Switching of Na+, K+-ATPase isoforms by salinity and prolactin in the gill of a cichlid fish

We identified and investigated the changes in expression of two gill Na(+), K(+)-ATPase α-subunit isoforms (α-1a and α-1b) in relationship with salinity acclimation in a cichlid fish, Mozambique tilapia. Transfer of freshwater (FW)-acclimated fish to seawater (SW) resulted in a marked reduction in α-1a expression within 24?h and a significant increase in α-1b expression with maximum levels attained 7 days after the transfer. In contrast, transfer of SW-acclimated fish to FW induced a marked increase in α-1a expression within 2 days, while α-1b expression decreased significantly after 14 days. Hypophysectomy resulted in a virtual shutdown of α-1a mRNA expression in both FW- and SW-acclimated fish, whereas no significant effect was observed in α-1b expression. Replacement therapy by ovine prolactin (oPrl) fully restored α-1a expression in FW-acclimated fish, while cortisol had a modest, but significant, stimulatory effect on α-1a expression. In hypophysectomized fish in SW, replacement therapy with oPrl alone or in combination with cortisol resulted in a marked increase in α-1a mRNA to levels far exceeding those observed in sham-operated fish. Expression of α-1b mRNA was unaffected by hormone treatment either in FW-acclimated fish or in SW-acclimated fish. The mRNA expression of fxyd-11, a regulatory Na(+), K(+)-ATPase subunit, was transiently enhanced during both FW and SW acclimation. In hypophysectomized fish in FW, oPrl and cortisol stimulated fxyd-11 expression in a synergistic manner. The clear Prl dependence of gill α-1a expression may partially explain the importance of this hormone to hyperosmoregulation in this species.     

Osmoregulatory action of PRL,GH, and cortisol in the gilthead seabream (Sparus aurata L)

The osmoregulatory actions of ovine prolactin (oPRL), ovine growth hormone (oGH), and cortisol were tested in the euryhaline gilthead seabream Sparus aurata. Acclimated to sea water (SW, 40 ppt salinity, 1000 mOsm/kg H(2)O) or brackish water (BW, 5 ppt, salinity, 130 mOsm/kg H(2)O), injected every other day for one week (number of injections, 4) with saline (0.9% NaCl), oPRL (4 microg/g body weight), oGH (4 microg/g body weight) or cortisol (5 microg/g body weight), and transferred from SW to BW or from BW to SW 24h after the last injection. Fish were sampled before and 24h after transfer. Gill Na(+), K(+)-ATPase activity, plasma osmolality, plasma ions (sodium and chloride), plasma glucose, and muscle water moisture were examined. SW-adapted fish showed higher gill Na(+), K(+)-ATPase activity, plasma osmolality, and plasma ions levels than BW-adapted fish. Transfer from SW to BW decreased plasma osmolality and ions levels after 24h, while transfer from BW to SW increased these parameters, whereas gill Na(+),K(+)-ATPase activity was unaffected. oPRL treatment significantly decreased gill Na(+),K(+)-ATPase activity and increased plasma osmolality and ions in SW- and BW-adapted fish. This treatment minimizes loss of osmolality and ions in plasma after transfer to BW and increased these values after transfer to SW. No significant changes were observed in gill Na(+),K(+)-ATPase activity, plasma osmolality, and plasma ions in oGH-treated group with respect to saline group before or after transfer from SW to BW or from BW to SW. Treatment with cortisol induced, in SW-adapted fish, a significant increase of gill Na(+),K(+)-ATPase activity and decrease of plasma osmolality and plasma ions. In BW-adapted fish this treatment induced a significant increases in gill Na(+),K(+)-ATPase activity, plasma osmolality, and plasma ions. After transfer to SW cortisol-treated fish had higher plasma osmolality than the saline group. Our results support the osmoregulatory role of PRL in the adaptation to hypoosmotic environment in the gilthead seabream S. aurata. Further studies will be necessary to elucidate the osmoregulatory role of GH in this species. Cortisol results suggest a "dual osmoregulatory role" of this hormone in S. aurata.     

