Corticosteroid regulation of Na,K-ATPase α1-isoform expression in Atlantic salmon gill during smolt development

The proposed mineralocorticoid-like signalling axis in teleost fish, consisting of the hormone 11-deoxycorticosterone (DOC) and a mineralocorticoid receptor (MR), has recently challenged our conception of cortisol being the only osmoregulatory corticosteroid in teleost fish. This paper aimed at comparing the osmoregulatory role of DOC with that of cortisol during the pre-adaptive development of SW-tolerance, smoltification, in Atlantic salmon. Using an in vitro gill block incubation system, the effect of DOC and cortisol in the gill was investigated from January to September, using Na⁺,K⁺-ATPase α-subunit isoforms α-1a and α-1b mRNA levels as targets for regulation by the hormones. Cortisol and DOC both conferred significant up-regulation of α-1a and α-1b mRNA levels at specific time-points during smoltification. However, the effect of cortisol and DOC on α-subunit isoforms varied seasonally between isoforms and hormones. The maximum induction of α-1a was 3- to 4-fold compared to controls whereas a 2-fold induction was observed for α-1b. The pattern and capacity of stimulation of α-1a through smoltification were similar for cortisol and DOC, whereas cortisol had an enhanced capacity to stimulate α-1b as compared to DOC. Even though there was no demonstrable change in cortisol or DOC sensitivity in the gill, the magnitude of the hormonal effects were seasonally dependent. This is the first report of DOC-induced effects on osmoregulatory targets in fish, thus indicating a role for DOC and MR signalling in 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.     

Effects of short-term acid and aluminum exposure on the parr-smolt transformation in Atlantic salmon (Salmo salar): disruption of seawater tolerance and endocrine status

Episodic acidification resulting in increased acidity and inorganic aluminum (Al_i) is known to interfere with the parr-smolt transformation of Atlantic salmon (Salmo salar), and has been implicated as a possible cause of population decline. To determine the extent and mechanism(s) by which short-term acid/Al exposure compromises smolt development, Atlantic salmon smolts were exposed to either control (pH 6.7–6.9) or acid/Al (pH 5.4–6.3, 28–64 μg l⁻¹ Al_i) conditions for 2 and 5 days, and impacts on freshwater (FW) ion regulation, seawater (SW) tolerance, plasma hormone levels and stress response were examined. Gill Al concentrations were elevated in all smolts exposed to acid/Al relative to controls confirming exposure to increased Al_i. There was no effect of acid/Al on plasma ion concentrations in FW however, smolts exposed to acid/Al followed by a 24 h SW challenge exhibited greater plasma Cl⁻ levels than controls, indicating reduced SW tolerance. Loss of SW tolerance was accompanied by reductions in gill Na⁺,K⁺-ATPase (NKA) activity and Na⁺,K⁺,2Cl⁻ (NKCC) cotransporter protein abundance. Acid/Al exposure resulted in decreased plasma insulin-like growth factor (IGF-I) and 3,3′,5′-triiodo-l-thyronine (T₃) levels, whereas no effect of treatment was seen on plasma cortisol, growth hormone (GH), or thyroxine (T₄) levels. Acid/Al exposure resulted in increased hematocrit and plasma glucose levels in FW, but both returned to control levels after 24 h in SW. The results indicate that smolt development and SW tolerance are compromised by short-term exposure to acid/Al in the absence of detectable impacts on FW ion regulation. Loss of SW tolerance during short-term acid/Al exposure likely results from reductions in gill NKA and NKCC, possibly mediated by decreases in plasma IGF-I and T₃.     

Changes in gene expression for GH/PRL/SL family hormones in the pituitaries of homing chum salmon during ocean migration through upstream migration

Gene expression for growth hormone (GH)/prolactin (PRL)/somatolactin (SL) family hormones in the pituitaries of homing chum salmon were examined, because gene expression for these hormones during ocean-migrating phases remains unclear. Fish were collected in the winter Gulf of Alaska, the summer Bering Sea and along homing pathway in the Ishikari River-Ishikari Bay water system in Hokkaido, Japan in autumn. The oceanic fish included maturing adults, which had developing gonads and left the Bering Sea for the natal river by the end of summer. The absolute amounts of GH, PRL and SL mRNAs in the pituitaries of the maturing adults in the summer Bering Sea were 5- to 20-fold those in the winter Gulf of Alaska. The amount of GH mRNA in the homing adults at the coastal seawater (SW) areas was smaller than that in the Bering fish, while the amount of PRL mRNA remained at the higher level until fish arrived at the Ishikari River. The gill Na⁺,K⁺-ATPase activity in the coastal SW fish and the plasma Na⁺ levels in the brackish water fish at the estuary were lowered to the levels that were comparable to those in the fresh water (FW) fish. In conclusion, gene expression for GH, PRL and SL was elevated in the pituitaries of chum salmon before initiation of homing behavior from the summer Bering Sea. Gene expression for GH is thereafter lowered coincidently with malfunction of SW adaptability in the breeding season, while gene expression for PRL is maintained high until forthcoming FW adaptation.     

