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.     

Changes in tissue free amino acid contents, branchial Na+/K+ -ATPase activity and bimodal breathing pattern in the freshwater climbing perch, Anabas testudineus (Bloch), during seawater acclimation

This study aimed to examine effects of short- or long-term acclimation to brackish water or seawater on the climbing perch, Anabas testudineus, which is an aquatic air-breathing teleost living typically in freshwater. A. testudineus exhibits hypoosmotic and hypoinoic osmoregulation; the plasma osmolality, [Na+] and [Cl-] of fish acclimated to seawater were consistently lower than those of the external medium. However, during short-term (1 day) exposure to brackish water (15 per thousand) or seawater (30 per thousand), these three parameters increased significantly. There were also significant increases in tissue ammonia and urea contents, contents of certain free amino acids (FAAs) in the muscle, and rates of ammonia and urea excretion in the experimental fish. The accumulated FAAs might have a transient role in cell volume regulation. In addition, these results indicate that increases in protein degradation and amino acid catabolism had occurred, possibly providing energy for the osmoregulatory acclimation of the gills in fish exposed to salinity stress. Indeed, there was a significant increase in the branchial Na+/K+ -ATPase activity in fish exposed to seawater for a prolonged period (7 days), and the plasma osmolality, [Na+] and [Cl-] and the tissue FAA contents of these fish returned to control levels. More importantly, there was a significant increase in the dependence on water-breathing in fish acclimated to seawater for 7 days. This suggests for the first time that A. testudineus could alter its bimodal breathing pattern to facilitate the functioning of branchial Na+/K+ -ATPase for osmoregulatory purposes.     

Branchial osmoregulatory response to salinity in the gilthead sea bream, Sparus auratus

The branchial osmoregulatory response of gilthead sea bream (Sparus auratus L.) to short-term (2-192 hr) and long-term (2 weeks) exposure to different environmental salinities (5 per thousand, 15 per thousand, 25 per thousand, 38 per thousand and 60 per thousand) was investigated. A "U-shaped" relationship was observed between environmental salinity and gill Na+,K+ -ATPase activity in both long- and short-term exposure to altered salinity, with the increase in activity occurring between 24 and 96 hr after the onset of exposure. Plasma osmolality and plasma ions (sodium, chloride, calcium and potassium) showed a tendency to increase in parallel with salinity. These variables only differed significantly (P<0.05) in fish adapted to 60 per thousand salinity with respect to fish adapted to full-strength sea-water (SW). Plasma glucose remained unchanged whereas plasma lactate was elevated at 5 per thousand and 60 per thousand. Muscle water content (MWC) was significantly lower in fish adapted to 60 per thousand. Chloride cells (CC) were only present on the surface of the gill filaments and absent from the secondary lamellae. CC distribution was not altered by external salinity. However, the number and size of CC were significantly increased at salinity extremes (5 per thousand and 60 per thousand), whereas fish exposed to intermediate salinities (15 per thousand and 25 per thousand) had fewer and smaller cells. Furthermore, the CC of fish exposed to diluted SW became rounder whereas they were more elongated in fish in full-strength and hypersaline SW. This is consistent with previous reports indicating the existence of two CC types in euryhaline fish. At likely environmental salinities, gilthead sea bream show minor changes in plasma variables and the effective regulation of gill Na+,K+ -ATPase. However, at very low salinities both haemodilution and up-regulation of gill Na+,K+ -ATPase predict a poor adaptation most likely related to deficiency or absence of specific components of the CC important for ion xuptake.     

