Aquaporin (AQP) channels in the spiny dogfish, Squalus acanthias I: Characterization of AQP3 and AQP15 function and expression, and localization of the proteins in gill and spiral valve intestine.

Complementary DNAs (cDNAs) for two aquaporin water channel genes (AQP3 and AQP15) were amplified cloned and sequenced to initiate this study. Northern blot analysis was carried out to confirm the mRNA sizes of these AQP genes with AQP3 mRNA bands exhibiting sizes of 1.2 and 1.6 k bases and AQP15 had a mRNA band of 2.1 k bases. Northern blot analysis was also performed on kidney and esophagus total RNA samples from fish acclimated to 75%, 100% or 120% seawater (SW). The level of AQP15 mRNA expression was shown to significantly decrease following salinity acclimation from 100 to 120% SW. An opposite but non-significantly different trend was observed for AQP3 mRNA levels. Full length cDNAs were then used to generate AQP3 and AQP15 mRNAs for microinjection into Xenopus oocytes. Both AQP3- and AQP15- microinjected oocytes exhibited significantly elevated apparent water permeability compared to control oocytes at neutral pH. The apparent water permeability was mercury-inhibitable, significantly so in the case of AQP3. AQP3 microinjected oocytes showed pH sensitivity in their apparent water permeability, showing a lack of permeability at acidic pH values. The Carboxyl-terminal derived amino acid sequences of AQP3 and AQP15 were used to generate rabbit affinity-purified polyclonal antibodies. Western blots with the antibodies showed a band of 31.3 kDa for AQP3 in the kidney, with minor bands at 26, 24 and 21 kDa. For AQP15 a band of 26 kDa was seen in gill and kidney. Fainter bands at 28 and 24 kDa were also seen in the kidney. There was also some higher molecular weight banding. None of the bands were seen when the antibodies were pre- blocked with their peptide antigens. Immunohistochemical localization studies were also performed in the gill and spiral valve intestine. In the gill, AQP15 antibody staining was seen sporadically in the membranes of surface epithelial cells of the secondary lamellae. Tyramide amplification of signals was employed in the spiral valve intestine. Tyramide-amplified AQP3 antibody staining was observed in the basal membrane of the invaginated epithelial cell layer of secondary intestinal folds in luminal surface of either the side wall of the spiral valve intestine or in internal valve tissue 'flaps'. For the AQP15 antibody, tyramide-amplified staining was instead found on the apical and to a lesser extent the lateral membranes of the same invaginated epithelial cell layer. The localization of AQP3 and AQP15 in the spiral valve intestine suggests that a trans-cellular water absorption pathway may exist in this tissue.

Myo-inositol phosphate synthase expression in the European eel (Anguilla anguilla) and Nile tilapia (Oreochromis niloticus): effect of seawater acclimation.

A single MIPS gene (Isyna1/Ino1) exists in eel and tilapia genomes with a single myo-d-inositol 3-phosphate synthase (MIPS) transcript identified in all eel tissues, although two MIPS spliced variants [termed MIPS(s) and MIPS(l)] are found in all tilapia tissues. The larger tilapia transcript [MIPS(l)] results from the inclusion of the 87-nucleotide intron between exons 5 and 6 in the genomic sequence. In most tilapia tissues, the MIPS(s) transcript exhibits much higher abundance (generally >10-fold) with the exception of white skeletal muscle and oocytes, in which the MIPS(l) transcript predominates. SW acclimation resulted in large (6- to 32-fold) increases in mRNA expression for both MIPS(s) and MIPS(l) in all tilapia tissues tested, whereas in the eel, changes in expression were limited to a more modest 2.5-fold increase and only in the kidney. Western blots identified a number of species- and tissue-specific immunoreactive MIPS proteins ranging from 40 to 67 kDa molecular weight. SW acclimation failed to affect the abundance of any immunoreactive protein in any tissue tested from the eel. However, a major 67-kDa immunoreactive protein (presumed to be MIPS) found in tilapia tissues exhibited 11- and 54-fold increases in expression in gill and fin samples from SW-acclimated fish. Immunohistochemical investigations revealed specific immunoreactivity in the gill, fin, skin, and intestine taken from only SW-acclimated tilapia. Immunofluorescence indicated that MIPS was expressed within gill chondrocytes and epithelial cells of the primary filaments, basal epithelial cell layers of the skin and fin, the cytosol of columnar intestinal epithelial and mucous cells, as well as unknown entero-endocrine-like cells.

