Drinking and Water Handling in the Medaka Intestine: A Possible Role of Claudin-15 in Paracellular Absorption?

When euryhaline fish move between fresh water (FW) and seawater (SW), the intestine undergoes functional changes to handle imbibed SW. In Japanese medaka, the potential transcellular aquaporin-mediated conduits for water are paradoxically downregulated during SW acclimation, suggesting paracellular transport to be of principal importance in hyperosmotic conditions. In mammals, intestinal claudin-15 (CLDN15) forms paracellular channels for small cations and water, which may participate in water transport. Since two cldn15 paralogs, cldn15a and cldn15b, have previously been identified in medaka, we examined the salinity effects on their mRNA expression and immunolocalization in the intestine. In addition, we analyzed the drinking rate and intestinal water handling by adding non-absorbable radiotracers, 51-Cr-EDTA or 99-Tc-DTPA, to the water. The drinking rate was >2-fold higher in SW than FW-acclimated fish, and radiotracer experiments showed anterior accumulation in FW and posterior buildup in SW intestines. Salinity had no effect on expression of cldn15a, while cldn15b was approximately 100-fold higher in FW than SW. Despite differences in transcript dynamics, Cldn15a and Cldn15b proteins were both similarly localized in the apical tight junctions of enterocytes, co-localizing with occludin and with no apparent difference in localization and abundance between FW and SW. The stability of the Cldn15 protein suggests a physiological role in water transport in the medaka intestine.

Salinity-dependent expression of ncc2 in opercular epithelium and gill of mummichog (Fundulus heteroclitus).

Mummichogs (Fundulus heteroclitus) can tolerate abrupt changes in environmental salinity because of their ability to rapidly adjust the activities of ionocytes in branchial and opercular epithelia. In turn, the concerted expression of sub-cellular effectors of ion transport underlies adaptive responses to fluctuating salinities. Exposure to seawater (SW) stimulates the expression of Na+/K+/2Cl- cotransporter 1 (nkcc1) and cystic fibrosis transmembrane regulator (cftr) mRNAs in support of ion extrusion by SW-type ionocytes. Given the incomplete understanding of how freshwater (FW)-type ionocytes actually operate in mummichogs, the transcriptional responses essential for ion absorption in FW environments remain unresolved. In a subset of species, a 'fish-specific' Na+/Cl- cotransporter denoted Ncc2 (Slc12a10) is responsible for the uptake of Na+ and Cl- across the apical surface of FW-type ionocytes. In the current study, we identified an ncc2 transcript that is highly expressed in gill filaments and opercular epithelium of FW-acclimated mummichogs. Within 1 day of transfer from SW to FW, ncc2 levels in both tissues increased in parallel with reductions in nkcc1 and cftr. Conversely, mummichogs transferred from FW to SW exhibited marked reductions in ncc2 concurrent with increases in nkcc1 and cftr. Immunohistochemical analyses employing a homologous antibody revealed apical Ncc2-immunoreactivity in Na+/K+-ATPase-immunoreactive ionocytes of FW-acclimated animals. Our combined observations suggest that Ncc2/ncc2-expressing ionocytes support the capacity of mummichogs to inhabit FW environments.

Differential Expression and Localization of Branchial AQP1 and AQP3 in Japanese Medaka (Oryzias latipes).

Aquaporins (AQPs) facilitate transmembrane water and solute transport, and in addition to contributing to transepithelial water transport, they safeguard cell volume homeostasis. This study examined the expression and localization of AQP1 and AQP3 in the gills of Japanese medaka (Oryzias latipes) in response to osmotic challenges and osmoregulatory hormones, cortisol, and prolactin (PRL). AQP3 mRNA was inversely regulated in response to salinity with high levels in ion-poor water (IPW), intermediate levels in freshwater (FW), and low levels in seawater (SW). AQP3 protein levels decreased upon SW acclimation. By comparison, AQP1 expression was unaffected by salinity. In ex vivo gill incubation experiments, AQP3 mRNA was stimulated by PRL in a time- and dose-dependent manner but was unaffected by cortisol. In contrast, AQP1 was unaffected by both PRL and cortisol. Confocal microscopy revealed that AQP3 was abundant in the periphery of gill filament epithelial cells and co-localized at low intensity with Na+,K+-ATPase in ionocytes. AQP1 was present at a very low intensity in most filament epithelial cells and red blood cells. No epithelial cells in the gill lamellae showed immunoreactivity to AQP3 or AQP1. We suggest that both AQPs contribute to cellular volume regulation in the gill epithelium and that AQP3 is particularly important under hypo-osmotic conditions, while expression of AQP1 is constitutive.

