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Comparative Biochemistry and Physiology Part B 129 Ž2001. 575᎐586

Cloning and expression of two isoforms of guanylate cyclase C ž GC-C/ from the European eel ž Anguilla anguilla/ Muriel M. Comrie, Christopher P. Cutler, Gordon CrambU School of Biology, Bute Medical Buildings, Uni¨ ersity of St Andrews, St Andrews, Fife, KY16 9TS, UK Received 5 September 2000; received in revised form 10 November 2000; accepted 17 November 2000

Abstract Complementary DNA fragments for two isoforms of particulate guanylate cyclase C ŽGC-C. were cloned from the intestine of the European eel Ž Anguilla anguilla.. Both isoforms exhibited higher nucleotide and amino acid sequence homologies to members of the GC-C family from other species than the related guanylate cyclase A or B ŽGC-A or GC-B. isoforms from the eel. Northern blots indicated that probes for both isoforms, termed GC-C1 and GC-C2, selectively hybridised to 4.8-kb transcripts in the intestine and the kidney. Expression of the GC-C2 transcript in the intestine was increased by 100% following the transfer of yellow FW-acclimated eels to SW. Likewise developmental maturation of yellow eels into pre-migratory silver eels resulted in a significant increase Ž60%. in the intestinal expression of GC-C2. No changes in expression of GC-C2 were seen in the kidney under any condition. RT-PCR indicated that the GC-C2 isoform is only expressed in anterior and mid-gut segments in FW-acclimated yellow eels. However, expression is also extended to the posterior gut segment when yellow eels are acclimated to SW or following developmental transformation into silver eels. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Anguilla; Eel; Intestine; Nucleotide sequence; Guanylate cyclase; GC-C; RT-PCR

1. Introduction Cyclic GMP is an important intracellular second messenger produced by an ever-expanding family of receptor guanylate cyclases. The guanylate cyclases exist either as cytosolic or membrane-associated forms; the cytosolic forms are

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Corresponding author. Tel.: q44-1334-463530; fax: q441334-463600. E-mail address: [email protected] ŽG. Cramb..

heterodimers which are activated by nitric oxide or carbon monoxide and the membrane forms are single pass integral membrane proteins which are activated by various endogenous peptides. Peptide ligands that have been identified so far include the members of natriuretic peptide family which comprise atrial, brain and C-type natriuretic peptides ŽANP, BNP and CNP, respectively. and the guanylin peptide family, comprising guanylin itself, uroguanylin and the most recently discovered lymphoguanylin. These peptides mediate their actions by binding and activating

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M.M. Comrie et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 575᎐586

members of the particulate guanylate cyclase receptor family and are part of a regulatory signalling system operating in a variety of epithelial tissues, which contribute to ion and water homeostasis in mammals Žsee Krause et al., 1997a; Wedel and Garbers, 1997; Forte, 1999; Forte et al., 2000 for reviews.. In the majority of texts, the membrane associated guanylate cyclase receptors are termed guanylate cyclase A ŽGC-A; the physiological receptor of ANP and possibly also BNP., guanylate cyclase B ŽGC-B; the physiological receptor for CNP. and guanylate cyclase C ŽGC-C; the receptor for all members of the guanylin peptide family.. The final natriuretic peptide receptor subtype, known as NPR-C, refers to the ‘clearance receptor’ which is distinct from the GC isoforms having no intracellular guanylate cyclase catalytic domain ŽMaack et al., 1987; Duff and Olson, 1992; Takashima et al., 1995.. Although GC-A, GC-B and NPR-C clearance receptors have been isolated, cloned and characterised in a number of lower vertebrates including teleosts, there have been relatively few reports on GC-C type receptors in fish. The guanylin receptor ŽGC-C., first identified by Field et al. Ž1978. as an intestinal guanylate cyclase which was activated by E. coli heat stable enterotoxin ŽSTa ., was initially cloned and sequenced from rat intestinal mucosa ŽSchulz et al., 1990.. In common with other members of the membrane guanylate cyclase family, it has an N-terminal extracellular ligand-binding domain, a single-pass transmembrane domain, an intracellular protein kinase-like domain and a C-terminal cyclase catalytic domain ŽSchulz et al., 1990; Wedel and Garbers, 1997.. Initial binding studies with STa also revealed the presence of GC-C-like receptors in various epithelial tissues taken from the North American opossum including the kidney, trachea, gall bladder and with the strongest signal occurring in the intestinal tract ŽForte et al., 1988, 1989; Krause et al., 1990.. Since then, studies have demonstrated that GC-C is expressed in the intestine of all mammals examined to date including man ŽKrause et al., 1994. and also in the intestines of birds ŽKrause et al., 1995. and reptiles ŽKrause et al., 1997b.. Preliminary binding studies have indicated the presence of a GC-C homologue in the intestine of fish ŽForte et al., 1997. and a full length cDNA homologue of GC-C has very recently been sequenced from medaka Ž Oryzias latipes. embryos

