Tricellulin, occludin and claudin-3 expression in salmon intestine and kidney during salinity adaptation.

Molecular regulation of tight junctions in osmoregulatory epithelia of euryhaline fishes must be extensive during ontogeny and acclimation to salinity changes. In this study, five tight junction proteins were examined in Atlantic salmon (Salmo salar): tight junction associated tricellulin, occludin and claudin-3 isoforms (a, b, c). A survey of tissue distribution in freshwater (FW) salmon showed that tricellulin expression was highest in the intestine. Occludin was detected in tissues with importance for epithelial transport and the order of expression was gill>intestine>kidney. The three claudin-3 isoforms were expressed at highest level in kidney tissue. Transfer of juvenile FW salmon to seawater (SW) elevated intestinal tricellulin and occludin mRNA, and these transcripts were also elevated at the time of best SW-tolerance during the course of smoltification. In the kidney, expression of tricellulin and claudin-3 isoforms was elevated after SW-transfer and tricellulin, occludin, claudin-3a and -3b increased in March before the peak smolt stage. In the gill, none of the examined tight junction proteins were impacted by SW-transfer. The data suggest that expression of tricellulin and occludin is dynamically involved in reorganization of intestinal epithelium and possibly changed paracellular permeability during SW-acclimation. The increased renal tricellulin and claudin-3 expression in SW suggests a role in remodeling of the kidney during SW-acclimation.

Functional characterization and localization of a gill-specific claudin isoform in Atlantic salmon.

Claudins are the major determinants of paracellular epithelial permeability in multicellular organisms. In Atlantic salmon (Salmo salar L.), we previously found that mRNA expression of the abundant gill-specific claudin 30 decreases during seawater (SW) acclimation, suggesting that this claudin is associated with remodeling of the epithelium during salinity change. This study investigated localization, protein expression, and function of claudin 30. Confocal microscopy showed that claudin 30 protein was located at cell-cell interfaces in the gill filament in SW- and fresh water (FW)-acclimated salmon, with the same distribution, overall, as the tight junction protein ZO-1. Claudin 30 was located at the apical tight junction interface and in cell membranes deeper in the epithelia. Colocalization with the α-subunit of the Na(+)-K(+)-ATPase was negligible, suggesting limited association with mitochondria-rich cells. Immunoblotting of gill samples showed lower claudin 30 protein expression in SW than FW fish. Retroviral transduction of claudin 30 into Madin-Darby canine kidney cells resulted in a decreased conductance of 19%. The decreased conductance correlated with a decreased permeability of the cell monolayer to monovalent cations, whereas permeability to chloride was unaffected. Confocal microscopy revealed that claudin 30 was expressed in the lateral membrane, as well as in tight junctions of Madin-Darby canine kidney cells, thereby paralleling the findings in the native gill. This study suggests that claudin 30 functions as a cation barrier between pavement cells in the gill and also has a general role in cell-cell adhesion in deeper layers of the epithelium.

Aquaporin expression dynamics in osmoregulatory tissues of Atlantic salmon during smoltification and seawater acclimation.

Osmotic 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.

Effects of cortisol, growth hormone and prolactin on gill claudin expression in Atlantic salmon.

We recently showed that a series of tight junction proteins of the claudin family are regulated in the gill of salmon during salinity acclimation. The aim of the present study was to investigate the role of cortisol, growth hormone (GH) and prolactin (PRL) on regulation of expression of these isoforms. Experiments on primary cultures of gill tissue showed that cortisol stimulates claudin 10e, 27a and 30 mRNA levels while no significant effects were observed on claudin 28a and 28b. The associated receptor signalling pathway was examined using glucocorticoid and mineralocorticoid receptor antagonists RU486 and spironolactone, respectively. The observed in vitro responses were blocked by RU486, suggesting the involvement of a glucocorticoid type receptor. Injections of FW salmon with cortisol increased the expression of claudin 10e, 27a, and 30 but did not affect claudin 28a and 28b significantly. While GH had no effect on its own, the combination of GH and cortisol reduced claudin 28b levels. Injection of SW salmon with PRL selectively increased the expression of claudin 28a but had no effect on the other examined isoforms. The data shows that FW- (27a and 30) and SW-induced (10e) claudins are all stimulated by cortisol while the major osmoregulatory hormones GH and PRL had no effect on these salinity sensitive isoforms. This suggests that other hormones and/or osmotic conditions interact with cortisol to determine claudin composition in the gill.