The role of cortisol and growth hormone in seawater adaptation and development of hypoosmoregulatory mechanisms in sea trout parr (Salmo trutta trutta)

The role of growth hormone (GH) and cortisol in the development of hypoosmoregulatory mechanisms in sea trout parr, Salmo trutta trutta, was investigated by injecting freshwater (FW) yearlings every second day with saline, ovine growth hormone (oGH, 2.0 micrograms/g), cortisol (hydrocortisone hemisuccinate, 8.0 micrograms/g), or oGH + cortisol for a maximum of 14 days. Subgroups of the treated fish were transferred to three-fourths seawater (SW) after 7 or 15 days of treatment and the effects on plasma Na+, Cl-, muscle water content, gill Na+/K(+)-ATPase activity, and gill interlamellar chloride cell density were examined. In FW, gill Na+/K(+)-ATPase chloride cell density, and chloride cell apical to basal length increased by all hormone treatments, most significant by oGH + cortisol treatment. Plasma ions and muscle water content were unaffected in FW. Both SW transfers resulted in considerable mortality (50%) in control fish, whereas few cortisol-treated and no GH-treated or GH + cortisol-treated fish died. Plasma Na+ and Cl- levels increased dramatically (greater than 50%) in control fish and muscle water content decreased (8%) on Day 2 after both transfers. All hormone-treated groups regulated plasma ions and muscle water significantly better than controls in SW, indicating the physiological significance of the treatment. Notably, the oGH + cortisol-treated fish showed only insignificant changes in ion-osmotic homeostasis after SW transfer, suggesting a synergistic effect of the two hormones. It is concluded that treatment with the two hormones increases the salinity tolerance of sea trout parr at a developmental stage where FW life is obligatory.     

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.     

Pituitary hormone mRNA expression in European sea bass Dicentrarchus labrax in seawater and following acclimation to fresh water

The mRNA expression of pituitary prolactin (prl), growth hormone (gh), somatolactin (sl), proopiomelanocortin (pomc), and gonadotropins (gthI and gthII) was quantified by real-time PCR, in sea bass, Dicentrarchus labrax, adapted for 1 month to seawater (SW) or freshwater (FW). In addition, IGF-I (igfI) mRNA expression in liver and branchial Na+/K+ -ATPase activity were determined. L17 ribosomal protein (rpL17) and elongation factor 1alpha (ef1alpha) were validated as reference genes in real-time PCR in the experimental context. The real-time PCR assays were validated for the different hormone genes considered. Expression of pituitary pomc, gthI, gthII, gh, and liver igfI was not significantly different between FW and SW fish. Pituitary prlwas 4.5-foldhigher in FWthan in SW, whereas pituitary sl was 1.8-fold higher in SW- compared with FW-adapted fish. Gill Na+/K+ -ATPase specific activity was 2.3-fold higher in FW sea bass compared with SW fish. Plasma cortisol levels were 6.5-fold lower in SW- than in FW-adapted specimens. The results are discussed in relation to the osmoregulatory strategy of this euryhaline SW species, which displays features that do not fit present models based on salmonids and FWeuryhaline teleosts.     

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.     

Osmoregulatory and metabolic changes in the gilthead sea bream Sparus auratus after arginine vasotocin (AVT) treatment