ŽMantoku et al., 1999.. The possibility that a GC-C receptor could be present in the intestine of teleost fish was of significant interest. If such a guanylate cyclase receptor did exist, this would suggest the presence of endogenous guanylin-like peptides in teleosts which may have important osmoregulatory functions in euryhaline species such as the eel. This paper reports the work carried out attempting to isolate and clone GC-C cDNA from the European eel Ž Anguilla anguilla..

2. Material and methods 2.1. Fish Yellow and migrating silver eels Ž250᎐750 g body wt.. from the River Tay catchment area ŽLunan Burn and Butterstone Loch. were obtained from private suppliers in Blairgowrie, Perthshire. The eels were maintained without feeding in a 12-h lightrdark cycle at ambient temperature, in tanks at the Gatty Marine Laboratory, St Andrews. After the eels had been transported to the Gatty Marine Lab they were placed in freshwater tanks to recover from any stress for at least 4 days before being acclimated to either fresh water ŽFW; 0᎐10 mOsmolrkg. or sea water ŽSW; 960᎐1020 mOsmolrkg. for a further 3 weeks. Fish were decapitated and pithed before the removal of tissues. Epithelial scrapings were taken for all intestinal samples whereas the whole kidney was sampled for RNA extractions. Unless otherwise stated all general chemicals were Analar grade supplied either by BDH Laboratories, Poole, Dorset or from Sigma Aldrich Ltd., Poole, Dorset. Specific reagents and kits were supplied as indicated. 2.2. Isolation of RNA Total RNA was isolated by a modification of the high salt precipitation method of Chomczynski and Mackey Ž1995.. Tissues to be investigated were dissected as rapidly as possible from eels, rinsed in ice cold phosphate buffered saline and homogenised in 5 vol. of ‘Solution D’ w4 M guanidinium isothiocyanate, 25 mM sodium citrate, 0.5% Žvrv. sarkosyl and 50 mM 2-mercaptoethanolx using a Polytron PT 10 homogeniser ŽKinematica Ltd.. set at position 5 for 20᎐30 s.