Interactive effects of ambient acidity and salinity on thyroid function during acidic and post-acidic acclimation of air-breathing fish (Anabas testudineus Bloch)

The interactive effects of ambient acidity and salinity on thyroid function are less understood in fish particularly in air-breathing fish. We, therefore, examined the thyroid function particularly the osmotic and metabolic competences of freshwater (FW) and salinity-adapted (SA; 20 ppt) air-breathing fish (Anabas testudineus) during acidic and post-acidic acclimation, i.e., during the exposure of fish to either acidified water (pH 4.2 and 5.2) for 48 h or clean water for 96 h after pre-exposure. A substantial rise in plasma T₄ occurred after acidic exposure of both FW and SA fish. Similarly, increased plasma T₃ and T₄ were found in FW fish kept for post-acidic acclimation and these suggest an involvement of THs in short-term acidic and post-acidic acclimation. Water acidification produced significant hyperglycaemia and hyperuremia in FW fish but not in SA fish. The SA fish when kept for post-acclimation, however, produced a significant hypouremia. In both FW and SA fish, gill Na⁺, K⁺-ATPase activity decreased but kidney Na⁺, K⁺-ATPase activity increased upon acidic acclimation. During post-acidic acclimation, gill Na⁺, K⁺-ATPase activity of the FW fish showed a rise while decreasing its activity in the SA fish. Similarly, post-acidic acclimation reduced the Na⁺, K⁺-ATPase activity of intestine but elevated its activity in the liver of SA fish. A higher tolerance of the SA fish to water acidification was evident in these fish as they showed tight plasma and tissue mineral status due to the ability of this fish to counteract the ion loss. In contrast, FW fish showed more sensitivity to water acidification as they loose more ions in that medium. The positive correlations of plasma THs with many tested metabolic and hydromineral indices of both FW and SA fish and also with water pH further confirm the involvement of THs in acidic and post-acidic acclimation in these fish. We conclude that thyroid function of this fish is more sensitive to environmental acidity than ambient salinity and salinity interference nullifies the toxic effect of water acidification.     

Cortisol promotes and integrates the osmotic competence of the organs in North African catfish (Clarias gariepinus Burchell): Evidence from in vivo and in situ approaches

The short-term in situ and long-term in vivo effects of cortisol were examined in North African catfish (Clarias gariepinus) to identify how this major corticosteroid integrates the osmotic competence of fish organs. In the in situ approach, the hydromineral effects of cortisol perfusion (75-300 ng ml⁻¹) for 20 min were tested and the indices of hydromineral and metabolic regulations were measured in our in vivo experimental fish after three alternate intraperitoneal cortisol injections (40 and 200 ng g⁻¹ body mass) for 5 days. Na⁺, K⁺-ATPase activity, a measure of cellular osmotic competence, responded to in situ and in vivo cortisol treatments. In situ cortisol delivery increased the Na⁺, K⁺-ATPase activity in the gill (P < 0.001) and kidney (P < 0.001) but decreased (P < 0.01) in the liver and showed no effect on intestine. In vivo cortisol treatment, on the contrary, increased Na⁺, K⁺-ATPase activity in the gills (P < 0.01), intestine (P < 0.05) and liver (P < 0.01) but decreased (P < 0.05) in the kidney. As expected, plasma cortisol increased (P < 0.001) with increasing doses of cortisol injections which produced direct effects on the metabolites and the mineral contents including the elevations of glucose (P < 0.05), lactate (P < 0.05) and Mg²⁺ (P < 0.05) and reductions of urea (P < 0.05), Na⁺ (P < 0.05) and K⁺ (P < 0.05) in the plasma. A decline of triiodothyronine (P < 0.01) occurred in the catfish after in vivo cortisol treatment and that implies a direct cortisol action on the homeostatic integration in this fish. Evidence is thus presented that in catfish cortisol regulates the whole body hydromineral and metabolite homeostasis by promoting and integrating the osmotic and metabolic functions of the multiple organ systems including liver.     

