Fatty acid and lipid class composition in eyes and brain from teleosts and elasmobranchs

The effect of 17beta-estradiol (E(2)) on osmoregulatory performance was examined in the euryhaline killifish, Fundulus heteroclitus. Fish were injected once with 1, 2 and 5 microg g(-1) E(2) and, 6 h after injection, transferred from 1 ppt seawater (SW) to full strength SW (40 ppt) or from SW to 1 ppt SW. In another set of experiments, fish were injected four times on alternate days with 2 microg g(-1) E(2) and then, 6 h after the last injection, transferred from 1 ppt SW to SW or from SW to 1 ppt SW. Fish were sampled 18 h after transfer (i.e., 24 h post-injection), and plasma osmolality, Na(+) and Cl(-) concentration and gill K(+)-pNPPase activity (a reflection of the sodium pump) were examined. Transfer from 1 ppt SW to SW resulted in significantly increased plasma osmolality, but did not affect gill K(+)-pNPPase activity. A single dose of E(2) (1, 2 and 5 microg g(-1)) prior to transfer from 1 ppt SW to SW increased plasma osmolality and decreased gill K(+)-pNPPase activity in a dose-dependent manner. Prolonged treatment with E(2) increased plasma osmolality and decreased gill K(+)-pNPPase activity in 1 ppt SW-adapted fish. Transfer of fish thus treated from 1 ppt SW to SW increased plasma osmolality and did not alter gill K(+)-pNPPase activity. Transfer from SW to 1 ppt SW had no significant effect on plasma osmolality or gill K(+)-pNPPase activity. Only the highest single dose of E(2) (5 microg g(-1)) prior to transfer from SW to 1 ppt SW decreased gill K(+)-pNPPase activity. Prolonged treatment with 2 microg g(-1) E(2) decreased gill K(+)-pNPPase activity only following transfer from SW to 1 ppt SW. The results substantiate an inhibitory action of E(2) on hypoosmoregulatory capacity in this euryhaline teleost.     

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.     

Pseudobranch and gill Na(+), K(+)-ATPase activity in juvenile chinook salmon,Oncorhynchus tshawytscha: developmental changes and effects of growth hormone,cortisol and seawater transfer

The teleost pseudobranch is a gill-like structure often fused to the anterior of the opercular cavity. Pseudobranch cells are mitochondria rich and have high levels of Na(+), K(+)-ATPase activity. In this study, pseudobranch Na(+), K(+)-ATPase activity in juvenile chinook salmon (Oncorhynchus tshawytscha) was compared to gill Na(+), K(+)-ATPase activity, a known marker of parr-smolt transformation, in three experiments. In two stocks of New Zealand chinook salmon, pseudobranch Na(+), K(+)-ATPase activity was found to significantly increase during development. At these times gill Na(+), K(+)-ATPase activity was also elevated. Pseudobranch Na(+), K(+)-ATPase activity did not increase 10 days after transfer from fresh water to 34 ppt seawater, a treatment that resulted in a twofold increase in gill Na(+), K(+)-ATPase activity. Cortisol (50 microg/g) and ovine growth hormone (5 microg/g) implants had no effect on pseudobranch Na(+), K(+)-ATPase activity in underyearling chinook salmon, while gill Na(+), K(+)-ATPase activity was stimulated by each hormone. In yearling chinook salmon, only cortisol stimulated pseudobranch Na(+), K(+)-ATPase activity 14 days post-implantation. It was concluded that the pseudobranch differs from the gill in terms of the regulation of Na(+), K(+)-ATPase activity and a role during adaptation to seawater is likely to be limited.     

