Regulation of expression of two aquaporin homologs in the intestine of the European eel: effects of seawater acclimation and cortisol treatment

Complementary DNAs encoding homologs of the mammalian aquaglyceroporins (termed AQPe) and aquaporin-1 isoforms (termed AQP1) were isolated from the European eel. The AQP amino acid sequences share 35-54% identity with other known human AQPs. Although AQPe mRNA expression was approximately equivalent along the entire length of the gut, AQP1 expression was the highest in the posterior/rectal segment. Seawater (SW) acclimation increased AQP1 mRNA abundance by 5- and 17-fold in the anterior, 14- and 23-fold in the mid-, and 9- and 7-fold in the posterior/rectal gut regions of yellow and silver eels, respectively. SW acclimation had an effect on AQPe mRNA expression only in the midintestine of silver eels, where a small but significant 1.7-fold increase in abundance was measured. Western blots using an eel AQP1-specific antibody identified the presence of a major immunoreactive 28-kDa protein, primarily within the posterior/rectal segment. A 3-wk SW transfer induced an increase in AQP1 protein abundance in all intestinal segments, with the posterior/rectal region still expressing protein levels approximately 40- and 8-fold higher than the anterior and midsegments, respectively. Strong AQP1 immunofluorescence was detected within the vascular endothelium in both freshwater (FW)- and SW-acclimated eels and in the epithelial apical brush border in the posterior/rectal gut regions of SW-acclimated eels. Cortisol infusion into FW eels had no effect on intestinal AQPe mRNA expression but induced increases in AQP1 mRNA and protein levels. These results provide evidence for the presence of a SW-induced and steroid-regulated AQP water channel pathway within the intestine of the European eel.     

Two isoforms of the Na+/K+/2Cl- cotransporter are expressed in the European eel (Anguilla anguilla)

Two cDNA isoforms of the NKCC1 secretory cotransporter have been isolated from the European eel. The NKCC1a isoform exhibited mRNA expression in a wide range of tissues in a similar fashion to mammals, whereas NKCC1b was expressed primarily in the brain. The effect of freshwater (FW) to seawater (SW) transfer on NKCC1a expression was dependent on the developmental stage. In non-migratory yellow eels, NKCC1a mRNA expression in the gill was transiently up-regulated 4.3-fold after 2 days but also subsequently by 2.5-6-fold 3 weeks after SW transfer. Gill NKCC1a expression was localised mainly in branchial chloride cells of SW acclimated yellow eels. In contrast to yellow eels, NKCC1a mRNA abundance was not significantly different following SW acclimation in silver eel gill. NKCC1a mRNA abundance decreased in the kidney following SW acclimation and this may correlate with lower tubular ion/fluid secretion and urine flow rates in SW teleosts. Kidney NKCC1a mRNA expression in silver eels was also significantly lower than in yellow eels, suggesting some pre-acclimation of mRNA levels. NKCC1a mRNA was expressed at similar low levels in the middle intestine of FW- and SW-acclimated yellow or silver eels, suggesting the presence of an ion secretory mechanism in this gut segment.     

Differential expression of absorptive cation-chloride-cotransporters in the intestinal and renal tissues of the European eel (Anguilla anguilla)

Duplicate pairs of isoforms of each of the NKCC2 and the NCC absorptive cation-chloride-cotransporters have been isolated from the European eel. As with mammalian NKCC2, NKCC2alpha isoform mRNA expression was restricted to renal tissues, whereas NKCC2beta isoform expression was present in intestine and urinary bladder. Similar to mammalian NCC, NCCalpha mRNA expression was also found in the kidney, whereas, expression of NCCbeta mRNA was found at low levels in a number of tissues but particularly in intestine. Following 3 weeks of transfer of yellow or silver (adult life stages) eels from freshwater (FW) to seawater (SW), renal mRNA expression of NKCC2alpha did not change whereas NCCalpha expression was reduced although only significantly in silver eels. This suggests that any changes in renal sodium chloride re-absorption in SW-acclimated fish may be due to decreased NCCalpha cotransporter activity rather than the result of suppression of NKCCalpha cotransporter activity. Intestinal mRNA expression of NKCC2beta generally increased following SW acclimation, although maximal increases occurred later in yellow (7 days) than silver (2 days) eels. Average levels of NKCC2beta mRNA abundance in the middle intestine were 89% of those in the anterior, and this was reduced to 44% (of the level in the anterior intestine) in posterior intestine/rectum. Expression of NCCbeta was only found in the posterior intestine/rectum. Together these results suggest intestinal sodium chloride absorption may switch from occurring via NKCCbeta to NCCbeta as imbibed fluid travels down the intestine and the concentration of luminal potassium decreases.     

