Teleost chloride cell. II. Autoradiographic localization of gill Na,K- ATPase in killifish Fundulus heteroclitus adapted to low and high salinity environments

The specific binding and inhibitory action of (3H)ouabain were employed to localize transport Na,K-ATPase in the euryhaline teleost gill, a NaCl-transporting osmoregulatory tissue in which both enzyme activity and transepithelial transport vary with environmental salinity. In killifish fully adapted to 10%, 100%, or 200% seawater, the gills were internally perfused and externally irrigated in situ. After suitable internal or external exposure to (3H)ouabain, individual gill arches were excised for Na,K-ATPase assay, measurement of radiolabel binding, or quantitative high-resolution autoradiography. Internal exposure to 50 muM ouabain resulted in essentially complete enzyme inhibition, and binding paralleled the increases in enzyme activity at higher salinities; in contrast, external exposure gave minimal and erratic results consistent with leakage of external ouabain into interstitial fluid. (3H)Ouabain autoradiographs demonstrated that, irrespective of exposure or salinity, most of the gill binding was associated with chloride cell. These cells increased in size and number with salinity and, at the subcellular level, the distribution pattern for bound ouabain was always identical to that for the amplified basal-lateral (tubular system) membrane. The combined physiologicmorphologic results constitute final direct proof that chloride cells are the primary site of gill Na,K-ATPase. More important, they provide convincing evidence for unexpected increases in basal-lateral enzyme at higher salinities and thus raise a fundamental objection to the long-postulated role of the Na pump in secretory NaCl transport.     

Ouabain inhibition of gill Na-K-ATPase: relationship to active chloride transport.

Ouabain circulating in blood inhibits Na-K-ATPase in the gills of seawater eels at a concentration similar to that necessary for inhibition in vitro. By contrast, a much higher concentration is required when ouabain is applied to the exterior of the gill. Inhibition by external ouabain occurs only when the drug gains access to the circulation of the fish, as evidenced by simultaneous inhibition of Na-K-ATPase in the kidney. These results suggest that the Na-K-ATPase of gill chloride cells faces inward, lining intracytoplasmic tubular channels continuous with the extracellular fluid. Inhibition of gill Na-K-ATPase by ouabain in intact salt water eels results in almost complete inhibition of the efflux of both Na+ and Cl-. The efflux is tritiated water was much less reduced, to 60% of normal. Since chloride is actively transported outward across the gill of seawater teleosts, it is suggested that active chloride transport is coupled to Na-K-ATPase. A neutral sodium chloride carrier is postulated that is energized by the movement of sodium from extracellular fluid down its electrochemical gradient into the chloride cell.     

Ambient salinity modulates the expression of sodium pumps in branchial mitochondria-rich cells of Mozambique tilapia,Oreochromis mossambicus

Na,K-ATPase (sodium pumps) provide the primitive driving force for ion transport in branchial epithelial cells. Immunoblots of epithelial homogenates of both seawater (SW)- and freshwater (FW)-adapted tilapia gills as well as rat brain homogenate, a positive control, revealed one major band with a molecular weight of about 100 kDa. SW-adapted tilapia gills possessed larger (about 2-fold) amounts of sodium pumps compared with FW-adapted tilapia gills. (3)H-ouabain binding representing functional binding sites of Na,K-ATPase was also higher (about 3.5-fold) in gills of SW-adapted tilapia compared to that of FW-adapted fish. Moreover, specific activities of SW fish were higher (about 2-fold) than those of FW fish. Double labeling of Na,K-ATPase and Con-A, a fluorescent marker of MR cells, in tilapia gills followed by analysis with confocal microscopy showed that sodium pumps were localized mainly in MR cells, including the SW type and different FW types. Although more-active expression of Na,K-ATPase was demonstrated in gills of SW-adapted tilapia, no significant differences in densities of apical openings of MR cells were found between SW- and FW-adapted fish. These results indicate that, during salinity challenge, tilapia develop more "functional" Na,K-ATPase in SW-type MR cells to meet physiological demands.     