Acute changes in gill Na+-K+-ATPase and creatine kinase in response to salinity changes in the euryhaline teleost, tilapia (Oreochromis mossambicus)

Some freshwater (FW) teleosts are capable of acclimating to seawater (SW) when challenged; however, the related energetic and physiological consequences are still unclear. This study was conducted to examine the changes in expression of gill Na(+)-K(+)-ATPase and creatine kinase (CK) in tilapia (Oreochromis mossambicus) as the acute responses to transfer from FW to SW. After 24 h in 25 ppt SW, gill Na(+)-K(+)-ATPase activities were higher than those of fish in FW. Fish in 35 ppt SW did not increase gill Na(+)-K(+)-ATPase activities until 1.5 h after transfer, and then the activities were not significantly different from those of fish in 25 ppt SW. Compared to FW, the gill CK activities in 35 ppt SW declined within 1.5 h and afterward dramatically elevated at 2 h, as in 25 ppt SW, but the levels in 35 ppt SW were lower than those in 25 ppt SW. The Western blot of muscle-type CK (MM form) was in high association with the salinity change, showing a pattern of changes similar to that in CK activity; however, levels in 35 ppt SW were higher than those in 25 ppt SW. The activity of Na(+)-K(+)-ATPase highly correlated with that of CK in fish gill after transfer from FW to SW, suggesting that phosphocreatine acts as an energy source to meet the osmoregulatory demand during acute transfer.     

Cl- uptake mechanism in freshwater-adapted tilapia (Oreochromis mossambicus)

In this study, the correlation between Cl(-) influx in freshwater tilapia and various transporters or enzymes, the Cl(-)/HCO(3)(-) exchanger, Na(+),K(+)-ATPase, V-type H(+)-ATPase, and carbonic anhydrase were examined. The inhibitors 2x10(-4) M ouabain (a Na(+),K(+)-ATPase inhibitor), 10(-5) M NEM (a V-type H(+)-ATPase inhibitor), 10(-2) M ACTZ (acetazolamide, a carbonic anhydrase inhibitor), and 6x10(-4) M DIDS (a Cl(-)/HCO(3)(-) exchanger inhibitor) caused 40%, 60%-80%, 40%-60%, and 40%-60% reduction in Cl(-) influx of freshwater tilapia, respectively. The inhibitor 2x10(-4) M ouabain also caused 50%-65% inhibition in gill Na(+),K(+)-ATPase activity. Western blot results showed that protein levels of gill Na(+),K(+)-ATPase, V-type H(+)-ATPase, and carbonic anhydrase in tilapia acclimated in low-Cl(-) freshwater were significantly higher than those acclimated to high-Cl(-) freshwater. Based on these data, we conclude that Na(+),K(+)-ATPase, V-H(+)-ATPase, the Cl(-)/HCO(3)(-) exchanger, and carbonic anhydrase may be involved in the active Cl(-) uptake mechanism in gills of freshwater-adapted tilapia.     

Osmoregulation and expression of ion transport proteins and putative claudins in the gill of southern flounder (Paralichthys lethostigma)

The southern flounder is a euryhaline teleost that inhabits ocean, estuarine, and riverine environments. We investigated the osmoregulatory strategy of juvenile flounder by examining the time-course of homeostatic responses, hormone levels, and gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein expression after salinity challenge. Transfer of freshwater (FW)-acclimated flounder to sea water (SW) induced an increase in plasma osmolality and cortisol and a decrease in muscle water content, plasma insulin-like growth factor I (IGF-I) and hepatic IGF-I mRNA, all returning to control levels after 4 days. Gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein levels were elevated in response to SW after 4 days. Transfer of SW-acclimated flounder to FW reduced gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein, increased plasma IGF-I, but did not alter hepatic IGF-I mRNA or plasma cortisol levels. Gill claudin-3 and claudin-4 immunoreactive proteins were elevated in FW versus SW acclimated flounder. The study demonstrates that successful acclimation of southern flounder to SW or FW occurs after an initial crisis period and that the salinity adaptation process is associated with changes in branchial expression of ion transport and putative tight junction claudin proteins known to regulate epithelial permeability in mammalian vertebrates.     

