Effect of Cd(2+) and Hg(2+) on the activity of Na(+)/K(+)-ATPase and Mg(2+)-ATPase adsorbed on polystyrene microtiter plates.

In the present study a polystyrene microtiter plate was tested as a support material for synaptic plasma membrane (SPM) immobilization by adsorption. The adsorption was carried out by an 18-h incubation at +4 degrees C of SPM with a polystyrene matrix, at pH 7.4. Evaluation of the efficiency of the applied immobilization method revealed that 10% protein fraction of initially applied SPM was bound to the support and that two SPM enzymes, Na(+)/K(+)-ATPase and Mg(2+)-ATPase, retained 70-80% activity after the adsorption. In addition, adsorption stabilizes Na(+)/K(+)-ATPase and Mg(2+)-ATPase, since the activities are substantial 3 weeks after the adsorption. Parallel kinetic analysis showed that adsorption does not alter significantly the kinetic properties of Na(+)/K(+)-ATPase and Mg(2+)-ATPase and their sensitivity to and mechanism of Cd(2+)- or Hg(2+)-induced inhibition. The only exception is the "high affinity" Mg(2+)-ATPase moiety, whose affinity for ATP and sensitivity toward Cd(2+) were increased by the adsorption. The results show that such system may be used as a practical and comfortable model for the in vitro toxicological investigations.     

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.     

Phospholemman expression is high in the newborn rabbit heart and declines with postnatal maturation

Phospholemman (PLM) is a small sarcolemmal protein that modulates the activities of Na(+)/K(+)-ATPase and the Na(+)/Ca(2+) exchanger (NCX), thus contributing to the maintenance of intracellular Na(+) and Ca(2+) homeostasis. We characterized the expression and subcellular localization of PLM, NCX, and the Na(+)/K(+)-ATPase alpha1-subunit during perinatal development. Western blotting demonstrates that PLM (15kDa), NCX (120kDa), and Na(+)/K(+)-ATPase alpha-1 (approximately 100kDa) proteins are all more than 2-fold higher in ventricular membrane fractions from newborn rabbit hearts (1-4-day old) compared to adult hearts. Our immunocytochemistry data demonstrate that PLM, NCX, and Na(+)/K(+)-ATPase are all expressed at the sarcolemma of newborn ventricular myocytes. Taken together, our data indicate that PLM, NCX, and Na(+)/K(+)-ATPase alpha-1 proteins have similar developmental expression patterns in rabbit ventricular myocardium. Thus, PLM may have an important regulatory role in maintaining cardiac Na(+) and Ca(2+) homeostasis during perinatal maturation.     

Functional characterization of a NapA Na(+)/H(+) antiporter from Thermus thermophilus

Na(+)/H(+) antiporters are ubiquitous membrane proteins and play an important role in cell homeostasis. We amplified a gene encoding a member of the monovalent cation:proton antiporter-2 (CPA2) family (TC 2.A.37) from the Thermus thermophilus genome and expressed it in Escherichia coli. The gene product was identified as a member of the NapA subfamily and was found to be an active Na(+)(Li(+))/H(+) antiporter as it conferred resistance to the Na(+) and Li(+) sensitive strain E. coli EP432 (DeltanhaA, DeltanhaB) upon exposure to high concentration of these salts in the growth medium. Fluorescence measurements using the pH sensitive dye 9-amino-6-chloro-2-methoxyacridine in everted membrane vesicles of complemented E. coli EP432 showed high Li(+)/H(+) exchange activity at pH 6, but marginal Na(+)/H(+) antiport activity. Towards more alkaline conditions, Na(+)/H(+) exchange activity increased to a relative maximum at pH 8, where by contrast the Li(+)/H(+) exchange activity reached its relative minimum. Substitution of conserved residues D156 and D157 (located in the putative transmembrane helix 6) with Ala resulted in the complete loss of Na(+)/H(+) activity. Mutation of K305 (putative transmembrane helix 10) to Ala resulted in a compromised phenotype characterized by an increase in apparent K(m) for Na(+) (36 vs. 7.6 mM for the wildtype) and Li(+) (17 vs. 0.22 mM), In summary, the Na(+)/H(+) antiport activity profile of the NapA type transporter of T. thermophilus resembles that of NhaA from E. coli, whereas in contrast to NhaA the T. thermophilus NapA antiporter is characterized by high Li(+)/H(+) antiport activity at acidic pH.     

