Christopher P. Cutler

PRIMARY SEQUENCE, TISSUE SPECIFICITY AND EXPRESSION OF THE NA+, K+-ATPASE ALPHA 1 SUBUNIT IN THE EUROPEAN EEL (ANGUILLA ANGUILLA)

The entire cDNA nucleotide sequence of the Na+,K(+)-ATPase alpha 1 isoform was cloned from the gills of the European eel (Anguilla anguilla) by a PCR based method. The amino acid sequence translated from the sequence shared 89.4 and 85.6% homology respectively with previously published Na+,K(+)-ATPase alpha subunit sequences from elasmobranch (Torpedo californica) and teleost (Catostomus commersoni) fish. The size of Na+,K(+)-ATPase alpha 1 mRNA transcripts in eel tissues was demonstrated to be 3.5 kb, except in the ovary where a 3.7 kb transcript existed. Na+,K(+)-ATPase alpha 1 mRNA was present at some level in all tissues investigated with the exception of cardiac and skeletal muscle where no Na+,K(+)-ATPase alpha 1 mRNA was detectable. The level of branchial Na+,K(+)-ATPase alpha 1 mRNA increased after the adaptation of freshwater eels to normal or double concentration seawater.

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.

Regulation of Na,K-ATPase by PLMS, the Phospholemman-like Protein from Shark MOLECULAR CLONING, SEQUENCE, EXPRESSION, CELLULAR DISTRIBUTION, AND FUNCTIONAL EFFECTS OF PLMS

In Na,K-ATPase membrane preparations from shark rectal glands, we have previously identified an FXYD domain-containing protein, phospholemman-like protein from shark, PLMS. This protein was shown to associate and modulate shark Na,K-ATPase activity in vitro. Here we describe the complete coding sequence, expression, and cellular localization of PLMS in the rectal gland of the shark Squalus acanthias. The mature protein contained 74 amino acids, including the N-terminal FXYD motif and a C-terminal protein kinase multisite phosphorylation motif. The sequence is preceded by a 20 amino acid candidate cleavable signal sequence. Immunogold labeling of the Na,K-ATPase α-subunit and PLMS showed the presence of α and PLMS in the basolateral membranes of the rectal gland cells and suggested their partial colocalization. Furthermore, through controlled proteolysis, the C terminus of PLMS containing the protein kinase phosphorylation domain can be specifically cleaved. Removal of this domain resulted in stimulation of maximal Na,K-ATPase activity, as well as several partial reactions. Both the E1∼P → E2-P reaction, which is partially rate-limiting in shark, and the K+ deocclusion reaction, E2(K) → E1, are accelerated. The latter may explain the finding that the apparent Na+ affinity was increased by the specific C-terminal PLMS truncation. Thus, these data are consistent with a model where interaction of the phosphorylation domain of PLMS with the Na,K-ATPase α-subunit is important for the modulation of shark Na,K-ATPase activity.

Molecular physiology of osmoregulation in eels and other teleosts: the role of transporter isoforms and gene duplication.

This review focuses on recent developments in the molecular biology of ion and water transporter genes in fish and the potential role of their products in osmoregulation in both freshwater and seawater environments. In particular details of isoforms of various ATPases, co-transporters, exchangers and ion channels in the eel as well as other teleost species are described. Many of the teleost transporter isoforms discovered so far, appear to occur as twin or duplicate copies compared to their homologous counterparts in higher vertebrates, although these duplicate isoforms often have distinct tissue-specific and developmental stage-dependent expression patterns. The possible meaning of this information will be examined in relation to the fish genome duplication debate.

Cloning and expression of three aquaporin homologues from the European eel (Anguilla anguilla): effects of seawater acclimation and cortisol treatment on renal expression

Background information. The European eel (Anguilla anguilla) is able to osmoregulate over a wide range of environmental salinities from FW (freshwater) to hyperconcentrated SW (seawater). Successful acclimation is associated with strict regulation of ion and water transport pathways within key osmoregulatory epithelia to enable animals to survive the dehydrating or oedematous conditions. These observations suggested that homologues of the AQP (aquaporin) water channel family were expressed in the eel and that these proteins may contribute to the water transport and osmoregulation in all euryhaline teleosts.

