Seasonal changes in androgen levels in stream- and hatchery-reared Atlantic salmon parr and their relationship to smolting

In stream-reared Atlantic salmon Salmo salar , plasma androgens were significantly greater in mature male parr than immature males and females in October, but had declined by January and did not differ significantly from immature fish throughout the spring. Immature fish in March were significantly larger and had greater gill Na⁺, K⁺-ATPase activity than their previously mature counterparts. Bimodal growth distribution was seen in hatchery-reared Atlantic salmon and a proportion of the male fish in the lower mode matured. Plasma testosterone (T) and 11-ketotestosterone (11-KT) were significantly elevated from September to December in mature male (1+ year) parr. In January, plasma androgens had declined in mature males and did not differ significantly from immature fish. By May all the hatchery fish were large enough to smolt and a proportion of the previously mature males had increased gill Na⁺, K⁺-ATPase activity. Therefore elevated androgens in the previous autumn do not prevent smolting. Parr with higher plasma T and 11-KT in April and May, that are presumably beginning to mature, had lower gill Na⁺, K⁺-ATPase activity, indicating that future maturation and associated increases in androgens may inhibit smolting.     

Smolting and seasonal variation in the smolt characteristics of one- and two-year-old Saimaa landlocked salmon under fish farm conditions

Silvering of the skin, reduced condition factor, elevated gill Na⁺, K⁺-ATPase activity and well-developed capacity to regulate the osmotic and ionic balance in sea water were observed in 1 and 2 year old hatchery-reared Saimaa landlocked salmon Salmo salar m. sebago during April-June. Loss of hypoosmoregulatory ability and gill Na⁺K⁺-ATPase activity was observed earlier in 2 year than 1 year old fish. Coincident with changes associated with smolting both age groups showed diminished osmoregulatory capacity in fresh water. Slow growth during May-June may also be attributed to osmoregulatory difficulties in fresh water. These results support the suggestion that the developmental changes at smolting are seasonal and unrelated to any salinity changes and the idea of smolting as evidence of maladaptation of the fish to fresh water.     

Genetic differences in physiology, growth hormone levels and migratory behaviour of Atlantic salmon smolts

Out of five strains of Atlantic salmon Salmo salar of 1+ years released upstream of a fyke net in the River Gudenaa in 1996, three, Lagan, Ätran and Corrib, migrated immediately, 50% of the recaptured fish reaching the net in 3–6 days. Burrishoole and Conon fish migrated with a 15–19 day delay. Smolt development in 1997 at the hatchery showed a spring surge in gill Na⁺, K⁺-ATPase activity in all strains which was correlated with increased seawater tolerance. Differences in the timing of gill enzyme development matched the observed migration pattern well. Lagan, Ätran and Corrib strains reached high enzyme activity earlier than the Burrishoole and Conon strains, and strains with delayed enzyme development and migration showed a delayed regression of seawater tolerance compared with the early strains. Inter-strain differences in plasma growth hormone profiles could not be related to the observed patterns of Na⁺, K⁺-ATPase and seawater tolerance development. The study gives evidence of genetic influence on the timing and intensity of smolting and subsequent migration in Atlantic salmon.     

Plasma ionic regulation and gill Na+/K+-ATPase changes during rapid transfer to sea water of yearling rainbow trout, Salmo gairdneri: time course and seasonal variation

Danish rainbow trout, Salmo gairdneri Richardson, (40–65 g) were transferred to 28%o sea water at intervals throughout the early spring and summer. Gill Na⁺/K⁺-ATPase of fish kept in fresh water surged distinctly during May. Simultaneously, a body silvering occurred and plasma concentrations of Cl⁻, Na⁺ and total thyroxine (T4) decreased. The seawater transfer-induced adaptive response in plasma electrolytes comprised a biphasic change, i.e., an adjustive peak phase and a regulatory phase lasting for 2 days and 1 week after transfer, respectively. Further, gill Na⁺/K⁺-ATPase activity increased to a new level after an initial lag phase of 2–3 days, but electrolyte regulation was mostly initiated prior to the adaptive change in ATPase activity. In spite of increasing pre-transfer freshwater Na⁺/K⁺-ATPase activity during the spring, the electrolyte peak level, the degree of muscle dehydration and the mortality of fish transferred to sea water increased from April to July. The apparent uncoupling of freshwater Na⁺/K⁺-ATPase activity and plasma electrolyte regulation in sea water is discussed in relation to smelting and prediction of hypo-osmoregulatory performance.     