Enhanced expression and release of C-type natriuretic peptide in freshwater eels

C-type natriuretic peptide (CNP) is recognized as a paracrine factor acting locally in the brain and periphery. To assess the role of CNP in teleost fish, a cDNA encoding a CNP precursor was initially cloned from the eel brain. CNP message subsequently detected by ribonuclease protection assay, using the cDNA as probe, was most abundant in the brain followed by liver, gut, gills, and heart. Expression was generally higher in freshwater (FW) than in seawater (SW) eels, but not in the brain. Plasma CNP concentration measured by a newly developed homologous radioimmunoassay for eel CNP was higher in FW than in SW eels. The CNP concentration was also higher in the heart of FW eels but not in the brain. These results show that CNP is abundantly synthesized in peripheral tissues of FW eels and secreted constitutively into the circulation. Therefore, CNP is a circulating hormone as well as a paracrine factor in eels. Together with our previous demonstration that CNP-specific receptor expression is enhanced in FW eels, it appears that CNP is a hormone important for FW adaptation. Because atrial NP (ANP) promotes SW adaptation in eels, CNP and ANP, despite high sequence identity, appear to have opposite effects on environmental adaptation of the euryhaline fish.     

Cloning and Expression of Guanylin from the European eel (Anguilla anguilla)

Extracts of intestinal epithelia from the European eel (Anguilla anguilla) stimulated cGMP production in the T84 human colon carcinoma cell line which suggested the presence of a guanylin-like peptide in this teleost fish. Degenerate oligonucleotide primers were subsequently used in RT-PCR resulting in the amplification, cloning, and sequencing of two cDNAs which represent possible 5' spliceoforms of an eel homologue of the mammalian peptide, guanylin. Northern blotting indicated that the main site of expression of the eel peptide is in the intestine with much lower signals also detected in the kidney. Intestinal expression of guanylin mRNA is up-regulated in both nonmigratory "yellow" and the more sexually mature, migratory "silver" eels following acclimation to the seawater environment. These results suggest that this peptide signalling system may play a role in osmoregulation in euryhaline teleost fish during migration between the marine and freshwater environments.     

The role of aquaporin 3 in teleost fish

The aquaporin isoform, AQP3 has now been identified in a number of different teleost fish species, with additional DNA sequence information on AQP3 genes in further fish species available in genome databases. In zebrafish (Danio rerio), the AQP3 gene is present as two duplicate isoforms resulting from a teleostean fish genome-wide duplication. A further splicoform/isoform has also been identified in rainbow trout (Oncorhynchus mykiss). The identification of these AQP3 isoforms in other fish species is consequently explored. The role of AQP3 in physiological/osmoregulatory processes, in various teleost organs is then described. In teleost gill, AQP3 is expressed in 'chloride' cells, and in some species, in other epithelial cell types, where it may have a number of different functions including the prevention of dehydration. In eel esophagus, immunohistochemistry shows that AQP3 is expressed in surface epithelial cells in the anterior esophagus, but in mucus cells within the epithelium of the posterior esophagus. In eel intestine, AQP3 is found in macrophage-like cells and probably plays no part in osmoregulatory processes. In the rectum, as in the posterior esophagus AQP3 is expressed in mucus cells. In eel kidney, AQP3 is expressed in a subset of renal tubules, and localizes to the apical pole of tubule cells. There is no apparent change in the location or protein abundance of renal AQP3 following the acclimation of eels from freshwater to seawater.     

Feeding habitat and silvering stage affect lipid content and fatty acid composition of European eel Anguilla anguilla tissues