In absence of local adaptation, plasticity and spatially varying selection rule: a view from genomic reaction norms in a panmictic species (Anguilla rostrata).

BACKGROUND: American eel (Anguilla rostrata) is one of the few species for which panmixia has been demonstrated at the scale of the entire species. As such, the development of long term local adaptation is impossible. However, both plasticity and spatially varying selection have been invoked in explaining how American eel may cope with an unusual broad scope of environmental conditions. Here, we address this question through transcriptomic analyses and genomic reaction norms of eels from two geographic origins reared in controlled environments. RESULTS: The null hypothesis of no difference in gene expression between eels from the two origins was rejected. Many unique transcripts and two out of seven gene clusters showed significant difference in expression, both at time of capture and after three months of common rearing. Differences in expression were observed at numerous genes representing many functional groups when comparing eels from a same origin reared under different salinity conditions. Plastic response to different rearing conditions varied among gene clusters with three clusters showing significant origin-environment interactions translating into differential genomic norms of reaction. Most genes and functional categories showing differences between origins were previously shown to be differentially expressed in a study comparing transcription profiles between adult European eels acclimated to different salinities. CONCLUSIONS: These results emphasize that while plasticity in expression may be important, there is also a role for local genetic (and/or epigenetic) differences in explaining differences in gene expression between eels from different geographic origins. Such differences match those reported in genetically distinct populations in other fishes, both in terms of the proportion of genes that are differentially expressed and the diversity of biological functions involved. We thus propose that genetic differences between glass eels of different origins caused by spatially varying selection due to local environmental conditions translates into transcriptomic differences (including different genomic norms of reaction) which may in turn explain part of the phenotypic variance observed between different habitats colonized by eels.

Seawater acclimation and inositol monophosphatase isoform expression in the European eel (Anguilla anguilla) and Nile tilapia (Orechromis niloticus).

Inositol monophosphatase (IMPA) is responsible for the synthesis of inositol, a polyol that can function as an intracellular osmolyte helping re-establish cell volume when exposed to hypertonic environments. Some epithelial tissues in euryhaline teleosts such as the eel and tilapia encounter considerable hyperosmotic challenge when fish move from freshwater (FW) to seawater (SW) environments; however, the roles played by organic osmolytes, such as inositol, have yet to be determined. Syntenic analysis has indicated that, as a result of whole genome- and tandem-duplication events, up to six IMPA isoforms can exist within teleost genomes. Four isoforms are homologs of the mammalian IMPA1 gene, and two isoforms are homologs of the mammalian IMPA2 gene. Although the tissue-dependent isoform expression profiles of the teleost isoforms appear to be species-specific, it was primarily mRNA for the IMPA1.1 isoform that was upregulated in epithelial tissues after fish were transferred to SW (up to 16-fold in eel and 90-fold in tilapia). Although up-regulation of IMPA1.1 expression was evident in many tissues in the eel, more substantial increases in IMPA1.1 expression were found in tilapia tissues, where SW acclimation resulted in up to 2,000-fold increases in protein expression, 16-fold increases in enzyme activity and 15-fold increases in tissue inositol contents. Immunohistochemical studies indicated that the tissue and cellular distribution of IMPA1.1 protein differed slightly between eels and tilapia; however, in both species the basal epithelial cell layers within the skin and fin, and the branchial epithelium and interstitial cells within the kidney, exhibited high levels of IMPA1.1 protein expression.

Surviving in a toxic world: transcriptomics and gene expression profiling in response to environmental pollution in the critically endangered European eel.