Prolactin controls Na+,Cl- cotransporter via Stat5 pathway in the teleost gill.

In some freshwater fish species, the control of gill Na, Cl cotransporter (Ncc2b) by prolactin appears to be instrumental to ionic homeostasis. This study was carried out to examine the signaling pathways involved in prolactin-mediated salt retention using gill explants from Japanese medaka (Oryzias latipes). Ovine prolactin induced a concentration-dependent stimulation of ncc2b with significant effects of 10, 100 and 1000 ng of hormone per mL media (2-6 fold). To understand the molecular mechanisms mediating prolactin control of gill function, we analyzed effects on signaling pathways known to be involved in the hormones action in other systems, namely Stat5, Akt and Erk1/2. Their activation was examined in a time course and concentration response experiment. Prolactin (1 µg mL-1) induced a rapid phosphorylation (stimulation) of Stat5 (10 min) that reached a plateau after 30 min and was maintained for at least 120 min. The effect of prolactin on Stat5 phosphorylation was concentration-dependent (4-12 fold). No activation of Akt or Erk1/2 was observed in either experiment. The Stat5 activation was further investigated in localization studies that demonstrated strong nuclear expression of phosphorylated Stat5 in prolactin-treated gill ionocytes. Using specific inhibitors, we analyzed the signalling pathways mediating prolactin induction of gill ncc2b. Co-incubation experiments showed that Stat5 inhibition blocked prolactin's stimulation of ncc2b expression, while PI3K-Akt and Mek1/2-Erk1/2 pathway inhibitors had no effect. These findings show that ncc2b expression is dependent on prolactin's downstream activation of Stat5 and its subsequent nuclear translocation within branchial ionocytes.

claudin-10 isoform expression and cation selectivity change with salinity in salt-secreting epithelia of Fundulusheteroclitus.

To provide insight into claudin (Cldn) tight junction (TJ) protein contributions to branchial salt secretion in marine teleost fishes, this study examined cldn-10 TJ protein isoforms of a euryhaline teleost (mummichog; Fundulus heteroclitus) in association with salinity change and measurements of transepithelial cation selectivity. Mummichogs were transferred from freshwater (FW) to seawater (SW, 35‰) and from SW to hypersaline SW (2SW, 60‰) in a time course with transfer control groups (FW to FW, and SW to SW). FW to SW transfer increased mRNA abundance of cldn-10d and cldn-10e twofold, whilst cldn-10c and cldn-10f transcripts were unchanged. Transfer from SW to 2SW did not alter cldn-10d, and transiently altered cldn-10e abundance, but increased cldn-10c and cldn-10f fourfold. This was coincident with an increased number of single-stranded junctions (observed by transmission electron microscopy). For both salinity transfers, (1) cldn-10e mRNA was acutely responsive (i.e. after 24 h), (2) other responsive cldn-10 isoforms increased later (3-7 days), and (3) cystic fibrosis transmembrane conductance regulator (cftr) mRNA was elevated in accordance with established changes in transcellular Cl- movement. Changes in mRNA encoding cldn-10c and -10f appeared linked, consistent with the tandem repeat locus in the Fundulus genome, whereas mRNA for tandem cldn-10d and cldn-10e seemed independent of each other. Cation selectivity sequence measured by voltage and conductance responses to artificial SW revealed Eisenman sequence VII: Na+>K+>Rb+∼Cs+>Li+ Collectively, these data support the idea that Cldn-10 TJ proteins create and maintain cation-selective pore junctions in salt-secreting tissues of teleost fishes.

Prolactin and cortisol regulate branchial claudin expression in Japanese medaka.