Expression and distribution of Na, K-ATPase in gill and kidney of the spotted green pufferfish, Tetraodon nigroviridis, in response to salinity challenge

Freshwater (FW) spotted green pufferfish (Tetraodon nigroviridis) were transferred directly from a local aquarium to fresh water (FW; 0 per thousand ), brackish water (BW; 15 per thousand ), and seawater (SW; 35 per thousand ) conditions in the laboratory and reared for at least two weeks. No mortality was found. To investigate the efficient mechanisms of osmoregulation in the euryhaline teleost, distribution and expression of Na,K-ATPase (NKA) in gill and kidney of the pufferfish were examined and the osmolality, [Na+] and [Cl-] of the blood were assayed. The lowest levels of both relative protein abundance and activity were found to be exhibited in the BW group, and higher levels in the SW group than FW group. In all salinities, branchial NKA immunoreactivity was found in epithelial cells of the interlamellar region of the filament and not on the lamellae. Relative abundance of kidney NKA alpha-subunit, as well as the NKA activity, was found to be higher in the FW pufferfish than fish in BW or SW. Renal NKA appeared in the epithelial cells of distal tubules, proximal tubules, and collecting tubules, but not in glomeruli, in fish groups of various salinities. Plasma osmolality and chloride levels were significantly lower in FW pufferfish than those in BW and SW, whereas plasma sodium did not differ among the groups. Although identical distributions of NKA were found in either gill or kidney of FW-, BW- or SW-acclimated spotted green pufferfish, differential NKA expression in fish of various salinity groups was associated with physiological homeostasis (stable blood osmolality), and illustrated the impressive osmoregulatory ability of this freshwater and estuarine species in response to salinity challenge.     

Adaptive shifts in osmoregulatory strategy and the invasion of freshwater by brachyuran crabs: evidence from Dilocarcinus pagei (Trichodactylidae)

To evaluate putative adaptive changes underpinning the invasion of freshwater by the Brachyura, this investigation examines anisosmotic extra and isosmotic intracellular osmoregulatory capabilities in Dilocarcinus pagei, a neotropical, hololimnetic crab, including its embryonic and juvenile phases. All ontogenetic stages show a remarkable ability to survive a high salinity medium (25 per thousand, 750 mOsm/kg H2O, 350 mm Na+, 400 mM Cl-). Adults hyper-regulate hemolymph osmolality up to isosmoticity at 744 mOsm kg/H2O (24 per thousand), [Na+] and [Cl-] becoming isoionic at 449 (22 per thousand) and 256 mM (16 per thousand), respectively. Hemolymph (420+/-39 mOsm/kg H2O) and urine (384+/-44 mOsm/kg H2O) are isosmotic in adults held in freshwater, and after 5-days exposure to 25 per thousand (787+/-9 mOsm/kg H2O and 777+/-43 mOs/kg H2O, respectively); D. pagei does not produce dilute urine. Total free amino acid (FAA) concentrations in embryos (14.9+/-1.2), juveniles (32.8+/-0.1) and adult muscle (10.9+/-2.1 mmol/kg wet weight) in freshwater are 30-fold less than in brackish/marine Crustacea, suggesting that FAA constitute a useful parameter to evaluate adaptation to freshwater. On acclimation to 25 per thousand, total FAA increase by approximately 100% in embryos and in adult muscle and nerve tissue and hemolymph, owing to large increases in proline, arginine and/or alanine. However, effective FAA contribution to intracellular osmolality increases only in embryos, from 3 to 4.5%. These findings suggest that gill-based, anisosmotic extracellular regulation has supplanted isosmotic intracellular regulatory mechanisms during the conquest of freshwater by the Brachyura, and indicate that D. pagei may be an old, well-adapted inhabitant of this biotope.     

Physiological response in the European flounder (Platichthys flesus) to variable salinity and oxygen conditions

