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Am J Physiol Regul Integr Comp Physiol 276: R490-R499, 1999;
0363-6119/99 $5.00
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Vol. 276, Issue 2, R490-R499, February 1999

Na+-K+-ATPase and Na+/Ca2+ exchange activities in gills of hyperregulating Carcinus maenas

Cedomil Lucu and Gert Flik

Department of Animal Physiology, Faculty of Science, University of Nijmegen, 6525 ED Nijmegen, The Netherlands; and Center for Marine Research, Institute Rudjer Boskovic, 52210 Rovinj, Croatia


    ABSTRACT
Top
Abstract
Introduction
Material and methods
Results
Discussion
References

Na+-K+-ATPase and Na+/Ca2+ exchange activities were studied in gills of Carcinus maenas in seawater (SW) and after transfer to dilute seawater (DSW). Carcinus hyperregulates its hemolymph osmolarity through active uptake of Na+, Cl-, and Ca2+. In DSW total Na+-K+-ATPase activity in posterior gills quadrupled; Na+/Ca2+ exchange specific activity was unaffected, and total activity increased 1.67-fold. Short-circuit current (Isc) in voltage-clamped posterior gill hemilamellae was -181 µA/cm2 in SW and -290 µA/cm2 in DSW and up to 90% ouabain sensitive; conductivity was similar in SW or DSW (42 and 46 mS/cm2, respectively) and representative of a leaky epithelium. The new steady state of hemolymph osmolarity 24 h after DSW transfer was preceded, already 3 h after transfer, by increased Na+-K+-ATPase but not Na+/Ca2+ exchange activity. Western blot analysis indicated that the amount of Na+-K+-ATPase protein had increased 2.1-fold in crabs acclimated 3 wk to DSW; however, 4 h after DSW transfer no difference in the amount of Na+-K+-ATPase protein was observed. After DSW transfer branchial cAMP content decreased. A negative correlation between branchial Na+-K+-ATPase activity and cAMP content points to rapid regulation of Na+-K+-ATPase through cAMP-dependent protein kinase A activity. Ca2+ transport may depend on the high-capacity Na+/Ca2+ exchanger coupled to the versatile sodium pump.

crab; sodium transport; ouabain; adenosine 3',5'-cyclic monophosphate


    INTRODUCTION
Top
Abstract
Introduction
Material and methods
Results
Discussion
References

THE SHORE CRAB Carcinus maenas (Decapoda, Crustacea) inhabits marine and brackish waters and is considered a euryhaline species. When in seawater (SW; osmolarity ~1,000 mosmol/l), its hemolymph osmolarity is always a little higher (e.g., 1,070 mosmol/l) than that of the surrounding water; however, in dilute seawater (DSW; for instance, 300 mosmol/l) the hemolymph osmolarity is strongly hyperregulated to ~600 mosmol/l (this study). To maintain the osmotic gradient over its integument, the shore crab actively absorbs Na+, Cl-, and Ca2+ from its environment (20, 22, 28, 41). In addition, the animal could decrease the ion permeability of its gills and other surfaces in contact with the water to prevent salt losses, but no such data are available in the literature. Interestingly, the conductivity reported for branchial epithelium of SW shore crab (40 mS/cm2; 26) is much smaller than that of the marine crab Uca tangeri (90 mS/cm2; 35), but higher than that (3 mS/cm2; 26) of the freshwater Chinese crab Eriocheir sinensis. These numbers for conductivity parallel the osmoregulatory capacity that is low in Uca, large in Eriocheir, and intermediate in Carcinus. The conductivity of the branchial epithelium of Carcinus is characteristic of a leaky epithelium and thus determined by the paracellular pathway (13).

