From the Ocean Research Institute, The University of Tokyo, Nakano, Tokyo 164-8639, Japan
Received for publication, March 26, 2003
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ABSTRACT |
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INTRODUCTION |
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Because of an aquatic life, fish provide unique opportunities for studies of body fluid regulation. When fish are in fresh water (FW), they suffer from an excess water load and salt loss that occurs across the gills. In seawater (SW), however, they face hypernatremia and hypovolemia (12). Accordingly, euryhaline fish that readily adapt to both media are able to reverse salt and water regulation when they move from FW to SW or vice versa. The intestine plays a pivotal role in this reversal. The intestine serves simply as a route of nutritional absorption when fish are in FW; however, it is subjected to dramatic morphological and biochemical (transporter molecules, etc.) changes during the course of SW adaptation and becomes a sole site of water uptake from the environment when fish are in SW (13, 14). Therefore, if the orally ingested water is not allowed to enter the intestine by esophageal ligation, fish in SW die in a few days from hypovolemia and hypernatremia (15). Because relative importance of the intestine changes so dramatically according to the environmental salinity, the intestine of euryhaline fish serves as a good model to analyze the role of osmoregulatory hormone such as guanylins.
The eel is a highly euryhaline species from which extensive data on its osmoregulation have been accumulated (16). It has been suggested that the buccal and intestinal epithelia of eels can sense Cl, which results in the regulation of drinking and intestinal absorption of water and NaCl (17, 18). It is thus likely that some humoral factors are released in response to changes in luminal Cl concentration to regulate these osmoregulatory processes. Atrial natriuretic peptide, a member of the natriuretic peptide family that utilizes cGMP as an intracellular messenger, like guanylin, is a possible candidate for the mediator because it potently inhibits drinking and intestinal NaCl absorption in eels (1921). However, the amount of atrial natriuretic peptide stored in the eel intestine appears to be too small to be the major humoral mediator released from the intestine (22). Thus, guanylins are more likely candidates as, in mammals, uroguanylin is shown to be secreted into the circulation and serves as an "intestinal natriuretic factor" mediating information transfer from the intestine to the kidney (13, 23).
In the present study, we attempted to identify guanylins in the intestine of the Japanese eel, Anguilla japonica. From a single eel, we isolated three distinct clones of guanylin-like peptides, which were named eel guanylin, uroguanylin, and renoguanylin. All three clones share the characteristics of guanylin peptides in terms of structure (two disulfide bonds) and tissue distribution (intestine and/or kidney). Furthermore, the mRNA expression of eel guanylin and uroguanylin was upregulated in the intestine and/or kidney during the course of adaptation to SW. Thus, we report here the presence of a novel guanylin family in the eel and further suggest that the eel guanylin family is likely to be intestinal paracrine/autocrine factors and/or endocrine hormones that are involved in adaptation of eels to hyperosmotic SW environment.
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EXPERIMENTAL PROCEDURES |
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RNA ExtractionAfter anesthesia in 0.1% (w/v) tricaine methanesulfonate (Sigma), the brain, gill, heart, liver, esophagus, stomach, anterior intestine, posterior intestine, kidney, head kidney, and spleen were isolated from eels and immediately frozen in liquid nitrogen. These organs include major tissues of guanylin/uroguanylin/lymphoguanylin expression in mammals (15, 7, 2426) and/or major osmoregulatory tissues in fish. The anterior and posterior intestines were separated at the clearly recognizable sphincter located distally. Total RNA was extracted using ISOGEN (Nippon Gene, Toyama, Japan).
cDNA CloningPoly(A)+ RNA was separated from total RNA of the posterior intestine of one SW eel using Oligotex-dT30 (Japan Synthetic Rubber, Tokyo, Japan). The cDNA pool was prepared from 0.5 µg Poly(A)+ RNA using SMART cDNA Library Construction kit (Clontech Laboratories, Palo Alto, CA). The partial cDNAs of three guanylin-like peptides (GLP-1, -2, and -3) were cloned by 3'-rapid amplification of cDNA ends (RACE) method using CDS III/3' primer and a designed sense primer: GUA-S1 or GUA-S7 (Table I). Amplification by PCR was performed using Ex Taq DNA polymerase (TaKaRa, Tokyo, Japan) at the following schedule: 94 °C for 30 s, then 35 cycles of 94 °C for 1 min, 68 °C for 30 s, and 72 °C for 1.5 min, and finally 72 °C for 7 min. After 3'-RACE, 5'-RACE was performed using 5' PCR primer and gene specific primers (GUA-A1 for GLP-1, GUA-A8 for GLP-2, and GUA-A3 for GLP-3) (Table I) on the same schedule except for annealing temperature (55 °C for GLP-1 and 3, and 65 °C for GLP-2). Finally, the full coding region was amplified using CDS III/3' primer and specific primers designed from the 5'-untranslated sequences: GUA-S2 for GLP-1, GUA-S8 for GLP-2, and GUA-S4 for GLP-3 (Table I). To increase the fidelity, the final amplification was performed with Advantage 2 DNA Polymerase Mix (Clontech) from non-amplified first strand cDNA. All amplified products were subcloned into pT7blue vector (Novagen, Madison, WI) and sequenced in a 310 DNA sequencer (PerkinElmer Life Sciences).