Failure to up-regulate gill Na+,K+-ATPase alpha-subunit isoform alpha1b may limit seawater tolerance of land-locked Arctic char (Salvelinus alpinus)

Many populations of Arctic char (Salvelinus alpinus) are land-locked, physically separated from the ocean by natural barriers and unable to migrate to sea like anadromous populations. Previous studies which experimentally transferred land-locked Arctic char to seawater report high mortality rates due to osmoregulatory failure and an inability to up-regulate gill Na(+),K(+)-ATPase activity. This study examined the mRNA expression of two recently discovered alpha-subunit isoforms of gill Na(+)K(+)-ATPase (alpha1a and alpha1b) during seawater exposure of land-locked Arctic char. mRNA levels of these gill Na(+),K(+)-ATPasealpha-subunit isoforms were compared to Na(+),K(+)-ATPase activity and protein levels and related to osmoregulatory performance. Land-locked Arctic char were unable to regulate plasma osmolality following seawater exposure. Seawater exposure did not induce an increase in gill Na(+),K(+)-ATPase activity or protein levels. Na(+),K(+)-ATPase isoform alpha1a mRNA quickly decreased upon exposure to seawater, while isoform alpha1b levels were unchanged. These results suggest the inability of land-locked Arctic char to acclimate to seawater is due a failure to up-regulate gill Na(+),K(+)-ATPase activity which may be due to their inability to increase Na(+),K(+)-ATPase alpha1b mRNA expression.     

Osmoregulatory action of 17beta-estradiol in the gilthead sea bream Sparus auratus

The osmoregulatory action of 17beta-estradiol (E2) was examined in the euryhaline teleost Sparus auratas. In a first set of experiments, fish were injected once with vegetable oil containing E2 (1, 2 and 5 microg/g body weight), transferred 12h after injection from sea water (SW, 38 ppt salinity) to hypersaline water (HSW, 55 ppt) or to brackish water (BW, 5 ppt salinity) and sampled 12h later (i.e. 24 h post-injection). In a second experiment, fish were injected intraperitoneally with coconut oil alone or containing E2 (10 microg/g body weight) and sampled after 5 days. In the same experiment, after 5 days of treatment, fish of each group were transferred to HSW, BW and SW and sampled 4 days later (9 days post-implant). Gill Na+,K+ -ATPase activity, plasma E2 levels, plasma osmolality, and plasma levels of ions (sodium and calcium), glucose, lactate, protein, triglyceride, and hepatosomatic index were examined. Transfer from SW to HSW produced no significant effects on any parameters assessed. E2 treatment did not affect any parameter. Transfer from SW to BW resulted in a significant decrease in plasma osmolality and plasma sodium but did not affect gill Na+,K+ -ATPase activity. A single dose of E2 attenuated the decrease in these parameters after transfer from SW to BW, but was without effect on gill Na+,K+ -ATPase activity. An implant of E2 (10 microg/g body weight) for 5 days significantly increased plasma calcium, hepatosomatic index, plasma metabolic parameters, and gill Na+,K+ -ATPase activity. In coconut oil-implanted (sham) fish, transfer from SW to HSW or BW during 4 days significantly elevated gill Na+,K+ -ATPase. Gill Na+,K+ -ATPase activity remained unaltered after transfer of E2-treated fish to HSW or BW. However, in E2-treated fish transferred from SW to SW (9 days in SW after E2-implant), gill Na+,K+ -ATPase activity decreased with respect to HSW- or BW-transferred fish. Shams transferred to HSW showed increased levels of lactate, protein, and trygliceride in plasma, while those transferred to BW only displayed increased trygliceride levels. E2-treated fish transferred to HSW showed higher protein levels without any change in other plasmatic parameters, while those transferred to BW displayed elevated plasma glucose levels but decreased osmolality and protein levels. These results substantiate a chronic stimulatory action of E2 on gill Na+,K+ -ATPase activity in the euryhaline teleost Sparus auratas.     