M.M. Comrie et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 575᎐586

Following homogenisation, total RNA was extracted by the sequential addition of 0.1 vol. 2 M sodium acetate, pH 4.0, 0.5 vol. water-saturated phenol and finally 0.2 vol. of chloroformrisoamyl alcohol Ž24:1 vrv.. Tubes were vortexed briefly between the addition of each solution and then centrifuged at 3954 = g for 30 min. at 4⬚C in a Beckman J6-MC centrifuge ŽBeckman Instruments Inc... The upper aqueous phase was carefully transferred to a fresh tube, and then 2.5 vol. of 2-propanol and 0.2 vol. of 1.2 M NaCl, 0.8 M sodium citrate, pH 7.0 was added sequentially with vortexing. The resulting solution was incubated at room temperature for 10 min before centrifugation at 3954 = g for 30 min. The supernatant was poured off and the pellet washed twice in 80% ethanol before drying under vacuum at room temperature for 5 min. After resuspension of the pellet in diethylpyrocarbonate ŽDEPC.treated water, diluted samples Ž1:100. were prepared and the absorbance was measured at 260 nm and 280 nm ŽPhilips PU 8620 UVrVISrNIR spectrophotometer. to estimate both the concentration and purity of the RNA samples. RNA samples from each extract were also run on denaturing formaldehyde gels and stained with ethidium bromide Žas detailed below. to ensure that no degradation of the RNA had occurred. 2.3. Cloning and sequencing Complementary DNA fragments for eel GC-C were amplified by the use of degenerate, inosinecontaining oligonucleotide primers in RT-PCR. Sequence information collected for all known mammalian guanylate cyclases was assessed before designing primers to regions exhibiting high conservation of amino acid homologies. Sense and anti-sense degenerate primers were used to amplify the initial fragments. When the nucleotide sequences of the initial fragments were determined, regions more 5⬘ or 3⬘ to these were subsequently amplified using combinations of other degenerate and specific primer sets. Finally, 5⬘ and 3⬘ amplifications of cDNA ends ŽRACE. were conducted using cDNA synthesised using Clontech’s ŽBasingstoke, UK. Marathon cDNA amplification kit as described previously ŽCutler et al., 2000.. Synthesis of first strand cDNA was carried out by using Superscript II reverse transcriptase ŽGibcoBRLrLife Technologies Ltd.. using total

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RNA extracted from the intestine of yellow SWacclimated eels according to the manufacturer’s instructions. Single strand cDNA was then used as template in PCR along with the primers designed to amplify different regions of eel GC-C. The following PCR conditions were routinely used. A 20 ␮l reaction would contain 0.025 vol. first strand cDNA reaction mix, 200 ␮M dNTPs, 5 ␮M sense and anti-sense primers and 0.625 units of Taq DNA polymerase ŽAmersham Pharmacia Biotech Ltd. or Biogene Ltd.. in a reaction buffer comprising 20 mM Tris᎐HCl, 50 mM KCl, 1.5 mM MgCl 2 , pH 8.4. Thermal cycle parameters were typically 92⬚C for 2 min followed by 40 cycles of 94⬚C for 4 s, 55⬚C for 30 s, and 72⬚C for 0.5᎐2 min Ždependent on expected fragment size. before a final extension time of 10 min at 72⬚C. Amplified fragments were either purified directly from the PCR mix using a Quick-Step PCR Purification System ŽEdge Biosystems Ltd.. or from an excised gel segment following electrophoresis on a 1᎐2% agarose gel in 1 = TAE w40 mM Tris base, 0.35% Žvrv. glacial acetic acid, 10 mM EDTA, pH 8.0x, using a Geneclean II DNA purification kit ŽAnachem Ltd., Luton, UK. both according to the manufacturer’s instructions. Purified fragments were blunt ended by incubation for 10 min at 72⬚C in the presence of Turbo Pfu polymerase, 200 ␮M dNTPs and Pfu incubation buffer according to the manufacturer’s ŽStratagene. instructions. Blunt-ended fragments were cloned into the plasmid vector pCR4 Blunt-TOPO and transfected into TOP10 competent cells according to the manufacturer’s ŽInvitrogen, Leek, The Netherlands. instructions. Positive colonies were identified by colony PCR and inserts purified using a Quick-Step PCR Purification System before sequencing using an ABI PRISM Big Dye Terminator Cycle Sequencing Reaction Kit ŽPE Applied Biosystems, Perkin Elmer Ltd.. as described previously ŽCutler et al., 2000.. Three clones of each cDNA fragment were sequenced in both directions and the overlapping sequences were determined and then combined using the GeneJockey II software package ŽBiosoft, Cambridge, UK.. 2.4. Northern blotting and analysis Individual mRNAs were separated using denaturing agarose gel electrophoresis. Total RNA Ž5 ␮g; as determined by absorbance at 260 nm. was