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Tissue DistributionTissue distribution of the three GLP mRNAs was determined by RT-PCR according to the previously described method (27). Total RNAs isolated from the tissues (5 µg each) were reverse-transcribed with Superscript First Strand Synthesis System (Invitrogen). PCR was performed using specific primers: GUA-S2 and GUA-A1 for GLP-1, GUA-S8 and GUA-A10 for GLP-2, and GUA-S4 and GUA-A3 for GLP-3 (Table I). To minimize the variation in amplification efficiency among the sequences, these specific primers were designed to adjust Tm values and target length (475, 438, and 419 bp for GLP-1, -2, and -3, respectively, which were visually distinguishable by length after electrophoresis). As an internal control, eel glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (GenBankTM accession number AB049458 [GenBank] ) was used. The PCR condition was as follows: 94 °C for 1 min, then 22 cycles of 94 °C for 1 min, 63 °C for 30 s and 72 °C for 1.5 min, and finally 72 °C for 7 min. The number of cycles for GLP-2 was increased to 26 because of its low level of expression. Amplified products were electrophoresed and detected after staining with ethidium bromide (EtBr). No signal was detected from the negative control without reverse-transcriptase.
Effects of Environmental Salinity on mRNA ExpressionGLP mRNA levels were compared between FW and SW eels in the osmoregulatory organs (intestine, kidney, and esophagus) and the liver. FW and SW eels (n = 6 in each case) were used to compare the expression after long-term adaptation (2 weeks) in each osmotic media. To examine the role of GLPs in short-term adaptation to SW, changes in the mRNA levels were examined in the intestine 0, 3, and 24 h after transfer of FW eels to SW (SW, 3 and 24 h) or to FW (time controls: FW, 0, 3, and 24 h) (n = 57 in each case).
RT-PCR was performed to examine the GLP expression level as the same conditions of tissue distribution except for PCR cycles (30 cycles) in uroguanylin of the anterior intestine. To test the reliability of this method, serially diluted template cDNAs (0.0080.512 µg) from the anterior intestine of a SW eel were amplified, and their signals were measured. It was demonstrated that the signal intensity was linear in this range over the dilution factor of 60 (data not shown). The current PCR conditions were designed carefully to make signals within the linear-range. The abundance of GLP mRNAs was shown as percentage to that of GAPDH mRNA in the experiment to examine long-term adaptation. In the short-term experiment, however, the expression of both eel GAPDH and -actin (GenBankTM accession number AB074846
[GenBank]
) increased in the intestine after SW transfer (see "Results"). Therefore, we did not normalize the values with those of GAPDH or
-actin as discussed under "Discussion." After electrophoresis and staining with EtBr, signal intensity was measured with an automatic image analyzer (FLA-2000, Fuji Film, Tokyo, Japan). For statistical analyses, Student's t test was applied except for highly variable cases where Mann-Whitney's U test was applied. Significance was determined at p < 0.05. All values are expressed as means ± S.E.
ImmunohistochemistryPredicted mature peptides in the C terminus of GLP-1 (15 amino acid residues) and GLP-2 and 3 (16 amino acid residues) were synthesized by Peptide Institute Inc. (Minoh, Osaka, Japan). The two disulfide bonds were correctly formed, and two topological stereoisomers produced were separated by reverse-phase high performance liquid chromatography (28).
Antisera were raised in two rabbits against synthetic GLP-1 conjugated to keyhole lympet hemocyanin. An antiserum for serotonin used to identify enterochromaffin cells was kindly provided by Dr. Ryogo Yui of Zenyaku Kogyo Co. Ltd., Tokyo, Japan (29). The specificity of the antisera for GLP-1 was tested by dot blot analysis using 1100 pmol of synthetic GLP-1, -2, and -3. The blotted membranes were preincubated in 0.05% Tween 20 in 10 mM PBS (pH 7.2) and incubated with the antiserum diluted to 500-, 1000-, and 3000-folds with 10 mM PBS for 2 h at room temperature. After rinsing in 10 mM PBS (pH 7.2), the membranes were stained by the avidin-biotin-peroxidase complex method (Vector Lab., Burlingame, CA). The specificity of the antiserum for serotonin was described in previous studies (29, 30).