Consensus exists that in euryhaline crabs such as Carcinus and the blue crab Callinectes sapidus, the posterior gills (nos. 5-9) play a more prominent osmoregulatory role during acclimation to DSW than the anterior gills (nos. 1-4). Indeed, in the posterior gills a higher abundance of striated cells (ion transporting cells) and, in these cells, a higher number of mitochondria associated with the basolateral plasma membrane domain and a higher Na+-K+-ATPase activity have been observed (5, 15, 17). The branchial epithelium of Crustacea is particularly rich in ouabain-sensitive Na+-K+-ATPase (10, 41). The Na+-K+-ATPase is key to a variety of energy-demanding transport processes (20, 41). In addition to the export proper of 3 Na+ in exchange for 2 K+ at the basolateral plasma membrane, the activity of this enzyme indirectly governs Na+/H+ exchange (21, 36, 45) as well as a Na+-K+-2Cl--cotransporter (30) in the apical membrane and secondary driven Na+/Ca2+ exchange in the basolateral plasma membrane (10). Accordingly, Na+-K+-ATPase activity is crucial in premolt and early postmolt animals to provide the sodium gradient for secondary calcium transport through the Na+/Ca2+ exchanger, a requirement for calcification of the exoskeleton (10, 42, 43). In isolated and perfused gills of both Carcinus and Callinectes, sodium influx was inhibited by serosal ouabain and thus depends on Na+-K+-ATPase activity (4, 22). Onken and Siebers (27) reported an 85% inhibition of short-circuit current (Isc) by ouabain in a Carcinus branchial hemilamellar preparation mounted in a Ussing chamber. Undoubtedly, the branchial Na+-K+-ATPase activity plays a pivotal role in the physiology of crabs.

On prolonged (weeks) exposure to DSW, the specific activity of Na+-K+-ATPase in euryhaline crab gills increases (25, 49). Neufeld and coworkers (25) suggested that Na+-K+-ATPase activity in Callinectes was enhanced by de novo synthesis of enzyme rather than by modulation of the existing enzyme. Biogenic amines were shown to stimulate phosphorylation of Na+-K+-ATPase of posterior gills of Eriocheir, but only in the presence of a source of cAMP-dependent protein kinase A (PKA; 46). Such data infer that regulation through rapid (de-) phosphorylation mechanisms does occur in crustacean species.

The apparent key role of Na+-K+-ATPase in crustacean osmoregulation and yet the scarcity of information available on mechanisms of modulation of this enzyme in general led us to investigate the following questions. Does acclimation to SW or DSW alter Na+-K+-ATPase and Na+/Ca2+ exchange activities in the gills of Carcinus by altered synthesis of enzyme or by modulation of existing enzymes? Is branchial Na+-K+-ATPase activity modulated directly after transfer to DSW, and, if so, is there a link with the cAMP level in the branchial epithelium?


    MATERIAL AND METHODS
Top
Abstract
Introduction
Material and methods
Results
Discussion
References

Animals

Shore crabs, Carcinus maenas, weighing 20-50 g were collected from the coast line of the Waddenzee (The Netherlands) and from the Baltic Sea (Kiel, Germany) from May to July, 1996, 1997, and 1998. In the experiments described here only intermolt male crabs were used. Animals were fed bovine heart twice a week. Artificial SW (salinity: 32per thousand ) was prepared by dissolving natural sea salt (Wimex, Krefeld, Germany) in deionized water. This SW was diluted with deionized water to obtain DSW (salinity: 10per thousand ). The animals were kept in the laboratory at 8-10°C under a natural light cycle.

Hemolymph Analyses

Hemolymph was collected by puncture of the hemolymph sacs at the base of the legs with the use of a tuberculine syringe fitted with a 23-gauge needle. Hemolymph thus collected was spun in an Eppendorf cup for 1 min at 9,000 g to remove cells. Next, the hemolymph osmolarity was determined on fresh samples with a Vogel osmometer calibrated with distilled water and a 300 mosmol/l standard. The remainder of the sample was quick frozen in solid CO2 and stored at -20°C for later ion analyses. Hemolymph Na content was determined by flame photometry; Cl-, Ca, and Mg were determined by commercial kits (Sigma). Ionized calcium in hemolymph and water was determined with a Radiometer calcium analyzer.

