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.     

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.     

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.     

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.     

Gill Na(+),K(+)-ATPase and osmoregulation in the estuarine crab, Chasmagnathus granulata Dana, 1851 (Decapoda, Grapsidae)

Some kinetic properties of gill Na(+),K(+)-ATPase of the estuarine crab, Chasmagnathus granulata, and its involvement in osmotic adaptation were analyzed. Results suggest the presence of different Na(+),K(+)-ATPase isoforms in anterior and posterior gills. They have different affinities for Na(+), but similar affinity values for K(+), Mg(2+), ATP and similar enzymatic profiles as a function of temperature of the incubation medium. Ouabain concentrations which inhibit 50% of enzyme activity were also similar in the two types of gills. Enzyme activity and affinity for Na(+) are higher in posterior gills than in anterior ones. Furthermore, affinities of Na(+),K(+)-ATPase of posterior gills for Na(+) and K(+) were similar to or higher than those of gills or other structures involved in the osmoregulation in several euryaline decapod crustaceans. Acclimation to low salinity was related to a significant increase in the maximum Na(+), K(+)-ATPase activity, mainly in posterior gills. On the other hand, crab acclimation to high salinity induced a significant decrease in maximum enzyme activity, both in anterior and posterior gills. These results are in accordance to the osmoregulatory performance showed by C. granulata in diluted media, and point out the major role of posterior gills in the osmoregulation of this species.     

The effect of temperature on juvenile Mozambique tilapia hybrids (Oreochromis mossambicus x O. urolepis hornorum) exposed to full-strength and hypersaline seawater

The effects of temperature on the salinity tolerance of Mozambique-Wami tilapia hybrids (Oreochromis mossambicus x O. urolepis hornorum) were investigated by transferring 35 g/l, 25 degrees C-acclimated fish to 35, 43, 51 or 60 g/l salinity at 15, 25 or 35 degrees C for 24 h, and by assaying gill tissue for branchial Na(+), K(+)-ATPase activity at the three temperatures after acclimating the fish to 15, 25 or 35 degrees C for 2 weeks. Tilapia survived all salinities at 25 and 35 degrees C; however, at 15 degrees C, mortality was 85.7% and 100% in the 51 g/l and 60 g/l groups, respectively. There was a significant interaction between temperature and salinity, as plasma osmolality, [Na(+)] and [Cl(-)] were significantly increased at 51 and 60 g/l salinity in 35 degrees C water (P<0.001). Additionally, muscle water content was significantly reduced at 43 g/l, 15 degrees C relative to pre-transfer values (P<0.001). Branchial Na(+), K(+)-ATPase activity was reduced at 15 degrees C regardless of acclimation temperature, and 25 degrees C-acclimated gill tissue did not show an increase in activity when assayed at 35 degrees C. Results indicate that the effects of a combined temperature-salinity transfer on plasma osmolality and ion concentrations, as well as muscle water content, are greater than when either challenge is given alone. Additionally, branchial Na(+), K(+)-ATPase activity is altered when assayed at varying temperatures; in the case of 15 degrees C, regardless of acclimation temperature. Our enzyme activity data may indicate the presence of a high temperature isoform of branchial Na(+), K(+)-ATPase enzyme.     

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.     

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.     

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.     

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.     

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.     

Behavioural adaptations of the fiddler crabs Uca vocans borealis (crane) and Uca lactea lactea (De Haan) for coexistence on an intertidal shore

The distribution and coexistence of the fiddler crabs U. vocans borealis and U. lactea lactea was investigated in the upper shore of a sandy beach with respect to particle size, water content and total organic carbon content of the sediments. A clear segregation of habitats between the two species was apparent. U. lactea lactea were only found in sediments with significantly lower total organic carbon content. U. lactea lactea had a higher ratio for the size of third maxilliped to body size than U. vocans borealis. There was no statistical difference in the median sediment particle size of the habitats where both species were found on the study shore. U. lactea lactea was larger on shores where U. vocans borealis were absent. Smaller U. vocans borealis individuals occupied sediments with higher water content and finer particles. Smaller individuals of U. vocans borealis also possessed fewer spoon-tipped setae on second maxilliped than their larger conspecifics, which were found mostly on coarser sediments. U. lactea lactea spent significantly more time on the surface than in the burrow during low tide when compared with U. vocans borealis. Both Uca species exhibited similar desiccation resistance. U. lactea lactea tends to keep its frontal region in close contact with sediments to maintain moisture when being exposed to air.     

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.     

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.     

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.     