Physiological mechanisms involved in acclimation to variable salinity and oxygen levels and their interaction were studied in European flounder. The fish were acclimated for 2 weeks to freshwater (1 per thousand salinity), brackish water (11 per thousand) or full strength seawater (35 per thousand) under normoxic conditions (water Po(2) = 158 mmHg) and then subjected to 48 h of continued normoxia or hypoxia at a level (Po(2) = 54 mmHg) close to but above the critical Po(2). Plasma osmolality, [Na(+)] and [Cl(-)] increased with increasing salinity, but the rises were limited, reflecting an effective extracellular osmoregulation. Muscle water content was the same at all three salinities, indicating complete cell volume regulation. Gill Na(+)/K(+)-ATPase activity did not change with salinity, but hypoxia caused a 25% decrease in branchial Na(+)/K(+)-ATPase activity at all three salinities. Furthermore, hypoxia induced a significant decrease in mRNA levels of the Na(+)/K(+)-ATPase alpha1-subunit, signifying a reduced expression of the transporter gene. The reduced ATPase activity did not influence extracellular ionic concentrations. Blood [Hb] was stable with salinity, and it was not increased by hypoxia. Instead, hypoxia decreased the erythrocytic nucleoside triphosphate content, a common mechanism for increasing blood O(2) affinity. It is concluded that moderate hypoxia induced an energy saving decrease in branchial Na(+)/K(+)-ATPase activity, which did not compromise extracellular osmoregulation.     

A critical analysis of transepithelial potential in intact killifish (Fundulus heteroclitus) subjected to acute and chronic changes in salinity

We investigated the in vivo salinity-dependent behavior of transepithelial potential (TEP) in Fundulus heteroclitus (3-9 g) using indwelling coelomic catheters, a technique which was validated against blood catheter measurements in a larger species (Opsanus beta; 35-70 g). In seawater (SW)-acclimated killifish, TEP was +23 mV (inside positive), but changed to -39 mV immediately after transfer to freshwater (FW). Acute transfer to dilute salinities produced a TEP profile, which rapidly attenuated as salinity increased (0, 2.5, 5 and 10% SW), with cross-over to positive values between 20 and 40% SW, and a linear increase thereafter (60, 80 and 100% SW). TEP response profiles were also recorded after acute transfer to comparable dilutions of 500 mmol L(-1) NaCl, NaNO3, Na gluconate, choline chloride, N-methyl-D-glutamate (NMDG) chloride, or 1,100 mosmol kg(-1) mannitol. These indicated high non-specific cation permeability and low non-specific anion permeability without influence of osmolality in SW-acclimated killifish. While there was a small electrogenic component in high salinity, a Na+ diffusion potential predominated at all salinities due to the low P Cl/P Na (0.23) of the gills. The very negative TEP in FW was attenuated in a linear fashion by log elevations in [Ca2+] such that P Cl/P Na increased to 0.73 at 10 mmol L(-1). SW levels of [K+] or [Mg2+] also increased the TEP, but none of these cations alone restored the positive TEP of SW-acclimated killifish. The very negative TEP in FW attenuated over the first 12 h of exposure and by 24-30 h reached +3 mV, representative of long-term FW-acclimated animals; this reflected a progressive increase in P Cl/P Na from 0.23 to 1.30, probably associated with closing of the paracellular shunt pathway. Thereafter, the TEP in FW-acclimated killifish was unresponsive to [Ca2+] (also to [K+], [Mg2+], or chloride salts of choline and NMDG), but became more positive at SW levels of [Na+]. Killifish live in a variable salinity environment and are incapable of gill Cl(-) uptake in FW. We conclude that the adaptive significance of the TEP patterns is that changeover to a very negative TEP in FW will immediately limit Na+ loss while not interfering with active Cl(-) uptake because there is none. Keeping the shunt permeability high for a few hours means that killifish can return to SW and instantaneously re-activate their NaCl excretion mechanism.     

Acute toxicity of ammonia and its effects on the haemolymph osmolality, ammonia-N, pH and ionic composition of early juvenile mud crabs, Scylla serrata (Forskål)