Membranes

Na+-K+-ATPase activity in gill homogenate or a partly purified basolateral plasma membrane fraction was determined as described earlier (10). The anterior (nos. 1-4) and posterior (nos. 5-9) pairs of gills were excised separately and homogenized on ice in a hypotonic buffer (10 ml/g fresh gills). The hypotonic buffer contained (in mmol/l) 12.5 NaCl, 1 HEPES, 1 DTT, 0.5 EDTA, and the protease inhibitor aprotinin (10 Trypsin inhibiting units/l of homogenization buffer). The isolated gills were homogenized in a Dounce homogenization device by 25 strokes and then filtered through cheese cloth (mesh 100 µm). The filtered homogenate was centrifuged at 500 g for 15 min, and the supernatant was taken and spun for 30 min at 10.000 g. The supernatant was then centrifuged at 50,000 g for 30 min, and the pellet was collected. The pellet was resuspended by 25 strokes with the Dounce device in a resealing solution, which consisted of (in mmol/l) 0.05 beta -mercaptoethanol, 0.02 EDTA, and 0.5 Tris · HCl (pH 8.5) in the same volume as that of the original homogenate. The suspension was kept on ice for 30 min, incubated for 15 min at 37°C in a water bath, and shaken vigorously at 5-min intervals. After cooling to 0°C, the suspension was homogenized once more by 20 strokes, centrifuged at 10,000 g, and the supernatant finally centrifuged at 50,000 g for 30 min. The pelletized membranes were taken up in a buffer containing (in mmol/l) 150 NaCl, 0.8 MgCl2, and 20 HEPES/Tris (pH 7.4) to a final protein concentration of 1.5 to 3 mg/ml BSA equivalents and consisted of 55% resealed vesicles and 45% leaky membrane fragments (10). Saponin (20 µg/mg membrane protein) was used to permeabilize the resealed membranes to optimize substrate accessibility for the intrinsic Na+-K+-ATPase.

Assays

Protein. Protein content of homogenate or membrane suspension was estimated by Bradford's procedure (BioRad, catalog no. 500-0002), using BSA as a reference.

Na+-K+-ATPase. Na+-K+-ATPase activity was assayed in triplicate by incubating 10 µl membrane suspension (permeabilized with saponin to obtain optimal substrate accessibility) with 500 µl assay solution A or E. A contains (in mmol/l) 100 NaCl, 5.0 MgCl, 0.1 EDTA, 15 imidazol/histidine (pH 7.5), 3 Na2ATP, and 12.5 KCl. E is composed as A, but KCl is omitted and ouabain (1.0 mmol/l) is added. The difference in phosphate released from ATP in solutions A and E reflects the ouabain-sensitive, K+-dependent Na+-K+-ATPase activity. After incubation in a thermostatted bath at 37°C for 15 min, the reaction was stopped by addition of 1 ml ice-cold 8.6% trichloroacetic acid. Liberated phosphate was quantitated colorimetrically by addition of 1 ml of 1.14% (wt/vol) ammonium heptamolybdate in sulphuric acid (36.3 ml concentrated sulfuric acid/liter distilled water), supplemented immediately before use with FeSO4 · 7H2O (9.2 g/100 ml). After a 30-min incubation the change in absorbance at 700 nm was recorded.

Na+/Ca2+ exchange. Na+/Ca2+ exchange activity was assayed as described before (10), with some modifications (extended ion concentration ranges in kinetic studies). In short, plasma membrane vesicles obtained from posterior gills were equilibrated in uptake buffer containing NaCl as the major osmolyte and 2.5 mmol/l KCl plus 5 µg/ml valinomycin (Sigma) to voltage clamp the membranes. Ca2+ kinetics of Na+/Ca2+ exchange activity were determined by assaying the difference in calcium taken up when such vesicles were transferred to a medium containing either 300 mmol/l NaCl (blank) or 300 mmol/l KCl and varying Ca2+ levels (0.1, 0.5, 1, 2.5, 5, 10, and 25 µmol/l). Na+ kinetics were determined by equilibrating vesicles in varying concentrations of NaCl (5, 25, 50, 100, 200, 300 mmol/l; substituting NaCl with KCl to maintain constant osmotic pressure) and saturating levels of Ca2+. The assay time was 5-15 s, yielding initial velocity of the carrier; the assay temperature was 37°C. The reaction was stopped by addition of 1 ml of ice-cold isotonic buffer containing 0.5 mmol/l EDTA. The stopped reaction was filtered over Schleicher and Schuell ME25 nitrocellulose filters (pore size 0.45 µm). After filtration, the filters were washed with two rinses of 3 ml of the same stop buffer. 45Ca was used as tracer, and its activity in the vesicles collected on the filters was assessed by liquid scintillation counting using a Wallac 1410 apparatus.