Branchial Na,K-ATPase and osmoregulation in the purple shore crab,Hemigrapsus nudus (Dana)

The purple shore crab, Hemigrapsus nudus, controls its hemolymph osmolality over a wide range of external salinities: it is a strong hyperosmoregulator in 25%, 50% and 75% sea water (SW) and is isosmotic in 100% SW. The role of branchial sodium + potassium-activated, magnesium-requiring adenosine triphosphatase (NA, K-ATPase) in osmoregulation was investigated by assaying enzyme-specific activity (SEA) in gills from crabs acclimated for 14 d in the four sea water media. Assay conditions were characterized for optimal ESA with crude homogenates of gills; ion and cofactor requirements were found to be similar to those of other crustacean Na, K-ATPases. Branchial ESA was highest in crabs acclimated for 2 weeks in 50% SW and was significantly correlated with the osmotic gradient across the body wall in 50%, 75% and 100% SW. Gills 6, 7 and 8 had the highest ESA in all media and possessed approximately 70% of the total branchial Na, K-ATPase activity, but all gills showed significant, approximately twofold increases of ESA in 50% SW compared with values in 100% SW. The time courses of increased branchial Na, K-ATPase ESA and decreased hemolymph osmotic pressure in crabs transferred from 100% SW to 50% SW are consistent with both increased in vivo activity of existing enzyme in the short term and a longer-term synthesis of new enzyme by the gills which is measured by our in vitro assay.     

Gill Na,K-ATPase in the spiny lobster Palinurus elephas and other marine osmoconformers. Adaptiveness of enzymes from osmoconformity to hyperregulation

Haemolymph inorganic osmolyte changes and Na,K-ATPase activities in trichobranchiate and epipodite tissues were examined in the spiny lobster Palinurus elephas gradually acclimated from seawater (SW; 38 ppt, salinity; 1291 mOsmol/l) down to dilute seawater (DSW; 20 ppt, salinity; 679 mOsmol/l). During acclimation to DSW haemolymph was only transiently hypoosmotic, becoming isosmotic to the medium over a 24-h period of acclimation. Na,K-ATPase specific activities in homogenates of the trichobranchiate gills from SW- and DSW-acclimated spiny lobsters were in the range of 2-3 μmol Pi/h/mg protein and were not significantly different. It has also been confirmed for the marine stenohaline crustaceans Maja crispata and Dromia personata that gill Na,K-ATPase maintains the same level of specific activity in SW- and DSW-acclimated crabs. The saponin-treated fraction of Na,K-ATPase activity in trichobranchiate gills was 67-89% and epipodites 63-64% over the native homogenates' activity and no differences in enzyme activities upon saponin treatment between SW- and DSW-acclimated spiny lobsters were found. Recovery of 6% and enrichment factor (1.6) of Na,K-ATPase in partially purified plasma membrane fractions of epipodites was relatively low and not different in SW- and DSW-acclimated spiny lobsters. In the hemiepipodite, negative short-circuit current was in the range from -16.7 to -22.7 μA cm(-2) and conductance varied in the range of 205-290 mS cm(-2), values which were not significantly different in spiny lobsters residing in SW or DSW. Very high conductance suggests leakiness of the hemiepipodite epithelium-cuticular complex. In contrast to the group of euryhaline hyperosmoregulating Crustacea in which activation of the specific activity of Na,K-ATPase upon acclimation to dilute seawater occurs, in marine osmoconformers there is no activation of the enzyme in dilute seawater. Based on the literature data and our own results, we have reported a correlation coefficient of 0.65 between specific activity of Na,K-ATPase and the sodium gradient (mmol Na/l; haemolymph-seawater ) between 12 species of osmoconforming and osmoregulating Crustacea. During evolution, hyperosmoregulating Crustacea have achieved internal osmolyte gradients generated by Na,K-ATPase and lowering the gill surface permeability. However these adaptive characteristics are not present in marine osmoconforming Crustacea, restraining them to migrate in the brackish water habitats.     