The current study was conducted to determine the LC(50) value of ammonia-N as well as the effects of acute exposure to elevated ammonia on the haemolymph osmolality, ionic composition, ammonia-N and pH levels of early juvenile mud crabs, Scylla serrata. The results show that early S. serrata juveniles have a high 96-h LC(50) value of 95.35 mg/L ammonia-N (6.81 mg/L NH(3)-N) or 6.80 mmol/L total ammonia-N (0.486 mmol/L NH(3)-N). Following a 96-h exposure, the haemolymph osmolality and K(+) levels of the surviving crabs remained unaltered (p>0.05) at all ammonia-N concentrations, while the haemolymph Na(+) and Ca(2+) were significantly lower (p<0.05) for the crabs exposed to 5.710 and 7.138 mmol/L ammonia-N. While the haemolymph ammonia-N levels of the crabs significantly increased (p<0.01) with increasing external ammonia-N concentrations, the haemolymph ammonia-N of the crabs remained below the external ammonia-N concentrations. The haemolymph pH of the crabs significantly increased between 0.714 and 4.283 mmol/L total ammonia-N. However, at 5.710 mmol/L total ammonia-N the haemolymph pH dropped and was not significantly different (p>0.05) from that of the control crabs which coincided with significantly lower (p<0.05) haemolymph Na(+) and Ca(2+) levels. These physiological responses may explain the high ammonia tolerance of early S. serrata juveniles.     

Arginine vasotocin, isotocin and melatonin responses following acclimation of gilthead sea bream (Sparus aurata) to different environmental salinities

Gilthead sea bream (Sparus aurata) is a euryhaline species with a capacity to cope with demands in a wide range of salinities and thus is a perfect model-fish to study osmoregulatory responses to salinity-adaptive processes and their hormonal control. Immature sea bream acclimated to different salinities, i.e. SW (38 per thousand), LSW (5 per thousand) and HSW (55 per thousand), were kept at 18 degrees C under natural photoperiod. Arginine vasotocin (AVT) and isotocin (IT) in plasma and pituitary were determined by HPLC. Plasma melatonin (Mel) was assayed by RIA. Plasma osmolality, ion concentrations (Na(+), K(+), Ca(2+), Cl(-)) and Na(+),K(+)-ATPase activity in gill were measured. A steady increase in plasma AVT, along with increasing water salinity was observed. Pituitary IT concentration in HSW-acclimated fish was significantly higher than that in LSW group. AVT/IT secretory system of sea bream does appear to be involved in the mechanism of long-term acclimation to different salinities. The distinct roles and control mechanisms of both nonapeptides are suggested. Plasma Mel was significantly higher in LSW compared with both HSW and SW groups. Data indicate that the changes in Mel level are linked to osmoregulation. Further studies are required to elucidate a complex role of AVT, IT and Mel in sea bream osmoregulation.     

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.     

Effects of intracellular and extracellular concentrations of Ca2+, K+, and Cl- on the Na+-dependent Mg2+ efflux in rat ventricular myocytes

Intracellular Mg2+ concentration ([Mg2+]i) was measured in rat ventricular myocytes with the fluorescent indicator furaptra (25 degrees C). After the myocytes were loaded with Mg2+, the initial rate of decrease in [Mg2+]i (initial Delta[Mg2+]i/Deltat) was estimated upon introduction of extracellular Na+, as an index of the rate of Na+-dependent Mg2+ efflux. The initial Delta[Mg2+]i/Deltat values with 140 mM [Na+]o were essentially unchanged by the addition of extracellular Ca2+ up to 1 mM (107.3+/-8.7% of the control value measured at 0 mM [Ca2+]o in the presence of 0.1 mM EGTA, n=5). Intracellular loading of a Ca2+ chelator, either BAPTA or dimethyl BAPTA, by incubation with its acetoxymethyl ester form (5 microM for 3.5 h) did not significantly change the initial Delta[Mg2+]i/Deltat: 115.2+/-7.5% (seven BAPTA-loaded cells) and 109.5+/-10.9% (four dimethyl BAPTA loaded cells) of the control values measured in the absence of an intracellular chelator. Extracellular and/or intracellular concentrations of K+ and Cl- were modified under constant [Na+]o (70 mM), [Ca2+]o (0 mM with 0.1 mM EGTA), and membrane potential (-13 mV with the amphotericin-B-perforated patch-clamp technique). None of the following conditions significantly changed the initial Delta[Mg2+]i/Deltat: 1), changes in [K+]o between 0 mM and 75 mM (65.6+/-5.0% (n=11) and 79.0+/-6.0% (n=8), respectively, of the control values measured at 140 mM [Na+]o without any modification of extracellular and intracellular K+ and Cl-); 2), intracellular perfusion with K+-free (Cs+-substituted) solution from the patch pipette in combination with removal of extracellular K+ (77.7+/-8.2%, n=8); and 3), extracellular and intracellular perfusion with K+-free and Cl--free solutions (71.6+/-5.1%, n=5). These results suggest that Mg2+ is transported in exchange with Na+, but not with Ca2+, K+, or Cl-, in cardiac myocytes.     