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M.M. Comrie et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 575᎐586

resuspended in 1 = MOPS Ž20 mM 3-w N-morpholinox-propanesulfonic acid, 8 mM sodium acetate, 1 mM EDTA, pH 7.8. containing 12.5 M formamide and 2.2 M formaldehyde and then denatured at 65⬚C for 15 min and snap cooled on ice before adding 0.1 vol. of 5% ‘Loading Dyes’ Ž0.025% bromophenol blue, 0.025% xylene cyanol and 50% glycerol; all wrv.. Samples Ž30᎐100 ␮l. were then and loaded onto the agarose gel᎐1.2% agarose wrv ŽBiogene Ltd.., 1 = MOPS containing 6.7% Žvrv. formaldehyde-and electrophoresed at 135 V Ž5 Vrcm. ŽPharmacia EPS 500r400 power pack, Pharmacia Ltd.. for 1.5᎐2 h in 1 = MOPS. After electrophoresis, gels were stained for 30 min in 0.1 M ammonium acetate, 5 ␮grml ethidium bromide before destaining for 1᎐2 h in several changes of 0.1 M ammonium acetate before viewing on the UV transilluminator ŽUVT-20M Trans-luminator, Herolab.. The relative amounts of RNA loaded onto each lane were assessed using the combined intensity levels of ethidium bromide stained 18S and 28S ribosomal RNA bands as quantified using a gel documentation and analysis system ŽSyngene, Cambridge, UK.. Variations in the amounts of RNA loaded onto each lane were determined and used to adjust the final radioactive signals obtained from the hybridisation per normalised microgram total RNA. Methods associated with the electroblotting of separated RNA onto nylon filters and the hybridisation of blots using 32 P-labelled probes were as detailed previously ŽCutler et al., 1995.. Blots were hybridised under highly stringent conditions at 48⬚C in a solution containing 20 mM NaH 2 PO4rNa 2 HPO4 , 1 M NaCl, 1 mM EDTA, 3% SDS, 50% formamide, 10 = Denhardts Ž0.2% polyvinylpyrrolidone, 0.2% Ficoll, 0.2% BSA., pH 7.4 supplemented with 0.5 mgrml sonicated, denatured calf thymus DNA and 0.5 mgrml baker’s yeast RNA. Blots were washed with increasing stringency with the final two washes each of 20 min in a buffer comprising 0.1% SDS and 0.1= SSC Ž0.015 M NaCl, 1.5 mM sodium citrate, pH 7.0. at 48⬚C. Quantitative analysis of 32 P-labelled GC-C DNA probe hybridising to filters was determined by electronic autoradiography using the radioactive counts collected over a set period of time as detected by an Instant Imager ŽCanberra Packard, Meriden, CA.. Statistical analysis was performed using StatView 4.01 software package ŽAbacus Concepts, Berke-

ley, CA. using ANOVA followed by Fisher’s PLSD post analyses of significance.