Branchial expression patterns of claudin isoforms in Atlantic salmon during seawater acclimation and smoltification.

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.

Rapid modulation of Na+/K+-ATPase activity in osmoregulatory tissues of a salmonid fish.

The effects of cyclic AMP on Na+/K+-ATPase activity were studied in the gill and kidney of the euryhaline brown trout Salmo trutta using two different experimental approaches. In the first series of experiments, in situ Na+/K+-ATPase activity was analyzed by measuring the ouabain-sensitive uptake of non-radioactive rubidium (Rb+) into gill cells and blocks of gill and kidney tissue. Rubidium uptake was linear for at least 30 min and was significantly inhibited by 1 mmol x l(-1) ouabain. Several agents presumed to increase the intracellular cyclic AMP concentration inhibited ouabain-sensitive Rb+ uptake in both gill (0.5 and 2 mmol x l(-1) dibutyryl-cyclic AMP, 1 mmol x l(-1) theophylline, 10 micromol x l(-1) forskolin and 10 micromol x l(-1 )isoproterenol) and kidney (10 micromol x l(-1) forskolin) tissue from freshwater-acclimated fish. In a separate series of experiments, ATP hydrolase activity was assayed in a permeabilised gill membrane preparation after incubation of tissue blocks with 10 micromol x l(-1 )forskolin. Forskolin elevated gill cyclic AMP levels 40-fold, inhibited maximal enzymatic Na+/K+-ATPase activity (Vmax) in gill tissue from both freshwater- and seawater-acclimated fish and reduced the apparent K+ affinity in the gills of seawater-acclimated fish, demonstrating that the effects are mediated through modifications of the enzyme itself. The protein phosphatase inhibitors okadaic acid and cyclosporin A did not affect forskolin-induced inhibition of Na+/K+-ATPase activity, indicating that forskolin-mediated modulation was stable for the duration of assay. We suggest that cyclic-AMP-mediated phosphorylation through protein kinases may underlie the rapid modulation of Na+/K+-ATPase activity in the osmoregulatory tissues of euryhaline teleosts.

Vacuolar-type H(+)-ATPase and Na+, K(+)-ATPase expression in gills of Atlantic salmon (Salmo salar) during isolated and combined exposure to hyperoxia and hypercapnia in fresh water.

Changes in branchial vacuolar-type H(+)-ATPase B-subunit mRNA and Na+, K(+)-ATPase alpha- and beta-subunit mRNA and ATP hydrolytic activity were examined in smolting Atlantic salmon exposed to hyperoxic and/or hypercapnic fresh water. Pre-smolts, smolts, and post-smolts were exposed for 1 to 4 days to hyperoxia (100% O2) and/or hypercapnia (2% CO2). Exposure to hypercapnic water for 4 days consistently decreased gill vacuolar-type H(+)-ATPase B-subunit mRNA levels. Salmon exposed to hyperoxia had either decreased or unchanged levels of gill B-subunit mRNA. Combined hyperoxia + hypercapnia decreased B-subunit mRNA levels, although not to the same degree as hypercapnic treatment alone. Hyperoxia generally increased Na+, K(+)-ATPase alpha- and beta-subunit mRNA levels, whereas hypercapnia reduced mRNA levels in presmolts (beta) and smolts (alpha and beta). Despite these changes in mRNA levels, whole tissue Na+, K(+)-ATPase activity was generally unaffected by the experimental treatments. We suggest that the reduced expression of branchial vacuolar-type H(+)-ATPase B-subunit mRNA observed during internal hypercapnic acidosis may lead to reduction of functional V-type H(+)-ATPase abundance as a compensatory response in order to minimise intracellular HCO3- formation in epithelial cells.