Effects of dietary soybean meal and photoperiod cycle on osmoregulation following seawater exposure in Atlantic salmon smolts

Atlantic salmon Salmo salar juveniles were fed either fishmeal-based diets (FM) or diets in which soybean meal (SBM) partly replaced the FM from first feeding on. The fish were kept at continuous daylight during the juvenile stage. During the last 3 weeks before reaching 100 g body mass, all fish were subjected to 12L:12D. Starting at 100 g body mass, groups of 60 fish from each feeding background were subjected to continuous light for 12 weeks (short winter), or a square-wave photoperiod cycle to stimulate parr to smolt transformation with 8L:16D during the first 6 weeks, and then continuous light during the last 6 weeks (long winter). After the 12 weeks, 20 fish from each treatment were subjected to 0, 24 or 96 h seawater exposure at a water salinity of 34. Hypo-osmoregulatory ability at seawater exposure was assessed by mortality, intestinal pathology, plasma ion concentrations and osmolality, gill Na⁺/K⁺-ATPase activity and element concentrations in the cytoplasm of distal intestinal enterocytes using X-ray microanalysis. The hypo-osmoregulatory capacity was higher in fish kept at short winter than at long winter, apparently due to more rapid development of gill Na⁺/K⁺-ATPase activity. Fish fed SBM suffered typical soybean meal-induced histological alterations of the distal intestine and apparent reductions in digestive function in the more proximal gastrointestinal regions. The net osmoregulatory capacity of these fish was maintained, as indicated by higher gill Na⁺/K⁺-ATPase activity and lower plasma Na⁺, Ca²⁺ and osmolality compared to the FM-fed fish. Thus, feeding SBM did not impair the hypo-osmoregulatory ability of the Atlantic salmon following seawater exposure.     

Salinity effects on oxygen consumption, gill Na+, K+-ATPase and ion regulation in juvenile coho salmon

The metabolic response of juvenile coho salmon Oncorhynchus kisutch to different salinities was examined, using whole-animal oxygen consumption rates and gill Na⁺, K⁺-ATPase activities as indicators of osmoregulatory energetics. Coho salmon smolts were acclimated to fresh water (FW), isosmotic salinity (ISO, 10‰) and sea water (SW, 28‰) and were sampled for up to 6 weeks for plasma levels of cortisol, glucose and ions (Na⁺, K⁺, Cl⁻), gill Na⁺, K⁺-ATPase activity and oxygen consumption rates. Following an initial adjustment period, plasma constituents in SW fish returned to near-FW values, indicating that the fish were acclimated to SW by day 21. Gill Na⁺, K⁺-ATPase activities on days 21 and 42 were lowest in ISO, higher in FW and highest in SW. This result is consistent with the idea that less energy would be required to maintain ion balance in an isosmotic environment, where the ionic gradients between extracellular fluid and water would be minimal. Oxygen consumption rates of swimming fish (1 body length s⁻¹), however, did not differ significantly between the three test salinities after 6 weeks. The results of this study suggest that the metabolic response of juvenile salmonids to changes in salinity is dependent on life-history stage (e.g. fry v . smolt), and that oxygen consumption rates do not necessarily reflect osmoregulatory costs.     