Several gill claudin (Cldn) tight junction proteins in Japanese medaka are regulated by salinity (cldn10 paralogs and cldn28b), while others are constitutively expressed (cldn27a, cldn28a and cldn30c). The role of the endocrine system in this regulation has yet to be understood. The in vitro effects of cortisol and prolactin on cldn expression in gill explant cultures were investigated in medaka. ncc2b and cftr were used as markers of specific ionocytes associated with freshwater- and seawater-acclimation, respectively. Concentration-response experiments were performed by overnight incubation with 0, 0.1, 1 and 10µgmL-1 cortisol or 0, 0.01, 0.1 and 1µgmL-1 ovine prolactin. Cortisol significantly up-regulated cftr, ncc2b, cldn10 paralogs, cldn27a and cldn30c from 1.2- to 5-fold control levels at 10µgmL-1. Cortisol had no effect on cldn28a and cldn28b. Prolactin had a concentration-dependent effect, decreasing expression of cftr (1µgmL-1, 2.2-fold) while increasing ncc2b (from 0.1µgmL-1, 6-7-fold). Prolactin up-regulated expression of 3 cldns: cldn28b (0.1 and 1µgmL-1), cldn10c and cldn10f (1µgmL-1), with up to 2-, 2.5- and 2-fold of control level, respectively. A combination experiment with both hormones showed that they act in synergy on cldn28b and have an additive effect on cftr, ncc2b, cldn10c and cldn10f. Our results showed that cortisol and prolactin are essential to maintain the expression of specific branchial claudins. This work also provides evidence that both hormones act directly on gill of medaka to modulate determinants of paracellular ion movement.

Regulation of gill claudin paralogs by salinity, cortisol and prolactin in Mozambique tilapia (Oreochromis mossambicus).

In euryhaline teleosts, reorganization of gill tight junctions during salinity acclimation involves dynamic expression of specific claudin (Cldn) paralogs. We identified four transcripts encoding Cldn tight junction proteins in the tilapia gill transcriptome: cldn10c, cldn10e, cldn28a and cldn30. A tissue distribution experiment found cldn10c and cldn10e expression levels in the gill to be 100-fold higher than any other tissues examined. cldn28a and cldn30 levels in the gill were 10-fold greater than levels in other tissues. Expression of these genes in Mozambique tilapia was examined during acclimation to fresh water (FW), seawater (SW), and in response to hormone treatments. Transfer of tilapia from FW to SW elevated cldn10c and cldn10e, while cldn28a and cldn30 were stimulated following transfer from SW to FW. In hypophysectomized tilapia transferred to FW, pituitary extirpation induced reduced expression of cldn10c, cldn10e and cldn28a; these effects were mitigated equally by either prolactin or cortisol replacement. In vitro experiments with gill filaments showed that cortisol stimulated expression of all four cldns examined, suggesting a direct action of cortisol in situ. Our data indicate that elevated cldn10c and cldn10e expression is important during acclimation of tilapia to SW possibly by conferring ion specific paracellular permeability. On the other hand, expression of cldn28a and cldn30 appears to contribute to reorganization of branchial epithelium during FW acclimation. Hormone treatment experiments showed that particular FW- and SW-induced cldns are controlled by cortisol and prolactin.

Does Japanese medaka (Oryzias latipes) exhibit a gill Na(+)/K(+)-ATPase isoform switch during salinity change?

Some euryhaline teleosts exhibit a switch in gill Na(+)/K(+)-ATPase (Nka) α isoform when moving between fresh water (FW) and seawater (SW). The present study tested the hypothesis that a similar mechanism is present in Japanese medaka and whether salinity affects ouabain, Mg(2+), Na(+) and K(+) affinity of the gill enzyme. Phylogenetic analysis classified six separate medaka Nka α isoforms (α1a, α1b, α1c, α2, α3a and α3b). Medaka acclimated long-term (>30 days) to either FW or SW had similar gill expression of α1c, α2, α3a and α3b, while both α1a and α1b were elevated in SW. Since a potential isoform shift may rely on early changes in transcript abundance, we conducted two short-term (1-3 days) salinity transfer experiments. FW to SW acclimation induced an elevation of α1b and α1a after 1 and 3 days. SW to FW acclimation reduced α1b after 3 days with no other α isoforms affected. To verify that the responses were typical, additional transport proteins were examined. Gill ncc and nhe3 expression were elevated in FW, while cftr and nkcc1a were up-regulated in SW. This is in accordance with putative roles in ion-uptake and secretion. SW-acclimated medaka had higher gill Nka V max and lower apparent K m for Na(+) compared to FW fish, while apparent affinities for K(+), Mg(2+) and ouabain were unchanged. The present study showed that the Japanese medaka does not exhibit a salinity-induced α isoform switch and therefore suggests that Na(+) affinity changes involve altered posttranslational modification or intermolecular interactions.