Time-course changes in the expression of Na+, K+-ATPase in gills and pyloric caeca of brown trout (Salmo trutta) during acclimation to seawater.

Changes in protein and mRNA expression of Na(+),K(+)-ATPase in gills and pyloric caeca of brown trout were investigated on a detailed time course after transfer from freshwater to 25 ppt seawater (SW). A transient deflection in plasma osmolality and muscle water content lasting from 4 h until day 3 was followed by restoration of hydromineral balance from day 5 onward. Gills and pyloric caeca responded to SW transfer by increasing Na(+),K(+)-ATPase activity from days 5 and 3, respectively, onward. In both tissues, this response was preceded by an increase in alpha-subunit Na(+), K(+)-ATPase mRNA as early as 12 h posttransfer. The similarity of the response in these two organs suggests that they both play significant physiological roles in restoring hydromineral balance after abrupt increase in salinity. Further, SW transfer induced a slight, though significant, increase in primary gill filament Na(+), K(+)-ATPase immunoreactive (NKIR) cell abundance. This was paralleled by a marked (50%) decrease in secondary lamellar NKIR cell abundance after less than 1 d in SW. Thus, SW acclimation in brown trout is characterised by a lasting decrease in overall NKIR cell abundance in the gill. We propose that SW transfer stimulates Na(+),K(+)-ATPase enzymatic activity within individual chloride cells long before (<1 d) it becomes apparent in measurements of whole-gill homogenate enzymatic activity. This is supported by the early stabilisation (12 h) of hydromineral balance.

Endocrine control of Na+,K+-ATPase and chloride cell development in brown trout (Salmo trutta): interaction of insulin-like growth factor-I with prolactin and growth hormone.

A 2-factorial (3x3) injection experiment was used to investigate the effect and interaction between different hormones on the initial phase of seawater (SW) acclimation in brown trout (Salmo trutta). Each fish was given 4 injections on alternate days in freshwater (FW). Factor 1 was either saline, 2 micrograms ovine prolactin (oPRL)/g, or 2 micrograms ovine growth hormone (oGH)/g. Factor 2 was either 0, 0. 01, or 0.1 mirograms recombinant human insulin-like growth factor-I (rhIGF-I)/g. In each of the 9 treatment groups, half of the fish were subjected to a 48-h SW-challenge test, and the remaining fish were sham-transferred to FW one day after the last injection. Hypo-osmoregulatory performance was increased by GH and impaired by PRL treatment as judged by changes in plasma osmolality, [Na+], [Cl-], total [Mg] and muscle water content (MWC) after SW transfer. IGF-I reduced plasma osmolality after transfer to SW but had no effect on plasma total [Mg] or MWC. The effects of the two factors on plasma osmolality, [Na+], [Cl-], and MWC were additive. In sham-transferred fish, GH and IGF-I, alone and in combination, stimulated Na+,K+-ATPase alpha-subunit mRNA (alpha-mRNA) content in the gill. This was paralleled by an overall increase in gill Na+, K+-ATPase activity in fish treated with 0.01 micrograms IGF-I/g. Simultaneous administration of PRL completely inhibited the increase in gill alpha-mRNA observed in the IGF-I-injected groups. Combination of GH and IGF-I did not further affect the alpha-mRNA level relative to the single hormone-injected groups. There was an overall decrease in Na+,K+-ATPase activity in pyloric caeca and middle intestine by the low dose and both doses of IGF-I respectively. No effect was observed in the posterior intestine. PRL and GH treatments did not affect enzyme activity in any intestinal segment. Both doses of IGF-I increased Na+,K+-ATPase-immunoreactive (NKIR) cell density in gill primary filaments. PRL and GH had no effect on primary filament NKIR cell density. GH and both doses of IGF-I reduced secondary lamellar NKIR cell density, whereas PRL had no effect. The main conclusion is that IGF-I and GH induce an overall redistribution of NKIR cells away from the secondary lamella onto the primary filament of FWacclimated trout. This is associated with an overall increased alpha-mRNA level in the gill, which may reflect an increased expression within individual NKIR cells in the primary filament. PRL completely abolished the IGF-I stimulation of alpha-mRNA levels, suggesting a desensitisation of the gill tissue to IGF-I, which may explain the overall anti-SW adaptive effect of PRL.