水体盐度对中华绒螯蟹成体雄蟹渗透压调节和生理代谢的影响

为研究长期不同水体盐度对中华绒螯蟹(Eriocheir sinensis以下简称河蟹)成体雄蟹渗透压调节和生理代谢的影响, 在不同水体盐度条件下(0、6、12和18)对河蟹雄体进行为期60d的养殖实验, 并分别检测其渗透调节及生理代谢相关指标。结果显示: (1)血清渗透压、Na+、Mg2+和Cl-含量随水体盐度上升而显著上升(P0.05), K+和Ca2+含量有上升趋势, 但各盐度组差异不显著(P0.05); 无论何组雄蟹, 其血清渗透压均显著高于对应的水体渗透压; (2)0组雄蟹后鳃Na+/K+-ATP酶活性显著高于其他组(P0.05), 其他各组间差异不显著(P0.05); (3)就血清生理代谢指标而言, 12组雄蟹血清中甘油三酯(TG)含量显著高于其他组(P0.05), 而尿酸(UA)、葡萄糖(Glu)、丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性相对较低; 所有组尿素(Urea)、碱性磷酸酶(ALP)含量差异不显著(P0.05); (4)就肝胰腺生理代谢指标而言, 6组肝胰腺MDA含量和-谷氨酰转肽酶(-GT)活力最低, 12组酸性磷酸酶(ACP)和-GT活性显著高于其他盐度组(P0.05)。因此, 适当提高水体盐度可提高河蟹成体雄蟹的血清渗透压及其主要离子含量, 同时降低其后鳃中Na+/K+-ATP酶活性。肝胰腺和血清代谢指标暗示12盐度组雄体的代谢水平相对较低, 具有较强的免疫性能和抗氧化能力。     

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.     

Handling of ferric iron by branchial and intestinal epithelia of climbing perch (Anabas testudineus Bloch)

With a view to test how the branchial and intestinal tissues of fish, the two sites of metal acquisition, utilize the water-borne ferric [Fe(III)] iron and whether the accumulation of this form of iron influences cellular Na/K gradient in these tissues, the gills and intestines of climbing perch adapted to freshwater (FW) and acclimated to dilute seawater (20 ppt; SW) were analyzed for ouabain-sensitive Na+, K+-ATPase activity, Fe and electrolyte contents after loading a low (8.95 microM) or high dose (89.5 microM) of Fe(III) iron in the water. The SW gills showed higher levels of total Fe after treating with 8.95 microM of Fe(III) iron which was not seen in the FW gills. Na+, K+-ATPase activity, reflecting Na/K pump activity, showed an increase in the FW gills and not in the SW gills. Substantial increase in the branchial Na and K content was observed in the SW gills, but the FW gills failed to show such effects after Fe(III) loading. The total Fe content was declined in the FW intestine but not in the SW intestine. Water-borne Fe(III) iron decreased the activity of Na+, K+-ATPase in the SW intestine while not changing its activity in the FW intestine. The Na and K content in the FW intestine did not respond to Fe(III) iron exposure but showed a reduction in its Na levels in the SW intestine. The moisture content in the gills and intestines of both the FW and SW perch remained unaffected after Fe(III) loading. In FW fish, the plasma Na levels were decreased by a low dose of Fe(III) iron, though a high dose of Fe(III) iron was required in the SW fish for such an effect. Overall, the results for the first time provide evidence that gills act as a major site for Fe(III) iron absorption and accumulation during salinity acclimation which depends on a high cellular Na/K gradient.     