Lipids, particularly fatty acids (FAs), are major sources of energy and nutrients in aquatic ecosystems and play key roles during vertebrate development. The European eel Anguilla anguilla goes through major biochemical and physiological changes throughout its lifecycle as it inhabits sea- (SW), and/or brackish- (BW) and/or freshwater (FW) habitats. With the ultimate goal being to understand the reasons for eels adopting a certain life history strategy (FW or SW residency vs. ‘habitat shifting’), we explored differences in lipid content and FA composition of muscle, liver and eyes from eels collected across Norwegian SW, BW and FW habitats, and at different lifecycle stages (yellow to silver). FW and SW eels had a higher lipid content overall compared to BW eels, reflecting differences in food availability and life history strategies. SW eels had higher proportions of certain monounsaturated FAs (MUFAs; 18:1n-9, 20:1n-9), and of the essential polyunsaturated FAs 20:5n-3 (eicosapentaenoic acid, EPA) and 22:6n-3 (docosahexaenoic acid) than FW eels, reflecting a marine-based diet. In contrast, the muscle of FW eels had higher proportions of 18:3n-3, 18:2n-6 and 20:4n-6 (arachidonic acid), as is typical of FW organisms. MUFA proportions increased in later stage eels, consistent with the hypothesis that the eels accumulate energy stores prior to migration. In addition, the decrease of EPA with advancing stage may be associated with the critical role that this FA plays in eel sexual development. Lipid and FA information provided further understanding of the habitat use and overall ecology of this critically endangered species.     

Salinity regulates claudin mRNA and protein expression in the teleost gill

The teleost gill carries out NaCl uptake in freshwater (FW) and NaCl excretion in seawater (SW). This transformation with salinity requires close regulation of ion transporter capacity and epithelial permeability. This study investigates the regulation of tight-junctional claudins during salinity acclimation in fish. We identified claudin 3- and claudin 4-like immunoreactive proteins and examined their expression and that of select ion transporters by performing Western blot in tilapia (Oreochromis mossambicus) gill during FW and SW acclimation. Transfer of FW tilapia to SW increased plasma osmolality, which was corrected after 4 days, coinciding with increased gill Na+-K+-ATPase and Na+-K+-2Cl(-) cotransporter expression. Gill claudin 3- and claudin 4-like proteins were reduced with exposure to SW. Transfer to FW increased both claudin-like proteins. Immunohistochemistry shows that claudin 3-like protein was localized deep in the FW gill filament, whereas staining was found apically in SW gill. Claudin 4-like proteins are localized predominantly in the filament outer epithelial layer, and staining appears more intense in the gill of FW versus SW fish. In addition, tilapia claudin 28a and 30 genes were characterized, and mRNA expression was found to increase during FW acclimation. These studies are the first to detect putative claudin proteins in teleosts and show their localization and regulation with salinity in gill epithelium. The data indicate that claudins may be important in permeability changes associated with salinity acclimation and possibly the formation of deeper tight junctions in FW gill. This may reduce ion permeability, which is a critical facet of FW osmoregulation.     

Water balance in the european eel (Anguilla anguilla L.). Role of the oesophagus in the utilisation of drinking water and a study of the osmotic permeability of the gills (author's transl)]

10 New experimental devices are described which allow chonic measurements of drinking rate and osmotic gill permeability in the eel. 20 The oesophagus of the seawater (SW) silver eel plays a role in osmoregulation. It decreases the concentration of Cl- and Na+ of the ingested SW without losing water in the serosal to mucosal direction. This allows for immediate water absorption in the intestine and decreases the quantity of ions actively absorbed by the intestine. In the freshwater (FW) silver eel, the oesophagus is impermeable to water, Cl- and Na+. The ionic impermeability exists only in the serosal to mucosal direction. A mucosal to serosal permeability to Cl- and Na+ exists in the FW oesophagus receiving hypertonic drinking water, this promotes seawater adaptation. 30 The osmotic gill permeability, measured in vivo in the silver eel, is very low in FW and decreases slightly in SW. Thus, the silver eel has an osmotic gill permeability preadapted to SW life. The kinetics of FW to SW adaptation are described.     

Tissue Distribution, Effects of Salinity Acclimation, and Ontogeny of Aquaporin 3 in the Marine Teleost, Silver Sea Bream (Sparus sarba)

The purpose of the present study was to ascertain the tissue-specific expression of the water channel protein, aquaporin 3 (AQP3), during salinity acclimation and larval development of silver sea bream (Sparus sarba). A cDNA fragment encoding aquaporin 3 (aqp3) from silver sea bream gill was cloned and from the deduced amino acid sequence a polyclonal antibody was prepared. AQP3 was found to be present in gill, kidney, liver, brain, heart, and spleen but not in whole blood. The abundance of AQP3 was significantly highest in gills of hypoosmotic (6 ppt) and isoosmotic (12 ppt) acclimated sea bream when compared to seawater (33 ppt) and hypersaline (50 ppt)- acclimated sea bream. Spleen tissue also displayed significantly high levels of AQP3 protein in hypoosmotic and isoosmotic salinities whereas the AQP3 abundance in brain, liver, heart, and kidney remained unchanged across the range of salinities tested. The ontogenetic profile of AQP3 was also investigated from developing sea bream larvae and AQP3 was first detected at 14 days posthatch (dph) and increased steadily up to 28–46 dph. In conclusion, this study has demonstrated that AQP3 expression is modulated in gill and spleen tissue of salinity acclimated sea bream and that it can be detected relatively early during larval development.     