Effects of insulin-like growth factor-I and cortisol on Na+, K+-ATPase expression in osmoregulatory tissues of brown trout (Salmo trutta).

The effect of recombinant bovine IGF-I (rbIGF-I) on hypo-osmoregulatory ability and the effect of rbIGF-I and cortisol (F) alone and in combination on Na+,K+-ATPase expression in fresh water (FW) acclimated brown trout (Salmo trutta) were examined in two experiments. In Experiment 1, fish were given three injections of saline or 0.01 or 0.1 microgram rbIGF-I/g, respectively, and subjected to a 24-h 25 ppt seawater (SW) challenge test 24 h after the last injection. Fish treated with 0.01 and 0.1 microgram rbIGF-I/g had better hypo-osmoregulatory ability than control fish as judged by their higher level of muscle water content and lower plasma osmolality after 24 h exposure to 25 ppt SW. Compared with control fish, gill Na+,K+-ATPase activity was unchanged 24 h after the first injection at either dose but significantly stimulated after three injections of either dose of rbIGF-I. In Experiment 2, fish were given three injections of saline, 0.1 microgram rbIGF-I/g, 4 microgram F/g, or 0.1 microgram rbIGF-I + 4 microgram F/g and sampled in FW 24 h after the last injection. IGF-I and F had additive stimulatory effects on Na+,K+-ATPase activity and alpha-subunit Na+,K+-ATPase mRNA levels in the gill. Injections of IGF-I and F alone and in combination increased Na+,K+-ATPase-immunoreactive (NKIR) cell number in the primary gill filament but had no effect on secondary lamellar NKIR cell number. NKIR cells were abundant in kidney tubules, pyloric ceca, and posterior intestine, but Na+,K+-ATPase enzyme activity was unaffected by treatment with F and/or IGF-I in these tissues. F but not rbIGF-I increased in vitro fluid transport capacity in the posterior intestine. In addition to confirming an overall SW-adaptive effect of rbIGF-I and F in FW-acclimated S. trutta, the study suggests the effect to be associated with stimulation of chloride cell development and Na+,K+-ATPase expression in the gill. The study indicates that the stimulatory effects of the two hormones on Na+,K+-ATPase expression are additive, highly organ specific, and restricted to the primary filament epithelium of the gill.

Osmoregulation and salinity effects on the expression and activity of Na+,K(+)-ATPase in the gills of European sea bass, Dicentrarchus labrax (L.).

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 alpha-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 alpha-subunit Na+,K(+)-ATPase mRNA level. Thus increased expression of alpha-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.

[Results after surgery of incisional hernias]. / Resultater efter operation for hernia incisionalis.

The purpose of this paper is to describe the subjective results after operation for ventral hernia. Patients operated for ventral hernia in the period from January 1, 1988 to December 31, 1992 were sent a questionnaire with a response rate of 87%. Forty-five percent had lasting discomfort, 30% were absent due to illness more than eight weeks after the operation and 22% were not able to go to work. These factors increase with the number of hernia operations. Regardless of the number of operations, about 70% of the patients wanted a new operation should the hernia recur. It is therefore important to give the patient an objective and detailed information to prevent from unrealistic expectations of the results of ventral hernia operation.