Dexamethasone modulates the activity of the eel branchial Na+/K+ATPase in both chloride and pavement cells

In fish, gills actively accumulate ions in freshwater (FW) with Na+ absorption taking place at the level of pavement cells, and excrete monovalent ions, mainly Na+ and Cl-, through the chloride cells in sea water (SW). The Na+/K+ATPase plays a crucial role in the functionality of osmoregulatory cells and we showed previously that angiotensin II modulates its activity in the eel gill (1). We here show the effects of synthetic steroid dexamethasone (DEX) on the activity of Na+/K+ATPase in both gill pavement and chloride cells from FW- and SW-adapted animals. Results showed that in the chloride cells 100 nM DEX provoked a significant increment in the activity of Na+/K+ATPase in both SW- and FW-adapted animals. This effect was greatest at 2 hours in SW, and at 6 hours in FW. The increment in the activity of the Na+/K+ATPase was dose-dependent in both environmental adaptations. Conversely, in pavement cells from FW-adapted eels 100 nM DEX decremented the activity of Na+/K+ATPase (4-fold reduction after 6 hour incubations), while in SW, DEX increased the enzyme activity of about 25% at 2 hours, and of about 55% at 6 hours. These results are consistent with the different physiological roles that pavement and chloride cells have in the two different adaptive conditions.     

The expression of gill Na,K-ATPase in milkfish,Chanos chanos,acclimated to seawater,brackish water and fresh water

Juvenile milkfish Chanos chanos (Forssk?l, 1775) were transferred from a local fish farm to fresh water (FW; 0 per thousand ), brackish water (BW; 10 per thousand, 20 per thousand ) and seawater (SW; 35 per thousand ) conditions in the laboratory and reared for at least two weeks. The blood and gill of the fish adapted to various salinities were analyzed to determine the osmoregulatory ability of this euryhaline species. No significant difference was found in plasma osmolality, sodium or chloride concentrations of milkfish adapted to various salinities. In FW, the fish exhibited the highest specific activity of Na, K-ATPase (NKA) in gills, while the SW group was found to have the lowest. Relative abundance of branchial NKA alpha-subunit revealed similar profiles. However, in contrary to other euryhaline teleosts, i.e. tilapia, salmon and eel, the naturally SW-dwelling milkfish expresses higher activity of NKA in BW and FW. Immunocytochemical staining has shown that most Na, K-ATPase immunoreactive (NKIR) cells in fish adapted to BW and SW were localized to the filaments with very few on the lamellae. Moreover, in FW-adapted milkfish, the number of NKIR cells found on the lamellae increased significantly. Such responses as elevated NKIR cell number and NKA activity are thought to improve the osmoregulatory capacity of the milkfish in hyposaline environments.     

Thyroid status alters gill ionic metabolism and chloride cell morphology as evidenced by scanning electron microscopy in a teleost Anabas testudineus (Bloch): short and long term in vivo study

Gill is the main organ of osmotic regulation in teleosts and chloride cells are the sites of ion transport across gill epithelium. Thyroid hormones are implicated in the regulation of osmotic balance in teleosts also. Treatment with 6-propyl thiouracil (6-PTU) inhibited the membrane bound enzyme Na+K+ ATPase in the gill while triiodothyronine (T3) injection stimulated it in a short-term in vivo study in the teleost Anabas testudineus. Na+, K+ and Ca2+ ions were also decreased in the 6-PTU treated fish and the T3 treatment increased their concentrations in the gill lamellae. The gill morphology also changed according to the thyroid status in the long term study. 6-PTU treatment altered the typical serrated morphology of the gill lamellae, while the T3 treatment reversed it. T3 injection increased the density of pavement and chloride cells as evidenced by scanning electron microscopy. The results demonstrate that physiological status of the thyroid influences gill Na+ pump activity and chloride cell morphological changes. Further, the study suggests a regulatory role of T3 on gill ions (Na+, K+ and Ca2+), Na+K+ and Ca2+ ATPase activity and the different gill cell types in A. testudineus.     

