2

eel GC-C1

Eel uroguanylin

Eel guanylin Eel renoguanylin

Fig. 6. Changes in gene expression of guanylins (a, b and c) and GC-Cs (d and e) after transfer of freshwater (FW) eels to seawater (SW) for 2 weeks. Plaincolumn, FW–FW transfer (time controls); filled column, FW–SW transfer. *P < 0.05; **P < 0.01. Modified from Yuge et al. (2003, 2006).


COS cells, and the affinity of three eel guanylins to eachreceptor was examined in terms of the cGMP productivity(Yuge et al., 2006). All guanylins stimulated cGMPproduction in a dose-dependent manner but eel ANP failedto have such effect. These data provide functional supportfor the assignment of these proteins as eel GC-Cs.The potency order for cGMP production was uroguany-lin > guanylin P renoguanylin for GC-C1, and guany-lin P renoguanylin > uroguanylin for GC-C2. Thedistinct ligand selectivity is consistent with the low homol-

ogy (53%) of the ECD of GC-C1 and GC-C2 comparedwith intracellular domains (74–90%). Interestingly, STa, aprofound stimulator for cGMP production in mammalianGC-C, failed to increase cGMP production in eel GC-Cs.This may be related to the low similarity of seven consecu-tive amino acid residues (Ser410-Lys416) near the MSD,which are suggested to be the site of STa binding (Hase-gawa and Shimonishi, 2005). Marshall et al. (2002) showedthat ion and fluid secretion occur in the killifish intestineafter combined stimulation of cellular Ca2+ and cyclic


nucleotides (cAMP and cGMP), which presumably aids inpurging toxic intestinal bacterial flora. However, two eelGC-Cs did not respond to STa by cGMP production.

Both GC-C1 and GC-C2 genes were expressed in the ali-mentary tracts and kidney of eels. Whereas GC-C1 tran-scripts were almost exclusively found in the intestine,GC-C2 transcripts were identified also in other segmentsof the alimentary tracts such as esophagus and stomach(Yuge et al., 2006). The expression of the GC-C1 andGC-C2 gene was enhanced in the intestine of SW-adaptedeels compared with FW fish (Fig. 6) but not in other tis-sues. Therefore, both hormone and receptor genes aremore abundantly expressed in SW eels than in FW eels.However, the expression of the receptor genes wasunchanged for 24 h after transfer of eels to SW, showingslower response of the receptors to salinity changes thanthat of ligands. In view of the coordinated increases inthe guanylin and GC-C genes in SW eel intestine, the eelguanylin–GC-C system seems to play a pivotal role inSW adaptation as a local paracrine and/or luminocrineregulator.

4.3. Evolution of guanylin–GC-C system

Both guanylin and its receptor have been found in mam-mals and teleost species and their presence is suggested inother, non-mammalian tetrapod species. Thus, the guany-lin family may be a phylogenetically old hormonal systemthat already existed before divergence of ray-finned fishand lobe-finned fish about 400 million years ago (Naruseet al., 2004). It is intriguing to examine which of the guany-lin peptides holds an ancestral nature and when they werediversified into multiple peptides.

The guanylin and uroguanylin genes are localized in tan-dem in the close proximity on chromosome 1 in humansand chromosome 4 in mice (see Forte, 2004). We alsofound that two guanylin genes exist in tandem on the samechromosome of two species of pufferfish and zebrafish inthe genome database (Fig. 3b). This similarity suggests thatthe two guanylin genes were formed by tandem duplicationbefore the divergence of teleost and tetrapod lineages. Con-sistently, the molecular phylogenetic analysis showed thatguanylin and uroguanylin are clustered, respectively, in tel-eosts and mammals (Fig. 5). This may indicate that bothguanylin and uroguanylin existed before divergence ofray-finned fishes and tetrapods. It is not known how thethird guanylin, renoguanylin, is generated in the eel, butthe structural and functional similarity and phylogeneticproximity of renoguanylin and guanylin indicate thatrenoguanylin originated from guanylin rather recently.Knowing that additional whole genome duplication (3R)occurred only in the teleost lineage (Christoffels et al.,2004), the linkage analysis showed that two medaka GC-C genes are generated by the 3R duplication step (Iioet al., 2005). Thus, it is possible that the renoguanylin genewas produced from guanylin at the 3R step and is stillretained in the eel. Linkage analysis is not possible with

renoguanylin in medaka as it is undetectable in the genomedatabase of this teleost species.