Osmoregulatory actions of growth hormone and prolactin in an advanced teleost.

To date, growth hormone (GH) is known to contribute to seawater adaptation only in salmonid fishes (primitive Euteleostei). Accordingly, the effects of homologous GH and two forms of homologous prolactin (PRL177 and PRL188) on hypoosmoregulatory ability and gill Na+,K(+)-ATPase activity in a more advanced euryhaline cichlid fish, the tilapia (Oreochromis mossambicus), were examined. Following adaptation of hypophysectomized fish to 25% seawater for 3 weeks, fish were given four injections of hormone or vehicle. They were then exposed to 100% seawater for 12 hr and examined for changes in plasma osmolality. Tilapia GH (0.02 and 0.2 microgram/g) significantly improved the ability of tilapia to decrease plasma osmolality following transfer to full-strength seawater, in a dose-related manner. Growth hormone treatment also significantly stimulated gill Na+,K(+)-ATPase activity (0.5 microgram/g). Both tilapia PRLs (PRL177 and PRL188) increased plasma osmolality in 100% seawater and reduced gill Na+,K(+)-ATPase activity, the effects induced by PRL188 being more significant than those by PRL177. Thus, GH may be involved in seawater adaptation of tilapia, a species belonging to the most advanced teleost super-order (Acanthopterygii), whereas both PRLs in tilapia are not involved in seawater adaptation.

Somatotropic actions of the homologous growth hormone and prolactins in the euryhaline teleost, the tilapia, Oreochromis mossambicus.

It is increasingly clear that growth hormone (GH) has growth-promoting effects in fishes, which are mediated in part by the insulin-like growth factor (IGF)-I. Growth-promoting actions of prolactin (PRL) have been reported in higher vertebrates, but are less well established in teleosts. We examined the effects of injecting homologous GH or the two homologous tilapia PRLs (tPRL177 and tPRL188) on the in vitro incorporation of [35S] sulfate (extracellular matrix synthesis) and [3H]thymidine (DNA synthesis) by ceratobranchial cartilage explants and on IGF-I mRNA levels in tilapia liver. Tilapia GH (tGH) and tPRL177 stimulated sulfate uptake at the highest doses examined. Thymidine incorporation was stimulated by tPRL177. tPRL188 was without these effects. Consistent with its somatotropic actions, tGH elevated IGF-I mRNA levels in the liver. tPRL177 also elevated liver IGF-I levels. Consistent with the previously described osmoregulatory actions of GH and PRL in teleosts, we observed that tGH elevated and tPRL177 and tPRL188 lowered levels of gill Na+,K+-ATPase activity. High-affinity, low-capacity binding sites for tGH in the tilapia liver were identified. tPRL177 binds with lower affinity than tGH to these sites but can displace 125I-labeled tGH from its receptor. The ability of tPRL177 to displace tGH was similar to that of ovine GH. tPRL188 did not displace 125I-labeled tGH binding. Collectively, this work suggests that tPRL177 may possess somatotropic actions similar to tGH, but only in freshwater tilapia where tPRL177 levels are sufficiently high for it to act as a competitive ligand for GH receptors.

[Incisional hernia. A study of morbidity, mortality and bed utilization in a Danish county]. / Hernia incisionalis. En opgørelse af morbiditet, mortalitet og sengedagsforbrug i et dansk amt.

Four hundred and four patients operated for ventral hernia during a five year period were reviewed with special emphasis on morbidity, mortality and number of days in hospital. The patients were divided into 357 patients who had been operated for ventral hernia only once (A) and 47 patients who had been operated more than once for ventral hernia (B). There were no significant differences between the two groups regarding to ages or preoperative condition. The postoperative complications were 21% in group A and 12% in group B, although this was not statistically significant. Of those who suffered a complication after the operation, 76% had a competing illness prior to the operation. The overall mortality was 0.5%. The patients were hospitalized for a total of 4846 days. In view of the results of this material and of the high recurrence rate demonstrated in the literature, a restrictive attitude towards operations for ventral hernia is recommended.