The Lineage-Specific Evolution of Aquaporin Gene Clusters Facilitated Tetrapod Terrestrial Adaptation

A major physiological barrier for aquatic organisms adapting to terrestrial life is dessication in the aerial environment. This barrier was nevertheless overcome by the Devonian ancestors of extant Tetrapoda, but the origin of specific molecular mechanisms that solved this water problem remains largely unknown. Here we show that an ancient aquaporin gene cluster evolved specifically in the sarcopterygian lineage, and subsequently diverged into paralogous forms of AQP2, -5, or -6 to mediate water conservation in extant Tetrapoda. To determine the origin of these apomorphic genomic traits, we combined aquaporin sequencing from jawless and jawed vertebrates with broad taxon assembly of >2,000 transcripts amongst 131 deuterostome genomes and developed a model based upon Bayesian inference that traces their convergent roots to stem subfamilies in basal Metazoa and Prokaryota. This approach uncovered an unexpected diversity of aquaporins in every lineage investigated, and revealed that the vertebrate superfamily consists of 17 classes of aquaporins (Aqp0 - Aqp16). The oldest orthologs associated with water conservation in modern Tetrapoda are traced to a cluster of three aqp2-like genes in Actinistia that likely arose >500 Ma through duplication of an aqp0-like gene present in a jawless ancestor. In sea lamprey, we show that aqp0 first arose in a protocluster comprised of a novel aqp14 paralog and a fused aqp01 gene. To corroborate these findings, we conducted phylogenetic analyses of five syntenic nuclear receptor subfamilies, which, together with observations of extensive genome rearrangements, support the coincident loss of ancestral aqp2-like orthologs in Actinopterygii. We thus conclude that the divergence of sarcopterygian-specific aquaporin gene clusters was permissive for the evolution of water conservation mechanisms that facilitated tetrapod terrestrial adaptation.

The effects of dietary sodium loading on the activity and expression of Na, K-ATPase in the rectal gland of the European Dogfish (Scyliorhinus canicula)

cDNA fragments of both the alpha- and beta-subunits of the Na, K-ATPase and a cDNA fragment of the secretory form of Na-K-Cl cotransporter from the European dogfish (Scyliorhinus canicula) were amplified and cloned using degenerate primers in RT-PCR. These clones were used along with a sCFTR cDNA from the related dogfish shark, Squalus acanthias to characterise the expression of mRNAs for these ion transporters in the dogfish rectal gland subsequent to an acute feeding episode. Following a single feeding event where starved dogfish were fed squid portions (20 g squid/kg fish), there was a delayed and transient 40-fold increase in the activity of Na, K-ATPase in crude rectal gland homogenates. Increases in enzyme activity were apparent 3 h after the feeding event and peaked at 9 h before returning to control values within 24 h. These increases in activity were accompanied by small and transient decreases in plasma sodium and chloride concentrations lasting up to 3 days. Significant increases in the expression of mRNAs for alpha- and beta-subunits of the Na, K-ATPase, the Na-K-Cl cotransporter and CFTR chloride channel were detected but not until 1-2 days after the feeding event. It is concluded that the transient increase in Na, K-ATPase activity is not attributable to increases in the abundance of alpha- and beta-subunit mRNAs but must be associated with some, as yet unknown, post-transcriptional activation mechanism.