Role of the GH-IGF-1 system in Atlantic salmon and rainbow trout postsmolts at elevated water temperature.

A comparative experiment with Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) postsmolts was conducted over 35 days to provide insight into how growth, respiration, energy metabolism and the growth hormone (GH) and insulin-like growth factor 1 (IGF-1) system are regulated at elevated sea temperatures. Rainbow trout grew better than Atlantic salmon, and did not show reduced growth at 19 °C. Rainbow trout kept at 19 °C had increased blood hemoglobin concentration compared to rainbow trout kept at 13 °C, while salmon did not show the same hemoglobin response due to increased temperature. Both species showed reduced length growth and decreased muscle glycogen stores at 19 °C. Circulating IGF-1 concentration was higher in rainbow trout than in Atlantic salmon, but was not affected by temperature in either species. Plasma IGF-binding protein 1b (IGFBP-1b) concentration was reduced in Atlantic salmon reared at 19 °C after 15 days but increased in rainbow trout at 19 °C after 35 days. The igfbp1b mRNA level in liver showed a positive correlation to plasma concentrations of glucose and IGFBP-1b, suggesting involvement of this binding protein in carbohydrate metabolism at 19 °C. At this temperature muscle igfbp1a mRNA was down-regulated in both species. The muscle expression of this binding protein correlated negatively with muscle igf1 and length growth. The plasma IGFBP-1b concentration and igfbp1b and igfbp1a expression suggests reduced muscle igf1 signaling at elevated temperature leading to glucose allostasis, and that time course is species specific due to higher thermal tolerance in rainbow trout.

Sexual maturation and changes in water and salt transport components in the kidney and intestine of three-spined stickleback (Gasterosteus aculeatus L.).

Mature three-spined stickleback males use spiggin threads secreted from their kidney to glue together nest material. This requires strongly hypertrophied renal proximal tubular cells, which compromises renal osmoregulatory function during the breeding period. Experimental evidence suggests that the intestine takes over hypotonic fluid secretion at that stage but the mechanism is unexplored. To unravel the molecular mechanism we analyzed and compared transcript levels of several membrane proteins involved in water and salt transport in intestinal and renal tissues, in non-mature males (NM), mature males (MM), and mature females (MF). Aquaporin paralogs aqp1a, -3a, -8aa, -8ab, -10a, and -10b, two Na(+),K(+)-ATPase alpha-1 subunit isoforms (nka547, nka976), Na(+),K(+),2Cl(-)-, and Na(+),Cl(-)-cotransporters (nkcc1a, nkcc2, ncc), the cystic fibrosis transmembrane conductance regulator (cftr) and two claudin isoforms (cldn2, cldn15a) were expressed in the intestine and kidney in all groups. There were no differences in aqp and cldn expression between intestines of NM and MM; nkcc2 was lower and nka levels tended to be higher in intestines of MM than in NM. In the kidney, aqp1 and aqp8ab levels were lower in MM than in NM, whereas aqp3a, nkcc1a, cldn15a, and spiggin were markedly elevated. This was accompanied by marked hypertrophy of kidney tubules in MM. The data support an altered kidney function in terms of water handling in mature males, whereas there was no support for modified trans-epithelial water permeability or salt-secretory activity in the intestine of mature males. Salt-absorptive activity in the intestine may, however, be down-regulated during male maturation.

Functional dynamics of claudin expression in Japanese medaka (Oryzias latipes): Response to environmental salinity.