Cloning and characterization of a Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene of the moderately halophilic bacterium <Emphasis Type="Italic">Halobacillus aidingensis</Emphasis> AD-6<Superscript>T</Superscript>

A gene encoding a Na(+)/H(+) antiporter was obtained from the genome of Halobacillus aidingensis AD-6(T), which was sequenced and designated as nhaH. The deduced amino acid sequence of the gene was 91% identical to the NhaH of H. dabanensis, and shared 54% identity with the NhaG of Bacillus subtilis. The cloned gene enable the Escherichia coli KNabc cell, which lack all of the major Na(+)/H(+) antiporters, to grow in medium containing 0.2 M NaCl or 10 mM LiCl. The nhaH gene was predicted to encode a 43.5 kDa protein (403 amino acid residues) with 11 putative transmembrane regions. Everted membrane vesicles prepared from E. coli KNabc cells carrying NhaH exhibited Na(+)/H(+) as well as Li(+)/H(+) antiporter activity, which was pH-dependent with the highest activity at pH 8.0, and no K(+)/H(+) antiporter activity was detected. The deletion of hydrophilic C-terminal amino acid residues showed that the short C-terminal tail was vital for Na(+)/H(+) antiporter activity.     

Importance for absorption of Na+ from freshwater of lysine, valine and serine substitutions in the alpha1a-isoform of Na,K-ATPase in the gills of rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar)

In the gills of rainbow trout and Atlantic salmon, the alpha1a- and alpha1b-isoforms of Na,K-ATPase are expressed reciprocally during salt acclimation. The alpha1a-isoform is important for Na(+) uptake in freshwater, but the molecular basis for the functional differences between the two isoforms is not known. Here, three amino acid substitutions are identified in transmembrane segment 5 (TM5), TM8 and TM9 of the alpha1a-isoform compared to the alpha1b-isoform, and the functional consequences are examined by mutagenesis and molecular modeling on the crystal structures of Ca-ATPase or porcine kidney Na,K-ATPase. In TM5 of the alpha1a-isoform, a lysine substitution, Asn783 --> Lys, inserts the epsilon-amino group in cation site 1 in the E(1) form to reduce the Na(+)/ATP ratio. In the E(2) form the epsilon-amino group approaches cation site 2 to force ejection of Na(+) to the blood phase and to interfere with binding of K(+). In TM8, a Asp933 --> Val substitution further reduces K(+) binding, while a Glu961 --> Ser substitution in TM9 can prevent interaction of FXYD peptides with TM9 and alter Na(+) or K(+) affinities. Together, the three substitutions in the alpha1a-isoform of Na,K-ATPase act to promote binding of Na(+) over K(+) from the cytoplasm, to reduce the Na(+)/ATP ratio and the work done in one Na,K pump cycle of active Na(+) transport against the steep gradient from freshwater (10-100 microM: Na(+)) to blood (160 mM: Na(+)) and to inhibit binding of K(+) to allow Na(+)/H(+) rather than Na(+)/K(+) exchange.     