The anterior intestine of SW eels was fixed by immersion in 4% paraformaldehyde in 10 mM PBS (pH 7.4) for 24 h at 4 °C, soaked in 30% sucrose in 10 mM PBS overnight, and embedded in Tissue-Tek O.C.T. compound (Sakura Finetechnical Co. Ltd., Tokyo, Japan) at 20 °C. Cryosections were cut at 20 µm and collected onto gelatincoated slides. The sections were incubated sequentially with: 1) 2% normal goat serum (NGS) for2hat room temperature, 2) the antiserum for GLP-1 diluted at 1:1000 with 10 mM PBS (pH 7.4) containing 0.02% keyhole lympet hemocyanin, 0.2% bovine serum albumin, 2% NGS, and 0.02% sodium azide, or the antiserum for serotonin diluted at 1:15000 with the same buffer for 40 h at 4 °C, 3) goat anti-rabbit IgG labeled with fluorescein isothiocyanate (Zymed Laboratories Inc., San Francisco, CA) diluted at 1:200 for GLP-1 and 1:300 for serotonin with 10 mM PBS (pH 7.4) containing 2% NGS for 2 h at room temperature. The sections were observed under a fluorescence microscope (OLYMPUS, Tokyo, Japan).
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RESULTS |
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A potential cleavage site for the signal peptide may be located between amino acid positions 20 and 21 (31). Comparing the potential mature sequences at the C terminus, GLP-1 and -3 had an aromatic amino acid (phenylalanine) at position 9 as in mammalian guanylins (tyrosine or phenylalanine), whereas GLP-2 had asparagine as in mammalian uroguanylin (Fig. 3). GLP-1 sequence ended with a cysteine residue as in all mammalian guanylins thus far identified, whereas GLP-2 and -3 had an additional residue (leucine) at the C terminus as did some mammalian uroguanylins (leucine or serine) (Fig. 3). Based on the sequence characteristics and sites of mRNA expression (see below), we named GLP-1 eel guanylin and GLP-2 eel uroguanylin (Fig. 1). GLP-3 was a novel peptide that shared the structural characteristics of both guanylin and uroguanylin. Because the expression of GLP-3 in the kidney relative to the intestine was more abundant than uroguanylin (see below), we named the new peptide "renoguanylin" (Fig. 1).
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Tissue DistributionThe RT-PCR applied to various eel tissues showed that eel guanylin was expressed only in the intestine, and no expression was detectable in other tissues including the alimentary tract (stomach and esophagus) (Fig. 4). Eel uroguanylin was expressed in the kidney, liver, and esophagus as well as the intestine (Fig. 4). However, because the overall expression of uroguanylin was low, four more PCR cycles were required to intensify the signal to the level of guanylin and renoguanylin. Eel renoguanylin was abundantly expressed in the kidney in addition to the intestine (Fig. 4). Its expression in the kidney relative to the intestine was much greater than that of uroguanylin. Judging from the result of dilution experiment, the negative tissues produced less than 1/100 of mRNA compared with the intestine (data not shown).
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Cellular Localization of Guanylin in Eel IntestineThe dot blot analysis revealed that the antiserum for eel guanylin has only a weak cross-reactivity with eel renoguanylin (less than 1/20) and has no cross-reactivity with eel uroguanylin (data not shown). In the intestine of SW eels, guanylin immunoreactivity was localized exclusively in some goblet cells (Fig. 5A). The signal was distributed ubiquitously in the cytoplasm but was absent in the mucus of the goblet cells. In some mammalian species, positive guanylin signals were reported in the enterochromaffin cells (33). However, no immunoreactive guanylin was identified in the enterochromaffin cells that were identified by the presence of serotonin immunoreactivity (Fig. 5B) (29).
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Comparison of Expression of Guanylins between FW and SW EelsThe mRNA expression of guanylin and uroguanylin was profoundly up-regulated in both anterior and posterior intestine of SW eels, but that of renoguanylin was enhanced only in the anterior intestine (Figs. 6, 7, 8). The up-regulation was more profound in the anterior intestine than in the posterior intestine of SW eels. In the kidney, the mRNA expression of uroguanylin, but not of renoguanylin, was up-regulated in SW eels (Figs. 7 and 8). The expression level of uroguanylin in the liver and esophagus was not different between FW and SW eels (Fig. 7, D and E).