Salinity regulation of 13 claudin paralogs was investigated in osmoregulatory organs of euryhaline Japanese medaka. They were identified by blast-search in the medaka genome database based on representation in osmoregulatory organs of other teleosts. Our hypothesis was that, because of their sequence similarities to mammalian orthologs previously characterized as barrier- and ion-selective channel-forming proteins, these paralogs would respond to salinity according to expected modulation of osmoregulatory function. Cldn10c, -10d, -10e, -10f, -27a, -28a, -28b and -30c had 4- to 100-fold higher expression in gill than other examined organs. Two splice variants of cldn10b were predominantly expressed in kidney, while cldn15a, -15b and -25 were found mainly in intestine. In gills, cldn27a, -28a, -28b and -30c did not change between fresh water (FW) and seawater (SW)-acclimated fish, while cldn10c, -10d, -10e, and -10f were most abundant in SW. Short-term SW transfer induced up-regulation of cldn10 gill paralogs after 1 day, decrease in cldn28b and no difference for cldn27a, -28a and -30c. The reverse pattern was observed after FW transfer of SW medaka. Intestinal cldn15a and -25 did not differ between FW and SW fish. However, cldn15b was 10-fold higher in FW than SW, suggesting a role in functional modulation of the intestine related to water and salt transport. In kidney, cldn10bs were elevated in SW fish, suggesting a role in paracellular ion transport in the marine nephron. Based on in silico analysis, most gill Cldn10s were predicted to form cation pores, whereas Cldn27a, 28a, 28b and 30c may increase epithelial resistance.

Transepithelial resistance and claudin expression in trout RTgill-W1 cell line: effects of osmoregulatory hormones.

In the present study, we examined the trout gill cell line RTgill-W1 as a possible tool for in vitro investigation of epithelial gill function in fish. After seeding in transwells, transepithelial resistance (TER) increased until reaching a plateau after 1-2 days (20-80Ω⋅cm(2)), which was then maintained for more than 6 days. Tetrabromocinnamic acid, a known stimulator of TER via casein kinase II inhibition, elevated TER in the cell line to 125% of control values after 2 and 6h. Treatment with ethylenediaminetetraacetic acid induced a decrease in TER to <15% of pre-treatment level. Cortisol elevated TER after 12-72 h in a concentration-dependent manner, and this increase was antagonized by growth hormone (Gh). The effects of three osmoregulatory hormones, Gh, prolactin, and cortisol, on the mRNA expression of three tight junction proteins were examined: claudin-10e (Cldn-10e), Cldn-30, and zonula occludens-1 (Zo-1). The expression of cldn-10e was stimulated by all three hormones but with the strongest effect of Gh (50-fold). cldn-30 expression was stimulated especially by cortisol (20-fold) and also by Gh (4-fold). Finally, zo-1 was unresponsive to hormone treatment. Western blot analysis detected Cldn-10e and Cldn-30 immunoreactive proteins of expected molecular weight in samples from rainbow trout gills but not from RTgill-W1 cultures, possibly due to low expression levels. Collectively, these results show that the RTgill-W1 cell layers have tight junctions between cells, are sensitive to hormone treatments, and may provide a useful model for in vitro study of some in vivo gill phenomena.

Aquaporin expression in the Japanese medaka (Oryzias latipes) in freshwater and seawater: challenging the paradigm of intestinal water transport?

We investigated the salinity-dependent expression dynamics of seven aquaporin paralogs (aqp1a, aqp3a, aqp7, aqp8ab, aqp10a, aqp10b and aqp11a) in several tissues of euryhaline Japanese medaka (Oryzias latipes). All paralogs except aqp7 and aqp10a had a broad tissue distribution, and several were affected by salinity in both osmoregulatory and non-osmoregulatory tissues. In the intestine, aqp1a, aqp7, aqp8ab and aqp10a decreased upon seawater (SW) acclimation in both long-term acclimated fish and during 1-3 days of the transition period. In the gill, aqp3a was lower and aqp10a higher in SW than in freshwater (FW). In the kidney no aqps were affected by salinity. In the skin, aqp1a and aqp3a were lower in SW than in FW. In the liver, aqp8ab and aqp10a were lower in SW than in FW. Furthermore, six Na(+),K(+)-ATPase α-subunit isoform transcripts were analysed in the intestine but none showed a consistent response to salinity, suggesting that water transport is not regulated at this level. In contrast, mRNA of the Na(+),K(+),2Cl(-)-cotransporter type-2 strongly increased in the intestine in SW compared with FW fish. Using custom-made antibodies, Aqp1a, Aqp8ab and Aqp10a were localized in the apical region of enterocytes of FW fish. Apical staining intensity strongly decreased, vanished or moved to subapical regions, when fish were acclimated to SW, supporting the lower mRNA expression in SW. Western blots confirmed the decrease in Aqp1a and Aqp10a in SW. The strong decrease in aquaporin expression in the intestine of SW fish is surprising, and challenges the paradigm for transepithelial intestinal water absorption in SW fishes.