Effect of salinity on expression of branchial ion transporters in striped bass (Morone saxatilis)

The time course of osmoregulatory adjustments and expressional changes of three key ion transporters in the gill were investigated in the striped bass during salinity acclimations. In three experiments, fish were transferred from fresh water (FW) to seawater (SW), from SW to FW, and from 15-ppt brackish water (BW) to either FW or SW, respectively. Each transfer induced minor deflections in serum [Na+] and muscle water content, both being corrected rapidly (24 hr). Transfer from FW to SW increased gill Na+,K+-ATPase activity and Na+,K+,2Cl- co-transporter expression after 3 days. Abundance of Na+,K+-ATPase alpha-subunit mRNA and protein was unchanged. Changes in Na+,K+,2Cl- co-transporter protein were preceded by increased mRNA expression after 24 hr. Expression of V-type H+-ATPase mRNA decreased after 3 days. Transfer from SW to FW induced no change in expression of gill Na+,K+-ATPase. However, Na+,K+,2Cl- co-transporter mRNA and protein levels decreased after 24 hr and 7 days, respectively. Expression of H+-ATPase mRNA increased in response to FW after 7 days. In BW fish transferred to FW and SW, gill Na+,K+-ATPase activity was stimulated by both challenges, suggesting both a hyper- and a hypo-osmoregulatory response of the enzyme. Acclimation of striped bass to SW occurs on a rapid time scale. This seems partly to rely on the relative high abundance of gill Na+,K+-ATPase and Na+,K+,2Cl- co-transporter in FW fish. In a separate study, we found a smaller response to SW in expression of these ion transport proteins in striped bass when compared with the less euryhaline brown trout. In both FW and SW, NEM-sensitive gill H+-ATPase activity was negligible in striped bass and approximately 10-fold higher in brown trout. This suggests that in striped bass Na+-uptake in FW may rely more on a relatively high abundance/activity of Na+,K+-ATPase compared to trout, where H+-ATPase is critical for establishing a thermodynamically favorable gradient for Na+-uptake.     

Gill Na(+), K(+)-ATPase in a series of hyper-regulating gammarid amphipods: enzyme characterisation and the effects of salinity acclimation

The enzyme Na(+), K(+)-ATPase was investigated in the gills of selected hyper-regulating gammarid amphipods. Gill Na(+), K(+)-ATPase was characterised with respect to the main cation and co-factor concentrations for the freshwater amphipod Gammarus pulex. The optimum cation and co-factor concentrations for maximal gill Na(+), K(+)-ATPase activity in G. pulex were 100mM Na(+), 15mM K(+), 15mM Mg(2+) and 5mM ATP, at pH 7.2. The effects of salinity acclimation on gill Na(+), K(+)-ATPase activity and haemolymph sodium concentrations was investigated in selected gammarid amphipods from different salinity environments. Maximal enzyme activity occurred in all gammarids when acclimated to the most dilute media. This maximal activity coincided with the largest sodium gradient between the haemolymph and the external media. As the haemolymph/medium sodium gradient decreased, a concomitant reduction in gill Na(+), K(+)-ATPase activity occurred. This implicates the involvement of gill Na(+), K(+)-ATPase in the active uptake of sodium from dilute media in hyper-regulating gammarids.     

Wild Arctic char (Salvelinus alpinus) upregulate gill Na+,K+-ATPase during freshwater migration

The successful acclimation of eurhyhaline fishes from seawater to freshwater requires the gills to stop actively secreting ions and start actively absorbing ions. Gill Na(+),K(+)-ATPase is known to be an integral part of the active ion secretion model of marine fishes, but its importance in the active ion uptake model of freshwater fishes is less clear. This study, conducted in the high Arctic, examines gill Na(+),K(+)-ATPase regulation in wild anadromous arctic char returning to freshwater from the ocean. Gill Na(+),K(+)-ATPase activity, protein expression, and mRNA expression of Na(+),K(+)-ATPase isoforms alpha 1a and alpha 1b were monitored in arctic char at three points along their migration route to and from Somerset Island, Nunavut, Canada: out at sea (Whaler's Point), in seawater near the river mouth (Nat's Camp), and after entering the Union River. Arctic char collected from the Union River had more than twofold greater gill Na(+),K(+)-ATPase activity. This was associated with a significant increase (threefold) in Na(+),K(+)-ATPase isoform alpha 1a mRNA expression and a significant increase in plasma sodium and osmolality levels compared with seawater char. Compared with char sampled from Whaler's Point, Na(+),K(+)-ATPase isoform alpha 1b mRNA expression was decreased by approximately 50% in char sampled at Nat's Camp and the Union River. These results suggest that the upregulation of gill Na(+),K(+)-ATPase activity is involved in freshwater acclimation of arctic char and implicate a role for Na(+),K(+)-ATPase isoform alpha 1a in this process. In addition, we discuss evidence that arctic char go through a preparatory phase, or "reverse smoltification," before entering freshwater.     