Osmoregulation during the development of glass eels and elvers

Drinking rates in glass eels and elvers of the European eel increased with environmental acclimation salinity from 0·07±0·02 (FW) to 0·70±0·09 μl g-¹ h-¹ (SW) at month 1 and from 1·12±0·42 (FW) to 12·85±1·05 ± l g-¹ h-¹ (SW) at month 5. Drinking rates increased with time in both FW and SW groups. FW acclimated eels when challenged acutely with SW increased drinking rate rapidly immediately upon transfer (0–15 min) and the magnitude of this response increased with developmental time from month 1 to month 5.     

Effect of transfer to sea water and back to fresh water on the histological structure of the eel kidney

Summary A cytometrical study of the various segments of kidney tubules was performed on silver or silvering male eels during acclimation to sea water (SW) for 2 to 160 days and their return to fresh water (FW). Tubular epithelial cell height and nuclear area are markedly reduced during the first two days, and further reduced in the following period to reach a stable level around the 20th day. The glomerular size is slightly decreased. Phospholipids are less abundant and the brush borders become thinner. In a single eel kept for 9 months in pure SW slowly concentrated by evaporation (NaCl 45 g/l), cell and nuclear values were similar to those obtained in full strength SW after 160 days. Distal and collecting tubules exhibit maximal reactivity, that of the first proximal tubule being minimal.When eels kept for 20 days in SW are returned to FW, there is a rapid increase (48 h), sometimes with an overshoot, of all measured parameters, followed by a rapid decrease (5 days) and a normalization of the values in the distal and collecting tubules.The rapid response of eel kidney to salinity changes does not appear to be linked to cellular hydration as the various segments of the kidney tubules react differently. The data are discussed with respect to osmotic acclimation and in relation to variations of prolactin secretion. Prolactin (PRL) release during transfer to FW stimulates the renal tubules less strongly than mammalian PRL: while mitotic activity and differentiation of new nephrons are induced by ovine PRL treatment in intact or hypophysectomized FW eels (Olivereau and Lemoine, 1969b), no such events were detected during acclimation to this hypotonic medium, at least during the first ten days. This rather reflects insufficient PRL release than a different biological activity, despite immunological differences between fish and ovine prolactin.

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Résumé L'étude cytométrique (hauteur épithéliale et aire nucléaire) des divers segments du tube rénal, réalisée chez l'Anguille mâle argentée ou en cours d'argenture lors de l'adaptation à l'eau de mer (SW) montre une réduction marquée dès les premières 48 h en SW qui s'accuse légèrement pour atteindre un niveau stable vers le 20^e jour; la taille des glomérules est réduite. Les phospholipides sont moins abondants, la bordure en brosse est plus mince. Chez une Anguille conservée 9 mois en SW initialement normale et lentement concentrée par évaporation (NaCl 45 g/l), les valeurs obtenues sont peu différentes de celles après 160 jours en SW pure. La réactivité maximale s'observe au niveau du tube distal et du collecteur, celle du tube proximal l étant minimale.Le retour en eau douce (FW) provoque une rapide augmentation (2 jours), parfois excessive, de ces paramètres, suivie d'une baisse rapide (5 jours) et de la normalisation des valeurs du tube distal et collecteur (10 jours).Les réponses du rein aux changements de salinité, très rapides, ne paraissent pas liées à des processus de déshydratation ou hydratation cellulaire car elles affectent inégalement les divers segments du néphron. Elles sont discutées en fonction des variations des électrolytes plasmatiques et de la sécrétion hypophysaire de prolactine (PRL): sa décharge qui accompagne le retour en FW stimule moins intensément le tube rénal que l'injection de PRL ovine, elle ne provoque pas une intense activité mitotique ni une différenciation de nouveaux néphrons, au moins pendant les 10 premiers jours. Ce fait représente probablement plus une sécrétion insuffisante de PRL lors du retour en milieu hypotonique qu'une différence d'activité biologique, malgré des différences immunologiques entre prolactines ovine et de Poisson.