Nutritional status and growth hormone regulate insulin-like growth factor binding protein (igfbp) transcripts in Mozambique tilapia.

Growth in teleosts is controlled in large part by the activities of the growth hormone (Gh)/insulin-like growth factor (Igf) system. In this study, we initially identified igf-binding protein (bp)1b, -2b, -4, -5a and -6b transcripts in a tilapia EST library. In Mozambique tilapia (Oreochromis mossambicus), tissue expression profiling of igfbps revealed that igfbp1b and -2b had the highest levels of expression in liver while igfbp4, -5a and -6b were expressed at comparable levels in most other tissues. We compared changes in hepatic igfbp1b, -2b and -5a expression during catabolic conditions (28days of fasting) along with key components of the Gh/Igf system, including plasma Gh and Igf1 and hepatic gh receptor (ghr2), igf1 and igf2 expression. In parallel with elevated plasma Gh and decreased Igf1 levels, we found that hepatic igfbp1b increased substantially in fasted animals. We then tested whether systemic Gh could direct the expression of igfbps in liver. A single intraperitoneal injection of ovine Gh into hypophysectomized tilapia specifically stimulated liver igfbp2b expression along with plasma Igf1 and hepatic ghr2 levels. Our collective data suggest that hepatic endocrine signaling during fasting may involve post-translational regulation of plasma Igf1 via a shift towards the expression of igfbp1b. Thus, Igfbp1b may operate as a molecular switch to restrict Igf1 signaling in tilapia; furthermore, we provide new details regarding isoform-specific regulation of igfbp expression by Gh.

Functional characterization of water transport and cellular localization of three aquaporin paralogs in the salmonid intestine.

Intestinal water absorption is greatly enhanced in salmonids upon acclimation from freshwater (FW) to seawater (SW); however, the molecular mechanism for water transport is unknown. We conducted a pharmacological characterization of water absorption in the rainbow trout intestine along with an investigation of the distribution and cellular localization of three aquaporins (Aqp1aa, -1ab, and -8ab) in pyloric caeca, middle (M), and posterior (P) intestine of the Atlantic salmon. In vitro iso-osmotic water absorption (J(v)) was higher in SW than FW-trout and was inhibited by (mmol L(-1)): 0.1 KCN (41%), 0.1 ouabain (72%), and 0.1 bumetanide (82%) suggesting that active transport, Na(+), K(+)-ATPase and Na(+), K(+), 2Cl(-)-co-transport are involved in establishing the driving gradient for water transport. J(v) was also inhibited by 1 mmol L(-1) HgCl(2), serosally (23% in M and 44% in P), mucosally (27% in M), or both (61% in M and 58% in P), suggesting involvement of both apical and basolateral aquaporins in water transport. The inhibition was antagonized by 5 mmol L(-1) mercaptoethanol. By comparison, 10 mmol L(-1) mucosal tetraethylammonium, an inhibitor of certain aquaporins, inhibited J(v) by 20%. In the presence of glucose, mucosal addition of phloridzin inhibited water transport by 20%, suggesting that water transport is partially linked to the Na(+)-glucose co-transporter. Using polyclonal antibodies against salmon Aqp1aa, -1ab, and -8ab, we detected Aqp1aa, and -1ab immunoreactivity in the brush border and sub-apical region of enterocytes in all intestinal segments. The Aqp8ab antibody showed a particularly strong immunoreaction in the brush border and sub-apical region of enterocytes throughout the intestine and also stained lateral membranes and peri-nuclear regions though at lower intensity. The present localization of three aquaporins in both apical and lateral membranes of salmonid enterocytes facilitates a model for transcellular water transport in the intestine of SW-acclimated salmonids.