The influence of arginine vasotocin (AVT) on osmoregulation and metabolism in gilthead sea bream Sparus auratus was evaluated by two experimental approaches. In the first, seawater (SW, 36 ppt)-acclimatized fish were injected intraperitoneally with vehicle (vegetable oil) or two doses of AVT (0.5 and 1 microg/g body weight). Twenty-four hours later, eight fish from each group were sampled; the remaining fish were transferred to low saline water (LSW, 6 ppt, hypoosmotic test), SW (transfer control), and hypersaline water (HSW, 55 ppt, hyperosmotic test). After another 24h (48-h post-injection), fish were sampled. The only significant effect observed was the increase of sodium levels in AVT-treated fish transferred to HSW. In the second experiment, fish were injected intraperitoneally with slow-release vegetable oil implants (mixture 1:1 of coconut oil and seeds oil) alone or containing AVT (1 microg/g body weight). After 3 days, eight fish from each group were sampled; the remaining fish were transferred to LSW, SW, and HSW as above, and sampled 3 days later (i.e. 6 days post-injection). In the AVT-treated group transferred from SW to SW, a significant increase vs. control was observed in gill Na(+),K(+)-ATPase activity. Kidney Na(+),K(+)-ATPase activity decreased in the AVT-treated group transferred to LSW and no changes were observed in the other groups. These osmoregulatory changes suggest a role for AVT during hyperosmotic acclimation based on changes displayed by gill Na(+),K(+)-ATPase activity. AVT treatment increased plasma cortisol levels in fish transferred to LSW and HSW. In addition, AVT treatment affected parameters of carbohydrate, lipid, amino acid, and lactate metabolism in plasma and tissues (gills, kidney, liver, and brain). The most relevant effects were the increased potential of liver for glycogen mobilization and glucose release resulting in increased plasma levels of glucose in AVT-treated fish transferred to LSW and HSW. These changes may be related to the energy repartitioning process occurring during osmotic adaptation of S. auratus to extreme environmental salinities and could be mediated by increased levels of cortisol in plasma.     

Prolactin gene expression and gill chloride cell activity in fugu Takifugu rubripes exposed to a hypoosmotic environment

To evaluate a possible involvement of prolactin (PRL) in low-salinity tolerance of a marine pufferfish Takifugu rubripes, or fugu, gene-expression profiles of PRL in the pituitary and PRL receptor (PRLR) in the osmoregulatory organs were investigated in fish exposed to 25%-dilute seawater (SW). Following transfer from full-strength (100%) SW to 25% SW, plasma osmolality and Na(+) and Cl(-) levels were slightly decreased on day 1, which were restored on days 3 and 7. Expression levels of PRL mRNA in the pituitary was significantly increased in response to 25% SW transfer, which was in sharp contrast with a remarkable decrease in growth hormone (GH) mRNA levels. These profiles suggest that PRL and GH are involved in hyper- and hypoosmoregulation, respectively, as is the case with euryhaline teleosts. Expression levels of PRLR mRNA in the gill and intestine were not significantly different from the initial levels, whereas, PRLR mRNA expression in the kidney was significantly higher on day 7 than the initial levels. Although transfer to 25% SW did not affect the average size of Na(+)/K(+)-ATPase-immunoreactive chloride cells in the gills, both size and density of apical openings of chloride cells became significantly smaller after transfer to 25% SW. These findings suggest that the possible hypoosmotic action of PRL is mediated by PRLR expressed in the osmoregulatory organs, and that low-salinity tolerance of fugu may involve reduction of an ion-secreting function of gill chloride cells. To further evaluate long-term effects of the low-salinity environment on growth and osmoregulation, fugu were raised in 25% and 100% SW for a prolonged period of 8 weeks. They grew similarly in 25% and 100% SW, and there was no significant difference in body weight and standard length at any weekly sampling point. The plasma osmolality was maintained at about 345mOsm/kg.H(2)O in both media, whereas the gill Na(+)/K(+)-ATPase activity was significantly lower in 25% SW than 100% SW. Gene expression of PRL in the pituitary was higher in 25% SW than in 100% SW; conversely, expression levels of GH were lower in 25% SW than in 100% SW. These findings support a hyperosmotic action of PRL and a hypoosmotic, rather than growth-promoting, action of GH in this marine teleost.     