Branchial chamber tissues in two caridean shrimps: the epibenthic Palaemon adspersus and the deep-sea hydrothermal Rimicaris exoculata

The structure of the epithelia of the branchial chamber organs (gills, branchiostegites, epipodites) and the localization of the Na(+),K(+)-ATPase were investigated in two caridean shrimps, the epibenthic Palaemon adspersus and the deep-sea hydrothermal Rimicaris exoculata. The general organization of the phyllobranchiate gills, branchiostegites and epipodites is similar in P. adspersus and in R. exoculata. The gill filaments are formed by a single axial epithelium made of H-shaped cells with thin lateral expansions and a basal lamina limiting hemolymph lacunae. In P. adspersus, numerous ionocytes are present in the epipodites and in the inner-side of the branchiostegites; immunofluorescence reveals their high content in Na(+),K(+)-ATPase. In R. exoculata, typical ionocytes displaying a strong Na(+),K(+)-ATPase specific fluorescence are observed in the epipodites only. While the epipodites and the branchiostegites appear as the main site of osmoregulation in P. adspersus, only the epipodites might be involved in ion exchanges in R. exoculata. In both species, the gill filaments are mainly devoted to respiration.     

Behavioural adaptations of the fiddler crabs Uca vocans borealis (crane) and Uca lactea lactea (De Haan) for coexistence on an intertidal shore

The distribution and coexistence of the fiddler crabs U. vocans borealis and U. lactea lactea was investigated in the upper shore of a sandy beach with respect to particle size, water content and total organic carbon content of the sediments. A clear segregation of habitats between the two species was apparent. U. lactea lactea were only found in sediments with significantly lower total organic carbon content. U. lactea lactea had a higher ratio for the size of third maxilliped to body size than U. vocans borealis. There was no statistical difference in the median sediment particle size of the habitats where both species were found on the study shore. U. lactea lactea was larger on shores where U. vocans borealis were absent. Smaller U. vocans borealis individuals occupied sediments with higher water content and finer particles. Smaller individuals of U. vocans borealis also possessed fewer spoon-tipped setae on second maxilliped than their larger conspecifics, which were found mostly on coarser sediments. U. lactea lactea spent significantly more time on the surface than in the burrow during low tide when compared with U. vocans borealis. Both Uca species exhibited similar desiccation resistance. U. lactea lactea tends to keep its frontal region in close contact with sediments to maintain moisture when being exposed to air.     

Adaptive branchial mechanisms in the sturgeon Acipenser naccarii during acclimation to saltwater

Variations of Na(+)/K(+)-ATPase activity and fatty-acid composition in the gills of the sturgeon Acipenser naccarii subjected to progressive acclimation to full seawater (35 ppt) were determined in relation to the hypo-osmoregulatory capacity of this species in the hyperosmotic medium. Blood samples were taken and gills arches were removed at intermediate salinity levels between 0 and 35 ppt and after 20 days at constant salinity (35 ppt). Plasma osmolality and Na(+)/K(+)-ATPase activity increased significantly with growing environmental salinity. Total saturated fatty acids (SFAs) decreased, while total polyunsaturated fatty acids (PUFAs) increased significantly with increasing salinity due mainly to changes in n-3 PUFAs (20:5n-3 and 22:6n-3). The n-3/n-6 ratio increased significantly during the acclimation process. The results show a direct relationship between salinity, increased gill Na(+)/K(+)-ATPase activity and ultrastructural changes of the gill chloride cells. Changes in the fatty-acid composition in gills of A. naccarii during progressive acclimation to full seawater suggest that variations of gill fatty acids may also have a role in osmoregulatory mechanisms.     

AS160 associates with the Na+,K+-ATPase and mediates the adenosine monophosphate-stimulated protein kinase-dependent regulation of sodium pump surface expression

The Na(+),K(+)-ATPase is the major active transport protein found in the plasma membranes of most epithelial cell types. The regulation of Na(+),K(+)-ATPase activity involves a variety of mechanisms, including regulated endocytosis and recycling. Our efforts to identify novel Na(+),K(+)-ATPase binding partners revealed a direct association between the Na(+),K(+)-ATPase and AS160, a Rab-GTPase-activating protein. In COS cells, coexpression of AS160 and Na(+),K(+)-ATPase led to the intracellular retention of the sodium pump. We find that AS160 interacts with the large cytoplasmic NP domain of the α-subunit of the Na(+),K(+)-ATPase. Inhibition of the activity of the adenosine monophosphate-stimulated protein kinase (AMPK) in Madin-Darby canine kidney cells through treatment with Compound C induces Na(+),K(+)-ATPase endocytosis. This effect of Compound C is prevented through the short hairpin RNA-mediated knockdown of AS160, demonstrating that AMPK and AS160 participate in a common pathway to modulate the cell surface expression of the Na(+),K(+)-ATPase.