Differential effects of cortisol and 11-deoxycorticosterone on ion transport protein mRNA levels in gills of two euryhaline teleosts, Mozambique tilapia (Oreochromis mossambicus) and striped bass (Morone saxatilis).

The role of cortisol as the only corticosteroid in fish osmoregulation has recently been challenged with the discovery of a mineralocorticoid-like hormone, 11-deoxycorticosterone (DOC), and necessitates new studies of the endocrinology of osmoregulation in fish. Using an in vitro gill explant incubation approach, DOC-mediated regulation of selected osmoregulatory target genes in the gill was investigated and compared with that of cortisol in two euryhaline teleosts, Mozambique tilapia (Oreochromis mossambicus) and striped bass (Morone saxatilis). The effects were tested in gills from both fresh water (FW)- and seawater (SW)-acclimated fish. Both cortisol and DOC caused an up-regulation of the Na(+),K(+)-ATPase α1 subunit in SW-acclimated tilapia but had no effect in FW-acclimated fish. Cortisol conferred an increase in Na(+),K(+),2Cl(-) cotransporter (NKCC) isoform 1a transcript levels in FW- and SW-acclimated tilapia, whereas DOC had a stimulatory effect only in SW-acclimated fish. Cortisol had no effect on NKCC isoform 1b mRNA levels at both salinities, while DOC stimulated this isoform in SW-acclimated fish. In striped bass, cortisol conferred an up-regulation of Na(+),K(+)-ATPase α1 and NKCC transcript levels in FW- and SW-acclimated fish, whereas DOC resulted in down-regulation of these transcripts in FW-acclimated fish. It was also found that both corticosteroids may rapidly (30 min) alter the mitogen-activated protein kinase signalling pathway in gill, inducing phosphorylation of extracellular signal-regulated kinase 1 (ERK1) and ERK2 in a salinity-dependent manner. The study shows a disparate organisation of corticosteroid signalling mechanisms involved in ion regulation in the two species and adds new evidence to a role of DOC as a mineralocorticoid hormone in teleosts.

Dynamic gene expression of GH/PRL-family hormone receptors in gill and kidney during freshwater-acclimation of Mozambique tilapia.

In teleosts, prolactin (PRL) and growth hormone (GH) act at key osmoregulatory tissues to regulate hydromineral balance. This study was aimed at characterizing patterns of expression for genes encoding receptors for the GH/PRL-family of hormones in the gill and kidney of Mozambique tilapia (Oreochromis mossambicus) during freshwater (FW)-acclimation. Transfer of seawater (SW)-acclimated tilapia to FW elicited rapid and sustained increases in plasma levels and pituitary gene expression of PRL177 and PRL188; plasma hormone and pituitary mRNA levels of GH were unchanged. In the gill, PRL receptor 1 (PRLR1) mRNA increased markedly after transfer to FW by 6h, while increases in GH receptor (GHR) mRNA were observed 48 h and 14 d after the transfer. By contrast, neither PRLR2 nor the somatolactin receptor (SLR) was responsive to FW transfer. Paralleling these endocrine responses were marked increases in branchial gene expression of a Na+/Cl- cotransporter and a Na+/H+ exchanger, indicators of FW-type mitochondrion-rich cells (MRCs), at 24 and 48 h after FW transfer, respectively. Expression of Na+/K+/2Cl- cotransporter, an indicator of SW-type MRCs, was sharply down-regulated by 6h after transfer to FW. In kidney, PRLR1, PRLR2 and SLR mRNA levels were unchanged, while GHR mRNA was up-regulated from 6h after FW transfer to all points thereafter. Collectively, these results suggest that the modulation of the gene expression for PRL and GH receptors in osmoregulatory tissues represents an important aspect of FW-acclimation of tilapia.

FXYD-11 associates with Na+-K+-ATPase in the gill of Atlantic salmon: regulation and localization in relation to changed ion-regulatory status.