Nitrophenylphosphate as a tool to characterize gill Na, K-ATPase activity in hyperregulating Crustacea

The kinetic properties of a gill Na(+), K(+)-ATPase from the freshwater shrimp Macrobrachium olfersii were studied using p-nitrophenylphosphate (PNPP) as a substrate. Sucrose gradient centrifugation of the microsomal fraction revealed a single protein fraction that hydrolyzed PNPP. The Na(+), K(+)-ATPase hydrolyzed PNPP (K(+)-phosphatase activity) obeying Michaelis-Menten kinetics with K(M)=1.72+/-0.06 mmol l(-1) and V(max)=259.1+/-11.6 U mg(-1). ATP was a competitive inhibitor of K(+)-phosphatase activity with a K(i)=50.1+/-2.5 micromol l(-1). A cooperative effect for the stimulation of the enzyme by potassium (K(0.5)=3.62+/-0.18 mmol l(-1); n(H)=1.5) and magnesium ions (K(0.5)=0.61+/-0.02 mmol l(-1), n(H)=1.3) was found. Sodium ions had no effect on K(+)-phosphatase activity up to 1.0 mmol l(-1), but above 80 mmol l(-1) inhibited the original activity by approximately 75%. In the range of 0-10 mmol l(-1), sodium ions did not affect stimulation of the K(+)-phosphatase activity by potassium ions. Ouabain (K(i)=762.4+/-26.7 micromol l(-1)) and orthovanadate (K(i)=0.25+/-0.01 micromol l(-1)) completely inhibited the K(+)-phosphatase activity, while thapsigargin, oligomycin, sodium azide and bafilomycin were without effect. These data demonstrate that the activity measured corresponds to that of the K(+)-phosphatase activity of the Na(+), K(+)-ATPase alone and suggest that the use of PNPP as a substrate to characterize K(+)-phosphatase activity may be a useful technique in comparative osmoregulatory studies of Na(+), K(+)-ATPase activities in crustacean gill tissues, and for consistent comparisons with well known mechanistic properties of the vertebrate enzyme.     

Regulation of renal Na(+),K(+)-ATPase and ouabain-sensitive H(+),K(+)-ATPase by the cyclic AMP-protein kinase A signal transduction pathway

We investigated the effect of the cyclic AMP-protein kinase A (PKA) signalling pathway on renal Na(+),K(+)-ATPase and ouabain-sensitive H(+),K(+)-ATPase. Male Wistar rats were anaesthetized and catheter was inserted through the femoral artery into the abdominal aorta proximally to the renal arteries for infusion of the investigated substances. Na(+),K(+)-ATPase activity was measured in the presence of Sch 28080 to block ouabain-sensitive H(+),K(+)-ATPase and improve specificity of the assay. Dibutyryl-cyclic AMP (db-cAMP) administered at a dose of 10(-7) mol/kg per min and 10(-6) mol/kg per min increased Na(+),K(+)-ATPase activity in the renal cortex by 34% and 42%, respectively, and decreased it in the renal medulla by 30% and 44%, respectively. db-cAMP infused at 10(-6) mol/kg per min increased the activity of cortical ouabain-sensitive H(+),K(+)-ATPase by 33%, and medullary ouabain-sensitive H(+),K(+)-ATPase by 30%. All the effects of db-cAMP were abolished by a specific inhibitor of protein kinase A, KT 5720. The stimulatory effect on ouabain-sensitive H(+),K(+)-ATPase and on cortical Na(+),K(+)-ATPase was also abolished by brefeldin A which inhibits the insertion of proteins into the plasma membranes, whereas the inhibitory effect on medullary Na(+),K(+)-ATPase was partially attenuated by 17-octadecynoic acid, an inhibitor of cytochrome p450-dependent arachidonate metabolism. We conclude that the cAMP-PKA pathway stimulates Na(+),K(+)-ATPase in the renal cortex as well as ouabain-sensitive H(+),K(+)-ATPase in the cortex and medulla by a mechanism requiring insertion of proteins into the plasma membrane. In contrast, medullary Na(+),K(+)-ATPase is inhibited by cAMP through a mechanism involving cytochrome p450-dependent arachidonate metabolites.     