3. Results Primer pairs used in PCR for amplification of eel GC-C fragments are shown in Table 1. Initial PCR experiments using degenerate Primer Pair 1 resulted in the amplification of a 529 nucleotide fragment of an eel homologue of GC-C ŽFig. 1a.. This cDNA was termed GC-C1 as subsequent attempts to amplify a 5⬘-extended fragment using degenerate Primer Pair 2 resulted in the isolation and cloning of another cDNA fragment of 1.4 kb, which encoded a second GC-C isoform, now termed GC-C2. As the original GC-C1 fragment failed to amplify in this second primer set, it is likely that the sense primer of Primer Pair 2 failed to hybridise to the GC-C1 cDNA under the conditions used. Likewise, Primer Pair 1 did not amplify GC-C2, again probably as a result of the low homology of the sense primer to the GC-C2 sequence. Despite using a number of other sense primers andror altering annealing temperatures andror incorporating different concentrations of formamide and DMSO to overcome potential GrC rich regions specific to this gene, no extended GC-C1 fragment was amplified. The reasons for this are presently unknown. The GC-C2 isoform was extended towards the 5⬘ end by the use of Primer Pair 3, where a third degenerate sense primer raised to a region within the extracellular domain was used in conjunction with a specific anti-sense primer. Finally a truncated 3⬘ end to GC-C2 was isolated using a specific sense primer along with the Marathon AP1 primer and the Marathon cDNA ŽPrimer Pair 4.. The 3⬘ end of the gene could not be extended due to the constant appearance of a palindromic sequence at the truncated 3⬘ end of the fragment. Possible reasons for this are currently being investigated. No 5⬘ RACE product was amplified under the conditions used. The nucleotide and putative amino acid sequences of both isoforms are shown in Fig. 1a,b. Both isoforms are members of the GC-C family of particulate guanylate cyclases with the GC-C2 cDNA fragment extending from amino acid 155 to amino acid 1067 of the human GC-C sequence Žde Sauvage et al., 1991.. Amino acid homologies within the intracellular region of the protein, where we have sequence information for

Primer pair 1 Sense:

5⬘-

GTT

TGA

CCA T

AAA G

AAA G

CGA T

AAC GTG

ITA C

CAT T

GGA

CAC T

Anti-sense:

5⬘-

GTA G

TTI C

ACI C

GTA G

TCI C

CCA G

AAI C

AAA GG

CAA G

TA

-3⬘

Primer pair 2 Sense:

5⬘-

CTI T C

AGI C C

CAA G

AAA G

AAA G

TGG

TCI C

CAC T

ATA C

CC

-3⬘

Anti-sense:

5⬘-

GTA G

TTI C

ACI C

GTA G

TCI C

CCA G

AAI C

AAA GG

CAA G

TA

-3⬘

Primer pair 3 Sense:

5⬘-

GCI C

GGI C

AGC T

TTC T

GGI C

CCI TTC

TCI C

TGC T

GAC T

CA T

-3⬘

Anti-sense:

5⬘-

GTC

CTT

CCT

CTG

GTC

CTC

A

-3⬘

Primer pair 4 (3⬘ RACE) Sense:

5⬘-

GGA

GAC

GAT

CGG

TGA

TGC

G

-3⬘

Anti-sense ŽAP1.:

5⬘-

CCA

TCC

TAA

TAC

GAC

TCA

CTA

TAG

GGC

-3⬘

a

-3⬘

Primer pairs were used in PCR amplifications as outlined in Section 2 using single-stranded cDNA prepared from RNA extracted from the intestine of SW-acclimated yellow eels. Deoxyribonucleotides are A, adenosine; C, cytidine; G, guanosine; T, thymidine; I, inosine. Wobbles in primer sequence are indicated with ᎐.

M.M. Comrie et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 575᎐586

Table 1 Primer sets used for the amplification of GC-C1r2 fragments by RT-PCRa

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M.M. Comrie et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 575᎐586

Table 2 Amino acid homologies between GC-C1, GC-C2 and other particulate guanylate cyclases a Speciesrisoform Eel GC-C1 Eel GC-C2 Medaka GC-C Frog GC-C Rat GC-C Guinea pig GC-C Pig GC-C Human GC-C Eel GC-A Eel GC-B

GC-C1 Ž%.

GC-C2 Ž%.