Based on the data obtained in mammals, Forte (2004)proposed that uroguanylin is an ancestral molecule of theguanylin family because it has higher affinity to GC-C thanguanylin and seems to have more essential roles from theintegrative viewpoint. In the genome database of a proto-chordate (Ciona intestinalis), we could not detect a guany-lin-like sequence. Molecular data are required incartilaginous fish and cyclostomes to elucidate the evolu-tionary history of the guanylin family in vertebrates. Froman evolutionary viewpoint, it is intriguing to note that eelGC-Cs have lost affinity to STa produced by the enterotox-igenic E. coli. It is possible that bacteria acquired theguanylin gene during parasitic enteric life by horizontalgene transfer, but no evolutionary vestige appears toremain in the sequence of STa.

5. Biological action of guanylins on eel intestine

Molecular biological and biochemical studies show thateels have a complex guanylin system compared with mam-mals, and the expression of both ligand and receptor genesare enhanced in the intestine after SW acclimation. Thus, itis likely that guanylins play important roles in water andion metabolism in the intestine of SW eels. To evaluatethe role, the effect of homologous guanylins on ion trans-port was examined electrophysiologically in the SW eelintestine using Ussing chambers (Yuge and Takei, inpress). Since the effect of guanylin on short circuit current(Isc) was minimal at the anterior intestine, the effects wereexamined in the middle and posterior intestine where mostwater absorption occurs (Loretz, 1995; Ando et al., 2003;Aoki et al., 2003). Both segments exhibited serosa-negativepotential difference. The application of guanylin on theserosal side of intestinal epithelia had little effect on Isc ineither segment. However, all three guanylins applied tothe mucosal side decreased Isc dose-dependently at 10�8–10�6 M and reversed the current at 10�7–10�6 M. Thus,guanylins may inhibit NaCl absorption in the middle andposterior segments of SW eel intestine. The effect of guany-lin was mimicked by 8Br-cGMP. The reversal of Isc isunique for guanylins and was not observed with anotherpotent inhibitory hormone, ANP (Ando et al., 1992; Loretzand Takei, 1997). However, ANP is 1–2 orders of magni-tude more potent than guanylins when applied to the sero-sal side of SW eel intestine. The inhibitory effect ofguanylin was more potent in the middle intestine than inthe posterior intestine, as expected from the more abun-dant expression of GC-C genes in the former (Yugeet al., 2006). No significant difference was detected in thepotency among three guanylins in the middle intestine,but guanylin was more potent than uroguanylin in the pos-terior intestine. This coincides well with more brisk expres-sion of the GC-C2 gene in the posterior intestine.

As the middle intestine was highly responsive to guany-lin, the mechanism of guanylin action was analyzed in this

3Na+

2K+

Na+

K+

2Cl-

(mucosal) (serosal)

Na+

Cl-

K+

Cl-

HCO3-

Cl-

HCO3-

K+

K+

Cl-

Cl-

Na+ Na+

Cl-,HCO3

-NPPB

Bumet

HCTZ

DIDS DIDS

NPPB

ENaC VDSCAmil TTX

CFTR

AE

NCC

NKCC

AE

Cl-channel

Fig. 7. Summary of inhibitors used to block the transporters or channels that exist on the apical or basolateral membrane of epithelial cells of SW teleostintestine. Drugs were applied to the mucosal and/or serosal side in the Ussing chamber. Only mucosal application of bumetanide or NPPB was effective inblocking guanylin effect. Amil, amiloride; TTX, tetrodotoxin. For other abbreviations of transporters and drugs, see text.