Immunolocalization of Na+/K(+)-ATPase in mitochondrion-rich cells of the atlantic hagfish (Myxine glutinosa) gill

Using a monoclonal antibody for the alpha-subunit of the Na+/K(+)-ATPase, DASPEI (a vital mitochondria dye), and confocal laser scanning microscopy, the presence of Na+/K(+)-ATPase in mitochondrion-rich cells of the hagfish gill was confirmed. In addition, the level of Na+/K(+)-ATPase expression in the hagfish gill was compared to that of fishes with different osmoregulatory strategies (little skate, Raja erinacea and mummichog, Fundulus heteroclitus). Immunocytochemistry detected a high density of columnar cells expressing Na+/K(+)-ATPase in the afferent filamental epithelium. Positive cells were also found in the lamellar epithelium but at a much lower density. The distribution of DASPEI staining was similar to that of the Na+/K(+)-ATPase antibody, indicating that the enzyme is expressed in mitochondrion-rich cells. Immunoblot analysis confirmed the specificity of the antibody for the 97 kDa alpha-subunit of the enzyme. The immunoreactive band intensity for the Atlantic hagfish was similar to that of the little skate, but less than half that of the full-strength seawater mummichog. These results are discussed in relation to gill function in early craniates.     

Effects of environmental salinity and temperature on osmoregulatory ability, organic osmolytes, and plasma hormone profiles in the Mozambique tilapia (Oreochromis mossambicus)

The Mozambique tilapia, Oreochromis mossambicus, is capable of surviving a wide range of salinities and temperatures. The present study was undertaken to investigate the influence of environmental salinity and temperature on osmoregulatory ability, organic osmolytes and plasma hormone profiles in the tilapia. Fish were acclimated to fresh water (FW), seawater (SW) or double-strength seawater (200% SW) at 20, 28 or 35 degrees C for 7 days. Plasma osmolality increased significantly as environmental salinity and temperature increased. Marked increases in gill Na(+), K(+)-ATPase activity were observed at all temperatures in the fish acclimated to 200% SW. By contrast, Na(+), K(+)-ATPase activity was not affected by temperature at any salinity. Plasma glucose levels increased significantly with the increase in salinity and temperature. Significant correlations were observed between plasma glucose and osmolality. In brain and kidney, content of myo-inositol increased in parallel with plasma osmolality. In muscle and liver, there were similar increases in glycine and taurine, respectively. Glucose content in liver decreased significantly in the fish in 200% SW. Plasma prolactin levels decreased significantly after acclimation to SW or 200% SW. Plasma levels of cortisol and growth hormone were highly variable, and no consistent effect of salinity or temperature was observed. Although there was no significant difference among fish acclimated to different salinity at 20 degrees C, plasma IGF-I levels at 28 degrees C increased significantly with the increase in salinity. Highest levels of IGF-I were observed in SW fish at 35 degrees C. These results indicate that alterations in gill Na(+), K(+)-ATPase activity and glucose metabolism, the accumulation of organic osmolytes in some organs as well as plasma profiles of osmoregulatory hormones are sensitive to salinity and temperature acclimation in tilapia.     