The Na(+)-K(+)-ATPase is the primary electrogenic component driving transepithelial ion transport in the teleost gill; thus regulation of its level of activity is of critical importance for osmotic homeostasis. In the present study, we examined the dynamics of the gill-specific FXYD-11 protein, a putative regulatory subunit of the pump, in Atlantic salmon during seawater (SW) acclimation, smoltification, and treatment with cortisol, growth hormone, and prolactin. Dual-labeling immunohistochemistry showed that branchial FXYD-11 is localized in Na(+)-K(+)-ATPase immunoreactive cells, and coimmunoprecipitation experiments confirmed a direct association between FXYD-11 and the Na(+)-K(+)-ATPase α-subunit. Transfer of freshwater (FW)-acclimated salmon to SW induced a parallel increase in total α-subunit and FXYD-11 protein expression. A similar concurrent increase was seen during smoltification in FW. In FW fish, cortisol induced an increase in both α-subunit and FXYD-11 abundance, and growth hormone further stimulated FXYD-11 levels. In SW fish, prolactin induced a decrease in FXYD-11 and α-subunit protein levels. In vitro cortisol (18 h, 10 µg/ml) stimulated FXYD-11, but not FXYD-9, mRNA levels in gills from FW and SW salmon. The data show that Na(+)-K(+)-ATPase expressed in branchial mitochondrion-rich cells is accompanied by FXYD-11, and that regulation of the two proteins is highly coordinated. The demonstrated association of FXYD-11 and α-subunit strengthens our hypothesis that FXYD-11 has a role in modulating the pump's kinetic properties. The presence of putative phosphorylation sites on the intracellular domain of FXYD-11 suggests the possibility that this protein also may transmit external signals that regulate Na(+)-K(+)-ATPase activity.

Developmental profile of claudin-3, -5, and -16 proteins in the epithelium of chick intestine.

Proteins in the claudin family are a main component of tight junctions and form a seal that modulates paracellular transport in intestinal epithelium. This research tests the hypothesis that claudins 3, 5, and 16 will appear in the epithelium of embryonic intestine during functional differentiation. Immunohistochemistry is utilized to explore the developmental patterns of claudin-3, -5, and -16 proteins in the epithelium of embryonic chick intestine from 9 days prior to hatching through the early post-hatch period. These claudin proteins either changed their cellular localization or first appeared around the time of hatching. After hatching, claudin-3 expression was prominent in basal-lateral regions of the epithelium along the entire villus, but was absent from crypts. Claudin-5 was expressed most strongly in the crypt and lower villus epithelium within junctional complexes, whereas immunostaining of claudin-16 was localized within goblet cells of the upper villus region. The relative mRNA levels of claudin-3, -5, and -16 showed similar patterns; transcript levels rose between 18 and 20 days of development, then dropped by 2 days post-hatch. Results of this work indicate that the claudin proteins assume their final locations within the epithelium around the time of hatching, suggesting that in addition to their known barrier and fence functions within tight junctions, these claudins may have additional roles in the differentiation and/or physiological function of chick intestine. The localization of claudin-16 to goblet cells and its distribution in the more mature cells of the upper villus region suggest an unexpected role in goblet cell maturation and mucus secretion.

Claudin-15 and -25b expression in the intestinal tract of Atlantic salmon in response to seawater acclimation, smoltification and hormone treatment.

In seawater fishes, osmotic homeostasis requires uptake of ions and water in the intestine and these processes are governed by the combined trans- and paracellular pathways. The current study examined mRNA expression of two tight junction proteins (claudin-15 and -25 b) predominantly expressed in the intestine of Atlantic salmon. We examined the response in pyloric caecae, middle and posterior intestine to seawater challenge, during smoltification and after injection with osmoregulatory hormones. Seawater (SW) transfer elevated levels of claudin-15 and -25 b while no change was induced throughout the smolt stage. In freshwater, cortisol and growth hormone inhibited claudin-15 expression in the two anterior segments. Claudin-25 b was elevated in all intestinal segments by growth hormone, while cortisol had an inhibitory effect. In seawater, prolactin and cortisol inhibited claudin expression. The data suggest that claudin expression is involved in the reorganisation of intestinal epithelium and possibly change paracellular permeability during SW acclimation. The lack of preparatory change during smoltification suggests that this process is not completed during smolt development. The effects of the tested hormones cannot explain the sum of changes induced by salinity, which, like the smoltification data, suggests the importance of additional factors and possibly contact with the imbibed SW per se.