BACKGROUND: Genomic and transcriptomic approaches have the potential for unveiling the genome-wide response to environmental perturbations. The abundance of the catadromous European eel (Anguilla anguilla) stock has been declining since the 1980s probably due to a combination of anthropogenic and climatic factors. In this paper, we explore the transcriptomic dynamics between individuals from high (river Tiber, Italy) and low pollution (lake Bolsena, Italy) environments, which were measured for 36 PCBs, several organochlorine pesticides and brominated flame retardants and nine metals. RESULTS: To this end, we first (i) updated the European eel transcriptome using deep sequencing data with a total of 640,040 reads assembled into 44,896 contigs (Eeelbase release 2.0), and (ii) developed a transcriptomic platform for global gene expression profiling in the critically endangered European eel of about 15,000 annotated contigs, which was applied to detect differentially expressed genes between polluted sites. Several detoxification genes related to metabolism of pollutants were upregulated in the highly polluted site, including genes that take part in phase I of the xenobiotic metabolism (CYP3A), phase II (glutathione-S-transferase) and oxidative stress (glutathione peroxidase). In addition, key genes in the mitochondrial respiratory chain and oxidative phosphorylation were down-regulated at the Tiber site relative to the Bolsena site. CONCLUSIONS: Together with the induced high expression of detoxification genes, the suggested lowered expression of genes supposedly involved in metabolism suggests that pollution may also be associated with decreased respiratory and energy production.

Cloning, tissue distribution and sub-cellular localisation of phospholipase C X-domain containing protein (PLCXD) isoforms.

Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes comprise a small family of receptor-regulated phosphodiesterases that control many cellular processes by the regulation of cytosolic calcium and/or the activity of several protein kinases. To date, six distinct classes of PI-PLC are known to exist in mammals. Here we characterise a seventh class of PI-PLC, which contains only the catalytic X domain in its structure, termed phospholipase C X-domain containing protein (PLCXD). At least three tissue-specific PLCXD isoforms exist in humans, comprising hPLCXD-1, hPLCXD-2 and hPLCXD-3, with hPLCXD-2 exhibiting three C-terminal spliceforms (2.1, 2.2 and 2.3). Specific amino acids known to be essential for the catalytic function of PI-PLCs were found to be conserved in all three human PLCXDs and over-expression of hPLCXD-1, 2.1 and 3 in the HeLa cell line increased endogenous PI-PLC activity. Human PLCXD isoforms exhibited tissue-specific expression profiles in mice and humans and immunocytochemistry revealed distinct sub-cellular localisations when over-expressed in human cultured cell lines. These novel proteins may therefore possess fundamental, and as yet uncharacterised roles in cell physiology.

Expression and Localization of Aquaporin 1a in the Sea-Bass (Dicentrarchus labrax) during Ontogeny.

The successful establishment of a species in a given habitat depends on the ability of each of its developing stages to adapt to the environment. In order to understand this process we have studied the adaptation of a euryhaline fish, the sea-bass Dicentrarchus labrax, to various salinities during its ontogeny. The expression and localization of Aquaporin 1a (AQP1a) mRNA and protein were determined in different osmoregulatory tissues. In larvae, the sites of AQP1a expression are variable and they shift according to age, implying functional changes. In juveniles after metamorphosis (D32-D48 post-hatch, 15-25 mm) and in pre-adults, an increase in AQP1a transcript abundance was noted in the digestive tract, and the AQP1a location was observed in the intestine. In juveniles (D87-D100 post-hatch, 38-48 mm), the transcript levels of AQP1a in the digestive tract and in the kidney were higher in sea water (SW) than at lower salinity. These observations, in agreement with existing models, suggest that in SW-acclimated fish, the imbibed water is absorbed via AQP1a through the digestive tract, particularly the intestine and the rectum. In addition, AQP1a may play a role in water reabsorption in the kidney. These mechanisms compensate dehydration in SW, and they contribute to the adaptation of juveniles to salinity changes during sea-lagoon migrations. These results contribute to the interpretation of the adaptation of populations to habitats where salinity varies.