Effect of hypermethioninemia on some parameters of oxidative stress and on Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase activity in hippocampus of rats

In the present study we investigated the effect of chronic administration of methionine, a metabolite accumulated in many inherited pathological conditions such as methionine adenosyltransferase deficiency and homocystinuria, on some parameters of oxidative stress, namely thiobarbituric acid reactive substances (TBARS), catalase activity and total thiol content, as well as on Na⁺,K⁺-ATPase activity in rat hippocampus. For chronic treatment, rats received subcutaneous injections of methionine (1.34–2.68 μmol/g of body weight), twice a day, from the 6th to the 28th day of age and controls received saline. Animals were killed 12 h after the last injection. Results showed that chronic hypermethioninemia significantly increased TBARS, decreased Na⁺,K⁺-ATPase activity but did not alter catalase and total thiol content. Since chronic hypermethioninemia altered TBARS and Na⁺,K⁺-ATPase activity at 12 h after methionine administration, we also investigated the effect of acute administration of this amino acid on the same parameters studied after chronic methionine administration. For acute treatment,29-day-old rats received one single injection of methionine (2.68 μmol/g of body weight) or saline and were killed 1, 3 or 12 h later. Results showed that rats subjected to acute hypermethioninemia presented a reduction of Na⁺,K⁺-ATPase activity and an increase in TBARS when the animals were killed at 3 and 12 h, but not at 1 h, after methionine administration. These data indicate that hypermethioninemia increases lipid peroxidation which may, at least partially, explain the effect of methionine on the reduction in Na⁺,K⁺-ATPase activity. If confirmed in human beings, our findings could suggest that the induction of oxidative stress and the inhibition of Na⁺,K⁺-ATPase activity caused by methionine might contribute to the neurophysiopathology observed in patients with severe hypermethioninemia.     

Alterations of Na/K-ATPase function in caveolin-1 knockout cardiac fibroblasts

Recent studies have demonstrated that the Na⁺/K⁺-ATPase is not only an ion pump, but also a membrane receptor that confers the ligand-like effects of cardiotonic steroids (CTS) such as ouabain on protein kinases and cell growth. Because CTS have been implicated in cardiac fibrosis, this study examined the role of caveolae in the regulation of Na⁺/K⁺-ATPase function and CTS signaling in cardiac fibroblasts. In cardiac fibroblasts prepared from wild-type and caveolin-1 knockout [Cav-1(−/−)] mice, we found that the absence of caveolin-1 did not affect total cellular amount or surface expression of Na⁺/K⁺-ATPase α1 subunit. However, it did increase ouabain-sensitive ⁸⁶Rb⁺ uptake. While knockout of caveolin-1 increased basal activities of Src and ERK1/2, it abolished the activation of these kinases induced by ouabain but not angiotensin II. Finally, ouabain stimulated collagen synthesis and cell proliferation in wild type but not Cav-1(−/−) cardiac fibroblasts. Thus, we conclude that caveolae are important for regulating both pumping and signal transducing functions of Na⁺/K⁺-ATPase. While depletion of caveolae increases the pumping function of Na⁺/K⁺-ATPase, it suppresses CTS-induced signal transduction, growth, and collagen production in cardiac fibroblasts.     

Ambient salinity modifies the action of triiodothyronine in the air-breathing fish Anabas testudineus Bloch: Effects on mitochondria-rich cell distribution, osmotic and metabolic regulations

The hydromineral and metabolic actions of thyroid hormone on osmotic acclimation in fish is less understood. We, therefore, studied the short-term action of triiodothyronine (T₃), the potent thyroid hormone, on the distribution and the function of gill mitochondria-rich (MR) cells and on the whole body hydromineral and metabolic regulations of air-breathing fish (Anabas testudineus) adapted to either freshwater (FW) or acclimated to seawater (SA; 30 g L⁻¹). As expected, 24 h T₃ injection (100 ng g⁻¹) elevated (P < 0.05) plasma T₃ but classically reduced (P < 0.05) plasma T₄. The higher Na⁺, K⁺-ATPase immunoreactivity and the varied distribution pattern of MR cells in the gills of T₃-treated FW and SA fish, suggest an action of T₃ on gill MR cell migration, though the density of these cells remained unchanged after T₃ treatment. The ouabain-sensitive Na⁺, K⁺-ATPase activity, a measure of hydromineral competence, showed increases (P < 0.05) in the gills of both FW and SA fish after T₃ administration, but inhibited (P < 0.05) in the kidney of the FW fish and not in the SA fish. Exogenous T₃ reduced glucose (P < 0.05) and urea (P < 0.05) in the plasma of FW fish, whereas these metabolites were elevated (P < 0.05) in the SA fish, suggesting a modulatory effect of ambient salinity on the T₃-driven metabolic actions. Our data identify gill MR cell as a target for T₃ action as it promotes the spatial distribution and the osmotic function of these cells in both fresh water and in seawater. The results besides confirming the metabolic and osmotic actions of T₃ in fish support the hypothesis that the differential actions of T₃ may be due to the direct influence of ambient salinity, a major environmental determinant that alters the osmotic and metabolic strategies of fish.     