Effects of salinity, hypophysectomy, and prolactin on whole-animal transepithelial potential in the tilapia Oreochromis mossambicus

We have examined whether two recently isolated forms of tilapia (Oreochromis mossambicus) prolactin exert similar effects on osmoregulatory physiology. The effects of salinity, hypophysectomy, and replacement therapy with tilapia prolactins on whole-animal transepithelial potential (TEP), gill Na+, K+-ATPase activity, and plasma ions were determined. When intact fish adapted to 25% seawater (SW) were transferred to different salinities, TEP reached a steady state after 10 hr; TEP increased with increasing salinity from fresh water (FW) to 75% SW but was stable from 75 to 125% SW. Plasma osmolality, [Na+], and [Cl-] of these fish 24 hr after salinity change showed that fish in 100 and 125% SW had greater osmotic perturbation than those transferred to lower salinities. Following a 5-day recovery period in 25% SW, hypophysectomized fish transferred to FW for 10 hr had significantly lower TEP and plasma ion levels than either sham-operated fish or intact fish under the same conditions. Injection of hypophysectomized fish with "small" prolactin (tPRL177), "large" prolactin (tPRL188), or a combination of both (0.5 micrograms/g body weight) 22 hr and again 20 min prior to transfer from 25% SW to FW, restored TEP and plasma ion levels to those of sham-operated fish. Neither prolactin affected the TEP or plasma ions of sham-operated (intact) fish. Hypophysectomized fish had lower gill Na+,K+-ATPase activity than sham-operated fish in FW, but prolactin injections as described above did not affect gill Na+,K+-ATPase activity in either hypophysectomized or sham-operated fish. Our results indicate that the two forms of prolactin are indistinguishable with regard to several aspects of tilapia osmoregulation.     

Glucocorticoid receptor upregulation during seawater adaptation in a euryhaline teleost,the tilapia (Oreochromis mossambicus)

Cortisol is an important seawater (SW) osmoregulatory hormone in the Mozambique tilapia (Oreochromis mossambicus), a highly euryhaline cichlid able to live in environments ranging from fresh water (FW) to salinities well in excess of full-strength seawater. Previous studies indicate that cortisol may promote SW adaptation by increasing gill chloride cell differentiation, Na(+)/K(+)-ATPase activity and subsequent excretion of excess salt following seawater acclimation. Despite cortisol's widely accepted role as a SW-adapting hormone, cortisol receptor regulation during SW acclimation is not well understood. The purpose of these studies was to determine whether the intracellular glucocorticoid receptor (GR) might be regulated in a manner consistent with cortisol's actions in SW adaptation. Saturation radioligand binding assays were conducted on gill cytoplasm preparations from fish sampled 4 and 24h and 4 and 14 days after transfer from FW to 2/3 SW or FW (control). Affinity (K(d)) of the gill GR remained constant over the timecourse, while numbers of receptors (B(max)) in SW fish were significantly elevated compared with controls at 24h and 4 days after transfer. Plasma osmolality was higher in fish transferred to SW for 24h, 4 days, and 14 days compared with those animals moved to FW. Plasma cortisol levels and hepatic cortisol binding remained constant between SW and FW fish throughout the timecourse of the salinity challenge. These studies indicate that seawater acclimation is accompanied by a specific upregulation of intracellular GR numbers in gill tissue. The lack of increase in circulating cortisol following SW adaptation may reflect enhancement of clearance of the steroid. It appears that an increase in cortisol receptors, which is closely associated with the rise in blood osmotic pressure that accompanies SW exposure, is an important component of cortisol's ability to promote SW adaptation in the tilapia.     

Relationships between gill Na(+),K(+)-ATPase activity and endocrine and local insulin-like growth factor-I levels during smoltification of masu salmon (Oncorhynchus masou)

We established profiles of insulin-like growth factor (IGF)-I mRNA in the liver, gill and white muscle and circulating IGF-I during smoltification of hatchery-reared masu salmon, and compared with that of gill Na(+),K(+)-ATPase (NKA) activity. Gill NKA activity peaked in May and dropped in June. Liver igf1 mRNA was high in March and decreased to low levels thereafter. Gill igf1 increased from March, maintained its high levels during April and May and decreased in June. Muscle igf1 mRNA levels were relatively high during January and April when water temperature was low. Serum IGF-I continuously increased from March through June. Serum IGF-I during March and May showed a positive correlation with NKA activity, although both were also related to fish size. These parameters were standardized with fork length and re-analyzed. As a result, serum IGF-I and gill igf1 were correlated with NKA activity. On the other hand, samples from desmoltification period (June) that had high serum IGF-I levels and low NKA activity disrupted the relationship. Expression of two IGF-I receptor (igf1r) subtypes in the gill decreased in June, which could account for the disruption by preventing circulating IGF-I from acting on the gill and retaining it in the blood. The present study suggests that the increase in gill NKA activity in the course of smoltification of masu salmon was supported by both endocrine and local IGF-I, and the decrease during desmoltification in freshwater was due at least in part to the down-regulation of gill IGF-I receptors.     