Plasma thyroxine and cortisol profiles and gill and kidney Na+/K+-ATPase and SDH activities during acclimation of the catfish Heteropneustes fossilis (bloch) to higher salinity, with special reference to the effects of exogenous cortisol on hypo-osmoregulatory ability of the catfish

When the stenohaline catfish Heteropneustes fossilis was transferred from fresh water (FW) to 30% seawater (SW), the Na(+)/K(+)-ATPase activity significantly increased in the kidney, while in gills it remained more or less constant. A reverse pattern was observed for succinic dehydrogenase (SDH) activity inasmuch as it significantly increased in gills and remained unchanged in the kidney. Plasma osmolality significantly increased within 3 days of transfer to 30% SW and remained significantly higher throughout the duration of experiment. These results suggest that catfish gills may not be able to reverse their function from salt uptake in FW to salt excretion at higher salinity, and that the elimination of monovalent as well as divalent ions is performed by the kidney but not the gills. The significant decline in plasma cortisol (F) levels following transfer to higher salinity may not be due to reduced production but rather to an enhanced utilization and clearance rate, a conclusion supported by the fact that exogenous administration of cortisol acetate (FA) resulted in significant increases in branchial and renal Na(+)/K(+)-ATPase in FW and 30% SW. FA also improved the plasma osmotic regulatory ability of the catfish, possibly due to a change in branchial function from salt-absorption to salt excretion, as was evident from a significant increase in branchial Na(+)/K(+)-ATPase activity in the fish in 30% SW pretreated with FA for 5 days. Consistently higher levels of plasma thyroxine (T4) following transfer to higher salinity suggest the involvement of this hormone at higher salinity.     

Changes in the levels of chloride cells and (Na+ + K+)-dependent ATPase in the gills of yellow and silver eels adapting to seawater.

Changes were measured in the numbers of chloride cells and the levels of (Na+ + K+)-DEPENDENT ATPase in the gills of immature, yellow eels and mature, silver eels during adaptation from freshwater to seawater. The percentage of chloride cells in yellow eels more than doubled after six days in seawater; at this time the specific activity and concentration of (Na+ + K+)-dependent ATPase in gills start to increase in parallel to reach maxima after two weeks that are 2.5 times the starting values. It is concluded that adaptation of yellow eels to seawater involves an increase in the numbers of chloride cells in gills as well as an increased amount of (Na+ + K+)-dependent ATPase per chloride cell. Mature silver eels in freshwater had essentially the same numbers of chloride cells and the same specific activity of the enzyme in the gills as yellow eels fully adapted to seawater. Transferring silver eels to seawater did not alter the percentage of chloride cells in gills although the level of (Na+ + K+)-dependent ATPase and its specific activity increased slightly. Thus, although the silver eel is better prepared for life in seawater than the yellow eel, it still has to attain an increased level of (Na+ + K+)-dependent ATPase in its chloride cells to be fully adapted to seawater.     