Famotidine,a histamine-2-receptor antagonist,inhibits the increase in rat gastric H+/K+-ATPase mRNA induced by intravenous infusion of gastrin 17 and histamine

We examined the effects of gastrin and histamine on rat gastric H⁺/K⁺-ATPase, the enzyme responsible for H⁺ secretion, gene expressionin vivo. Gastrin 17 (G 17) or histamine dihydrochloride (histamine) was continuously infused through the femoral vein of anesthetized rats. Gastric H⁺/K⁺-ATPase mRNA levels were measured using northern blot analysis. Infusion of G 17 and histamine increased the H⁺/K⁺-ATPase mRNA level significantly compared with basal control level or vehicle control level (P<0.01). However, pretreatment with famotidine, a potent histamine-2 (H₂)-receptor antagonist, inhibited the increase of rat gastric H⁺/K⁺-ATPase mRNA following G 17 and histamine infusion. These findings indicate that both histamine and G 17 increase expression of H⁺/K⁺-ATPase mRNA by activating H₂ receptor on the parietal cell.     

Abundant expression of sodium-potassium-activated adenosinetriphosphatase alpha 1 subunit in corpus luteum of porcine ovary

Follicular development is accompanied by the accumulation of follicular fluid. During corpus luteum formation, follicular fluid is diminished and antrum is replaced by lutein cells. These dynamic changes in fluid distribution suggest the existence of control mechanism of fluid transport and membrane permeability. One of the major factors regulating membrane permeability is the sodium-potassium-activated adenosinetriphosphatase (Na⁺-K⁺-ATPase). To elucidate the possible involvement of Na⁺-K⁺-ATPase in follicular growth and luteinization, immunohistochemical localization of Na⁺-K⁺-ATPase α1 subunit and enzyme activity in porcine ovary were investigated. In primordial follicles, Na⁺-K⁺-ATPase α1 subunit immunostaining was localized only in the oocyte and the surrounding stromal cells. In preantral follicles, immunostaining for Na⁺-K⁺-ATPase α1 subunit became apparent in granulosa and theca cells. As the follicle matured, the staining intensity in the oocyte, theca, and granulosa cells increased, which corresponded with the enzyme activity. Na⁺-K⁺-ATPase α1 subunit immunostaining became most abundant in granulosa and theca lutein cells in corpus luteum, and decreased in the regressing corpus luteum. Enzyme activity in corpus luteum was significantly higher than that in the follicles. This is the first study indicating that Na⁺-K⁺-ATPase α1 subunit expression is augmented in granulosa cells by follicular growth and most abundant in lutein cells in the corpus luteum, suggesting its possible involvement in corpus luteum formation.     

Isoform-specific alterations in cardiac and erythrocyte Na ,K-ATPase activity induced by norepinephrine

Background: Myocardial Na⁺,K⁺-ATPase activities are decreased in congestive heart failure because of an increase in plasma norepinephrine levels, but it is difficult to monitor the activities in the clinical setting.Methods and Results: This study investigated whether erythrocyte Na⁺,K⁺-ATPase activity can reflect myocardial enzyme activity and whether isoform-specific alterations occur in the presence of catecholamine. Na⁺,K⁺-ATPase activity was measured by the colorimetric method by using the left ventricular myocardium and erythrocytes prepared from eight rabbits given norepinephrine for 7 days and from eight control rabbits that received saline. The protein levels of total catalytic subunit and α₁ - or α₃-isoform of Na⁺,K⁺-ATPase were determined by Western blot analysis. Na⁺,K⁺-ATPase activity was lower in both myocardium and erythrocytes from norepinephrine-treated rabbits than control rabbits (P < .01 and P < .01, respectively). There was a close correlation in Na⁺,K⁺-ATPase activity between myocardium and erythrocytes (r = .963). Total catalytic subunit protein level was lower in myocardium from norepinephrine-treated rabbits than control rabbits, but the α₁-isoform level was similar between the two groups. The α₃-isoform level was lower in norepinephrinetreated rabbits than control rabbits. In erythrocytes, α₁-isoform was lower in norepinephrinetreated rabbits than control rabbits.Conclusions: Na⁺,K⁺-ATPase activity in myocardium could be reflected in erythrocyte membrane, although there was a difference in isoform-specific regulation between the two.     

