SUMMARY This study examines changes in gill Na+,K+-ATPase(NKA) α- and β-subunit isoforms,Na+,K+,2Cl- cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR I and II) in anadromous and landlocked strains of Atlantic salmon during parr-smolt transformation, and after seawater (SW) transfer in May/June. Gill NKA activity increased from February through April, May and June among both strains in freshwater (FW),with peak enzyme activity in the landlocked salmon being 50% below that of the anadromous fish in May and June. Gill NKA-α1b, -α3,-β1 and NKCC mRNA levels in anadromous salmon increased transiently, reaching peak levels in smolts in April/May, whereas no similar smolt-related upregulation of these transcripts occurred in juvenile landlocked salmon. Gill NKA-α1a mRNA decreased significantly in anadromous salmon from February through June, whereas α1a levels in landlocked salmon, after an initial decrease in April, remained significantly higher than those of the anadromous smolts in May and June. Following SW transfer, gill NKA-α1b and NKCC mRNA increased in both strains, whereas NKA-α1a decreased. Both strains exhibited a transient increase in gill NKA α-protein abundance, with peak levels in May. Gill α-protein abundance was lower in SW than corresponding FW values in June. Gill NKCC protein abundance increased transiently in anadromous fish, with peak levels in May, whereas a slight increase was observed in landlocked salmon in May,increasing to peak levels in June. Gill CFTR I mRNA levels increased significantly from February to April in both strains, followed by a slight,though not significant increase in May and June. CFTR I mRNA levels were significantly lower in landlocked than anadromous salmon in April/June. Gill CFTR II mRNA levels did not change significantly in either strain. Our findings demonstrates that differential expression of gill NKA-α1a,-α1b and -α3 isoforms may be important for potential functional differences in NKA, both during preparatory development and during salinity adjustments in salmon. Furthermore, landlocked salmon have lost some of the unique preparatory upregulation of gill NKA, NKCC and, to some extent, CFTR anion channel associated with the development of hypo-osmoregulatory ability in anadromous salmon.

SUMMARYOsmotic balance in fish is maintained through the coordinated regulation of water and ion transport performed by epithelia in intestine, kidney and gill. In the current study, six aquaporin (AQP) isoforms found in Atlantic salmon (Salmo salar) were classified and their tissue specificity and mRNA expression in response to a hyperosmotic challenge and during smoltification were examined. While AQP-1a was generic, AQP-1b had highest expression in kidney and AQP-3 was predominantly found in oesophagus, gill and muscle. Two novel teleost isoforms, AQP-8a and -8b, were expressed specifically in liver and intestinal segments, respectively. AQP-10 was predominantly expressed in intestinal segments, albeit at very low levels. Transfer from freshwater (FW) to seawater (SW) induced elevated levels of intestinal AQP-1a, -1b and -8b mRNA, whereas only AQP-8b was stimulated during smoltification. In kidney, AQP-1a, -3 and -10 were elevated in SW whereas AQP-1b was reduced compared with FW levels. Correspondingly, renal AQP-1a and -10 peaked during smoltification in April and March, respectively, as AQP-1b and AQP-3 declined. In the gill, AQP-1a and AQP-3 declined in SW whereas AQP-1b increased. Gill AQP-1a and -b peaked in April, whereas AQP-3 declined through smoltification. These reciprocal isoform shifts in renal and gill tissues may be functionally linked with the changed role of these organs in FW compared with SW. The presence and observed dynamics of the AQP-8b isoform specifically in intestinal sections suggest that this is a key water channel responsible for water uptake in the intestinal tract of seawater salmonids.