segment using selective blockers of transporters and chan-nels that are known to be localized in the SW teleost intes-tine (Yuge and Takei, in press). Among blockers tested,mucosal application of 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), a blocker for Cl� channel includingCFTR, and bumetanide, an inhibitor for NKCC, inhibitedthe guanylin-induced decrease in Isc (Fig. 7). However,amiloride, a blocker for ENaC, 4,4 0-diisothiocyanatostil-bene-2,2 0-disulfonic acid (DIDS), a blocker for anionexchanger (AE), hydrochlorothiazide, a blocker of Na+–Cl�-cotransporter (NCC), and tetrodotoxin, a blocker forvoltage-dependent Na+ channel (VDSC), were withouteffects. The lack of inhibition by tetrodotoxin excludesthe possible mediation of nervous elements in the guanylineffect. On the other hand, bumetanide alone reversed Isc,showing that NKCC is a source of serosa-negative poten-tial difference at the distal intestine. Therefore, inhibitionof the guanylin effect by bumetanide may be non-specificdue to the deterioration of the whole transport processes.

Guanylin inhibited Isc in SW eel intestine after mucosalapplication but not after serosal application, showing thatGC-C1 and GC-C2 are localized on the mucosal side asshown by the mammalian GC-C (Forte, 2004). Further-more, high doses of guanylin reversed Isc from serosa-to-mucosa to mucosa-to-serosa direction. The majority ofnet NaCl absorption in SW fish intestine is achieved bythe absorptive NKCC2 located on the apical membraneacross the concentration gradient primarily energized byNa+, K+-ATPase (Ando et al., 1975; Musch et al., 1982;Palfrey and Rao, 1983; Trischitta et al., 1992; Schettinoand Lionetto, 2003). The accumulated Cl� ions in the epi-thelial cell are expelled onto the blood side via Cl� channelby intracellular negativity caused by Na+, K+-ATPase,while accumulated K+ ions are recycled into the intestinallumen and to the basolateral space via K+ channels for

proper functioning of apical NKCC and basolateral Na+,K+-ATPase. These processes via conductive channels giverise to serosa-negative transepithelial potential in SW fishintestine (Halm et al., 1985; Loretz, 1995). The basolateralK+–Cl�-cotransporter also appears to play a role in Cl�

absorption and K+ recycling (Schettino and Lionetto,2003). More importantly, as ingested SW contains muchlower concentration of K+ (10 mM) relative to Na+

(450 mM), luminal K+ must be supplemented via apicalK+ channels for active functioning of NKCC. It is possiblethat the blockade of NKCC by bumetanide reversed Isc,probably because NKCC is a basic element for serosa-neg-ativity across SW fish intestine and its blockade results inserosa positivity produced only by basolateral Na+, K+-ATPase as observed in mammalian colon (Kunzelmannand Mall, 2002; Geibel, 2005).

The electrophysiological difference in the intestine ofSW teleost and mammals reflects differences in function;the major function of the SW teleost intestine is fluidabsorption (Field et al., 1978; Loretz, 1987), while bothabsorption and secretion occur in the mammalian intestine.In the mammalian intestine, the baseline Isc is below zero(serosa-positive) and guanylins further decrease Isc. Thisindicates that active secretion of anions such as Cl� andHCO3

� occurs into the lumen via CFTR, which mayaccompany copious fluid secretion via water channels(Guba et al., 1996; Joo et al., 1998). It is not knownwhether fluid secretion also occurs in the SW eel intestinewhen Isc is reversed by guanylin. This issue will be dis-cussed in detail below (Section 6).