Effects of seawater acclimation on mRNA levels of corticosteroid receptor genes in osmoregulatory and immune systems in trout

Influence of environmental salinity on expression of distinct corticosteroid receptor (CR) genes, glucocorticoid receptor (GR)-1 and -2, and mineralcorticoid receptor (MR), was examined in osmoregulatory and hemopoietic organs and leucocytes of steelhead trout (Oncorhynchus mykiss). There was no significant difference in plasma cortisol levels between freshwater (FW)- or seawater (SW)-acclimated trout, whereas Na+, K+-ATPase was activated in gill of SW fish. Plasma lysozyme levels also showed a significant increase after acclimation to SW. In SW-acclimated fish, mRNA levels of GR-1, GR-2, and MR were significantly higher in gill and body kidney than those in FW. Head kidney and spleen showed no significant change in these CR mRNA levels after SW-acclimation. On the other hand, leucocytes isolated from head kidney and peripheral blood showed significant decreases in mRNA levels of CR in SW-acclimated fish. These results showed differential regulation of gene expression of CR between osmoregulatory and immune systems.     

Developmental differences in the responsiveness of gill Na+,K(+)-ATPase to cortisol in salmonids.

The ability of cortisol to increase gill Na+,K(+)-ATPase activity was examined in several salmonid species during development. Coho salmon (Oncorhynchus kisutch) parr were unresponsive to cortisol in vitro (10 micrograms/ml for 2 days) in November. Responsiveness was significant from January to March, peaking in January just prior to seasonal increases in gill Na+,K(+)-ATPase activity. Gill tissue became unresponsive to in vitro cortisol in April when in vivo gill Na+,K(+)-ATPase activity peaked. The ability of cortisol to stimulate gill, Na+,K(+)-ATPase activity in postemergent fry (2-3 months after hatching) was examined in chum (O. keta), chinook (O. tschawytscha), coho, and Atlantic salmon (Salmo salar). Initial levels of gill Na+,K(+)-ATPase activity were elevated in chum salmon, which normally migrate as fry. Cortisol (10 micrograms/ml for 4 days in vitro) increased gill Na+,K(+)-ATPase activity in chum salmon fry (48% above initial levels), had a limited but significant effect in chinook salmon fry, and had no effect in coho and Atlantic salmon fry. In an in vivo experiment, Atlantic salmon previously exposed to simulated natural photoperiod (SNP) and continuous light (L24) received four cortisol injections of 2 micrograms.g-1 every third day. SNP fish responded with increased gill Na+,K(+)-ATPase activity (+66%), whereas L24 fish were not affected. Atlantic salmon presmolts with initially low levels of gill Na+,K(+)-ATPase activity responded to cortisol in vitro, whereas smolts with initially high levels of gill Na+,K(+)-ATPase activity were unresponsive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of growth hormone and cortisol on Na(+)-K(+)-2Cl(-) cotransporter localization and abundance in the gills of Atlantic salmon.

The hormones responsible for the regulation of the teleostean gill Na(+)-K(+)-2Cl(-) cotransporter have not been elucidated. With Western blotting and immunocytochemistry, Na(+)-K(+)-2Cl(-) cotransporter abundance and localization were examined in the gills of Atlantic salmon (Salmo salar) following 2-week treatment with growth hormone (GH; 5.0 microg x g(-1)), cortisol (50 microg x g(-1)), and both hormones in combination (GH+cortisol). GH and cortisol treatments increased gill Na(+)-K(+)-2Cl(-) cotransporter abundance over levels seen in controls, and both hormones together (GH+cortisol) produced a greater effect than either hormone alone. Gill Na(+),K(+)-ATPase activity was also elevated by all three hormone treatments. Compared to controls, Na(+)-K(+)-2Cl(-) cotransporter immunoreactive chloride cells on the primary filament were greater in number and size following all three treatments. Although the number of immunoreactive chloride cells on the secondary lamellae did not differ among the treatment groups, GH+cortisol increased their size. These data indicate that GH and cortisol increase gill Na(+)-K(+)-2Cl(-) cotransporter abundance through chloride cell proliferation and differentiation in the gills of Atlantic salmon and are likely the hormones responsible for Na(+)-K(+)-2Cl(-) cotransporter regulation during smolting and seawater acclimation.     