Somatolactin mRNA expression during the parr–smolt transformation in hatchery-reared Atlantic salmon Salmo salar smolts

Expression of somatolactin (SL) mRNA was quantified during smoltification at 2 week intervals during January and February and weekly during March, April and May 2004 in a line-bred ranching stock of Atlantic salmon Salmo salar . Gene expression of SL (and a splice variant termed SL₁₈₀) was lower in fish sampled in the later weeks of smoltification than in those at the beginning, a pattern opposite to that of gill Na⁺ K⁺-ATPase activity.     

Cortisol stimulates hypo-osmoregulatory ability in Atlantic salmon, Salmo salar L.

Hypo-osmoregulatory ability in juvenile Atlantic salmon, Sulrno salur L., was improved by cortisol treatment. Implantation of a vegetable shortening pellet containing cortisol (50 mg kg⁻¹) resulted in elevated plasma cortisol titres. Maximum cortisol levels (160–170ngml⁻¹) were observed at days 6 and 12 after the implantation and dropped significantly by day 55. Cortisol-implanted fish in fresh water developed a twofold increase in gill Na⁺/K⁺-ATPase activity at days 6 and 12, and a threefold increase by day 55. Intestinal mucosa Na⁺/K⁺-ATPase activity was not affected by cortisol. Cortisol-implanted fish exposed to 28 ppt sea water for 48 h tended to show an improved ability to regulate their plasma osmolarity and reduce their ionic load. The osmo-regulatory ability attained at days 12 and 55 was further evaluated by exposing fish to 37 ppt sea water for 96 h. While all the control fish died relatively early in these tests, cortisol-implanted fish showed a clear reduction in their mortality rate. These results indicate that cortisol can induce biochemical and organismal changes during winter months that typify preadaptive events normally occurring in the spring.     

Changes in plasma thyroid hormone levels in pink salmon, Oncorhynchus gorbuscha, during their spawning migration in the Fraser River (Canada)

Plasma thyroid hormone levels were measured in pink salmon, Oncorhynchus gorbuscha , during their spawning migration in the Fraser River, British Columbia. The plasma levels of both l-thyroxine (T₄) and triiodo-l-thyronine (T₃) were significantly higher in males than in females. In both sexes the hormone levels were maintained, or increased somewhat, during the early stages of migration, but fell thereafter. In females the plasma T₄ and T₃ levels of salmon collected on the spawning grounds were at or below detectable levels of the assays. The changes in thyroid hormone levels are correlated with changes in plasma insulin, gonadotropin, gonadal steroid hormones, cortisol and vitellogenin levels measured in the same specimens.     

Glutamate Induces a Calcineurin-Mediated Dephosphorylation of Na+,K+-ATPase that Results in Its Activation in Cerebellar Neurons in Culture

Abstract: In primary cultures of cerebellar neurons glutamate neurotoxicity is mainly mediated by activation of the NMDA receptor, which allows the entry of Ca²⁺ and Na⁺ into the neuron. To maintain Na⁺ homeostasis, the excess Na⁺ entering through the ion channel should be removed by Na⁺,K⁺-ATPase. It is shown that incubation of primary cultured cerebellar neurons with glutamate resulted in activation of the Na⁺,K⁺-ATPase. The effect was rapid, peaking between 5 and 15 min (85% activation), and was maintained for at least 2 h. Glutamate-induced activation of Na⁺,K⁺-ATPase was dose dependent: It was appreciable (37%) at 0.1 µ M and peaked (85%) at 100 µ M . The increase in Na⁺,K⁺-ATPase activity by glutamate was prevented by MK-801, indicating that it is mediated by activation of the NMDA receptor. Activation of the ATPase was reversed by phorbol 12-myristate 13-acetate, an activator of protein kinase C, indicating that activation of Na⁺,K⁺-ATPase is due to decreased phosphorylation by protein kinase C. W-7 or cyclosporin, both inhibitors of calcineurin, prevented the activation of Na⁺,K⁺-ATPase by glutamate. These results suggest that activation of NMDA receptors leads to activation of calcineurin, which dephosphorylates an amino acid residue of the Na⁺,K⁺-ATPase that was previously phosphorylated by protein kinase C. This dephosphorylation leads to activation of Na⁺,K⁺-ATPase.     