cAMP. The cAMP level in posterior gills was estimated by RIA (Amersham), employing 125I-labeled cAMP and a highly sensitive and specific antibody. The concentration of cAMP in the sample is inversely proportional to the radioactivity in the immunocomplex and is quantified by interpolation from a standard curve. Freshly dissected posterior gills (30 to 50 mg fresh weight) were homogenized by 35 strokes in 1 ml 0.1 M HCl using a Dounce homogenization device. The suspension was centrifuged (13,000 g for 15 min), and 0.5 ml of the supernatant was diluted with an equal amount of 0.1 M HCl, quick frozen, and kept for no longer than 3 wk before measurement. Protein content was determined on the remainder of supernatant. Defrosted samples were centrifuged, and the supernatant was dried overnight in a speedvac centrifuge. To the dried samples 1 ml of assay buffer (A) as used for the Na+-K+-ATPase determinations was added, and the sample was vigorously vortexed. Samples were taken (50 µl) and diluted in 1,950 µl buffer in duplicate. To enhance the sensitivity of the RIA, the sample was acetylated by adding just above the sample surface 5 µl of a mixture of one part of acetic anhydride (10.58 mol/l) and two parts of triethylamine (7.21 mol/l). The samples were stored in the refrigerator for 1 h and, next, into each tube, 200 µl of RIA mixture (1 part 50 mM Na-acetate with 1 part of antibody and labeled 125I-cAMP) was added. The samples were vortexed and stored overnight at 4°C. The immunocomplex was separated from the free labeled cAMP by precipitation with polyethylene glycol (15% wt/vol) plus chicken egg albumin (2.4% wt/vol) through centrifugation at 4,000 rpm for 20 min at 4°C. The supernatant was discarded and radioactivity in the pelletized material was measured by a gamma counter (LKB Clinigamma). To establish correlation between branchial cAMP content and Na+-K+-ATPase activity, the rest of the same gill sampled for cAMP determination was processed and assayed for Na+-K+-ATPase activity.

Electrophysiology

For the measurement of branchial epithelial Isc and conductance, the posterior gills 7 or 8 were used. Lamellae were dissected to a hemilamellar preparation, which consists of a single cell layer supported by cuticle (27). The so-called dark area of the hemilamella, which is particularly rich in mitochondria-rich cells and located near the afferent blood vessel (5, 15, 17), was selected for the transepithelial measurements in a miniaturized (aperture 0.012 cm2) version of a Ussing chamber (27). The hemichambers were connected to automated voltage clamp equipment (Bioengineering, The University of Iowa) that allows continuous measurement of Isc. The transepithelial potential difference was measured by two calomel electrodes connected to the chambers through agar-KCl bridges (3 M KCl in 3% agar). The Isc is referred to as negative when current flows from the apical (cuticle side) to the basolateral (hemolymph) side. Tissue conductance was measured by recording the current resulting from unipolar voltage pulses (1 mV) imposed across the tissue. Both hemichambers were perfused with saline using a Watson-Marlow peristaltic pump (0.55 ml/min). The total resistance measured by unipolar voltage pulses was corrected for chamber resistance by substracting the resistance measured in a saline-filled chamber after the tissue installed had been pierced with a needle (on completion of the experiment); the resistance thus measured was 5.6 ± 0.2 Omega /cm2; values for current were corrected following Ohm's law. The saline used contained (in mmol/l) 235 NaCl, 5 KCl, 2 MgCl2, 4 CaCl2, 2 NaHCO3, 6 glucose, and 10 HEPES (pH was adjusted by Tris to 7.6). During a first period of 90 min, epithelium was superfused with saline on both sides. Then the serosal saline was replaced by ouabain (1 mmol/l) containing saline for 20 min, after which the serosal perfusate was switched again to normal saline.