The European sea bass, Dicentrarchus labrax, tolerates salinities ranging from freshwater (FW) to hypersaline conditions. In two experiments, we analysed changes in plasma ions, muscle water content (MWC), gill Na+,K+‐ATPase activity, and α‐subunit mRNA expression during the course of acclimation from 15 ppt salt water to FW or high salinity seawater (HSSW). In Experiment 1, fish (6.2 ± 1.1 g) were acclimated from 15 ppt to either FW, 5, 15, 25, 50, or 60 ppt SW and sampled after 10 days. Gill Na+,K+‐ATPase activity was stimulated in FW‐ and in 50 and 60 ppt SW‐groups relative to the 15 ppt control group. In Experiment 2, subgroups of fish (89 ± 7 g) were transferred from 15 ppt SW to FW or 50 ppt SW, and sampled 1, 2, 4, and 10 days later. Plasma osmolality, [Na+] and [Cl–] decreased in the FW‐group and increased in the HSSW‐group one day after transfer and lasting until day 10. This was accompanied by a pronounced increase in MWC in the FW‐group and an insignificant decrease in the HSSW‐group. The plasma [Na+]:[Cl–]‐ratio increased markedly in the FW‐group and decreased slightly in the HSSW‐group, suggesting acid‐base balance disturbances after transfer. Gill Na+,K+‐ATPase activity was unchanged in 15 ppt SW but doubled in FW‐ and HSSW‐groups after transfer. In both groups, this was preceded by a 2‐ to 5‐fold elevation of the gill α‐subunit Na+,K+‐ATPase mRNA level. Thus increased expression of α‐subunit mRNA is part of the molecular mechanism of both FW and SW acclimation in sea bass. Gill Na+,K+‐ATPase Na+‐, K+‐, and ouabain‐affinity were similar in fish acclimated to FW, 15 ppt, and HSSW, suggesting that identical isoforms of the catalytic subunit of the enzyme are expressed irrespective of salinity. J. Exp. Zool. 282:290–300, 1998. © 1998 Wiley‐Liss, Inc.

In euryhaline teleosts, permeability changes in gill epithelia are essential during acclimation to changed salinity. This study examined expression patterns of branchial tight junction proteins called claudins, which are important determinants of ion selectivity and general permeability in epithelia. We identified Atlantic salmon genes belonging to the claudin family by screening expressed sequence tag libraries available at NCBI, and classification was performed with the aid of maximum likelihood analysis. In gill libraries, five isoforms (10e, 27a, 28a, 28b, and 30) were present, and quantitative PCR analysis confirmed tissue-specific expression in gill when compared with kidney, intestine, heart, muscle, brain, and liver. Expression patterns during acclimation of freshwater salmon to seawater (SW) and during the smoltification process were examined. Acclimation to SW reduced the expression of claudin 27a and claudin 30 but had no overall effect on claudin 28a and claudin 28b. In contrast, SW induced a fourfold increase in expression of claudin 10e. In accord, a peak in branchial claudin 10e was observed during smoltification in May, coinciding with optimal SW tolerance. Smoltification induced no significant changes in expression of the other isoforms. This study demonstrates the expression of an array of salmon claudin isoforms and shows that SW acclimation involves inverse regulation, in the gill, of claudin 10e vs. claudin 27a and 30. It is possible that claudin 10e is an important component of cation selective channels, whereas reduction in claudin 27a and 30 may change permeability conditions in favor of the ion secretory mode of the SW gill.

The dynamics of branchial Na(+),K(+),2Cl(-) cotransporter (NKCC) and Na(+),K(+)-ATPase (NKA) expression were investigated in brown trout and Atlantic salmon during salinity shifts and the parr-smolt transformation, respectively. In the brown trout, Western blotting revealed that NKCC and NKA abundance increased gradually and in parallel (30- and ten-fold, respectively) after transfer to seawater (SW). The NKA hydrolytic activity increased ten-fold after SW-transfer. Following back-transfer to fresh water (FW), the levels of both proteins and NKA activity decreased. The NKCC immunostaining in the gill of SW-acclimated trout was strong, and mainly localized in large cells in the filament and around the bases of the lamellae. In FW-acclimated trout, immunostaining was less intense and more diffuse. Partial cDNAs of the secretory NKCC1 isoform were cloned and sequenced from both brown trout and Atlantic salmon gills. Two differently sized transcripts were detected by Northern blotting in the gill but not in other osmoregulatory tissues (kidney, pyloric caeca, intestine). The abundance in the gill of these transcripts and of the associated NKCC protein increased four- and 30-fold, respectively, during parr-smolt transformation. The abundance of NKA alpha-subunit protein also increased in the gill during parr-smolt transformation though to a lesser extent than enzymatic activity (2.5- and eight-fold, respectively). In separate series of in vitro experiments, cortisol directly stimulated the expression of NKCC mRNA in gill tissue of both salmonids. The study demonstrates the coordinated regulation of NKCC and NKA proteins in the gill during salinity shifts and parr-smolt transformation of salmonids.