Cell signaling and ion transport across the fish gill epithelium

A large array of circulating and local signaling agents modulate transport of ions across the gill epithelium of fishes by either affecting transport directly or by altering the size and distribution of transporting cells in the epithelium. In some cases, these transport effects are in addition to cardiovascular effects of the same agents, which may affect the perfusion pathways in the gill vasculature and, in turn, affect epithelial transport indirectly. Prolactin is generally considered to function in freshwater, because it is the only agent that allows survival of some hypophysectomized fish species in freshwater. It appears to function by either reducing branchial permeability, Na,K-activated ATPase activity, or reducing the density of chloride cells. Cortisol was initially considered to produce virtually opposite effects (e.g., stimulation of Na,K-activated ATPase and of chloride cell size and density), but more recent studies have found that this steroid stimulates ionic uptake in freshwater fishes, as well as the activity of H-ATPase, an enzyme thought to be central to ionic uptake. Thus, cortisol may function in both high and low salinities. Growth hormone and insulin-like growth factor appear to act synergistically to affect ion regulation in seawater fishes, stimulating both Na,K-activated ATPase and Na-K-2Cl co-transporter activity, and chloride cell size, independent of their effects on growth. Some of the effects of the GH-IGF axis may be via stimulation of the number of cortisol receptors. Thyroid hormones appear to affect seawater ion regulation indirectly, by stimulating the GH-IGF axis. Natriuretic peptides were initially thought to stimulate gill ionic extrusion, but recent studies have not corroborated this finding, so it appears that the major mode of action of these peptides may be reduction of salt loading by inhibition of oral ingestion and intestinal ionic uptake. Receptors for both arginine vasotocin and angiotensin have been described in the gill epithelium, but their respective roles and importance in fish ion regulation remains unknown. The gill epithelium may be affected by both circulating and local adrenergic agents, and a variety of studies have demonstrated that stimulation of alpha-adrenergic versus beta-adrenergic receptors produces inhibition or stimulation of active salt extrusion, respectively. Local effectors, such as prostaglandins, nitric oxide, and endothelin, may affect active salt extrusion as well as gill perfusion. Recent studies have suggested that the endothelin inhibition of salt extrusion is actually mediated by the release of both NO and prostaglandins. It is hoped that modern molecular techniques, combined with physiological measurements, will allow the dissection of the relative roles in ion transport across the fish gill epithelium of this surprisingly large array of putative signaling agents.     

Identification of a crab gill FXYD2 protein and regulation of crab microsomal Na,K-ATPase activity by mammalian FXYD2 peptide

This investigation discloses the recognition of an FXYD2 protein in a microsomal Na,K-ATPase preparation from the posterior gills of the blue crab, Callinectes danae, by a mammalian (rabbit) FXYD2 peptide specific antibody (γC(33)) and MALDI-TOF-TOF mass spectrometry techniques. This is the first demonstration of an invertebrate FXYD2 protein. The addition of exogenous pig FXYD2 peptide to the crab gill microsomal fraction stimulated Na,K-ATPase activity in a dose-dependent manner. Exogenous pig FXYD2 also considerably increased enzyme affinity for K(+), ATP and NH(4)(+). K(0.5) for Na(+) was unaffected. Exogenous pig FXYD2 increased the V(max) for stimulation of gill Na,K-ATPase activity by Na(+), K(+) and ATP, by 30% to 40%. The crab gill FXYD2 is phosphorylated by PKA, suggesting a regulatory function similar to that known for the mammalian enzyme. The PKA-phosphorylated pig FXYD2 peptide stimulated the crab gill Na,K-ATPase activity by 80%, about 2-fold greater than did the non-phosphorylated peptide. Stimulation by the PKC-phosphorylated pig FXYD2 peptide was minimal. These findings confirm the presence of an FXYD2 peptide in the crab gill Na,K-ATPase and demonstrate that this peptide plays an important role in regulating enzyme activity.     

Effects of salinity on chloride cells in the euryhaline cyprinodontid fish Rivulus marmoratus

Summary The ultrastructure and density of chloride cells in the gill, opercular epithelium, and opercular skin of the euryhaline self-fertilizing fish Rivulus marmoratus (Cyprinodontidae) were studied with electron and fluorescence microscopy. R. marmoratus raised from birth in 1, 50, 100, and 200% seawater were compared. Chloride cells from fish raised in each of the four salinities exhibited an invaginated pit structure at the apical crypt. Multicellular complexes were present in the 1% seawater group and in those fish raised in higher salinities where elaborate interdigitations were seen between cells. Chloride cells from gills of fish raised in 200% seawater had a significantly higher percentage of their cytoplasmic volume composed of mitochondria than did those from fish raised in 1% seawater (69.9% vs 37.4%). The opercular skin and opercular epithelium had the same density of chloride cells (4.2×10⁴-4.5×10⁴ chloride cells/cm²), and this number did not vary significantly with increased salinity. The opercular skin thus appears far more responsive to environmental salinity than the opercular epithelium. Chloride cells from the opercular epithelium of fish raised in 200% seawater were found to be 39% larger than those from fish raised in 1% seawater, whereas the chloride cells from the opercular skin of the 200% seawater group were 107% larger than those from the 1% seawater group.     