Dimethyl sulfoxide-induced conformational state of Na(+)/K(+)-ATPase studied by proteolytic cleavage

Effects of dimethyl sulfoxide (Me(2)SO) on substrate affinity for phosphorylation by inorganic phosphate, on phosphorylation by ATP in the absence of Na(+), and on ouabain binding to the free form of the Na(+)/K(+)-ATPase have been attributed to changes in solvation of the active site or Me(2)SO-induced changes in the structure of the enzyme. Here we used selective trypsin cleavage as a procedure to determine the conformations that the Na(+)/K(+)-ATPase acquires in Me(2)SO medium. In water or in Me(2)SO medium, Na(+)/K(+)-ATPase exhibited after partial proteolysis two distinct groups of fragments: (1) in the presence of 0.1 M Na(+) or 0.1 M Na(+) + 3 mM ADP (enzyme in the E1 state) cleavage produced a main fragment of about 76 kDa; and (2) in the presence of 20 mM K(+) (E2 state) a 58-kDa fragment plus two or three fragments of 39-41 kDa were obtained. Cleavage in Me(2)SO medium in the absence of Na(+) and K(+) exhibited the same breakdown pattern as that obtained in the presence of K(+), but a 43-kDa fragment was also observed. An increase in the K(+) concentration to 0.5 mM eliminated the 43-kDa fragment, while a 39- to 41-kDa doublet was accumulated. Both in water and in Me(2)SO medium, a strong enhancement of the 43-kDa band was observed in the presence of either P(i) + ouabain or vanadate, suggesting that the 43-kDa fragment is closely related to the conformation of the phosphorylated enzyme. These results indicate that Me(2)SO acts not only by promoting the release of water from the ATP site, but also by inducing a conformation closely related to the phosphorylated state, even when the enzyme is not phosphorylated.     

Carboxyl-terminal hydrophilic tail of a NhaP type Na+/H+ antiporter from cyanobacteria is involved in the apparent affinity for Na+ and pH sensitivity

Little information is available on the C-terminal hydrophilic tails of prokaryotic Na(+)/H(+) antiporters. To address functional properties of the C-terminal tail, truncation mutants in this domain were constructed. Truncation of C-terminal amino acid residues of NhaP1 type antiporter from Synechocystis PCC6803 (SynNhaP1) did not change the V(max) values, but increased the K(m) values for Na(+) and Li(+) about 3 to 15-fold. Truncation of C-terminal tail of a halotolerant cyanobacterium Aphanothece halophytica (ApNhaP1) significantly decreased the V(max) although it did not alter the K(m) values for Na(+). The C-terminal part of SynNhaP1 was expressed in E. coli and purified as a 16kDa soluble protein. Addition of purified polypeptide to the membrane vesicles expressing the C-terminal truncated SynNhaP1 increased the exchange activities. Change of Glu519 and Glu521 to Lys in C-terminal tail altered the pH dependence of Na(+)/H(+) and Li(+)/H(+) exchange activities. These results indicate that the specific acidic amino acid residues at C-terminal domain play important roles for the K(m) and the pH dependence of the exchange activity.     

Effects of palytoxin on cation occlusion and phosphorylation of the (Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>)-ATPase

Palytoxin (PTX) inhibits the (Na(+) + K+)-driven pump and simultaneously opens channels that are equally permeable to Na+ and K+ in red cells and other cell membranes. In an effort to understand the mechanism by which PTX induces these fluxes, we have studied the effects of PTX on: 1) K+ and Na+ occlusion by the pump protein; 2) phosphorylation and dephosphorylation of the enzyme when a phosphoenzyme is formed from ATP and from P(i); and 3) p-nitro phenyl phosphatase (p-NPPase) activity associated with the (Na+, K+)-ATPase. We have found that palytoxin 1) increases the rate of deocclusion of K+(Rb+) in a time- and concentration-dependent manner, whereas Na+ occluded in the presence of oligomycin is unaffected by the toxin; 2) makes phosphorylation from P(i) insensitive to K+, and 3) stimulates the p-NPPase activity. The results are consistent with the notion that PTX produces a conformation of the Na+, K(+)-pump that resembles the one observed when ATP is bound to its low-affinity binding site. Further, they suggest that the channels that are formed by PTX might arise as a consequence of a perturbation in the ATPase structure, leading to the loss of control of the outside "gate" of the enzyme and hence to an uncoupling of the ion transport from the catalytic function of the ATPase.     

