Gordon Cramb

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.

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

BackgroundGenomic 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.ResultsTo 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.ConclusionsTogether 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 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.

Actions of atrial natriuretic peptide (ANP) on cyclic nucleotide concentrations and phosphatidylinositol turnover in ventricular myocytes

Atrial natriuretic peptide (ANP) stimulates cGMP production in isolated rabbit ventricular myocytes incubated in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (1mM). Half maximal activation was found at 10(-8)M ANP. Cellular cGMP concentrations of around 0.6 pmol/10(6) cells were elevated 4-6 fold by ANP (10(-6)M), 3-4 fold by carbachol (1mM) and around 10 fold by sodium nitroprusside (1mM). ANP had no effect on basal or isoprenaline-stimulated cAMP concentrations or on basal or noradrenaline-stimulated turnover of phosphatidylinositol. From these results we conclude that ANP receptors, coupled to particulate guanylate cyclase, exist in cardiac ventricular muscle. This indicates that ANP may also have a physiological action on ventricular muscle contractility during volume expansion.

Selective lysosomal uptake and accumulation of the beta-adrenergic antagonist propranolol in cultured and isolated cell systems.

The beta adrenoreceptor antagonist propranolol is rapidly taken up and accumulated in various cultured cell lines. When incubated in the presence of low concentrations of propranolol (10(-9) M), Hela (non-differentiated epithelia), BC3H1 (smooth muscle) and MDCK (differentiated kidney epithelia) cell cultures take up (t1/2 = 4-10 min) and accumulate the drug such that the intracellular concentration is over 1000 times that in the incubation medium. The release of propranolol from the cells was slower (t1/2 = 22 min) than the rate of uptake but the dissociation was stimulated by the addition of 1 microM propranolol to the external medium (t1/2 = 9 min). Uptake, which is non-stereoselective, is dependent on pH and is inhibited by the lysosomotropic agents, NH4Cl, methylamine and chloroquine. At higher concentrations (greater than 10(6) M), uptake is accompanied by a visual swelling of intracellular acidic vesicles staining with acridine orange. These results suggest that propranolol, a basic amphiphilic amine, is accumulated within the lysosomes of these cells. Uptake was confined to these cultured cell systems with no chloroquine-sensitive propranolol uptake, being found in isolated rabbit ventricular myocytes, red blood cells or blood platelets. Although alprenolol and cyanopindolol competed with propranolol for uptake, isoprenaline, adrenaline, noradrenaline, phenylephrine, atenolol, practolol and salbutamol were not effective inhibitors. The possible consequences of this uptake and accumulation of propranolol by certain tissues is discussed in relation to the known actions of the drug, particularly during or after abrupt withdrawal from chronic applications.

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.

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

BackgroundAmerican 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.ResultsThe 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.ConclusionsThese 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.