A comparison of osmoregulatory responses in plasma and tissues of rainbow trout (Oncorhynchus mykiss) following acute salinity challenges

Euryhaline teleosts regulate their internal osmotic and ionic status across a wide range of external salinities. Studies often rely on measurements on plasma when osmoregulatory status is perturbed, whereas tissue measurements are used for small fish with limited blood volume. However, a direct comparison is lacking for plasma and various tissues. In the present study the relationships between plasma, white muscle and carcass were examined for a range of osmoregulatory variables in rainbow trout (Oncorhynchus mykiss) following challenge with an acute (24 h) transfer from freshwater to a hyper-osmotic salinity of either 25 or 35. Significant increases in plasma osmolality, [Na+], [Cl?], [Ca2+], and [Mg2+] were observed when salinity was increased, but plasma [K+] was unaffected. The water content of both tissues showed reciprocal changes to plasma osmolality. The carcass content of all ions measured showed a significant increase at the highest ambient salinity. In white muscle, Na+, K+ and Mg2+ showed significant increases with external salinity, but Cl? and Ca2+ were unaffected. Measurements from both tissues can provide reliable surrogates for most of the plasma osmoregulatory variables except Cl? and Ca2+ when using white muscle tissue. In the case of internal regulation of K+ both tissues provide sensitive and quantitatively similar indicators of environmental salinity disturbance, whereas plasma does not.     

Na+ gradient-dependent Mg2+ transport in smooth muscle cells of guinea pig tenia cecum

Thin strips of guinea pig tenia cecum were loaded with the Mg2+ indicator furaptra, and the indicator fluorescence signals measured in Ca2+-free condition were converted to cytoplasmic-free Mg2+ concentration ([Mg2+]i). Lowering the extracellular Na+ concentration ([Na+]o) caused a reversible increase in [Mg2+]i, consistent with the inhibition of Na+ gradient-dependent extrusion of cellular Mg2+ (Na+-Mg2+ exchange). Curve-fitting analysis indicated that the relation between [Na+]o and the rate of rise in [Mg2+], had a Hill coefficient of approximately 3, a [Na+]o at the half-maximal rate of rise of approximately 30 mM, and a maximal rate of 0.16 +/- 0.01 microM/s (mean +/- SE, n = 6). Depolarization with 56 mM K+ shifted the curve slightly toward higher [Na+]o without significantly changing the maximal rate, suggesting that the Na+-Mg2+ exchange was inhibited by depolarization. The maximal rate would correspond to a flux of 0.15-0.4 pmol/cm2/s, if cytoplasmic Mg2+ buffering power (defined as the ratio of the changes in total Mg2+ and free Mg2+ concentrations) is assumed to be 2-5. Ouabain (1-5 microM) increased the intracellular Na+ concentration, as assessed with fluorescence of SBFI (sodium-binding benzofuran isophthalate, a Na+ indicator), and elevated [Mg2+]i. In ouabain-treated preparations, removal of extracellular Na+ rapidly increased [Mg2+]i, with an initial rate of rise roughly proportional to the degree of the Mg2+ load, and, probably, to the Na+ load caused by ouabain. The enhanced rate of rise in [Mg2+]i (up to approximately 1 microM/s) could be attributed to the Mg2+ influx as a result of the reversed Na+-Mg2+ exchange. Our results support the presence of a reversible and possibly electrogenic Na+-Mg2+ exchange in the smooth muscle cells of tenia cecum.     

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.     