Phospholipase C-η2 is activated by elevated intracellular Ca(2+) levels.

Phospholipase C-η2 (PLCη2) is a novel enzyme whose activity in a cellular context is largely uncharacterised. In this study the activity of PLCη2 was examined via [(3)H]inositol phosphate release in COS7 cells expressing the enzyme. PLCη2 activity increased approximately 5-fold in response to monensin, a Na(+)/H(+) antiporter. This was significantly inhibited by CGP-37157 which implies that the effect of monensin was due, at least in part, to mitochondrial Na(+)/Ca(2+)-exchange. Direct activation of PLCη2 by <1µM Ca(2+) was confirmed in permeabilised transfected cells. The roles of the PH and C2 domains in controlling PLCη2 activity via membrane association were also investigated. A PH domain-lacking mutant exhibited no detectable activity in response to monensin or Ca(2+) due to an inability to associate with the cell membrane. Within the C2 domain, mutation of D920 to alanine at the predicted Ca(2+)-binding site dramatically reduced enzyme activity highlighting an important regulatory role for this domain. Mutation of D861 to asparagine also influenced activity, most likely due to altered lipid selectivity. Of the C2 mutations investigated, none altered sensitivity to Ca(2+). This suggests that the C2 domain is not responsible for Ca(2+) activation. Collectively, this work highlights an important new component of the Ca(2+) signalling toolkit and given its sensitivity to Ca(2+), this enzyme is likely to facilitate the amplification of intracellular Ca(2+) transients and/or crosstalk between Ca(2+)-storing compartments in vivo.

Differential timing of gene expression regulation between leptocephali of the two Anguilla eel species in the Sargasso Sea.

The unique life-history characteristics of North Atlantic catadromous eels have long intrigued evolutionary biologists, especially with respect to mechanisms that could explain their persistence as two ecologically very similar but reproductively and geographically distinct species. Differential developmental schedules during young larval stages have commonly been hypothesized to represent such a key mechanism. We performed a comparative analysis of gene expression by means of microarray experiments with American and European eel leptocephali collected in the Sargasso Sea in order to test the alternative hypotheses of (1) differential timing of gene expression regulation during early development versus (2) species-specific differences in expression of particular genes. Our results provide much stronger support for the former hypothesis since no gene showed consistent significant differences in expression levels between the two species. In contrast, 146 genes showed differential timings of expression between species, although the observed expression level differences between the species were generally small. Consequently, species-specific gene expression regulation seems to play a minor role in species differentiation. Overall, these results show that the basis of the early developmental divergence between the American and European eel is probably influenced by differences in the timing of gene expression regulation for genes involved in a large array of biological functions.

Aquaporin 4 is a Ubiquitously Expressed Isoform in the Dogfish (Squalus acanthias) Shark.

The dogfish ortholog of aquaporin 4 (AQP4) was amplified from cDNA using degenerate PCR followed by cloning and sequencing. The complete coding region was then obtained using 5' and 3' RACE techniques. Alignment of the sequence with AQP4 amino acid sequences from other species showed that dogfish AQP4 has high levels (up to 65.3%) of homology with higher vertebrate sequences but lower levels of homology to Agnathan (38.2%) or teleost (57.5%) fish sequences. Northern blotting indicated that the dogfish mRNA was approximately 3.2 kb and was highly expressed in the rectal gland (a shark fluid secretory organ). Semi-quantitative PCR further indicates that AQP4 is ubiquitous, being expressed in all tissues measured but at low levels in certain tissues, where the level in liver > gill > intestine. Manipulation of the external environmental salinity of groups of dogfish showed that when fish were acclimated in stages to 120% seawater (SW) or 75% SW, there was no change in AQP4 mRNA expression in either rectal gland, kidney, or esophagus/cardiac stomach. Whereas quantitative PCR experiments using the RNA samples from the same experiment, showed a significant 63.1% lower abundance of gill AQP4 mRNA expression in 120% SW-acclimated dogfish. The function of dogfish AQP4 was also determined by measuring the effect of the AQP4 expression in Xenopus laevis oocytes. Dogfish AQP4 expressing-oocytes, exhibited significantly increased osmotic water permeability (P(f)) compared to controls, and this was invariant with pH. Permeability was not significantly reduced by treatment of oocytes with mercury chloride, as is also the case with AQP4 in other species. Similarly AQP4 expressing-oocytes did not exhibit enhanced urea or glycerol permeability, which is also consistent with the water-selective property of AQP4 in other species.