Seasonal changes in ionoregulatory variables of the glass eel Anguilla anguilla following estuarine entry: comparison with resident elvers

In the present study, glass eels Anguilla anguilla in the Minho River estuary (41·5° N, 8·5° W) decreased in size (standard length, L _S and mass, M ) from the beginning (autumn) to the end of the sampling season (summer). On the other hand elvers increased in L _S and M from spring to summer and were significantly larger than glass eels in paired comparisons. Branchial Na⁺/K⁺-ATPase and vacuolar (V-type) proton ATPase ( in vitro activities), two important ion transporting pumps, did not show significant seasonal changes in either glass eels or elvers although in glass eels Na⁺/K⁺-ATPase (activity) expression was significantly higher than in elvers. In a single month comparison Na⁺/K⁺-ATPase branchial mRNA expression was also higher in glass eels as was the protein level expression of both Na⁺/K⁺-ATPase and NKCC (Na⁺:K⁺:2Cl⁻ co-transporter). Immunofluorescence microscopy indicated apical CFTR Cl⁻ channel labelling in Na⁺/K⁺-ATPase positive chloride cell in glass eels which was absent in elvers. Whole body sodium concentration and percentage water did not show significant seasonal differences in either glass eels or elvers although there were significant differences between these two groups during some months.     

Glutamate Uptake Stimulates Na+,K+-ATPase Activity in Astrocytes via Activation of a Distinct Subunit Highly Sensitive to Ouabain

Abstract: The excitatory amino acid glutamate was previously shown to stimulate aerobic glycolysis in astrocytes by a mechanism involving its uptake through an Na⁺-dependent transporter. Evidence had been provided that Na⁺,K⁺-ATPase might be involved in this process. We have now measured the activity of Na⁺,K⁺-ATPase in cultured astrocytes, using ouabain-sensitive ⁸⁶Rb uptake as an index. l -Glutamate increases glial Na⁺,K⁺-ATPase activity in a concentration-dependent manner with an EC₅₀ = 67 µ M . Both l - and d -aspartate, but not d -glutamate, produce a similar response, an observation that is consistent with an uptake-related effect rather than a receptor-mediated one. Under basal conditions, concentration-dependent inhibition of Na⁺,K⁺-ATPase activity in astrocytes by ouabain indicates the presence of a single catalytic site with a low affinity for ouabain ( K _0.5 = 113 µ M ), compatible with the presence of an α₁ isozyme. On stimulation with glutamate, however, most of the increased activity is inhibited by low concentrations of ouabain ( K _0.5 = 20 n M ), thus revealing a high-affinity site akin to the α₂ isozyme. These results suggest that astrocytes possess a glutamate-sensitive isoform of Na⁺,K⁺-ATPase that can be mobilized in response to increased neuronal activity.     

Expression of Na+, K+-ATPase α-Subunit in Animalized and Vegetalized Embryos of the Sea Urchin, Hemicentrotus pulcherrimus.

A marked increase in the Na⁺, K⁺-ATPase activity of sea urchin embryos occurred following an elevation of its mRNA level, revealed by Northern blotting analysis, in developmental period between the swimming blastula and the late gastrula stage. cDNA clone of Na⁺, K⁺-ATPase α-subunit, obtained from γgt10 cDNA library of sea urchin gastrulae, was digested with EcoRl ad Hindlll. The obtained 268 bp cDNA fragment, hybridized to a 4.6 Kb RNA, was used as probe for Northern blotting analysis. The level of Na⁺, K⁺-ATPase mRNA was higher in embryo-wall cell fraction isolated from late gastrulae (ectoderm cells) than the level in the bag fraction, containing mesenchyme cells (mesoderm cells) and archenteron (endoderm cells). The activity of Na⁺, K⁺-ATPase and the level of its mRNA were higher in animalized embryos obtained by pulse treatment with A23187 for 3 hr, starting at the 8–16 cell stage and were considerably lower in vegetalized embryos induced by 3 hr treatment with Li⁺ than that in normal embryos at the post gastrula corresponidng stage. Augmentation of Na⁺, K⁺-ATPase gene expression can be regarded as a marker for ectoderm cell differentiation at the post gastrula stage, which results from determination of cell fate in prehatching period.     