Osmoregulatory effects of hypophysectomy and homologous prolactin replacement in hybrid striped bass

The effects of ovine prolactin (oPRL) and striped bass prolactin (sbPRL; Morone saxatilis) on plasma osmolality, electrolyte balance, and gill Na(+),K(+)-ATPase activity were investigated in hypophysectomized (Hx), freshwater (FW)-acclimated, hybrid striped bass (M. saxatilisxMorone chrysops). They were kept in dilute (isoosmotic) seawater for about 10 days after surgery. Seven days after transfer to FW, Hx fish had lower plasma osmolality and lower levels of Na(+), Cl(-), and Ca(2+) than sham-operated and intact fish. Fish were injected four times with oPRL (1, 5, or 20 microg/g body mass), sbPRL (10 or 100 ng/g), or hormone vehicle (0.9% NaCl) at 48-h intervals (days 0, 2, 4, and 6) in FW and then sampled for blood plasma 24 h after the fourth injection (day 7). In Hx fish, oPRL (5 and 20 microg/g) and sbPRL (10 and 100 ng/g) were effective in maintaining plasma osmolality and levels of Na(+), Cl(-), and Ca(2+) above values seen in saline-injected controls. Hypophysectomy did not affect branchial Na(+),K(+)-ATPase activity, but enzyme activity was significantly reduced in Hx fish receiving oPRL (20 mug/g) or sbPRL (10 or 100 ng/g). These results indicate that PRL acts to maintain plasma osmotic and ionic balance in FW-adapted hybrid striped bass, and that this may involve downregulation of branchial Na(+),K(+)-ATPase activity.     

A kinetic study of the gill (Na+, K+)-ATPase, and its role in ammonia excretion in the intertidal hermit crab, Clibanarius vittatus

To better comprehend the role of gill ion regulatory mechanisms, the modulation by Na(+), K(+), NH(4)(+) and ATP of (Na(+), K(+))-ATPase activity was examined in a posterior gill microsomal fraction from the hermit crab, Clibanarius vittatus. Under saturating Mg(2+), Na(+) and K(+) concentrations, two well-defined ATP hydrolyzing sites were revealed. ATP was hydrolyzed at the high-affinity sites at a maximum rate of V=19.1+/-0.8 U mg(-1) and K(0.5)=63.8+/-2.9 nmol L(-1), obeying cooperative kinetics (n(H)=1.9); at the low-affinity sites, hydrolysis obeyed Michaelis-Menten kinetics with K(M)=44.1+/-2.6 mumol L(-1) and V=123.5+/-6.1 U mg(-1). Stimulation by Na(+) (V=149.0+/-7.4 U mg(-1); K(M)=7.4+/-0.4 mmol L(-1)), Mg(2+) (V=132.0+/-5.3 U mg(-1); K(0.5)=0.36+/-0.02 mmol L(-1)), NH(4)(+) (V=245.6+/-9.8 U mg(-1); K(M)=4.5+/-0.2 mmol L(-1)) and K(+) (V=140.0+/-4.9 U mg(-1); K(M)=1.5+/-0.1 mmol L(-1)) followed a single saturation curve and, except for Mg(2+), obeyed Michaelis-Menten kinetics. Under optimal ionic conditions, but in the absence of NH(4)(+), ouabain (K(I)=117.3+/-3.5 mumol L(-1)) and orthovanadate inhibited up to 67% of the ATPase activity. The inhibition studies performed suggest the presence of F(0)F(1), V- and P-ATPases, but not Na(+)-, K(+)- or Ca(2+)-ATPases as contaminants in the gill microsomal preparation. (Na(+), K(+))-ATPase activity was synergistically modulated by NH(4)(+) and K(+). At 20 mmol L(-1) K(+), a maximum rate of V=290.8+/-14.5 U mg(-1) was seen as NH(4)(+) concentration was increased up to 50 mmol L(-1). However, at fixed NH(4)(+) concentrations, no additional stimulation was found for increasing K(+) concentrations (V=135.2+/-4.1 U mg(-1) and V=236.6+/-9.5 U mg(-1) and for 10 and 30 mmol L(-1) NH(4)(+), respectively). This is the first report to detail ionic modulation of gill (Na(+), K(+))-ATPase in C. vittatus, revealing an asymmetrical, synergistic stimulation of the enzyme by K(+) and NH(4)(+), as yet undescribed for other (Na(+), K(+))-ATPases, and should provide a better understanding of NH(4)(+) excretion in pagurid crabs.     