A role for inositol monophosphatase 1 (IMPA1) in salinity adaptation in the euryhaline eel (Anguilla anguilla).

This study investigated the expression and tissue distribution of inositol monophosphatase (IMPA1) and characterized its role in salinity adaptation in the eel. The coding sequence of eel IMPA1 was determined and confirmed to be orthologous to the mammalian gene/enzyme by phylogenetic analysis and structural modeling. Quantitative real-time PCR and Western blot techniques indicated up to 17-fold increases in mRNA expression and 2-fold increases in protein abundance in major osmoregulatory tissues following transfer of fish to seawater (SW). This was accompanied by up to 5-fold increases in enzyme activity, and 1.8- and 3-fold increases in inositol contents within the gill and kidney, respectively. Immunohistological studies revealed that IMPA1 protein expression predominated in SW-acclimated fish within basal epithelial/epidermal layers of the gill, esophagus, intestine, skin, and fins. SW transfer also induced a 10-fold increase in inositol content in the fin. IMPA1 immunoreactivity was also identified in chondrocytes within the cartilagenous matrix of the gills and fins, as well as in clusters of interstitial cells surrounding the kidney tubules. The observed increases in expression of IMPA1 highlight a protective role for inositol within various eel tissues following SW acclimation. This constitutes an adaptive mechanism in teleost fish naturally exposed to hypertonic environments.

Identification of functionally segregated sarcoplasmic reticulum calcium stores in pulmonary arterial smooth muscle.

In pulmonary arterial smooth muscle, Ca(2+) release from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) may induce constriction and dilation in a manner that is not mutually exclusive. We show here that the targeting of different sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPases (SERCA) and RyR subtypes to discrete SR regions explains this paradox. Western blots identified protein bands for SERCA2a and SERCA2b, whereas immunofluorescence labeling of isolated pulmonary arterial smooth muscle cells revealed striking differences in the spatial distribution of SERCA2a and SERCA2b and RyR1, RyR2, and RyR3, respectively. Almost all SERCA2a and RyR3 labeling was restricted to a region within 1.5 microm of the nucleus. In marked contrast, SERCA2b labeling was primarily found within 1.5 microm of the plasma membrane, where labeling for RyR1 was maximal. The majority of labeling for RyR2 lay in between these two regions of the cell. Application of the vasoconstrictor endothelin-1 induced global Ca(2+) waves in pulmonary arterial smooth muscle cells, which were markedly attenuated upon depletion of SR Ca(2+) stores by preincubation of cells with the SERCA inhibitor thapsigargin but remained unaffected after preincubation of cells with a second SERCA antagonist, cyclopiazonic acid. We conclude that functionally segregated SR Ca(2+) stores exist within pulmonary arterial smooth muscle cells. One sits proximal to the plasma membrane, receives Ca(2+) via SERCA2b, and likely releases Ca(2+) via RyR1 to mediate vasodilation. The other is located centrally, receives Ca(2+) via SERCA2a, and likely releases Ca(2+) via RyR3 and RyR2 to initiate vasoconstriction.

Regulation of expression of the myo-inositol monophosphatase 1 gene in osmoregulatory tissues of the European eel Anguilla anguilla after seawater acclimation.