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Changes in Expression of Guanylins after Transfer of Eels from FW to SWThe mRNA expression of guanylin and uroguanylin did not change for3hinthe anterior intestine, but it increased at 24 h after transfer of eels from FW to SW (Fig. 9, IA and IIA). However, the expression of renoguanylin did not change for 24 h (Fig. 9, IIIA). Similar results were obtained in the posterior intestine (Fig. 9B) except for the increase of uroguanylin after 3 h (Fig. 9, IIB).
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Although there was no difference in GAPDH expression in the intestine between FW- and SW-adapted eels (Figs. 6, 7, 8), the expression increased for 24 h after transfer of FW eels to SW (Fig. 10, I). We examined another housekeeping gene, -actin, but its expression was also enhanced after SW transfer (Fig. 10, II). This may be due to the dramatic morphological changes that occur during the early phase of SW adaptation (34). Because specific changes in the expression of guanylins should be calculated independently of such hypertrophic and hyperplastic changes that accompany the expression of housekeeping genes, we showed the expression level of guanylins with absolute values without normalization with the housekeeping genes.
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DISCUSSION |
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In mammals, the kidney-specific activity of uroguanylin depends on the ninth amino acid residue. Because mammalian guanylin with an aromatic residue at this position is sensitive to a chymotrypsin-like endopeptidase located on the brush border of proximal tubules (1, 2, 35), filtered guanylin in the urine is quickly metabolized. In contrast, mammalian uroguanylin has asparagine at this position, which results in resistance to this enzyme. Thus, mature uroguanylin is abundantly excreted into the urine and can act on the renal tubules from the luminal side (36). In some uroguanylins, an additional leucine or serine residue is attached to the C-terminal cysteine. This addition is thought to inhibit the conversion of active peptide to a non-bioactive stereoisomer and thus stabilize the peptide in plasma (37). These structural characteristics reported in mammals are also conserved in eel guanylin and uroguanylin.
The tissue distribution of guanylin and uroguanylin also supports the current classification in eels. In mammals, guanylin is synthesized mainly in the intestine, whereas uroguanylin is produced more widely in the intestine, kidney, stomach, pancreas, and heart (13, 2426). Accordingly, the eel peptide that is expressed only in the intestine is guanylin, and those expressed more widely in various tissues is uroguanylin (Fig. 4). The third peptide, renoguanylin, was expressed almost exclusively in the intestine and kidney. In particular, its message in the kidney is more abundant than uroguanylin. Thus, the third peptide was named renoguanylin. It is possible that this novel peptide exists also in other vertebrate species. The uroguanylin of A. japonica is almost identical to the GLP of A. anguilla, which was identified for the first time in nonmammalian vertebrates (11).
In addition to the mature sequences located at the C terminus, the guanylin peptides have a conserved sequence in the prosegment across different species (see Fig. 2). It has been shown that synthetic mature guanylin of 15 amino acid residues is often mixed with a topological stereoisomer that is biologically inactive (38), but only biologically active peptide with accurate cysteine connectivity is produced in the presence of the N-terminal prosegment (28). The present results obtained in eels provide supportive evidence from an evolutionary viewpoint that the conserved prosegment is important for the accurate connectivity of disulfide bonds.
Guanylin-producing Cells in Eel IntestineImmunohistochemical data showed that guanylin is produced by the goblet cell. In mammals, guanylin immunoreactivity has been localized in the typical exocrine goblet cells and/or endocrine enterochromaffin cells of the intestine (33, 39). Perkins et al. (40) showed that in the rat intestine, guanylin is produced in the goblet cells and uroguanylin in the enterochromaffin cells using antisera raised against the prosegment of each peptide where similarity is very low. Because our guanylin antisera do not cross-react with uroguanylin, the current results support their data from the comparative viewpoint.
Goblet cells are mucus-secreting cells in the intestine (41). There are biochemically distinct subpopulations of goblet cells in which different types of mucins and other secretory products are synthesized. This coincides with the present study that only a small population of goblet cells are guanylin-immunoreactive. It is possible that guanylin is secreted into the lumen with mucus and regulate epithelial transport of water and ions. It is also possible that guanylin regulates mucus secretion in an autocrine fashion as suggested in the rat (42). The major function of mucus is to protect epithelial cells from luminal agents such as enterotoxins and various digestive enzymes (41). It is expected that in fish, mucus also serves to protect the cells from sudden changes in luminal osmotic pressure as expected after drinking of SW.