100 88 85 85 85 82 84 84

88 100 87 85 85 84 84 84

54 54

53 51

a Amino acid sequences were compared only for the intracellular region of the proteins for which sequence information is known for GC-C1 Žsee Fig. 1a,b..

both isoforms, indicates over 80% homology with all known GC-C proteins from other species, including the teleost fish medaka ŽMantoku et al., 1999.. Within this same region there is less than 55% amino acid homology to the related GC-A ŽKashiwagi et al., 1999. and GC-B ŽKatafuchi et al., 1994. receptors which have previously been cloned from the Japanese eel Ž Anguilla japonica. ŽTable 2.. Both GC-C1 and GC-C2 cDNAs when probed at high stringency in Northern blots hybridise to a 4.8-kb transcript in both intestinal and kidney RNA samples ŽFigs. 2 and 3.. The GC-C1 probe

was the 529 nucleotide fragment amplified by Primer Pair 1 and the GC-C2 probe was the 1.02-kb nucleotide fragment amplified by Primer Pair 3. Although the nucleotide homology between the GC-C1 probe and the GC-C2 mRNA sequence was very high, with 88% of the nucleotides being identical, the highly stringent conditions which were used for the hybridisation and washing Žsee Section 2. would severely restrict any cross-hybridisation between the probe and mRNA for the non-homologous isoform. Although not accurately quantified, expression of both isoforms in the intestine was estimated to be approximately one order of magnitude greater than that found in the kidney. This is similar to that reported in mammals where the intestine is also the tissue which exhibits highest levels of expression ŽKrause et al., 1997a.. The relative expression of the GC-C2 isoform was investigated further in both yellow and silver eels which had always been maintained in FW or which had been acclimated for 3 weeks to SW. Once the radioactive intensities of signals on the Northern blots had been corrected for total RNA loading Žas determined by the intensity of ethidium bromide staining of both the 18S and 28S ribosomal RNAs, as indicated in Section 2. the relative levels of mRNA abundance were determined ŽFig. 4.. With the intestinal samples, yellow eels acclimated to FW exhibited the lowest

Fig. 1. Ža. Interleaved nucleotide and amino acid sequences of GC-C1. Numbers on the left refer to the nucleotide sequence and those on the right, the amino acid sequence. This sequence has been submitted to the EMBL GenBank database ŽEuropean Bioinformatics Institute, Hinxton, UK. under the accession number AJ291611. Žb. Interleaved nucleotide and amino acid sequences of GC-C2. Numbers on the left refer to the nucleotide sequence and those on the right, the amino acid sequence. This cDNA fragment is equivalent to the region in human GC-C extending from amino acid 155 to 1067 Žde Sauvage et al., 1991.. The homologous region to GC-C1 extends from amino acid 611 to amino acid 785. This sequence has been submitted to the EMBL GenBank database ŽEuropean Bioinformatics Institute, Hinxton, UK. under the accession number AJ291612.

M.M. Comrie et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 575᎐586

levels of GC-C2 mRNA, however, after 3 weeks of SW acclimation expression levels were significantly higher, around twice that seen in the FW fish. SW-acclimated silver eels Žthe more sexually mature adult stage of the life cycle that is induced prior to the migration of fish back into the marine environment to breed. exhibited similar levels of

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expression to the yellow SW eels. Interestingly, FW silver eels also exhibited a significant, 1.6-fold higher level of expression of GC-C2 mRNA than their yellow eel counterparts. The extent of expression of the GC-C2 transcript along the length of the gut was examined using RT-PCR. Using Primer Pair 3, which only

Fig. 1. Ž Continued..

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M.M. Comrie et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 575᎐586

Fig. 1. Ž Continued..

Fig. 2. Northern blot showing the specific expression of GC-C1 in tissues isolated from FW- and SW-acclimated yellow eels. In all cases 5 ␮g of total RNA was loaded on each lane. Following hybridisation the blot was exposed to autoradiographic film for 6 h at y80⬚C. The estimated size of the GC-C1 transcript was 4.8 kb.

M.M. Comrie et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 575᎐586

recognises the GC-C2 transcript, a 1.0-kb fragment was routinely amplified when using RNA samples extracted from anterior, mid and posterior intestinal segments from all eel groups with the exception of the posterior gut of FWacclimated yellow eels ŽFig. 5.. Despite several attempts using different fish andror Primer Pair 2, no GC-C2 signals were ever obtained from this gut segment in this group of fish. Similar studies indicated that there was no significant change in the low levels of expression of GC-C2 in the kidney samples isolated from any of the experimental groups of fish ŽFig. 4..