Previous microarray studies in our laboratory identified a number of genes that were differentially expressed in "silver" eels after transfer from freshwater (FW) to seawater (SW). A group of genes, which are related to the synthesis, processing, and transport of certain known osmolytes in mammalian cells, have been identified. One gene implicated with osmolyte production is myo-inositol monophosphatase (IMPA1). The aim of this study was to compare the expression of IMPA1 in the major osmoregulatory tissues (intestine, gill, and kidney) as fish move between FW and SW environments. No difference in IMPA1 gene expression was observed in any tissues 6 h after eel transfer to SW; however, after 2 days acclimation, a 1.9- and a 2.5-fold increase in mRNA expression was found in kidney and gill, respectively. These elevated levels were maintained for up to 5 months (4.9- and 3.4-fold, respectively) after SW transfer. No IMPA1 mRNA expression was detected in the intestine. Western blot analysis confirmed the IMPA1 protein was upregulated in the gill, but no changes in protein abundance were detected in the kidney 5 months after SW transfer. Our studies have revealed a potential role for IMPA1 in salinity adaptation in the European eel.

Guanylin-like peptides, guanylate cyclase and osmoregulation in the European eel (Anguilla anguilla).

Three guanylin-like peptides, guanylin, uroguanylin and renoguanylin and two guanylate cyclase type C (GC-C) receptor isoforms were cloned and sequenced from the European eel (Anguilla anguilla). All peptides and both receptors (GC-C1 and GC-C2) were predominantly expressed within the intestine and kidney of both sexually immature yellow, and sexually maturing, migratory silver eels. The derived amino acid sequences for the pre-prohormones and guanylate cyclase isoforms had structural features in common with sequences previously reported for guanylin-like peptides and guanylate cyclases from teleost fish and other species in general. The highest sequence homologies for the prohormones were found within the active, 15-16 amino acid C-terminal peptide domain, whereas the guanylate cyclase receptors exhibited highest homology throughout the transmembrane domain and intracellular region of the protein comprising the kinase homology, oligomerisation/coiled-coil and catalytic domains. In both yellow and silver eels, seawater (SW) acclimation induced sustained increases in the expression of uroguanylin and GC-C1 mRNAs within the intestine but no significant changes were found in the abundance of mRNAs for guanylin, renoguanylin or GC-C2. Likewise there were no significant changes in expression of any of the prohormone or receptor mRNAs within the renal kidney following transfer to SW. The results suggest that uroguanylin and GC-C1 are key components of a cGMP signalling system that may play an important role within intestinal enterocytes for the regulation of salt and water absorption in the SW-acclimated eel.

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.

Transcriptomic approach to the study of osmoregulation in the European eel Anguilla anguilla.

In euryhaline teleosts, osmoregulation is a fundamental and dynamic process that is essential for the maintenance of ion and water balance, especially when fish migrate between fresh water (FW) and sea water (SW) environments. The European eel has proved to be an excellent model species to study the molecular and physiological adaptations associated with this osmoregulatory plasticity. The life cycle of the European eel includes two migratory periods, the second being the migration of FW eels back to the Sargasso Sea for reproduction. Various anatomical and physiological changes allow the successful transition to SW. The aim of this study was to use a microarray approach to screen the osmoregulatory tissues of the eel for changes in gene expression following acclimation to SW. Tissues were sampled from fish at selected intervals over a 5-mo period following FW/SW transfer, and RNA was isolated. Suppressive subtractive hybridization was used for enrichment of differentially expressed genes. Microarrays comprising 6,144 cDNAs from brain, gill, intestine, and kidney libraries were hybridized with appropriate targets and analyzed; 229 differentially expressed clones with unique sequences were identified. These clones represented the sequences for 95 known genes, with the remaining sequences (59%) being unknown. The results of the microarray analysis were validated by quantification of 28 differentially expressed genes by Northern blotting. A number of the differentially expressed genes were already known to be involved in osmoregulation, but the functional roles of many others, not normally associated with ion or water transport, remain to be characterized.

Aquaporin molecular characterization in the sea-bass (Dicentrarchus labrax): the effect of salinity on AQP1 and AQP3 expression.

