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,25-dihydroxyvitamin D3 in Atlantic salmon (Salmo salar)
1 National Institute of Nutrition and Seafood Research, PO Box 2029 Nordnes, N-5817, Bergen, Norway
2 Department of Animal Physiology, Faculty of Science, Radboud University Nijmegen, Toernooiveld 1 NL-6525ED, Nijmegen, The Netherlands
3 Division of Pediatrics, Department of Clinical and Molecular Medicine, University of Bergen, Haukeland University Hospital, N-5021, Bergen, Norway
(Requests for offprints should be addressed to G Flik; Email: g.flik{at}science.ru.nl)
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Abstract |
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Introduction |
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-hydroxylase (1OHase), leads to an increase in the plasma level of calcitriol (Brown et al. 1999). The vitamin D system appears to function in a similar fashion in fish. Indeed, all three major vitamin D metabolites are found in fish (Graff et al. 1999, Suzuki et al. 2000, Larsson et al. 2003) and the VDR has been sequenced in Japanese flounder (Suzuki et al. 2000), zebrafish (NM130919), and common carp (AJ784084 [GenBank] ). In European eel, Anguilla anguilla, calcitriol administration in vivo increased serum phosphate concentration (MacIntyre et al. 1976), and in American eel, Anguilla rostrata, it increased intestinal calcium absorption (Fenwick et al. 1984). This is interesting because in contrast to mammals, fish are not particularly dependent upon food for their calcium. The major fraction of calcium required by the fish is taken up directly from the water via the gills (Verbost et al. 1994) and the uptake rate is largely independent of the water calcium concentration. Dietary calcium load may decrease branchial uptake to avoid overload and to ensure the required net influx for homeostasis and growth. In addition, vitamin D is involved in phosphorus metabolism. Vielma & Lall (1998a) hypothesized about a negative relation between phosphorus concentration in the diet and 25(OH)D3 content in the liver of Atlantic salmon. A hyperphosphatemic effect of vitamin D has been described in eel, A. anguilla (Fenwick et al. 1984), catfish, Clarias batrachus (Swarup et al. 1984), and carp, Cyprinus carpio (Swarup et al. 1991).
Mineral absorption (including that of calcium and phosphorus) requires regulated (primary or secondary) active transport processes. In fish, the antihypercalcemic stanniocalcin (Wagner et al. 1998), the hypercalcemic calciotropes prolactin (PRL), and parathyroid hormone-related protein (PTHrP) dominate the regulation of calcium handling (Abbink et al. 2006); little is known about the presumed hypercalcemic role of calcitriol in fish. Seawater (SW) fish such as sea bream do express PTHrP, which was shown to exert hypercalcemic actions (Guerreiro et al. 2001, Abbink et al. 2006). The control of mineral homeostasis may be different in marine and freshwater (FW) environments (Brown et al. 1991, Larsson et al. 1995).
During smoltification, many physiological changes take place induced by various hormones. One of the most prominent changes during smoltification is the increase in levels of growth hormone (GH; Ágústsson et al. 2001, 2003). GH has an important role in stimulating growth and improving hypo-osmoregulatory ability, which is accomplished through a multitude of mechanisms. One of the most important mechanism is the increase in branchial Na+/K+-ATPase activity to provide the fish with SW-type chloride cells (McCormick 1996); isoform switch may be a second phenomenon in this adaptation. Ágústsson et al.(2003) measured an increase of both GH and PRL mRNA expression during smoltification. After SW transfer, GH mRNA increased; PRL mRNA expression decreased steeply. GH levels increase even further in SW, facilitating a period of fast growth for Atlantic salmon. This period of fast growth demands the supply of many nutrients, especially calcium and phosphorus for skeletal growth.
In this report, we have evaluated plasma vitamin D metabolite levels and quantified Atlantic salmon VDR (sVDR) expression in the three main tissues (gills, intestine, and kidney) involved in calcium and/or phosphorus homeostasis in Atlantic salmon, Salmo salar. We quantified plasma vitamin D metabolites and nuclear VDR mRNA levels in FW salmon, SW salmon, and intermediate stages during parr–smolt transformation.