ANP is considered as the most potent hormone thatinhibits ion transport across the teleost intestine whenapplied to the serosal side (O’Grady et al., 1985; Andoet al., 1992; Loretz and Takei, 1997). Unlike guanylin,however, ANP does not reverse Isc even at 10�6 M.


Furthermore, the IC50 of ANP was 1 · 10�9 M in SW eelintestine (Ando et al., 2003), which is 50-fold lower thanthat of guanylin (5 · 10�8 M) in the same species (Yugeand Takei, in press). The IC50 of guanylin in the eel is sim-ilar to that in mammals (Guba et al., 1996; Joo et al., 1998).This difference may be due to the fact that ANP is princi-pally a circulating hormone acting from the blood side,while guanylins are secreted into lumen and act locally.We reported that plasma ANP concentration in SW eelsis ca. 0.5 · 10�10 M (Kaiya and Takei, 1996).

The precise mechanism of ion transport is less known inthe teleost intestine than in the mammalian intestine. Themajor differences between teleosts and mammals are: (1)absorptive-type NKCC2 is present on the apical membraneof epithelial cells in SW teleosts, while secretory-typeNKCC1 exists on the basolateral membrane in mammals;(2) CFTR is localized on the basolateral side in the intes-tine of SW killifish (Marshall et al., 2002), but it is foundon the apical side in mammals. In the SW killifish, immu-noreactive CFTR is also localized on the apical membraneof 10–20% enterocytes that may be secretory-type, but it isabsent in the dominant absorptive-type enterocytes (Mar-shall et al., 2002). It is possible that guanylins act onlyon the secretory-type enterocytes if such classification isapplicable to the eel intestine.

In the case of ANP, the target transporter or channel isdifferent in teleosts and mammals: NKCC in the flounder(O’Grady et al., 1985) and ENaC or Na+-coupled glucosetransporter in mammals (Gonzalez Boac et al., 2000).However, guanylin seems to regulate CFTR in both tele-osts and mammals. In mammals, the inhibition of NKCC1by bumetanide also prevents guanylin action (Guba et al.,1996; Joo et al., 1998), which may be a secondary effectcaused by the paucity of intracellular Cl� for secretionthrough CFTR by the inhibition of NKCC1. As mentionedabove, the prevention of guanylin action by bumetanide inabsorptive-type SW eel intestine may not be a direct actionbut may be mediated by deterioration of the whole trans-port system via inhibition of NKCC2.

HCO

Ca2+/Mg2+ + HC

Cl-

Cl-Na+ K+ 2Cl-

K+ CFTR

(anterior) (mid

Fig. 8. Hypothetical action of guanylin on the CFTR in SW eel intestine. Guinduce parallel absorption of monovalent ions and water. Luminal HCO3

� maand precipitates CaCO3 and MgCO3 to decrease luminal fluid osmolality, fur

6. Future directions

Finally, the biological significance of guanylin actionsfor SW adaptation will be considered (Fig. 8). It seems thata possible guanylin-induced decrease in NaCl absorptionwould cause a reduction of water absorption, which is dis-advantageous for SW adaptation. Furthermore, if Cl�

secretion occurs, water may move in parallel into the lumenand further challenge SW adaptation. However, Cl� secre-tion into the intestinal lumen is necessary for proper func-tioning of NKCC2, which is the major driving force forwater absorption in SW teleost intestine. As SW containssimilar amounts of Cl� and Na+, if NKCC absorbs 2Cl�

and one Na+ from the luminal fluid, Cl� may be in short-age in the distal part and should be supplemented for con-tinuous functioning of NKCC2. Furthermore, fourosmolytes (Na+, K+ and 2Cl�) are absorbed in exchangeof two Cl� ions. Thus, Cl� secretion may result in anincrease in water absorption overall. K+ ions may be sup-plemented into the lumen easily via abundant Ba-sensitiveK+ channels on the apical membrane (Halm et al., 1985).In fact, Cl� secretion into the lumen is likely in SW fishintestine, since the decrease in Cl� concentration alongthe intestinal segment is much smaller than Na+ concentra-tion (Loretz, 1995), and since luminal fluid Cl� concentra-tion is higher (59 ± 8 mM) than Na+ concentration(4 ± 1 mM) in the posterior intestine of SW eels (Tsukadaet al., 2005). In addition to the activation of NKCC2,secreted Cl� can be used to mobilize AE that exchangesCl� with intracellular HCO3