Developmental and environmental regulation of chloride cells in young American shad, Alosa sapidissima

Location, abundance, and morphology of gill chloride cells were quantified during changes in osmoregulatory physiology accompanying early development in American shad, Alosa sapidissima. During the larval-juvenile transition of shad, gill chloride cells increased 3.5-fold in abundance coincident with gill formation, increased seawater tolerance, and increased Na(+),K(+)-ATPase activity. Chloride cells were found on both the primary filament and secondary lamellae in pre-migratory juveniles. Chloride cells on both the primary filament and secondary lamellae increased in abundance (1.5- to 2-fold) and size (2- to 2.5-fold) in juveniles held in fresh water from August 31 to December 1 (the period of downstream migration) under declining temperature. This proliferation of chloride cells was correlated with physiological changes associated with migration (decreased hyperosmoregulatory ability and increased gill Na(+),K(+)-ATPase activity). Increases in chloride cell size and number of fish in fresh water were delayed and of a lower magnitude when shad were maintained at constant temperature (24 degrees C). When juveniles were acclimated to seawater, chloride cell abundance on the primary filament did not (though size increased 1.5- to 2-fold), but cells on the secondary lamellae disappeared. Na(+),K(+)-ATPase was immunolocalized to chloride cells in both fresh water and seawater acclimated fish. The disappearance of chloride cells on the secondary lamellae upon seawater acclimation is evidence that their role is confined to fresh water. The proliferation of chloride cells in fresh water during the migratory-associated loss of hyperosmoregulatory ability is likely to be a compensatory mechanism for increasing ion uptake. J. Exp. Zool. 290:73-87, 2001.     

Gill Na+-K+-ATPase activity correlates with basolateral membrane lipid composition in seawater- but not freshwater-acclimated Arctic char (Salvelinus alpinus)

The successful migration of euryhaline teleost fish from freshwater to seawater requires the upregulation of gill Na+-K+-ATPase, an ion transport enzyme located in the basolateral membrane (BLM) of gill chloride cells. Following 39 days of seawater exposure, Arctic char had similar plasma sodium and chloride levels as individuals maintained in freshwater, indicating they had successfully acclimated to seawater. This acclimation was associated with an eightfold increase in gill Na+-K+-ATPase activity but only a threefold increase in gill Na+-K+-ATPase protein number, suggesting that other mechanisms may also modulate gill Na+-K+-ATPase activity. We therefore investigated the influence of membrane composition on Na+-K+-ATPase activity by examining the phospholipid, fatty acid, and cholesterol composition of the gill BLM from freshwater- and seawater-acclimated Arctic char. Mean gill BLM cholesterol content was significantly lower ( approximately 22%) in seawater-acclimated char. Gill Na+-K+-ATPase activity in individual seawater Arctic char was negatively correlated with BLM cholesterol content and positively correlated with %phosphatidylethanolamine and overall %18:2n6 (linoleic acid) content of the BLM, suggesting gill Na+-K+-ATPase activity of seawater-acclimated char may be modulated by the lipid composition of the BLM and may be especially sensitive to those parameters known to influence membrane fluidity. Na+-K+-ATPase activity of individual freshwater Arctic char was not correlated to any membrane lipid parameter measured, suggesting that different lipid-protein interactions may exist for char living in each environment.     

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.     

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.     

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.