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Materials and Methods |
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FW-adapted parr and SW-adapted adult Atlantic salmon (NLA strain, Aqua Gen AS, Trondheim, Sør Trøndelag, Norway) were obtained from Matre Aquaculture Research Centre (Institute of Marine Research, Bergen, Norway). Fish were anesthetized using 0.3 ml/l benzocaine, sampled for blood and subsequently killed by a blow to the head. Tissue was immediately sampled as described below. Fish were not fed 24 h before sampling.
Smolts (Bolaks strain, Tomre Fiskeanlegg AS, Eikelandsosen, Norway) were provided by the Institute of Marine Research, Bergen, Norway. Smolts were sampled prior to SW exposure, 7 days after transfer to a mixture of 50% FW and 50% SW, and 7 days after transfer to 100% SW. Fish were anesthetized, killed, and sampled similar to parr and adult.
Blood and tissue collection
Immediately after anesthesia blood was collected from the caudal vessels using heparized syringes fitted with 24-gauge needles; the blood was kept on ice. After centrifugation at 1811 g for 15 min at 4 °C, the plasma thus obtained was collected and stored at –80 °C until analysis for 25(OH)D3, 1,25(OH)2D3, and 24R,25(OH)2D3, as described by Aksnes (1980).
The fish were killed by spinal transection and gill, gut (the section posterior to the caecae) and kidney tissue were collected and stored in RNAlater (Ambion, Fostor City, CA, USA). A mid-piece of the left second gill arch of the fish was taken. The gut was flushed with saline to remove food and then stored in RNAlater. The peritoneal cavity was opened and the kidney exposed; a piece from the left side was sampled. Sampling of the parr and adult fish was carried out at about 2-h travel distance from the institute. To standardize the procedure, all samples were stored 24 h at 4 °C in RNAlater before they were frozen at –80 °C until RNA extraction.
Cloning and sequencing
To obtain the nucleotide sequence of the sVDR, two oligonucleotide primers, SSfw and SSrv (Table 1
) were designed based on data of two teleosts, the zebrafish (Brachydanio rerio) and Japanese flounder (Paralichthys olivaceus). A piece of the mid-intestine was homogenized in TRIzol reagent (Invitrogen) and RNA isolated according to the manufacturer’s instructions. One microgram RNA was reverse-transcribed with 300 ng random primers, 10 nmol dNTPs, 200 nmol, 10 U RNase inhibitor, and 200 U RT Superscript II (Gibco-BRL) for 50 min at 37 °C. PCR with the SSfw and SSrv primers on the cDNA yielded a partial sVDR sequence. The 5' and 3' ends of the sequence were obtained by rapid amplification of cDNA ends (RACE)-PCR (GeneRacer, Invitrogen) according to the manufacturer’s protocol, including the use of nested PCR. Gene-specific primers used for the RACE-PCR are found in Table 1.
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Phylogenetic analysis
Sequences of available vertebrate sVDR receptors were retrieved from the NCBI site (www.ncbi.nih.gov), using the GenBank database. Multiple-sequence alignment was carried out with ClustalWat the EMBL website (www.ebi.ac.uk/clustalw/) from the European Bioinformatics Institute. Phylogenetic and molecular evolutionary analyses were conducted using molecular evolutionary genetics analyses (MEGA) version 3.1 (Kumar et al. 2004) and a phylogenetic tree was constructed on the basis of amino acid difference (p-distance) with a neighbour-joining method with 1000 bootstrap replications.
Tissue distribution
Salmon from the NLA strain (Aqua Gen AS), smoltified 6 months prior to sampling, were used for the tissue distribution analysis of sVDR. Fresh samples of pyloric caecae, midgut, hindgut, head kidney, kidney, spleen, liver, ovary, muscle, heart, and brain were homogenized using MagNA Lyser Green Beads (Roche) with Trizol (Invitrogen). Skin, gill, and vertebra samples were flash-frozen in liquid N2 and homogenized in a mortar prior to Trizol extraction. Total RNA was purified using Trizol extraction and subjected to DNAse treatment (DNA-free, Ambion). RNA quantity and quality (A260/280) was assessed using a NanoDrop ND100 spectrophotometer (Saveen Biotech, Wilmington, DE, USA) and by gel electrophoresis using an Agilent 2100 Bioanalyzer (Agilent Technologies). cDNA and PCR products were prepared using the OneStep RT-PCR kit (Qiagen) with gene-specific primers for sVDR (OSVDRF and OSVDRR, Table 1
) and 18S (AJ427629
[GenBank]
). One microgram RNA was used in all reactions. Thermal cycling conditions were 30 min at 50 °C (RT), 15 min at 95 °C, 35 cycles with 40 s at 94 °C, 1 min at 55 °C, 1 min at 72 °C, and a final elongation step with 10 min at 72 °C. PCR products were separated on a 2% agarose gel at 80 V (0.16 A) for 1 h using ethidium bromide as a marker.