K+-Phosphatase activity of gill (Na+, K+)-ATPase from the blue crab, Callinectes danae: low-salinity acclimation and expression of the alpha-subunit

The kinetic properties of a microsomal gill (Na(+), K(+)) ATPase from the blue crab, Callinectes danae, acclimated to 15 per thousand salinity for 10 days, were analyzed using the substrate p-nitrophenylphosphate. The (Na(+), K(+))-ATPase hydrolyzed the substrate obeying Michaelian kinetics at a rate of V=102.9+/-4.3 U.mg(-1) with K(0.5)=1.7+/-0.1 mmol.L(-1), while stimulation by magnesium (V=93.7+/-2.3 U.mg(-1); K(0.5)=1.40+/-0.03 mmol.L(-1)) and potassium ions (V=94.9+/-3.5 U.mg(-1); K(0.5)=2.9+/-0.1 mmol.L(-1)) was cooperative. K(+)-phosphatase activity was also stimulated by ammonium ions to a rate of V=106.2+/-2.2 U. mg(-1) with K(0.5)=9.8+/-0.2 mmol.L(-1), following cooperative kinetics (n(H)=2.9). However, K(+)-phosphatase activity was not stimulated further by K(+) plus NH(4) (+) ions. Sodium ions (K(I)=22.7+/-1.7 mmol.L(-1)), and orthovanadate (K(I)=28.1+/-1.4 nmol.L(-1)) completely inhibited PNPPase activity while ouabain inhibition reached almost 75% (K(I)=142.0+/-7.1 micromol.L(-1)). Western blotting analysis revealed increased expression of the (Na(+), K(+))-ATPase alpha-subunit in crabs acclimated to 15 per thousand salinity compared to those acclimated to 33 per thousand salinity. The increase in (Na(+), K(+))-ATPase activity in C. danae gill tissue in response to low-salinity acclimation apparently derives from the increased expression of the (Na(+), K( (+) ))-ATPase alpha-subunit; phosphate-hydrolyzing enzymes other than (Na(+), K(+))-ATPase are also expressed. These findings allow a better understanding of the kinetic behavior of the enzymes that underlie the osmoregulatory mechanisms of euryhaline crustaceans.     

Brain Na+, K+-ATPase isoforms: different hypothalamus and mesencephalon response to acute desipramine treatment

We studied Na(+), K(+)-ATPase activity alpha isoforms by performing ouabain inhibition curves in rat hypothalamus and mesencephalon after acute administration of desipramine to rats. In hypothalamus, Ki values for high, intermediate and low affinity populations were 0.075x10(-9) M, 0.58x10(-6) M and 0.97x10(-3) M, with isoform distribution of 55%, 28% and 17%, respectively. In mesencephalon, Ki values for high, intermediate and low affinity populations were 1.80x10(-9) M, 0.56x10(-6) M and 0.21x10(-3) M, with isoform distribution of 28%, 46% and 21%, respectively. Three hours after acute administration of 10 mg/kg desipramine to rats, Na(+), K(+)-ATPase activity in hypothalamus increased significantly 54%, 39% and 51% as assayed respectively in the absence of ouabain or in the presence of 1x10(-9) M, or 5x10(-6) M ouabain, whereas only a trend was recorded in the presence of 1x10(-3) M ouabain. In such conditions, enzyme activity in mesencephalon increased significantly 73%, 54%, 30% and 271%, respectively. Present results showed that desipramine treatment enhances the activity of Na(+), K(+)-ATPase alpha isoforms in rat hypothalamus and mesencephalon, but the extent of this increase differs according to the isoform and the anatomical area studied, suggesting a differential enzyme regulation in response to noradrenergic stimulation.