Western Blotting

Posterior gills were fractionated using a Dounce homogenization device (20 strokes). The homogenate was filtered over cheese cloth (100 µm mesh), and the filtrate was spun for 5 min at 500 g to remove nuclei and cellular debris. Next the membranes were collected by centrifugation of the supernatant for 30 min at 50,000 g. The pelletized membranes were resuspended in a small volume of buffer containing (in mmol/l) 300 sucrose, 10 HEPES/Tris (pH 7.4), 2 DTT, 0.5 Na2EDTA, and 10 U/ml aprotinin. Protein was measured, and the suspension was brought to 2 mg/ml with the same buffer. Just before electrophoresis, 5 µl membrane suspension was mixed with 10 µl sample buffer (2 mM DTT, 0.5% bromophenol blue, 30% glycerol, 20 mM Tris · HCl; pH 6.8) and microwave treated (2 × 1 min at 600 W); this treatment was critical to extract a single immunoreactive species from the membrane preparation. Samples were run on 10% polyacrylamide slab gels; kaleidoscope prestained markers (Biorad no. 161 0324) were used as reference. After SDS-PAGE, proteins were electroblotted to nitrocellulose membranes (pore size 0.45 µm; Schleicher and Schuell, code 401196). After blocking the membranes with 3% low-fat coffee creamer plus 1% BSA and 0.1% gelatin, the proteins were probed with a mouse monoclonal antibody against the alpha -subunit of an avian Na+-K+-ATPase (Developmental Study Hybridoma Bank, The University of Iowa) for 1 h at room temperature and subsequently for 12-16 h at 4°C. Goat anti-mouse IgG-peroxidase conjugate was used to visualize the Na+-K+-ATPase epitope. Preparations were made of gills obtained from crabs in SW and from crabs residing in DSW either for 4 h or 3 wk.

Calculations and Statistics

Data are presented as mean values ± SE, unless otherwise indicated. Differences among groups were assessed by ANOVA and Student's t-test or the Mann-Whitney U test was used, where appropriate, as the follow-up test to determine the level of significance. Linear regression analysis was based on the least-squares method. Quantitation of Western blots was carried out determining the "adjusted volume" of immunoreactive protein bands (i.e., optical density multiplied with the area, OD × mm2) using Molecular Analyst Software (Biorad, Hercules, CA).


    RESULTS
Top
Abstract
Introduction
Material and methods
Results
Discussion
References

Acclimation

In crabs acclimated for 3 wk to DSW compared with those in SW, hemolymph osmolarity had decreased by 43%, from 1,076 to 615 mosmol/l; the sodium level by 37%, from 509 to 319 mmol/l; the chloride level by 40%, from 540 to 322 mmol/l; the total calcium level by 28%, from 12.7 to 9.2 mmol/l; the Ca2+ level by 25%, from 6.8 to 5.1 mmol/l; and the magnesium level by 55%, from 22.3 to 10.0 mmol/l (Table 1).

                              
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  		Table 1.   Osmolarity and sodium, chloride, calcium (total and ionic), and magnesium concentrations in ambient medium and hemolymph of Carcinus maenas acclimated to SW or DSW

In posterior gills of crabs acclimated to DSW, the Na+-K+-ATPase specific activity in crude homogenates had increased by 73% and the total amount of protein extracted increased by 67%, which resulted in a calculated 3.96-fold increase of total Na+-K+-ATPase activity. In a plasma membrane fraction a 98% increase in specific activity and a 60% increase in the total amount of protein recovered resulted in a 3.9-fold increase of total Na+-K+-ATPase activity (Table 2).

                              
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  		Table 2.   Na+-K+-ATPase activities in crude homogenate and in a plasma membrane fraction of posterior gills of Carcinus maenas in SW and in animals acclimated for at least 3 wk to DSW

The kinetics of Na+ dependency (Fig. 1A) and Ca2+ dependency (Fig. 1B) of Na+/Ca2+ exchange activity did not differ in plasma membranes obtained from SW or DSW crabs. With an increase in branchial protein of 67%, a 1.67-fold increase in total exchange activity is calculated (total activity = specific activity × total protein).