Real-time quantitative PCR
Expression levels of sVDR mRNA were assessed by the use of quantitative RT-PCR. Subsequent to Trizol extraction, RNA was DNase treated to remove possible traces of genomic DNA using the DNA-free kit (Ambion). Briefly, RNA was diluted with 0.1 volume DNase buffer and incubated with two units rDNase I for 30 min at 37 °C. To inactivate DNase, 0.1 volume of DNase Inactivation reagent was added and the sample incubated for 2 min at room temperature. RNA quantity and quality (A260/280) were assessed using an Eppendorf Biophotometer. The RNA was reverse-transcribed using the TaqMan Reverse Transcription Reagents kit (Applied Biosystems). Briefly, solutions were (final concentration): MgCl2 (5 mM), PCR buffer II (1 x volume), dNTPs (1 mM each), RNase Inhibitor (1 U/µl), random hexamers (18S) or oligo d(T)16 primers (2.5 µM), and MuLV Reverse Transcriptase (2.5 U/µl) in a total volume of 20 µl. After an initial step of 10-min incubation at 25 °C, reverse transcription was performed for 60 min at 48 °C followed by an inactivation step for 5 min at 95 °C and a decrease to 4 °C. For quantitative PCR analysis, 5 µl of five times diluted RT-mix was used as template in 25 µl amplification mixture, containing 12.5 µl SYBR Green Master Mix (Applied Biosystems) and 3 µl each primer (final concentration 600 nM). Primers were designed using Primer Express 2.0. The sequences of the primer sets used in the quantitative PCR for sVDR (QVDRfw and QVDRrv) and the housekeeping genes 18S (Q18Sfw and Q18Srv) and elongation factor 1-a (QEL1fw and QEL1rv) are found in Table 1. All primer sets had an efficiency >98% and all products showed a single melting transition. After an initial step at 95 °C for 10 min, a real-time quantitative PCR of 40 cycles was performed (GeneAmp 5700, Applied Biosystems), each cycle consisting of 15-s denaturation at 95 °C and 1-min annealing and extension at 60 °C. Cycle threshold values were determined and expression was quantified using the relative expression software tool (REST) program (Pfaffl et al. 2002). All samples were assayed in triplicate. Both housekeeping genes gave similar results. Results are presented here only relative to 18S expression.
Statistical analysis
Statistical analysis of sVDR mRNA expression was analyzed using the REST program (Pfaffl et al. 2002). Vitamin D metabolite data are expressed as means ± standard deviation (S.D.); differences among groups were assessed by ANOVA. Significance of differences was assessed by parametric (Student’s t-test) or non-parametric (Mann–Whitney U-test), tests where appropriate and P<0.05 was taken as fiducial limit.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Plasma levels of 1,25(OH)2D3 (Table 2) increase significantly (P<0.05) in FW smolt (1.19 ± 0.45 nM) when compared with FW parr (0.46 ± 0.22 nM). The highest plasma levels of 1,25(OH)2D3 (4.20 ± 1.00 nM) were found in the transition phase, when the fish were in 50% FW/50% SW, which is about nine times higher than the value for parr. Plasma levels of 1,25(OH)2D3 decrease again after transfer to 100% SW (0.98 ± 0.43 nM) and stay at this level in adult fish (0.96 ± 0.27 nM), a value two times higher than that observed in parr. No significant changes in plasma concentrations of either 25(OH)D3 or 24,25(OH)2D3 were found between any of the groups, although significant variations in values between individual fish within the same group occurred.