Role for calcium in the development of ovarial patency in Heliothis virescens

Insect oocytes sequester nutritive proteins from the hemolymph under the regulation by juvenile hormone (JH), in a process called patency. Here, a pharmacological approach was used to decipher the role for calcium in ovarial patency in the moth, Heliothis virescens. Follicular epithelial cells were exposed in calcium-free or calcium-containing media to JH I, JH II or JH III alone, or in combination with various inhibitors of signal transduction. Protein kinase inhibitors, Na(+)/K(+) -ATPase inhibitor, ouabain, an inhibitor of voltage-dependent calcium channels in plasma membrane, omega-Conotoxin MVII, endoplasmic reticulum (ER) Ca(2+) -ATPase inhibitor, thapsigargin, ER inositol 1,4,5-triphosphate receptor (IP(3)R) inhibitor, 2-ABP and ER ryanodine receptor (RyR) inhibitor, ryanodine, were used. The results of our study suggest that JH II evokes patency via protein kinase C-dependent signaling pathway, and activation of Na(+)/K(+) -ATPase, similar to JH III. Response to JH II and JH III predominantly relies upon external and internal calcium stores, using voltage-dependent calcium channels, IP(3)Rs and RyRs. In contrast, regulation of patency by JH I appears to be largely calcium independent, and the calcium-dependent component of the signaling pathway likely does not use IP(3)Rs, but RyRs only. The JH II, JH III and calcium-dependent component of JH I signaling pathway probably utilize calcium/calmodulin-dependent kinase II for activation of Na(+)/K(+) -ATPase.     

H+ extrusion by an apical vacuolar-type H+-ATPase in rat renal proximal tubules

The activity of Na+/H(+)-exchange and H(+)-ATPase was measured in the absence of CO2/HCO3 by microfluorometry at the single cell level in rat proximal tubules (superficial S1/S2 segments) loaded with BCECF [2'7'-bis(carboxyethyl)5-6-carboxyfluorescein- acetoxymethylester]. Intracellular pH (pHi) was lowered by a NH4Cl-prepulse technique. In the absence of Na+ in the superfusion solutions, pHi recovered from the acid load by a mechanism inhibited by 0.1 microM bafilomycin A1, a specific inhibitor of a vacuolar-type H(+)-ATPase. Readdition of Na+ in the presence of bafilomycin A1 produced an immediate recovery of pHi by a mechanism sensitive to the addition of 10 microM EIPA (ethylisopropylamiloride), a specific inhibitor of Na+/H+ exchange. The transport rate of the H(+)-ATPase is about 40% of Na+/H(+)-exchange activity at a similar pHi (0.218 +/- 0.028 vs. 0.507 +/- 0.056 pH unit/min. Pre-exposure of the tubules to 30 mM fructose, 0.5 mM iodoacetate and 1 mM KCN (to deplete intracellular ATP) prevented a pHi recovery in Na(+)-free media; readdition of Na+ led to an immediate pHi recovery. Tubules pre-exposed to Cl(-)-free media for 2 hr also reduced the rate of Na(+)-independent pHi recovery. In free-flow electrophoretic separations of brush border membranes and basolateral membranes, a bafilomycin A1-sensitive ATPase activity was found to be associated with the brush border membrane fraction; half maximal inhibition is at 6 x 10(-10) M bafilomycin A1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reverse Na(+)/Ca(2+)-exchange mediated Ca(2+)-entry and noradrenaline release in Na(+)-loaded peripheral sympathetic nerves