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  		Fig. 1.   Na+/Ca2+ exchange activity in plasma membrane vesicles of posterior gills of Carcinus maenas acclimated to saltwater (SW; black-triangle) or diluted SW (DSW; bullet ). A, sodium dependence; B, calcium dependence. Mean values for 6 individual preparations are given; error bars indicate SD.

Isc in hemilamellae increased by 60%, from -181 ± 38 µA/cm2 in SW crabs to -290 ± 31 µA/cm2 in DSW crabs. Ouabain (1 mmol/l) applied for 20 min to the serosal side (indicated by the sign of the Isc) resulted in 80 and 89% inhibition of the Isc current in SW and DSW crab gills, respectively. When ouabain was removed, after 90 min the Isc had restored to 72 and 83% of the current before application of ouabain, indicating reversible binding of the inhibitor to the sodium pump. The branchial conductance of the epithelium was comparable in the two groups (42 ± 5 mS/cm2 in SW and 46 ± 5 mS/cm2 in DSW) (P > 0.15, n = 8; Table 3).

                              
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  		Table 3.   Isc, Isc-ouabain, percentage inhibition by ouabain, and G in hemilamellae isolated from posterior gills (7th or 8th gill) of Carcinus maenas acclimated to SW or DSW

Western Blots

Western blots of gill membrane preparations revealed a protein with an apparent molecular radius of 100.4 ± 5.4 kDa (Fig. 6), a value in perfect agreement with the 104-kDa phosphorylated alpha -subunit of the branchial enzyme reported for Callinectes by Towle and coworkers (44). This particular protein was 2.1-fold (±0.3; P < 0.01; n = 6) more abundant in membranes of crabs kept for at least 3 wk in DSW than in SW crabs.

Transfer

After transfer to DSW, hemolymph osmolarity dropped from 1,076 ± 20 mosmol/l (n = 8) at the start of the experiment to ~800 mosmol/l after 4 h and eventually stabilized ~600 mosmol/l after 24 h and thereafter (Fig. 2).


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  		Fig. 2.   Changes in hemolymph osmolarity of Carcinus maenas after transfer to DSW. Mean values (±SD) are given for 6 animals per time point. Dotted lines, osmolarity of SW (top) and DSW (bottom).

In posterior gills, a significant increase in Na+-K+-ATPase specific activity (from 30 ± 6 to 50 ± 10 µmol Pi · h-1 · mg protein-1; n = 8, P < 0.01) was observed 4 h after transfer and thereafter (Fig. 3). The Na+-K+-ATPase specific activity in anterior gills of SW crabs was twofold lower (15 ± 4 µmol Pi · h-1 · mg protein-1; n = 8, P < 0.001) than in posterior gills, and in anterior gills activities had increased twofold and significantly as of 16 h after transfer to DSW (data not shown). The measurement of Isc in hemilamellae of animals shortly after transfer to DSW was not feasible because of the 2-3 h required to prepare the hemilamellar setup. Na+/Ca2+ exchange activity measured under conditions for maximum velocity (i.e., 300 mmol/l Na+ and 25 µmol/l Ca2+) did not differ and was 60 ± 5 and 67 ± 14 nmol Ca2+ · 15 s-1 · mg protein-1 (n = 4; P > 0.15) for crabs in SW and crabs 4 h after transfer to DSW, respectively.


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  		Fig. 3.   Na+-K+-ATPase specific activity (V) in homogenate of posterior gills of Carcinus maenas after transfer to DSW. Mean values for 6-8 animals per time point are given; error bars indicate SD. ** P < 0.01.

In another series of experiments, in which branchial Na+-K+-ATPase and cAMP content was assayed on the same tissue sample, Na+-K+-ATPase had increased significantly as early as 3 h after transfer, and cAMP content had decreased significantly after 6 h (Fig. 4). Na+-K+-ATPase activity and cAMP content are negatively correlated (Fig. 5): for SW crabs the slope of the regression line was less steep than for crabs exposed for 6 h to DSW.