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The full-length 1684 bp cDNA sVDR0 sequence contains an open reading frame encoding a protein of 423 amino acids (Fig. 1; EMBL database: accession number AJ780914
[GenBank]
). During 3' RACE, two isoforms of the sVDR0, viz. sVDR1 (AM238619
[GenBank]
, Fig. 2) and sVDR2 (AM238620
[GenBank]
, Fig. 3), were identified. We compared the sVDR0 sequence with that present in the zebrafish VDR genome and identified eight exons in the sVDR0 (start of each exon is boxed in gray in Fig. 1). In both spliced variants sVDR1 and sVDR2, the first two base pairs differed from the sVDR0 and are ‘gt’ (boxed in Figs 2 and 3), which is the characteristic signal for the start of an intron. The strongest evidence for intron retention is probably the place of the splicing site, which is exactly in between two exons. We therefore conclude that the isoforms sVDR1 and sVDR2 are both generated from the primary transcript by alternative splicing due to intron retention.
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The relationship between the salmon canonical sVDR0 and orthologs of other species is presented in a consensus phylogenetic tree (Fig. 5). VDRs are rather conserved in vertebrates (Fig. 4), indicated by relatively high sequence identities in the fish line (Danio rerio 89%; P. olivaceus type a 90%; type b 87%) and with that of Homo sapiens (70%). The proper clustering of the fish VDR sequences (indicated in gray) and separate from amphibia, reptiles, birds, and mammals is in line with the consensus about evolution of the vertebrates (Fig. 5).
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To assess the tissue distribution of the sVDR gene expression, we used RT-PCR (Fig. 6). The predicted 470 bp sVDR was found throughout the intestine, head kidney, kidney, pituitary, ovary, muscle, and gill. We could not detect sVDR expression in the spleen, heart, liver, and skin. Attempts to demonstrate sVDR in skeletal tissue failed, which we relate to the difficult extraction of RNA from this tissue. However, in other unpublished studies, we succeeded to obtain sVDR signal from both bone and scales. The midgut RT, which served as control for potential genomic DNA contamination, was negative. The preparation of cDNA was assumed to be, judged on the successful amplification of an 87 bp 18S fragment in all organs.
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Due to low expression of sVDR1 and sVDR2 (500–1000 times lower than sVDR0), we were not able to get reliable results on their quantitative expression. We therefore decided to design one primer set over an intron and at the beginning of the sequence to include all three transcripts sVDR0, sVDR1, and sVDR2 in our quantitative analyses. sVDR expression in the kidney (Table 3) is 2.8-fold down-regulated (P = 0.01) in FW smolt when compared with parr. During SW transfer, the sVDR is up-regulated to levels intermediate between parr and FW smolt. In adult SW fish, the expression of the sVDR is again down-regulated and threefold (P = 0.015) lower than that in parr, but not significantly different from FW smolt.
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The expression of sVDR in the gill of FW smolt is down-regulated 2.4-fold (P = 0.0015) when compared with parr. During SW transfer, sVDR expression is up-regulated to the same level as in parr. sVDR expression in adult fishes is significantly decreased (P<0.05) when compared with parr, 50% SW and SW smolt, but not significantly different from FW smolt.
In Figs 7–9, we plotted calcitriol plasma levels against sVDR mRNA expression in kidney, gill, and intestine respectively. Each graph shows six individual fish from groups ‘parr’, ‘FW smolt’, and ‘adult’. In all tissues, we see a negative correlation between calcitriol plasma levels and sVDR mRNA expression. Data on parr are clustering separately from FW smolt and adult fish.
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Discussion |
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VDRs in salmon
Intron retention has been demonstrated for a mouse VDR once before (Ebihara et al. 1996). This is the first report for such a phenomenon outside mammals, in a teleostean VDR. Intron retention of both the sVDR1 (intron 7) and sVDR2 (intron 6) occurs in the E/F domain and as a consequence both receptors are predicted to be impaired in retinoid X receptor (RXR) heterodimerization and ligand binding. The splicing of the murine VDR occurs at intron 8, and also in the E/F domain (Ebihara et al. 1996), and it was concluded that homodimer-spliced variants may compete at specific VDR responsive elements and by doing so prevent actions of the canonical VDR. We can only speculate on a similar role for the two spliced variants in salmon. However, we predict that the vast majority (>99%) of the sVDR mRNA consists of the canonical receptor sVDR0, making this transcript presumably the most important in Atlantic salmon.