Expression of Na(+)-K(+)-ATPase in the brown trout, Salmo trutta: in vivo modulation by hormones and seawater.

Expression of the Na(+)-K(+)-ATPase alpha-subunit was investigated in the gill and trunk kidney of Salmo trutta. Groups of freshwater (FW) fish were treated with various hormones [cortisol: 3 x 4.0 micrograms/g; recombinant salmon growth hormone (rsGH): 3 x 0.25 micrograms/g; salmon prolactin (sPRL): 3 x 0.25 micrograms/g; recombinant bovine insulin-like growth factor-I (rbIGF-I): 2 x 0.01 micrograms/g; or 2 x 0.1 micrograms/g] or transferred to 25 parts per thousand seawater (SW) and sampled after 1, 2, 3, and 50 days. Total RNA was analyzed by Northern blotting using Xenopus laevis Na(+)-K(+)-ATPase alpha-subunit cDNA as probe. The probe detected a 3.8-kb transcript. Relative to untreated FW control fish, the abundance of alpha-subunit Na(+)-K(+)-ATPase mRNA in gill tissue increased 1.7-to 2.5-fold after treatment with cortisol, rsGH, and rbIGF-I and after transfer to SW. Na(+)-K(+)-ATPase enzyme activity was also significantly stimulated in these groups, except at 0.01 micrograms/g rbIGF-I. sPRL was without effect. In the kidney, alpha-subunit mRNA level and Na(+)-K(+)-ATPase activity were unaffected by hormone treatment and SW transfer. The results indicate that an increased abundance of alpha-subunit mRNA is part of the molecular mechanism behind the increased gill Na(+)-K(+)-ATPase activity induced by SW transfer, cortisol, GH, and IGF-I.

Effects of environmental salinity on pituitary growth hormone content and cell activity in the euryhaline tilapia, Oreochromis mossambicus.

Studies were undertaken to determine whether several indicators of growth hormone (GH) cell activity, namely GH content, fine structure, and volume of the GH region, differ in the pituitaries of freshwater (FW) and seawater (SW) tilapia, Oreochromis mossambicus. Tilapia raised from the stage of yolk-sac absorption for 7 months in SW contain significantly more GH in their pituitaries than in those of fish reared in FW. Pituitary growth hormone content in tilapia raised in FW for 7 months and transferred to SW for 49 days is greater than that in sibling tilapia retained in FW. Conversely, GH content is significantly lower in the pituitaries of SW-reared tilapia transferred to FW for 49 days than that in the pituitaries from fish retained in SW. Likewise, the volume of the GH region and activity of the GH cells are enhanced in pituitaries from SW-reared tilapia over that seen in pituitaries from FW fish. Taken together, all data indicate heightened GH cell activity in SW-raised tilapia and suggest that GH may play a causal role in the greater growth rates observed in SW tilapia compared to FW fish and/or that GH may be involved in SW osmoregulation. The latter suggestion is supported, in part, by our observation that in vivo oGH treatment (2 micrograms/g body wt) stimulated gill Na+,K(+)-ATPase activity.

Physiology of seawater acclimation in the striped bass, Morone saxatilis (Walbaum).