Effects of cortisol and growth hormone on osmoregulation in pre- and desmoltified coho salmon (Oncorhynchus kisutch)

Salmonid species which undergo smoltification show a concurrent enhancement in saltwater (SW) osmoregulatory ability. This developmental change is marked by an increase in SW tolerance and gill Na+,K+-ATPase activity which appears to result, in part, from an increase in gill chloride cell density. Previous studies have suggested that cortisol and growth hormone (GH) may stimulate SW osmoregulatory mechanisms in salmonids. In this study, these hormones were examined for their ability to induce smoltification-associated osmoregulatory changes in pre- and desmoltified coho salmon (Oncorhynchus kisutch). Cortisol treatment for 12 days increased gill Na+,K+-ATPase activity in presmolts and gill residual (Na+,K+-independent) ATPase activity in both groups. Chloride cell density in presmolt primary and secondary lamellae and in desmolt secondary lamellae was increased as well. The rise in plasma sodium levels in fish transferred to SW was reduced only in desmolts. Treatment with bovine GH for 12-13 days increased gill Na+,K+-ATPase activity in presmolts and in desmolts. However, GH treatment in either group did not increase gill residual ATPase activity or alter plasma sodium levels in SW-transferred animals. Gill chloride cell density in presmolts also was unaffected (desmolts were not examined). Thus, both cortisol and GH are partially able to produce changes similar to those observed during smoltification. The contrasting effects of these hormones on gill chloride cell density and gill residual ATPase activity suggest that cortisol may stimulate chloride cell proliferation and/or differentiation, whereas GH may act specifically to increase gill Na+,K+-ATPase activity.     

Smoltification and seawater adaptation in coho salmon (Oncorhynchus kisutch): plasma calcium regulation, osmoregulation, and calcitonin

In order to examine the dynamics of ion regulation, osmoregulation, and plasma calcitonin during the parr-smolt transformation (smoltification), blood and gill tissue were collected from yearling coho salmon, Oncorhynchus kisutch, from February to October. Fish were kept in fresh water (FW) throughout this period. In addition, fish were exposed to seawater (SW) at the peak of smoltification in mid-April, and samples from these fish were collected until July. Plasma osmolality, gill Na+,K+-ATPase activity, plasma levels of calcitonin, and free and total calcium and magnesium were measured. SW adaptability of FW fish was assessed throughout the study by measurements of plasma osmolality following a 24-hr exposure to seawater. The greatest hypoosmoregulatory ability occurred in April-May, although SW-adapted fish had higher plasma osmolality than FW-adapted fish at all times. Gill Na+,K+-ATPase activity in FW-adapted fish increased from April to June and increased rapidly following exposure of fish to SW, and remained elevated in SW-adapted fish. Free plasma calcium and magnesium levels increased following SW exposure, but returned to prior levels within 1 week. Netting and confinement stress during sampling caused an increase in plasma osmolality and free calcium and magnesium levels in both FW- and SW-adapted fish. Changes in hypoosmoregulatory ability during smoltification and SW adaptation were correlated with changes in gill Na+,K+-ATPase activity. A sharp transitory peak in plasma calcitonin levels occurred early in smoltification (March) and in SW-adapted fish in June. Plasma calcitonin levels gradually increased in FW-adapted fish during the period of desmoltification. However, no change in plasma calcitonin levels occurred during SW-induced hypercalcemia, suggesting that the hormone does not play a major role in short-term plasma calcium regulation in coho salmon.