However, low mRNA levels do not, by default, exclude a role for the spliced variants and it is important to make some remarks on the nature of these variants. A common feature among the spliced variants known to us is that splicing always occurs in the E/F domain. Indeed, in common carp (C. carpio) VDR (AJ784084 [GenBank] ), we found two alternatively spliced variants that result from intron retention, again in the E/F domain. Cypriniformes (belonging to the Ostariophysi) and Salmoniformes (belonging to the Protacanthopterygi; Nelson 1994) share a common ancestor in the Cretaceous period some 130–140 million years ago. Teleosts and mammals shared a common ancestor 450 million years ago (Hoegg & Meyer 2005). The ‘survival’ of spliced variants during a significant time of evolution suggests a functional role for the variants.
A recently proposed conformational ensemble model for VDR activity by Norman (2006) describes that the chirality of calcitriol may translate into either slow classical genomic or more rapid responses previously assigned to a putative membrane receptor. The canonical salmon VDR and splice variant sVDR1 could function along the model proposed by Norman (2006); the splice variant sVDR2 lacks amino acids R274 and S278, two residues that are crucial for ligand stabilization and their absence could impair functionality of this receptor. As both splice variants are impaired in their heterodimerization with the RXR (Ebihara et al. 1996), genomic actions seem excluded, and rapid membrane-bound receptor activity remains a possibility. Indeed, it now appears that the VDR in some cells is associated with caveolae in or near the plasma membrane (Huhtakangas et al. 2006), which would give an anatomical argument for plasma membrane-associated VDR activity. Clearly, this puzzle is very complex and warrants further research.
From FW to SW: increasing calcitriol and decreasing sVDR
Plasma calcitriol levels more than doubled in SW adult salmon when compared with FW parr. Simultaneously, the mRNA expression of sVDR decreases in gill, intestine, and kidney. Three important considerations concerning the sVDR and its ligand calcitriol should be advanced before we can further hypothesize about the function of the vitamin D endocrine system in this salmon. First, the affinity of the sVDR to calcitriol is unknown and awaits transactivation studies. Secondly, we have no information on metabolic clearance and secretion rates or distribution space of calcitriol in FW and/or SW Atlantic salmon. Thirdly, both slow genomic responses as well as more rapid responses of calcitriol exist that require different regulation. This means we cannot solely depend on calcitriol and sVDR data to understand the calcitriol system completely.
As mentioned in the considerations above, rapid responses to calcitriol exist: within minutes after administration, calcitriol increases intestinal calcium transport and activates protein kinase C in FW fish (Nemere et al. 2000) and mammals (Boyan et al. 1999, Wali et al. 2003). Larsson et al.(2003) conducted binding studies to enterocyte membrane receptors for calcitriol in rainbow trout (Oncorhynchus mykiss), a close relative of salmon. They demonstrated that after acclimation to SW specific binding of calcitriol decreased, which they interpreted as a down-regulation of a putative membrane VDR due to a decreased need for short-term calcitriol-driven calcium transport. The longer-term calcitriol actions via the nuclear VDR have not been studied in salmon before. Increasing plasma calcitriol concentrations do not, by default, result in increased stimulation of vitamin D responsive elements. Studies in humans (Ebeling et al. 1992) and rats (Horst et al. 1990) have demonstrated that intestinal resistance to calcitriol exists and is related to aging. Despite increasing plasma calcitriol levels intestinal calcium absorption decreases, due to decreased intestinal VDR levels. This phenomenon contributes to age-related bone loss in humans and rats. As mentioned before, immediately following SW transfer, Atlantic salmon experience a growth spurt that requires large quantities of both calcium and phosphorus. It is unlikely and unwanted that decreasing levels of sVDR result in a decrease in calcium uptake. Larsson et al.(2003) furthermore found an increase in 24,25(OH)2D3 plasma levels and specific binding of 24,25(OH)2D3 to enterocyte membranes in rainbow trout after SW transfer. The time course in the experiment of Larsson was much shorter (1 week) and fish were transferred from FW to SW immediately; therefore the results from these different experiments cannot be compared directly. We did not find significant changes in 24,25(OH)2D3 plasma levels in this study. Especially, in the smolt groups the variation in 24,25(OH)2D3 plasma levels between fish within the same group was rather large. Apparently, SW exposure arouses the vitamin D system, but assessment of 24,25(OH)2D3 activity may require another experimental design. Indeed, our experiments primarily focussed on calcitriol.