4. Discussion The European eel, Anguilla anguilla, has been shown to express two isoforms of a gene homologue of the mammalian guanylin receptor, GC-C. Comparison of amino acid sequences throughout a region between the kinase homology domain and the catalytic domain, where sequence information is available for both isoforms, indicates a greater than 80% homology to the GC-Cs reported for other species including amphibians and mammals and the teleost fish, medaka. In contrast, comparison of the same region with sequences reported for the GC-A and GC-B receptors isolated from the Japanese eel results in under 55% amino acid homology. The results of Northern blotting indicate that both eel isoforms

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Žtermed GC-C1 and GC-C2. are approximately 4.8 kb in size and predominantly expressed in the intestine and to a lesser extent in the kidney of adult yellow eels. Using primers that were specific for the GC-C2 isoform, RT-PCR experiments concluded that expression of this isoform is limited to the anterior and mid regions of the intestine in FW-acclimated yellow eels. However, following transfer of fish to SW or following developmental maturation into silver eels, expression of GC-C2 is also detected in the posterior gut segments. The expression of a GC-C homologue in both the intestine and kidney of the eel indicates the presence of a guanylinrGC-C signal transduction system and a possible role in ion and fluid homeostasis in this species. Both human guanylin and STa have recently been shown to increase cyclic GMP production in the intestinal mucosa of several fish species ŽForte et al., 1997.. The possibility of an osmoregulatory role is also supported by the finding that expression of GC-C in the intestine is dependent on both the developmental stage of the fish and the salinity of the external environment. Studies using a cDNA probe to the extracellular domain of GC-C2 Žlikely to be highly isoform specific. clearly shows that mRNA for this isoform is increased by 60᎐100% in the intestine of SW-acclimated yellow and both FW- and SW-acclimated silver eels compared to FW-acclimated yellow eels. This may reflect the changes in expression observed in the posterior

Fig. 3. Northern blot showing the specific expression of GC-C2 in tissues isolated from FW- and SW-acclimated yellow eels. In all cases 5 ␮g of total RNA was loaded on each lane. Following hybridisation the blot was exposed to autoradiographic film for 3 h at y80⬚C. The estimated size of the GC-C2 transcript was 4.8 kb.

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Fig. 4. Quantification of radioactive signals from Northern blots. Radioactive counts were collected for intestinal and kidney blots and corrected for RNA loading as described in Section 2. Relative signal intensities are expressed independently for intestine Žgrey bars. and kidney Žblack bars. RNA samples. As specific activities of the GC-C2 probe varied and the radioactive counts were collected for different times for each blot, intestinal and kidney signals are not related. Statistical analysis was by one-way ANOVA and Fisher’s PLSD post hoc test. U Significant at the 5% level Ž P- 0.05., UU significant at the 1% level Ž P- 0.01. compared to YFW fish. Error bars indicate S.E. Ž n s 6..

gut segments by the PCR experiments, or may also be due to a general increase in expression throughout the entire length of the intestine.

Fig. 5. Results of RT-PCR using template cDNA synthesised from separate intestinal regions from eels of all four experimental groups. Lanes 1᎐12 are the PCR amplification products using Primer Pair 3 in anterior Žlanes 1, 4, 7 and 10., mid Žlanes 2, 5, 8 and 11. and posterior Žlanes 3, 6, 9 and 12. gut segments from yellow FW Žlanes 1᎐3., yellow SW Žlanes 4᎐6., silver FW Žlanes 7᎐9. and silver SW Žlanes 10᎐12. eels. The lane marked M refers to the DNA size standards. Expected GC-C2 fragment size is 1.02 kb.