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  		Fig. 4.   Na+-K+-ATPase (V, right x-axis, hatched bars) and cAMP content (left x-axis, open bars) in posterior gills of Carcinus maenas in SW (0 h after transfer) and after transfer to DSW. Mean values for 5-7 individual samples are given, error bars indicate SD. NS, not significant (P > 0.15). * P < 0.05, ** P < 0.01; *** P < 0.001.


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  		Fig. 5.   Correlation between cAMP content and Na+-K+-ATPase in posterior gills of Carcinus maenas in SW (bullet ) and DSW for 6 h (open circle ). For SW the regression was Na+-K+-ATPase (µmol Pi · h-1 · mg protein-1) = -0.38 cAMP (pmol/mg protein) + 27.86 (r2 = 0.84, P < 0.01), for DSW the regression was Na+-K+-ATPase (µmol Pi · h-1 · mg protein-1) = -0.709 cAMP (pmol/mg protein) + 32.40 (r2 = 0.81, P < 0.01).

Four hours after transfer of crabs from SW to DSW, no increase (1.2 ± 0.3-fold; P > 0.15, n = 8) in alpha -subunit (100.4-kDa band) expression was observed (Fig. 6).


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  		Fig. 6.   Western blot of Na+-K+-ATPase extracted from homogenates of posterior gills of Carcinus maenas in SW (lane 1), for 3 wk in DSW (lane 2), or for 4 h DSW (lane 3); apparent molecular radius of kaleidoscope markers (lane 4) are given on right. Blots were probed with a mouse monoclonal against chicken Na+-K+-ATPase (cytosolic epitope of alpha -subunit), recognizing a 100.4-kDa protein as the prominent molecular species.


    DISCUSSION
Top
Abstract
Introduction
Material and methods
Results
Discussion
References

Acclimation

The strong hyperregulation of its hemolymph osmolarity in DSW requires that the shore crab enhances active uptake of sodium, chloride, and calcium. As the ionic gradients between the hyperregulated hemolymph and ambient medium increase with decreasing ambient salinity, we predicted enhanced sodium pump activity in gills of animals in DSW. Indeed, the Isc, which is for the major part ouabain-sensitive and thus an Na+-K+-ATPase-dependent sodium current, had almost doubled in posterior gills of crabs in DSW. In line with these results we found an increase in total Na+-K+-ATPase activity as well as in alpha -subunit expression in these gills of crabs in DSW. The larger, fourfold, increase in enzyme activity and Na+-K+-ATPase alpha -subunit expression compared with the increase in Isc indicates that only part of the total enzyme present contributes to pumping. Thus the shore crab residing in DSW upregulates its branchial sodium pump activity by increasing the number of Na+-K+-ATPase copies through de novo synthesis to enhance the sodium motive force for hyperregulation.

The hyperregulation of hemolymph osmolarity in the shore crab in DSW is primarily based on a resetting of hemolymph Na+ and Cl- levels as these ions make up the bulk of the inorganic osmolytes in this compartment. Our electrophysiological and biochemical data provide compelling evidence that enhanced sodium pumping through ouabain-sensitive Na+-K+-ATPase activity in the basolateral plasma membrane compartment is the pivotal active transport process in this resetting. Na+ entry via the apical membrane in this tissue is indirectly coupled to the basolateral membrane sodium pump and pathways include an Na+/H+ exchange (36, 45), Na+-K+-2Cl- cotransport (30), or an Na+-conductive pathway coupled to a H+-extruding ATPase (30, 50). For the isolated perfused gill of the shore crab it was shown that amiloride affected both the transepithelial potential difference and the sodium influx from the water (21). Amiloride-sensitive, electrogenic 2Na+/H+ exchange was first demonstrated in membrane vesicles of crab gills (36), and, later, its specific structure was identified and sequenced (45). Cl- transport in this tissue is coupled to Na+ transport (22) and mediated through secondary active transport involving either apical Na+-K+-2Cl- cotransport and/or an Na+/H+-exchange phenomenon plus a Cl- channel (30, 37).