SALT INTAKE AND SENSITIVITY OF INTESTINAL AND RENAL NA+ -K+ ATPase TO INHIBITION BY DOPAMINE IN SPONTANEOUS HYPERTENSIVE AND WISTAR-KYOTO RATS

The present study evaluated the activity of jejunal Na⁺-K⁺-ATPase and its sensitivity to inhibition by dopamine in spontaneous hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats during low (LS), normal (NS) and high (HS) salt intake. Basal jejunal Na⁺-K⁺-ATPase activity in SHR on LS intake was higher than in WKY rats. Jejunal Na⁺-K⁺-ATPase activity in WKY rats, but not in SHR, on LS intake was significantly reduced (20% decrease) by dopamine (1 μM) and SKF 38393 (10nM), but not quinerolane (10 nM), this being antagonized the D₁ receptor antagonist (SKF 83566). Changing from LS to NS or HS intake in WKY rats increased basal jejunal Na⁺-K⁺-ATPase activity and attenuated the inhibitory effect of dopamine. In SHR, changing from LS to NS or HS intake increased basal jejunal Na⁺-K⁺-ATPase activity. Basal renal Na⁺-K⁺-ATPase activity in SHR on LS intake was similar to that in WKY rats and was insensitive to inhibition by dopamine . Changing from LS to NS or HS intake in WKY rats increased basal renal Na⁺-K⁺-ATPase activity without affecting the inhibitory effect of dopamine. In SHR, changing from LS to NS or HS intake failed to alter basal renal Na⁺-K⁺-ATPase activity. It is concluded that inhibition of jejunal Na⁺-K⁺ ATPase activity by D₁ dopamine receptor activation is dependent on salt intake in WKY rats, and SHR animals fail to respond to dopamine, irrespective of their salt intake.     

Intrastriatal Hypoxanthine Reduces Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase Activity and Induces Oxidative Stress in the Rats

The main objective of this study was to investigate the effects of a single intrastriatal injection of hypoxanthine, a metabolite accumulated in Lesch Nyhan disease and possibly involved in its neuropathology, on Na⁺,K⁺-ATPase activity, as well as on some parameters of oxidative stress, namely chemiluminescence (an index of lipid peroxidation), total radical-trapping antioxidant parameter—TRAP (an index of total antioxidant capacity of the tissue) and total thiol protein membrane content, in striatum, cerebral cortex and hippocampus of rats. Results show that hypoxanthine significantly decreased Na⁺,K⁺-ATPase activity and TRAP while increased chemiluminescence in all ipsislateral structures tested. However, no effect on total thiol protein membrane content was detected. We suggest that hypoxanthine induces oxidative stress in all cerebral structures studied (striatum, hippocampus and cerebral cortex) and that the reduction of Na⁺,K⁺-ATPase activity was probably mediated by reactive oxygen species.     

Mutation I810N in the α3 isoform of Na+,K+-ATPase causes impairments in the sodium pump and hyperexcitability in the CNS

In a mouse mutagenesis screen, we isolated a mutant, Myshkin (Myk), with autosomal dominant complex partial and secondarily generalized seizures, a greatly reduced threshold for hippocampal seizures in vitro, posttetanic hyperexcitability of the CA3-CA1 hippocampal pathway, and neuronal degeneration in the hippocampus. Positional cloning and functional analysis revealed that Myk/+ mice carry a mutation (I810N) which renders the normally expressed Na⁺,K⁺-ATPase α3 isoform inactive. Total Na⁺,K⁺-ATPase activity was reduced by 42% in Myk/+ brain. The epilepsy in Myk/+ mice and in vitro hyperexcitability could be prevented by delivery of additional copies of wild-type Na⁺,K⁺-ATPase α3 by transgenesis, which also rescued Na⁺,K⁺-ATPase activity. Our findings reveal the functional significance of the Na⁺,K⁺-ATPase α3 isoform in the control of epileptiform activity and seizure behavior.