Euryhaline fish possess the ability to compensate for environmental salinity changes through hydro-mineral regulation. A number of proteins have been studied in order to understand water and ion exchanges, known as fish osmoregulation. Sea-bass (Dicentrarchus labrax) cDNA sequences encoding a homologue of mammalian aquaporin (termed AQP1) and a homologue of mammalian aquaglyceroporin (termed AQP3) have been isolated and sequenced. The aquaporin amino acid sequences share respectively more than 60% and 65% identity with other known aquaporins. We have shown that salinity influences aquaporin expression levels in the gill, kidney and digestive tract, the main osmoregulatory organs. AQP1 may have a major osmoregulatory role in water transport in kidney and gut in SW-acclimated fish, whereas AQP3 could be implicated in gill water transport in FW-acclimated fish.

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.

Functional significance of the shark Na,K-ATPase N-terminal domain. Is the structurally variable N-Terminus involved in tissue-specific regulation by FXYD proteins?

The proteolytic profile after mild controlled trypsin cleavage of shark rectal gland Na,K-ATPase was characterized and compared to that of pig kidney Na,K-ATPase, and conditions for achieving N-terminal cleavage of the alpha-subunit at the T(2) trypsin cleavage site were established. Using such conditions, the shark enzyme N-terminus was much more susceptible to proteolysis than the pig enzyme. Nevertheless, the maximum hydrolytic activity was almost unaffected for the shark enzyme, whereas it was significantly decreased for the pig kidney enzyme. The apparent ATP affinity was unchanged for shark but increased for pig enzyme after N-terminal truncation. The main common effect following N-terminal truncation of shark and pig Na,K-ATPase is a shift in the E(1)-E(2) conformational equilibrium toward E(1). The phosphorylation and the main rate-limiting E(2) --> E(1) step are both accelerated after N-terminal truncation of the shark enzyme, but decreased significantly in the pig kidney enzyme. Some of the kinetic differences, like the acceleration of the phosphorylation reaction, following N-terminal truncation of the two preparations may be due to the fact that under the conditions used for N-terminal truncation, the C-terminal domain of the FXYD regulatory protein of the shark enzyme, PLMS or FXYD10, was also cleaved, whereas the gamma or FXYD2 of the pig enzyme was not. In the shark enzyme, N-terminal truncation of the alpha-subunit abolished association of exogenous PLMS with the alpha-subunit and the functional interactions were abrogated. Moreover, PKC phosphorylation of the preparation, which relieves PLMS inhibition of Na,K-ATPase activity, exposed the N-terminal trypsin cleavage site. It is suggested that PLMS interacts functionally with the N-terminus of the shark Na,K-ATPase to control the E(1)-E(2) conformational transition of the enzyme and that such interactions may be controlled by regulatory protein kinase phosphorylation of the N-terminus. Such interactions are likely in shark enzyme where PLMS has been demonstrated by cross-linking to associate with the Na,K-ATPase A-domain.

Sequence, circulating levels, and expression of C-type natriuretic peptide in a euryhaline elasmobranch, Carcharhinus leucas.

The present study has examined expression and circulating levels of C-type natriuretic peptide (CNP) in the euryhaline bull shark, Carcharhinus leucas. Complementary DNA and deduced amino acid sequence for CNP in C. leucas were determined by RACE methods. Homology of CNP amino acid sequence in C. leucas was high both for proCNP and for mature CNP when compared with previously identified elasmobranch CNPs. Mature CNP sequence in C. leucas was identical to that in Triakis scyllia and Scyliorhinus canicula. Levels of expression of CNP mRNA were significantly decreased in the atrium but did not change in either the brain or ventricle following acclimation to a SW environment. However, circulating levels of CNP significantly increased from 86.0+/-7.9 fmol ml(-1) in FW to 144.9+/-19.5 fmol ml(-1) in SW. The results presented demonstrate that changes in environmental salinity influences both synthesis of CNP from the heart and also circulating levels in C. leucas. Potential stimulus for release and modes of action are discussed.