Osmotic and ionic regulation in shore crabsCarcinus maenas inhabiting a tidal estuary

Shore crabsCarcinus maenas were exposed to salinities fluctuating according to the natural tidal rhythm. To this end they were maintained in net cages positioned in the estuarine waters of the river Elbe. The cages were lifted every hour, and between 8–12 specimens were analyzed for hemolymph concentrations of Na, K, Ca, Mg, and osmolality. The results obtained were compared with the respective data measured in external brackish water. In addition, the specific activity of Na−K-ATPase in a posterior gill was determined. Hemolymph Na and Mg as well as branchial Na−K-ATPase were also determined in crabs collected in the North Sea and the Baltic. The results show that inC. maenas living in salinities fluctuating with the tides by approx. 15‰ S, Na, K and Ca were hyperregulated, and Mg was effectively hyporegulated. The concentrations of all hemolymph ions and the activity of the Na−K-ATPase were kept constant over the whole tidal cycle. In Baltic crabs, Na was effectively hyperregulated and gill Na−K-ATPase was significantly elevated by a factor of ca 2 when compared with North Sea crabs. It is suggested that long-term hyperregulation of Na in constant salinities results from an increased number of Na−K-ATPase molecules which may change by synthesis or degradation following salinity stress. Constant hemolymph levels of hyperregulated Na in crabs inhabiting fluctuating brackish water are accomplished by activation of existing Na−K-ATPase by low Na and inhibition by higher ambient concentrations. This work is part of the first author's doctoral thesis submitted to the Department of Biology at the University of Hamburg.     

急性盐度胁迫对军曹鱼稚鱼渗透压调节的影响

研究了环境盐度急性胁迫对军曹鱼(Rachycentron canadum)稚鱼鳃Na⁺-K⁺ATPase(NKA)活性及血清渗透压、Na⁺、K⁺和Cl^-离子调节的影响.结果表明:将稚鱼从盐度37中直接转移至盐度0、5、15、25、37（对照）和45的水体中,12 h后仅盐度0处理出现死亡(死亡率100％).各处理鳃NKA活性和血清渗透压在最初3 h内出现一定波动,随后变化平稳.试验结束时(12 h), NKA活性与盐度梯度呈“U”型分布,盐度5处理酶活性显著高于其它处理(P<0.05),盐度15处理活性最低,而各处理的血清渗透压大小(293～399 mOsmol·kg^-1)与盐度呈正相关;在3～12 h内稚鱼血清Na⁺和Cl^-浓度随盐度升高而升高,但增幅较小,血清K⁺浓度则与盐度呈负相关;12 h稚鱼的等渗点为328.2 mOsm·kg^-1,相当于盐度11.48,而Na⁺、K⁺和Cl^-等离子点分别为155.2、6.16和137.1 mmol·L^-1,分别相当于盐度10.68、20.44及8.41.军曹鱼在生理上具有广盐性鱼类的“低渗环境高NKA活性”特征,有较强及迅速的渗透压和离子调节与平衡能力.     

Acclimation of S aurata to various salinities alters energy metabolism of osmoregulatory and nonosmoregulatory organs

The impact of different environmental salinities on the energy metabolism of gills, kidney, liver, and brain was assessed in gilthead sea bream (Sparus aurata) acclimated to brackish water [BW, 12 parts/thousand (ppt)], seawater (SW, 38 ppt) and hyper saline water (HSW, 55 ppt) for 14 days. Plasma osmolality and levels of sodium and chloride presented a clear direct relationship with environmental salinities. A general activation of energy metabolism was observed under different osmotic conditions. In liver, an enhancement of glycogenolytic and glycolytic potential was observed in fish acclimated to BW and HSW compared with those in SW. In plasma, an increased availability of glucose, lactate, and protein was observed in parallel with the increase in salinity. In gills, an increased Na+-K+-ATPase activity, a clear decrease in the capacity for use of exogenous glucose and the pentose phosphate pathway, as well as an increased glycolytic potential were observed in parallel with the increased salinity. In kidney, Na+-K+-ATPase activity and lactate levels increased in HSW, whereas the capacity for the use of exogenous glucose decreased in BW- and HSW- acclimated fish compared with SW-acclimated fish. In brain, fish acclimated to BW or HSW displayed an enhancement in their potential for glycogenolysis, use of exogenous glucose, and glycolysis compared with SW-acclimated fish. Also in brain, lactate and ATP levels decreased in parallel with the increase in salinity. The data are discussed in the context of energy expenditure associated with osmotic acclimation to different environmental salinities in fish euryhaline species.