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Pia Kiilerich, Karsten Kristiansen and Steffen S Madsen

Based on real-time RT-PCR, analysis of transcripts of selected ion-regulatory proteins (Na+, K+-ATPase α1a and α1b subunit, Na+, K+, 2Cl cotransporter, cystic fibrosis transmembrane conductance regulator (CFTR), and H+-ATPase B-subunit), the regulatory role of cortisol and the associated receptor signaling pathway (glucocorticoid (GR) versus mineralocorticoid (MR)) of cortisol was investigated in the salmon gill. Using a gill organ culture technique, the effect of cortisol with and without addition of specific hormone receptor antagonists (RU486 and spironolactone) was analyzed in gills from freshwater (FW) and seawater (SW) acclimated fish. The effect of cortisol was highly dependent on acclimation to salinity. In FW, cortisol stimulated the transcript levels of CFTR-I and Na+, K+-ATPase α1a and α1b. Addition of RU486 totally abolished the cortisol effects on CFTR-I and Na+, K+-ATPase α1b, suggesting that signaling was mediated via GR. Interestingly, both spironolactone and RU486 were able to inhibit the cortisol effect on Na+, K+-ATPase α1a indicating a role for both MR and GR in regulation of this target gene. In SW, cortisol increased the transcript levels of CFTR-I, CFTR-II, Na+, K+-ATPase α1a and α1b, and NKCC. Interestingly, the effect of cortisol on CFTR-I and Na+, K+-ATPase α1a was mediated through GR and MR respectively, while both GR and MR signaling were required in the regulation of CFTR-II and Na+, K+-ATPase α1b. In FW gills, GR1 and MR transcript levels were not significantly affected by cortisol. In SW gills, GR1 and MR transcripts were downregulated by cortisol; GR1 was regulated via the MR and MR regulation was mediated via GR.

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Steffen Søndergaard Madsen, Lars Nørholm Jensen, Christian Kølbæk Tipsmark, Pia Kiilerich and Russell John Borski

Effects of salinity and hormones on cystic fibrosis transmembrane conductance regulator (CFTR) and α-subunit Na+,K+-ATPase (α-NKA) mRNA (analysed by semi-quantitative PCR) and protein expression (analysed by western blotting and immunocytochemistry) were investigated in gills of striped bass. Freshwater (FW) to seawater (SW) transfer induced a disturbance in serum [Na+]. Gill CFTR protein, mRNA level and Na+,K+-ATPase activity were unaffected by SW transfer, whereas α-NKA mRNA increased after transfer. CFTR immunoreactivity was observed in large cells in FW and SW gill filaments at equal intensity. Cortisol decreased serum [Na+] in FW fish, but had no effect on gill Na+,K+-ATPase activity, α-NKA and CFTR mRNA levels. Incubation of gill tissue with cortisol (24 h, >0.01 μg/ml) and epidermal growth factor (EGF 10 μg/ml) decreased CFTR mRNA levels relative to pre-incubation and control levels. CFTR expression was unaffected by IGF-I (10 μg/ml). α-NKA mRNA levels decreased by 50% after 24 h control incubation; it was slightly stimulated by cortisol and unaffected by IGF-I and EGF. In isolated gill cells, phosphorylation of extracellular-regulated kinase (ERK) 1/2 was stimulated by EGF but not affected by IGF-I. This study is the first to report a branchial EGF response and to demonstrate a functional ERK 1/2 pathway in the teleost gill. In conclusion, CFTR and Na+,K+-ATPase are differentially regulated by salinity and hormones in gills of striped bass, despite the putative involvement of both in salt excretion.

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Pia Kiilerich, Christian K Tipsmark, Russell J Borski and Steffen S Madsen

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.

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Pia Kiilerich, Sylvain Milla, Armin Sturm, Claudiane Valotaire, Sylvie Chevolleau, Franck Giton, Xavier Terrien, Jean Fiet, Alexis Fostier, Laurent Debrauwer and Patrick Prunet

Cortisol and glucocorticoid receptors (GRs) play an important role in fish osmoregulation, whereas the involvement of the mineralocorticoid receptor (MR) and its putative ligand 11-deoxycorticosterone (DOC) is poorly investigated. In this study, we assessed the implication of DOC and MR in rainbow trout (Oncorhynchus mykiss) osmoregulation during hypo- and hypersaline acclimation in parallel with the cortisol–GR system. A RIA for DOC was developed to measure plasma DOC levels, and a MR-specific antibody was developed to localize MR protein in the gill, intestine, and kidney. This is the first study to report DOC plasma levels during salinity change and MR localization in fish osmoregulatory tissue. Corticosteroid receptor mRNA abundance was investigated in osmoregulatory tissue during salinity acclimation, and the effect of cortisol and DOC on ionic transporters gene expression was assayed using an in vitro gill incubation method. Differential tissue-, salinity-, and time-dependent changes in MR mRNA levels during both hyper- and hyposaline acclimations and the ubiquitous localization of MR in osmoregulatory tissue suggest a role for the MR in osmoregulation. Presumably, DOC does not act as ligand for MR in osmoregulation because there were no changes in plasma DOC levels during either freshwater–seawater (FW–SW) or SW–FW acclimation or any effect of DOC on gill ionic transporter mRNA levels in the gill. Taken together, these results suggest a role for MR, but not for DOC, in osmoregulation and confirm the importance of cortisol as a major endocrine regulator of trout osmoregulation.