Expression of Gill Vacuolar-Type H+-ATPase B Subunit, and Na+, K+-ATPase α1 and β1 Subunit Messenger RNAs in Smolting Salmo salar

Abstract Changes in gill vacuolar-type H+-ATPase B subunit, and Na+,K+-ATPase α and β subunit mRNA expression were examined during the course of smoltification in Salmo salar. We cloned and sequenced cDNA fragments of S. salar gill i) vacuolar-type H+-ATPase (V-H+-ATPase) B subunit, ii) Na+,K+ATPase α1 subunit, and iii) Na+,K+-ATPase β1 subunit, and used these as Northern blotting probes. During smoltification, the salmon showed a typical increase in gill Na+,K+-ATPase activity and improved hypo-osmo-regulatory ability as judged by their ability to regulate plasma [Cl−] in a 24-hr seawater challenge test (35 ppt). Gill Na+,K+-ATPase α1 and β1 subunit mRNA levels were regulated at a constant ratio during smoltification. Both transcripts were elevated during the build-up of gill Na+,K+-ATPase activity, underlining the importance of increased mRNA levels for increased enzyme activity. Gill V-H+-ATPase B subunit mRNA levels were high during the early phase of smoltification. These results support our hypothesis that gill V-H+-ATPase expression may be elevated during the early stages of smoltification in order to counter the effects of increased ionic efflux when in FW. The peak smolt stage was, however, characterized by simultaneously elevated gill Na+,K+ATPase expression and low V-H+-ATPase expression, and possibly ensures the complete transformation of the gill into a hypo-osmoregulatory organ and hence the development of optimal SW-tolerance of the smolt.

Expression and functional characterization of four aquaporin water channels from the European eel (Anguilla anguilla).

SUMMARY The European eel is a euryhaline teleost which has been shown to differentially up- and downregulate aquaporin (AQP) water channels in response to changes in environmental salinity. We have characterized the transport properties of four aquaporins localized to osmoregulatory organs – gill, esophagus, intestine and kidney. By sequence comparison these four AQP orthologs resemble human AQP1 (eel AQP1), AQP3 (eel AQP3) and AQP10 (AQPe). The fourth member is a duplicate form of AQP1 (AQP1dup) thought to arise from a duplication of the teleost genome. Using heterologous expression in Xenopus oocytes we demonstrate that all four eel orthologs transport water and are mercury inhibitable. Eel AQP3 and AQPe also transport urea and glycerol, making them aquaglyceroporins. Eel AQP3 is dramatically inhibited by extracellular acidity (91% and 69% inhibition of water and glycerol transport respectively at pH 6.5) consistent with channel gating by protons. Maximal water flux of eel AQP3 occurred around pH 8.2 – close to the physiological pH of plasma in the eel. Exposure of AQP-expressing oocytes to heavy metals revealed that eel AQP3 is highly sensitive to extracellular nickel and zinc (88.3% and 86.3% inhibition, respectively) but less sensitive to copper (56.4% inhibition). Surprisingly, copper had a stimulatory effect on eel AQP1 (153.7% activity of control). Copper, nickel and zinc did not affect AQP1dup or AQPe. We establish that all four eel AQP orthologs have similar transport profiles to their human counterparts, with eel AQP3 exhibiting some differences in its sensitivity to metals. This is the first investigation of the transport properties and inhibitor sensitivity of salinity-regulated aquaporins from a euryhaline species. Our results indicate a need to further investigate the deleterious effects of metal pollutants on AQP-containing epithelial cells of the gill and gastrointestinal tract at environmentally appropriate concentrations.

Primary sequence, tissue specificity and mRNA expression of the Na+,K+ -ATPase ?1 subunit in the European eel (Anguilla anguilla)

The entire amino acid coding sequence of the Na+,K+-ATPase β1 isoform was cloned from the gill of the European eel (Anguilla anguilla) by a PCR based method. The amino acid sequence translated from the nucleotide sequence shared 61.4 and 56.2% homology respectively with previously published Na+,K+-ATPase β1 isoform sequences from the clawed toad (Xenopus laevis) and the ray (Torpedo californica) an elasmobranch fish. The size of the Na+,K+-ATPase β1 mRNA transcript in eel tissues was demonstrated to be 2.35 Kb. Detectable levels of Na+,K+-ATPase β1 mRNA were found at some level in all tissues except liver and cardiac muscle. The level of branchial Na+,K+-ATPase β1 mRNA was observed to increase after the adaptation of fresh water eels to normal or double concentration sea water.