Functional Analysis and Tissue-Specific Expression of Drosophila Na+,K+-ATPase Subunits

Abstract: We have previously purified and characterized a nervous system-specific glycoprotein antigen from adult Drosophila heads, designated Nervana [nerve antigen (NRV)] and identified two separate genes coding for three different proteins. All three proteins share homology with the β subunits of Na⁺,K⁺-ATPase from various other species. In this study we have isolated a new Drosophila Na⁺,K⁺-ATPase α subunit cDNA clone (PSα; GenBank accession no. AF044974) and demonstrate expression of functional Na⁺,K⁺-ATPase activity when PSα mRNA is coinjected into Xenopus oocytes along with any of the three different Nrv mRNAs. Western blotting, RNase protection assays, and immunocytochemical staining of adult fly sections indicate that NRV2 is expressed primarily in the nervous system. Staining is most intense in the brain and thoracic ganglia and is most likely associated with neuronal elements. NRV1 is more broadly expressed in muscle and excretory tissue and also shows diffuse distribution in the nervous system. Similar to other species, Drosophila expresses multiple isoforms of Na⁺,K⁺-ATPase subunits in a tissue- and cell type-specific pattern. It will now be possible to use the advantages of Drosophila molecular and classical genetics to investigate the phenotypic consequences of altering Na⁺,K⁺-ATPase expression in various cell and tissue types.     

Pre‐migratory differentiation of wild brown trout into migrant and resident individuals

In February to March, wild brown trout Salmo trutta were captured by electrofishing in a natural watercourse (tributaries of the River Lille Aa, Denmark), individually tagged (Passive Integrated Transponders), and released. Representatives of the tagged brown trout were recaptured on the release sites in April by electrofishing and eventually caught in downstream smolt traps ('migrants') placed in the main river or by electrofishing ('residents') on the initial sites in June. Upon each capture, smolt appearance and body size were evaluated, and a non‐lethal gill biopsy was taken and used for Na⁺,K⁺‐ATPase analysis. Based on repetitive gill enzyme analysis in individual fish, a retrospective analysis of the rate of development in individual brown trout ultimately classified as migrants or residents was performed. Two months prior to migration, a bimodal morphological and physiological (gill Na⁺,K⁺‐ATPase) development concurred and was related to the subsequent differentiation into resident and migratory fractions of each population. This differentiation was unrelated to growth rate and body size of individual fish but skewed in favour of migratory females. Individuals destined to become migrants developed a smolt‐like appearance before the onset of migration and had higher rate of change of gill Na⁺,K⁺‐ATPase activity than fish remaining residents. The rate of change of gill Na⁺,K⁺‐ATPase activity was independent of the distance migrated to the trap (3–28 km). Thus in bimodal wild brown trout populations a major increase in enzyme activity takes place before migration is initiated and is a characteristic of migratory individuals only.     

Plasticity of smolting in spring chinook salmon: relation to growth and insulin-like growth factor-I

In two year classes of Willamette River spring chinook salmon, reared at the Willamette Hatchery, and two groups of Yakima River spring chinook salmon, one sampled from the Yakima River and the other reared in a hatchery, fish which had relatively high growth rates in the summer–autumn period smolted in the autumn as measured by increases in gill Na⁺ K⁺ AT Pase activity. In contrast, groups with relatively low growth rate did not smolt in the autumn. Plasma levels of insulin-like growth factor-I (IGF-I) showed discrete differences between groups, with high levels associated with increased gill Na⁺ K⁺ AT Pase activities. These results demonstrate that smolting is plastic in spring chinook salmon, occurring in the autumn or the spring. In addition, smolting appeared to be related to growth rate; however, the relationships shown were correlational and causal mechanisms were not elucidated. Yet, the results do indicate a relationship between growth, an endocrine growth factor and smolting, suggesting a mechanistic link between developmental plasticity and the environment mediated by the endocrine system.     