� (Grosell et al., 2001)(Fig. 8). As will be discussed below, active HCO3

� secre-tion occurs in the distal intestine of SW teleost fish (Wilsonet al., 2002).

In mammalian intestine, CFTR transports not only Cl�

but HCO3� to some extent (Guba et al., 1996; Seidler et al.,

1997). We have not measured changes in HCO3� secretion

yet, but it is possible that both Cl� and HCO3� secretion

are stimulated by guanylin through CFTR in SW eel intes-tine. In the SW teleost intestine, high concentration of

3-

CaCO3O3- =

MgCO3

HCO3-

dle) (posterior)

CFTR

anylin-induced secretion of Cl� via CFTR may activate apical NKCC2 toy be increased by Cl�=HCO3

� exchanger and by secretion through CFTR,ther facilitating water absorption.


HCO3� in the lumen plays an essential role in precipitating

divalent cations, Ca2+ and Mg2+, as carbonate complexes(Fig. 8). These cations in ingested SW are highly concen-trated in the distal intestine of SW fish (Wilson et al.,2002; Tsukada et al., 2005), since their absorption is onlymodest (ca. 10–20%) in the intestine despite high concen-trations in SW (50 mM Mg2+ and 10 mM Ca2+). This willcause an increase in luminal osmolality and inhibit waterabsorption. Therefore, if guanylin stimulates HCO3

� secre-tion and precipitates concentrated Ca2+ and Mg2+ in theposterior intestine, the decreased luminal fluid osmolalitymay increase water absorption, promoting SW adaptation.Guanylin-induced Cl� secretion may also stimulate HCO3

�

secretion through Cl�=HCO3� exchanger, which further

drives CaCO3 and MgCO3 precipitation (Grosell et al.,2001, 2005). In fact, luminal fluids are more alkaline inmarine teleost than in mammals by higher HCO3

� secre-tion, and its secretion is stimulated by luminal Ca2+ con-centration (Wilson et al., 2002). Further, luminal HCO3

�

stimulates NKCC and thus NaCl and water absorptionby the SW eel intestine (Ando, 1990). It is possible thatendogenous guanylin is involved in such local adjustmentsin the intestine of marine teleosts.

Taken together, we suggest a hypothesis that guanylinstimulates CFTR to supply Cl� ions to the luminal fluidand to mobilize NKCC2 located on the apical mem-brane, thereby increasing water absorption by the SWeel intestine. NKCC is a major transporter to energizewater absorption, but double the Cl� ions are trans-ported relative to Na+ ions from the luminal fluid, whichmay result in Cl� shortage. Furthermore, guanylin mayalso stimulate HCO3

� secretion into the lumen throughCFTR. HCO3

� interacts with concentrated Ca2+ andMg2+, resulting in white precipitates in the distal intes-tine of SW eels (Fig. 1). The precipitation decreasesluminal fluid osmolality and thus may promote waterabsorption. If such hypothesized processes are demon-strated in the future, the intestinal guanylin system in tel-eosts will be recognized as an important hormonalsystem for SW adaptation.

Acknowledgments

The authors thank Dr. Christopher A. Loretz for hiscritical reading of this manuscript. This series of workswere supported by Grant-in-Aid for Basic Research (A)(13304063 and 16207004) to Y.T. and by Research Fellow-ship for Young Scientists to S.Y. from the Japan Societyfor the Promotion of Science.

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The intestinal guanylin system and seawater adaptation in eels