Several experiments were performed to investigate the physiology of seawater acclimation in the striped bass, Morone saxatilis. Transfer of fish from fresh water (FW) to seawater (SW; 31-32 ppt) induced only a minimal disturbance of osmotic homeostasis. Ambient salinity did not affect plasma thyroxine, but plasma cortisol remained elevated for 24h after SW transfer. Gill and opercular membrane chloride cell density and Na(+),K(+)-ATPase activity were relatively high and unaffected by salinity. Average chloride cell size, however, was slightly increased (16%) in SW-acclimated fish. Gill succinate dehydrogenase activity was higher in SW-acclimated fish than in FW fish. Kidney Na(+), K(+)-ATPase activity was slightly lower (16%) in SW fish than in FW fish. Posterior intestinal Na(+),K(+)-ATPase activity and water transport capacity (Jv) did not change upon SW transfer, whereas middle intestinal Na(+),K(+)-ATPase activity increased 35% after transfer and was correlated with an increase in Jv (110%). As salinity induced only minor changes in the osmoregulatory organs examined, it is proposed that the intrinsic euryhalinity of the striped bass may be related to a high degree of "preparedness" for hypoosmoregulation that is uncommon among teleosts studied to data.

In-vitro effects of insulin-like growth factor-I on gill Na+,K(+)-ATPase in coho salmon, Oncorhynchus kisutch.

The effect of ovine GH (oGH) in vivo and recombinant bovine insulin-like growth factor-I (rbIGF-I) in vitro on gill Na+,K(+)-ATPase activity was investigated in two seasonal experiments conducted during the parr-smolt transformation period of coho salmon. In 1991, when fish were held under a photoperiod of 12 h light : 12 h darkness, the stimulatory effect of oGH (1 microgram/g) on gill Na+,K(+)-ATPase in vivo decreased at the time of expected parr-smolt transformation. Gill Na+,K(+)-ATPase from control fish was insensitive to rbIGF-I in vitro from February to June, whereas GH treatment induced sensitivity to rbIGF-I (100-1000 micrograms/l) in vitro in February and March, but not later in development. In 1992, when fish were held under natural conditions, oGH (4 micrograms/g) stimulated gill Na+,K(+)-ATPase in vivo from February to July. There was, however, of pronounced developmental change in sensitivity of gill Na+,K(+)-ATPase to rbIGF-I in vitro. In February, gills from control fish were insensitive, but oGH treatment in vivo induced sensitivity to rbIGF-I in vitro (100-1000 micrograms/l). In April and May, control fish were sensitive to rbIGF-I in vitro. This sensitivity was not further potentiated by oGH treatment in vivo. In June, gills from control or oGH-treated fish were not sensitive to rbIGF-I in vitro, but in July exogenous oGH again induced gill tissue sensitivity to rbIGF-I at 1000 micrograms/l. Both studies showed that rbIGF-I stimulates gill Na(+),K(+)-ATPase directly; an ability that may depend on priming by endogenous or exogenous GH. This supports the role of IGF-I as an endocrine mediator for GH action during parr-smolt transformation.

Opposite effects of 17 beta-estradiol and combined growth hormone-cortisol treatment on hypo-osmoregulatory performance in sea trout presmolts, Salmo trutta.

In March, sexually immature sea trout presmolts (Salmo trutta trutta) were injected every second day with saline, 2 micrograms 17 beta-estradiol (E2)/g, 2 micrograms ovine growth hormone (GH) + 6 micrograms cortisol (F)/g, or all three hormones (E2-GH-F) simultaneously. A SW-challenge test was performed after six injections. At the time of SW-transfer, high total plasma calcium levels in E2- and E2-GH-F-treated fish indicated activated vitellogenesis in these groups. All control, GH-F, and E2-GH-F-treated fish survived SW-transfer, whereas 43% of the E2-treated fish died after transfer. On Day 2 after transfer, there were marked differences among groups in their osmoregulatory response. Changes in ion-osmotic parameters (plasma Na+, Cl-, Mg2+, and total calcium and muscle water) indicated the following degree of osmotic stress: E2 greater than control greater than E2-GH-F greater than GH-F, which was inversely correlated with pretransfer gill Na+/K(+)-ATPase activity: GH-F greater than E2-GH-F greater than control greater than E2. On Day 7 after transfer there were no major differences among the groups with regard to plasma ions and muscle water content. The detrimental influence of elevated plasma E2 levels on hypo-osmo-regulatory physiology may indicate an important role of E2 during development.