Involvement of Na+,K+-ATPase in the Mitogenic Effect of Insulin-Like Growth Factor-I on Cultured Rat Astrocytes

Abstract: We have previously reported that insulin/insulin-like growth factor (IGF)-I induced the α1 isoform of Na⁺,K⁺-ATPase in cultured astrocytes. In this study the effects of insulin/IGF-I on Na⁺,K⁺-ATPase activity and cell proliferation were examined in astrocytes cultured under the various conditions, to test the possible involvement of the enzyme activity in the mitogenic action of IGF-I on astrocytes. Insulin increased Na⁺,K⁺-ATPase activity and stimulated cell proliferation in subconfluent astrocytes (cultured for 7–14 days in vitro). In contrast, these effects were not observed in confluent cells (cultured for 28 days). Furthermore, insulin stimulated neither the enzyme activity nor [³H]thymidine incorporation in astrocytes preincubated in fetal calf serum-free medium for 2 days (quiescent cells) and treated with dibutyryl cyclic AMP (differentiated cells). The increases in Na⁺,K⁺-ATPase activity and expression of the α1 mRNA preceded the mitogenic effect. ¹²⁵I-IGF-I binding experiment showed that all the cells used here had similar binding characteristics. The insulin-induced increase in enzyme activity was not affected by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), and it was observed even in Ca²⁺-free medium. The stimulation by IGF-I of [³H]thymidine incorporation was attenuated by ouabain and a low external K⁺ level. These findings suggest that stimulation of Na⁺,K⁺-ATPase activity is involved in the mitogenic action of IGF-I on cultured astrocytes.     

Impairment of Na+,K+-ATPase activity following enterotoxigenic Campylobacter jejuni infection: changes in Na+, Cl− and 3-O-methyl-D-glucose transport in vitro, in rat ileum

Abstract Unidirectional fluxes of Na⁺, Cl⁻ and 3-O-methyl-D-glucose (3-MG) were measured in vitro across Campylobacter jejuni live culture-infected and control rat ileal short-circuited tissues by the Using Chamber technique. Net secretion of Na⁺ and enhanced secretion of Cl⁻ ions was observed in the infected animals ( P < 0.001, n =6) as compared to the net absorption of Na⁺ and marginal secretion of Cl⁻ ions in the control animals. There was a significant decrease in the mucosal-to-serosal fluxes of 3-MG in C. jejuni -infected rat ileum. The specific Na⁺,K⁺-ATPase activity when measured biochemically in the membrane-rich fraction of enterocytes was found to be significantly lower (58%) in the infected group as compared to the control group ( P < 0.001). Our results therefore suggest that infection with an enterotoxigenic C. jejuni inhibits the Na⁺,K⁺-ATPase activity in rat enterocytes. The impairment of Na⁺,K⁺-ATPase activity thus appears to induce a secondary change in Na⁺,Cl⁻ and 3-MG transport in vitro in rat ileum.     

Inhibition of Rat Brain Microsomal Na+,K+-ATPase by S-Adenosylmethionine

Abstract: Rat brain microsomes were preincubated with S -adenosylmethionine (SAM), MgCl₂, and CaCl₂, then re-isolated, and the activity of Na⁺,K⁺-ATPase determined. SAM inhibited the Na⁺,K⁺-ATPase activity compared with microsomes subjected to similar treatment in the absence of SAM. A biphasic inhibitory effect was observed with a 50% decrease at a SAM concentration range of 0.4 μ M -3.2 μ M and a 70% reduction at a concentration range above 100 μ M . Inclusion of either S- adenosylhomocysteine or 3-deazaadenosine in the preincubations prevented the SAM inhibition of Na⁺,K⁺-ATPase activity. The inhibition by SAM appeared to be Mg²⁺- or Ca²⁺-dependent.