Phosphorus is also an important mineral for bone formation and its uptake is regulated by vitamin D (Cross et al. 1990). Like FW, SW contains normally little or no free phosphorus and the major source of phosphorus is the diet. Vielma & Lall (1998a,b) demonstrated that control of phosphorus homeostasis in Atlantic salmon is similar to that in vertebrates. Phosphorus absorption occurs via a regulated process in the intestine. The kidney contributes to phosphorus conservation to guarantee sufficient supply for bone formation (fish deposit apatites in their bone as do mammals; Flik et al. 1986). Phosphorus is a major waste product in aquaculture and phosphorus wasting is intentionally kept to a minimum. Fish feeds contain generally about as much phosphorus as the fish requires for homeostasis and growth, and might at some stages even be the key limiting diet component. A low-Pi diet leads to increased plasma levels of calcitriol, via stimulation of 1OHase activity. Conversely, increased concentrations of vitamin D3 in the diet lead to increased plasma Pi levels in rainbow trout (Avila et al. 1999). Dietary phosphorus requirement for Atlantic salmon has been studied (Vielma & Lall 1998a,b), but no data on vitamin D metabolites were included. Phosphorus absorption is not entirely regulated by the nuclear VDR. Low levels of 1,25(OH)2D3 elicit a rapid increase (4–8 min after administration) in Pi transport in the perfused duodenal loop of normal chicks (Nemere 1996). Such fast effects suggest a signal transduction pathway independent of genomic regulation. Moreover, it suggests a (direct) coupling of calcitriol and phosphorus transport mechanisms. Indeed, if this direct coupling exists in Atlantic salmon than the predicted enhanced need for phosphorus during the growth spurt of the salmon at sea would clearly benefit from higher plasma calcitriol levels to guarantee maximum phosphorus uptake and retention within physiological limits. With the differences in calcium and phosphorus availability and uptake routes mentioned above, the picture emerges of a dual uptake route for calcium (gills and intestine) and a single route for phosphorus (intestine). The ion-transporting cells in fish gills (chloride cells; Flik et al. 1996) and intestine (enterocytes) thus differ in their make-up for calcium uptake or combined calcium and phosphorus uptake respectively and it is interesting to elucidate the role of the vitamin D endocrine system in these transports.
However, we must realize that unlike mammals that are dependent upon their food for calcium, fish have access to an unlimited amount of calcium via the ambient water. In fish, it is phosphorus availability that is limited, and fast non-genomic responses in phosphorus uptake are therefore highly desirable. Further research is needed to investigate the role of the vitamin D endocrine system in short- and long-term phosphorus uptake in salmon.
Smoltification and vitamin D: pre-adaptation to SW
During smoltification, the fish prepares itself for life at sea and undergoes morphological as well as physiological changes. External stimuli like light trigger the smoltification process, and several hormones involved in the smoltification process (GH, PRL) have been identified (McCormick et al. 1987). On the basis of this study calcitriol can be added to the list of hormones involved or taking part in the smollification process. Compared with parr, calcitriol plasma level and sVDR mRNA expression in FW smolts significantly (P<0.05) increase and decrease respectively these levels are retained in SW adult fish. There is no significant difference (P>0.05) in calcitriol plasma levels and sVDR mRNA levels between FW smolt and SW adult salmon. This indicates that during smoltification, the calcitriol–sVDR axis in FW is changing to the SW status. Plasma calcitriol levels during SW transfer significantly fluctuate. They are highest in 50% SW smolts, about 3–4 times higher than FWor SW smolts. This is in line with observations in rainbow trout (Larsson et al. 2003). One day after SW transfer calcitriol plasma levels in trout peaked and decreased with time spent in SW.
Changes in sVDR mRNA level and plasma calcitriol in our study do substantiate a role for the vitamin D endocrine system in the physiology of Atlantic salmon. The observation of sVDR variants that are spliced in one of the key functional protein domains and expressed at mRNA level in both teleost and mammalian species warrants further research for a more complete understanding of the vitamin D endocrine system.
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Acknowledgements |
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Funding |
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References |
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Received in final form 19 March 2007
Accepted 20 March 2007
Made available online as an Accepted Preprint 20 March 2007
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