Whichever may be true, these results indicate a degree of pre-adaptation in FW-acclimated silver eels prior to their migration to the SW environment. In mammals activation of this peptiderreceptor signalling system is known to result in the secretion of both Cly and HCOy 3 into the intestinal lumen via the cystic fibrosis transmembrane conductance regulator ŽCFTR. or other functional chloride channels ŽCurrie et al., 1992; Forte et al., 1993; Hamra et al., 1993; Kita et al., 1994; Guba et al., 1996; Joo et al., 1998.. The presence of GC-C in both the eel and medaka, suggests that similar iono-regulatory systems are in operation within the teleost gut. However, questions may be asked as to why GC-C expression is up regulated in the intestine of the SW-acclimated eel when the major physiological role of this tissue, in terms of osmoregulation, is ion and water absorption? The possibility exists that this signalling system operates to antagonise salt and therefore water uptake across the intestine thus preventing system overload following drinking in SW thus modulating any sudden rises in plasma osmolality and blood volume. ANP has also been proposed to have similar actions, suppressing Naq, Cly and water uptake across the teleost intestine immediately after the initiation of the drinking response ŽAndo et al., 1992; Loretz and Takei, 1997.. In addition, the membrane permeant analogue of cyclic GMP, 8-Br cGMP, has also been shown to have potent inhibitory actions on short circuit current and Cly uptake across the isolated perfused eel intestine ŽTrischitta et al., 1996.. The possibility also exists that the eel GC-C signalling system is also involved to some extent in acid᎐base balance. Wilson et al. Ž1996. reported a substantial secretion of endogenous base ŽHCOy 3 and CO 32y . into the intestinal and rectal fluids of SW-acclimated O. mykiss resulting in an intraluminal pH of 8.9. Shedadeh and Gordon Ž1969. have also reported the high pH of rectal fluids in SW-acclimated O. mykiss and in addition had observed carbonate deposits within the intestinal mucosa. The secretion of carbonates and bicarbonates may mainly be associated with osmoregulation, mediating intestinal Cly uptake and the precipitation of divalent cations in the intestinal lumen, rather than having any direct actions on acid᎐base balance ŽWison and Tierney, 1999..

M.M. Comrie et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 575᎐586

Although low levels of GC-C2 mRNA expression were detected in the kidney using this same probe, no significant changes in expression were found between any of the experimental groups. These results suggest that the signalling system present in the kidney may not be directly involved with the other osmoregulatory changes associated with FWrSW transfer. However, the existence of GC-C receptors in this tissue suggests that an intestinalrkidney endocrine axis does exist, as has been recently suggested for mammals ŽForte et al., 2000 The presence of two isoforms of the eel homologue of GC-C is unique. No other isoforms for GC-C have been reported in any other species. The presence of two or more copies of various genes is not unusual with many groups reporting the presence of additional isoforms in various teleost species including the European eel ŽCutler et al., 1996, 2000.. This is probably the result of a gene duplication event that occurred early in telostean evolution. Whether these two isoforms of GC-C exhibit functional or regulatory differences will require further studies at both molecular and physiological levels. These results therefore suggest the presence of a guanylinruroguanylin-like peptide signalling system in the intestine and kidney of the European eel. The increase in GC-C2 expression in SW-acclimated fish suggests that this putative peptide signalling system is likely to be involved in the osmoregulatory adaptive response of the intestine when fish enter the SW environment. The finding that FW-acclimated silver eels also exhibit an increase in GC-C2 expression suggests that the physiological adaptations which accompany the developmental change from yellow to silver stages of the life cycle also include changes in the sensitivity of this peptide signalling system prior to fish moving into the SW environment. The true role of this receptor system in osmoregulation awaits further physiological investigation.

Acknowledgements These studies were supported by the Natural Environment Research Council ŽN.E.R.C... MMC is in receipt of an N.E.R.C. Studentship. G.C. would like to thank the Biochemical Society and The Royal Society for travel grants for the 4th. I.C.F.E. in Seattle.

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