Possible Osmoregulatory Role of Eel GuanylinsThe present study showed that the expression of eel guanylins was upregulated in the intestine after adaptation of eels to SW (Figs. 6, 7, 8). The up-regulation is particularly prominent in the anterior part where water and NaCl absorption most actively occur in SW eels.2 Thus, these peptides may play regulatory roles in this intestinal segment after adaptation to SW.
The expression of guanylin and uroguanylin did not increase for 3 h except for uroguanylin in the posterior intestine (Fig. 9, I and II). Renoguanylin expression did not change for 24 h (Fig. 9, III). These characteristics are markedly different from those of immediate-acting, osmoregulatory hormones that respond to salinity changes within a few hours (43). These results support the notion that guanylins may be involved primarily in the chronic phase of SW adaptation. Compared with guanylin and uroguanylin, renoguanylin appears to have functions different from the osmoregulation, although a small increase in renoguanylin gene expression occurred in SW-adapted eels.
In the kidney, the expression of uroguanylin mRNA increased in SW eels (Fig. 7C), suggesting that locally synthesized uroguanylin acts on the kidney to regulate urinary excretion of water and electrolytes as suggested in mammals (1, 2, 35, 36). However, renoguanylin expression did not change with environmental salinity (Fig. 8C), which also suggests its functions in the kidney other than osmoregulation. Intestinal uroguanylin may also be secreted into the circulation, filtered by the kidney glomerulus, and act on the tubule cells from the lumen in the eel, as demonstrated in mammals as an intestinal natriuretic factor (13, 23).
There are two reports showing that altered oral salt intake changes the expression of guanylin and uroguanylin in mammals, which may have occurred in the eel after drinking SW. In the rat distal colon, high sodium diet did not change the expression of guanylin, but low sodium diet down-regulates its expression (44). In the mouse kidney, however, high oral salt intake increases the expression of uroguanylin (45). Interestingly, uroguanylin expression was enhanced by high-salt loading via drinking water and not by salt content of the diet, probably because increased water drinking diluted high salt diets. In the current study, the expression of three guanylins was up-regulated in SW eels. Because eels drink copious SW to compensate water loss osmotically across the body surfaces (15, 17), they serve as a good model for oral salt loading. Furthermore, the salt loading occurs as a part of physiological processes for SW adaptation in fish. Therefore, eel guanylins may be involved in the physiological process that governs adaptation to high salinity environment. Recently, Kita et al., showed that plasma guanylin and uroguanylin are increased by saltloading to the rat intestine within 30 min using a reliable radioimmunoassay for linearized peptides, suggesting a quick response of guanylin and uroguanylin to an intestinal salt load. It is of interest to examine how plasma levels respond to a long-term salt loading.
At least three peptides, guanylin, uroguanylin, and renoguanylin, form the guanylin family in eels. Interestingly, a novel peptide, renoguanylin, appears to have functions different from the other two. Thus, it should be determined whether all members share a single receptor, GC-C, as suggested in mammals. In the opossum where lymphoguanylin was identified in addition to guanylin and uroguanylin, a new guanylyl cyclase-coupled receptor, OK-GC, was found (47), although it does not appear to be a lymphoguanylin-specific receptor (2, 47). Consistent with the presence of multiple ligands, two types of GC-C-like receptors have been partially cloned in the European eel (48). It should be determined whether the GC-C-like receptors are in fact receptors for the eel guanylin family by comparing the affinity to each peptide. It is also of interest to examine whether or not renoguanylin-like peptide is present in mammals and other vertebrate species.
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FOOTNOTES |
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* This work was supported in part by grants-in-aid for Scientific Research from the Japan Society for the Promotion of Science (A) (13304063) (to Y. T.) and for Creative Basic Research (12NP0201) from the Ministry of Education, Science, Sports and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Laboratory of Physiology, Ocean Research Inst., The University of Tokyo, 1-15-1 Minamidai, Nakano, Tokyo 164-8639, Japan. Tel.: 81-3-5351-6464; Fax: 81-3-5351-6463; E-mail: yuge{at}ori.u-tokyo.ac.jp.
1 The abbreviations used are: GC-C, guanylyl cyclase receptor C; FW, fresh water; SW, seawater; GLP, guanylin-like peptides; RACE, rapid amplification of cDNA ends; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EtBr, ethidium bromide; PBS, phosphate-buffered saline; GN, guanylin; UGN, uroguanylin; RGN, renoguanylin; LGN, lymphoguanylin.
2 T. Tsukada and Y. Takei, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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