[(3)H]noradrenaline ([(3)H]NA) released from sympathetic nerves in the isolated main pulmonary artery of the rabbit was measured in response to field stimulation (2Hz, 1ms, 60V for 3min) in the presence of uptake blockers (cocaine, 3 x10(-5)M and corticosterone, 5 x10(-5)M). The [(3)H]NA-release was fully blocked by the combined application of the selective and irreversible 'N-type' voltage-sensitive Ca(2+)-channel (VSCC)-blocker omega-conotoxin (omega-CgTx) GVIA (10(-8)M) and the 'non-selective' VSCC-blocker aminoglycoside antibiotic neomycin (3x10(-3)M). Na(+)-loading (Na(+)-pump inhibition by K(+)-free perfusion) was required to elicit further NA-release after blockade of VSCCs (omega-CgTx GVIA+neomycin). In K(+)-free solution, in the absence of functioning VSCCs (omega-CgTx GVIA+neomycin), the fast Na(+)-channel activator veratridine (10(-5)M) further potentiated the nerve-evoked release of [(3)H]NA. This NA-release was significantly inhibited by KB-R7943, and fully blocked by Ca(o)(2+)-removal. However, Li(+)-substitution was surprisingly ineffective. The non-selective K(+)-channel blocker 4-aminopyridine (4-AP, 10(-4)M) also further potentiated the nerve-evoked release of NA in K(+)-free solution. This potentiated release was concentration-dependently inhibited by KB-R7943, significantly inhibited by Li(+)-substitution and abolished by Ca(o)(2+)-removal. It is concluded that in Na(+)-loaded sympathetic nerves, in which the VSCCs are blocked, the reverse Na(+)/Ca(2+)-exchange-mediated Ca(2+)-entry is responsible for transmitter release on nerve-stimulation. Theoretically we suppose that the fast Na(+)-channel and the exchanger proteins are close to the vesicle docking sites.     

Intracellular pH regulation by Na(+)/H(+) exchange requires phosphatidylinositol 4,5-bisphosphate

The carrier-mediated, electroneutral exchange of Na(+) for H(+) across the plasma membrane does not directly consume metabolic energy. Nevertheless, acute depletion of cellular ATP markedly decreases transport. We analyzed the possible involvement of polyphosphoinositides in the metabolic regulation of NHE1, the ubiquitous isoform of the Na(+)/H(+) exchanger. Depletion of ATP was accompanied by a marked reduction of plasmalemmal phosphatidylinositol 4,5-bisphosphate (PIP(2)) content. Moreover, sequestration or hydrolysis of plasmalemmal PIP(2), in the absence of ATP depletion, was associated with profound inhibition of NHE1 activity. Examination of the primary structure of the COOH-terminal domain of NHE1 revealed two potential PIP(2)-binding motifs. Fusion proteins encoding these motifs bound PIP(2) in vitro. When transfected into antiport-deficient cells, mutant forms of NHE1 lacking the putative PIP(2)-binding domains had greatly reduced transport capability, implying that association with PIP(2) is required for optimal activity. These findings suggest that NHE1 activity is modulated by phosphoinositides and that the inhibitory effect of ATP depletion may be attributable, at least in part, to the accompanying net dephosphorylation of PIP(2).     

Inhibition of NTPDase, 5′-nucleotidase, Na/K-ATPase and acetylcholinesterase activities by subchronic treatment with Casearia sylvestris

The aqueous extract of Casearia sylvestris was tested in cortical membrane preparations. C. sylvestris was obtained commercially from two different sources, designated as Sample A and Sample B. The enzymes studied in this work were NTPDase-like, 5'-Nucleotidase, Na(+)/K(+)-ATPase and acetylcholinesterase (AChE). Adult rats received aqueous extracts from C. sylvestris in a dose of 20mg/kg body wt. daily for a 75-day-period, by oral administration (gavage). Our study showed that this treatment caused an inhibition of NTPDase-like activity with both, ATP (19.41% with Sample A and 25.03% with Sample B) and ADP (41.57% with Sample A and 31.20% with Sample B) as substrates. This treatment also caused an inhibition of 5'-nucleotidase activity (28.34% with Sample A and 31.46% with Sample B) and Na(+)/K(+)-ATPase (25.08% with Sample A and 24.81% with Sample B). The rate of acetylcholine degradation was reduced, as shown by the inhibition of AChE (31.65% and 26.74%, Samples A and B, respectively). These results suggest that extracts of C. sylvestris can cause neurochemical alterations in the purinergic and cholinergic systems of the central nervous system.     