     

Antidiuretic Effect of Eel ANP Infused at Physiological Doses in Conscious, Seawater-Adapted Eels, Anguilla japonica

Atrial natriuretic peptide (ANP) is known as a potent natriuretic/diuretic hormone in vertebrates. However, eel ANP infused at doses that did not alter arterial blood pressure (0.3-3.0 pmol/kg/min) decreased urine volume and increased urinary Na concentration in seawater (SW)-adapted eels but not in freshwater (FW)-adapted eels. The renal effects were dose-dependent and disappeared after infusate was switched back to a vehicle (0.9% NaCl). Urinary Na excretion (volume x Na concentration) did not change during ANP infusion. ANP infusion increased plasma ANP concentration, but the increase at the highest dose was still within those observed endogenously after injection of hypertonic saline. Urinary Mg and Ca concentrations increased during ANP infusion in SW eels, but urinary Ca excretion decreased in FW eels. Plasma Na concentration profoundly decreased during ANP infusion only in SW eels, suggesting that ANP stimulates Na extrusion via non-renal routes. These results indicate that ANP is a hormone which specifically extrudes Na ions and thereby promotes SW adaptation in the eel. This is in sharp contrast with mammals where ANP is a volume regulating hormone that extrudes both Na and water.     

Aquaporins in complex tissues. I.Developmental patterns in respiratory and glandular tissues of rat

Developmentalexpression of aquaporin water transport proteins is not well understoodin respiratory tract or secretory glands; here we define aquaporinprotein ontogeny in rat. Expression of aquaporin-3 (AQP3), AQP4, andAQP5 proteins occurs within 2 wk after birth, whereas AQP1 firstappears before birth. In most tissues, aquaporin protein expressionincreases progressively, although transient high-level expression isnoted in distal lung (AQP4 at postnatal day+2) and trachea (AQP5 at postnatalday +21 and AQP3 at postnatal day+42). In mature animals, AQP5 is abundant in distallung and salivary glands, AQP3 and AQP4 are present in trachea, andAQP1 is present in all of these tissues except salivary glands.Surprisingly, all four aquaporin proteins are highly abundant innasopharynx. Unlike AQP1, corticosteroids did not induce expression ofAQP3, AQP4, or AQP5 in lung. Our results seemingly implicate aquaporinsin proximal airway humidification, glandular secretion, and perinatalclearance of fluid from distal airways. However, the studies underscorea need for detailed immunohistochemical characterizations anddefinitive functional studies.

     

Expression of a duplicate Na,K-ATPase beta(1)-isoform in the European eel (Anguilla anguilla)

Recent studies on teleost fish have suggested that their genomes have undergone ancient polyploidization events resulting in the duplication of the genome. A duplicate copy of the Na,K-ATPase beta(1)-isoform (called beta(233)) has been identified in the European eel (Anguilla anguilla). The beta(233)-isoform shares high levels of nucleotide (74.8%) and amino acid (69.9%) homology with the eel beta(1)-subunit as well as other vertebrate beta(1)-sequences. Compared with the widely expressed beta(1)-isoform, expression of beta(233)-mRNA is mainly restricted to epithelial tissues. Seawater acclimation induced increases in beta(233)-mRNA levels in kidney, gill, and intestine of migratory "silver" but not the nonmigratory "yellow" adult eels, suggesting that the factors responsible for this upregulation are themselves developmentally regulated. Expression of a variably glycosylated 40- to 52-kDa beta(233)-protein in both gill "chloride" and intestinal epithelial cells suggests that the beta(233)-isoform of Na,K-ATPase may play an important functional role in the major osmoregulatory tissues of euryhaline fish such as the eel.     

Gonad differentiation and body growth in Anguilla anguilla L.

Using histological sections, the gonads of samples of yellow and silver eels of two populations were examined. The populations were previously analysed for growth and sex ratio. The histological structures observed are similar to those described in previous publications for the European eel, Anguilla anguilla and to those indicated for the Pacific eel, A. japonica . Well differentiated gonads are present in the silver eels. In the yellow eels, ranging in age from 0 + to 2 + years and from a length of 20 cm to that at which they become silver, undif-ferentiated and both sex gonads are found. Histological evidence is presented which suggests that the ovary, found even in young and small eels, is completely differentiated at a very early stage. The testis-like gonad of the yellow eel is a more primitive, and possibly reversible, gonad which differentiates completely at the beginning of sexual maturation and the change to the silver phase.