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

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

Regulation of the Na+/K+ ATPase by Klotho

Klotho-hypomorphic (Klotho(hm)) mice suffer from renal salt wasting and hypovolemia despite hyperaldosteronism. The present study explored the effect of Klotho on renal Na(+)/K(+) ATPase activity. According to immunohistochemistry and confocal microscopy Na(+)/K(+) ATPase protein abundance in isolated collecting ducts was lower in Klotho(hm) mice than in their wild type littermates (Klotho(+/+)). Analysis with dual electrode voltage clamp recording showed that expression of Klotho in Xenopus oocytes increased the Na(+)/K(+) ATPase pump current. Treatment of Xenopus oocytes with Klotho protein similarly increased the pump current. In conclusion, Klotho increases the membrane abundance and activity of the Na(+)/K(+) ATPase. Decreased Na(+)/K(+) ATPase activity could thus contribute to the volume-depletion of klotho(hm) mice.     

The effect of copper on osmoregulation in the freshwater amphipod Gammarus pulex

The influence of copper on osmoregulation in the freshwater amphipod Gammarus pulex was determined from the analysis of water permeability, haemolymph sodium concentration, sodium influx and gill Na(+)/K(+) ATPase and Mg(2+) ATPase activity. Exposure to nominal copper concentrations of 100 microg l(-1) or greater caused a significant reduction in both haemolymph sodium concentration and sodium influx within 4 h. Measurements of water permeability, expressed as the half-time of exchange of body water (t(1/2)), excluded structural gill damage as the cause of this fall in haemolymph sodium. Copper at 10 microg l(-1) or above in the assay solution significantly reduced gill Na(+)/K(+) ATPase activity. In contrast gill Mg(2+) ATPase activity was markedly less affected by copper. These differences in enzyme sensitivity were considered with respect to the potential mechanisms of copper toxicity.     

Ontogeny of salinity tolerance and hyper-osmoregulation by embryos of the intertidal crabs Hemigrapsus edwardsii and Hemigrapsus crenulatus (Decapoda, Grapsidae): survival of acute hyposaline exposure

The adults of Hemigrapsus edwardsii and Hemigrapsus crenulatus are euryhaline crabs and strong hyper-osmoregulators. Their embryos are carried externally attached to the abdominal pleopods of female crabs, where they are exposed to temporal and spatial changes in salinity associated with their intertidal and estuarine habitats. Although embryos lack the branchial and excretory organs responsible for adult osmoregulation, post-gastrula embryos were highly tolerant of exposure to hypo-osmotic sea water. Detached eggs (embryos+envelopes), of both species, at all developmental stages between gastrulation and hatching, exhibited 80-100% survival for periods up to 96 h in sea water (osmolality, 1050 mmol kg(-1)) and in dilutions to 50%, 10%, and 1%. Cleavage stages were less tolerant of dilution; H. edwardsii, <50% survived 24 h in 10% sea water; H. crenulatus <50% survived 6 h in 10% sea water. Post-gastrulation stages strongly hyper-osmoregulated but cleavage stages were hyper-osmoconformers (maintaining internal osmolality approximately 150 mmol kg(-1) above external). Osmoregulatory capacity was reduced just prior hatching, particularly in H. crenulatus, although salinity tolerance remained high. Gastrulation therefore marks a critical stage in the ontogeny of osmoregulation and salinity tolerance. Total Na+/K(+)-ATPase activity increased greatly during embryogenesis of H. crenulatus (undetectable in blastulae; gastrulae 0.31+/-0.05 pmol P(i) embryo(-1) min(-1); pre-hatching 16.4+/-1.0 pmol P(i) embryo(-1) min(-1)). Na+/K(+)-ATPase activity increased in embryos exposed to dilute sea water for 24 h implicating regulation of this transporter in a short-term acclimation response.