As in shore crabs, ~50% of the hemolymph calcium concentration was in the ionic form (Ca2+, the electrophysiologically important fraction), the hemolymph calcium level is far less hyperregulated than the levels of the monovalent osmolytes. This may explain why in this crab, when exposed to DSW, no adjustment was observed of the kinetics of its main Ca2+-transporting enzyme, the Na+/Ca2+ exchanger. The presumed operation of this carrier far below its maximum (as derived from in vitro kinetics and prevailing ionic conditions), its large capacity (10), and an actually lower Ca2+ gradient between animal and medium when moved from SW to DSW, all favor the idea that no adjustment of calcium carrier capacity is needed in DSW compared with SW.

Interestingly, magnesium must be excreted in both SW and DSW, as its level is always maintained far below ambient level. Whether magnesium regulation results primarily from limiting uptake from the environment, controlling excretion, or a combination of both processes is not known, but our data show that it is strictly hyporegulated in relation to ambient salinity. This crab should prove an excellent model to study Mg transporters, a class of transporters for which unfortunately no data are available for crustacean species.

The values for branchial epithelial conductivity in SW and DSW ranged from 42 to 46 mS/cm2; apparently the paracellular pathway, which determines this conductivity in a leaky epithelium (i.e., when electrical resistance <100 Omega /cm2; 13), is not readjusted to changes in ambient medium. External Ca2+ plays a key role in the paracellular permeability of leaky epithelia to water and ions (24) and the high levels of this ion in SW (10 mM) and DSW (3.9 mM) apparently suffice to maintain epithelial conductance constant. During the course of our experiments no changes were observed in epithelial conductivity, which we take to indicate that the quality of the preparations was constant. Under open-circuit conditions the transepithelial potential was 5.5 ± 2.0 mV (apical side positive), in compliance with data on an isolated perfused gill preparation (22), and thus our Ussing chamber measurements are realistic and reflect the in vivo situation. The values for Isc in SW crabs are in good agreement with previously published data for the same species (27, 30). The increase in Isc in crabs in DSW compared with those in SW resulted from an increase in ouabain-sensitive current, and this is direct evidence that the Na+-K+-ATPase pumping activity is specifically enhanced.

For euryhaline crabs, including Carcinus, it was reported that a few week acclimation to DSW increases Na+-K+-ATPase activity in posterior gills (25, 38, 49). Acclimation processes so far have been linked with ionocytes in the posterior gills that are particularly abundant close to the afferent blood vessel (5, 17). Although we focused our studies on posterior gills, we observed that also an increase in Na+-K+-ATPase activity in the anterior gills forms part of the adaptive response to a hypoosmotic challenge.

Branchial ionocytes in shore crab in DSW show clear signs of hyperplasia (5), and we here suggest on the basis of our biochemical data that this hyperplasia implies enhanced enzyme activity as well as enhanced turnover of Na+-K+-ATPase and increased membrane trafficking in this very active epithelium. An important implication of our results is that the estimate of total enzyme activity is a sensitive parameter for the response of the animal to the hyposmotic challenge, but that it overestimates the involvement of the enzyme in actual ion transport. The high Na+-K+-ATPase activities (30-50 µmol Pi · h-1 · mg prot-1) in crude homogenate of gills (these values compare for instance to an activity of 77.4 µmol Pi · h-1 · mg prot-1 reported for purified microsomal fractions of blue crab gills; 44) in all likelihood resulted from the use of saponin to unmask enzyme activity that is otherwise silent due to membrane resealing during homogenization of the tissue: both in crude homogenates and in plasma membrane fractions up to 50% of the Na+-K+-ATPase activity may go undetected (10).

Interestingly, Na+/Ca2+ exchange specific activity did not change when the crab was transferred to or kept in DSW. We have discussed before that this calcium transporter operates far below saturation and has a large overcapacity (10). Apparently these properties imply that this enzyme needs no adjustment; this conclusion is further corroborated by the notion that the gradient for Ca2+ between DSW and hemolymph is smaller than that between hemolymph and SW. The importance of the electrophysiological conditions at the level of the basolateral plasma membrane for the operation of this carrier, i.e., a guarantee that the transmembrane potential does not reach the reversal potential of the carrier bringing it in the import rather than export mode (34), seems to be secured by the adjustment of the sodium pump discussed above.

Transfer

The eventual stabilization of hemolymph osmolarity after acute transfer of shore crab to DSW is preceded by a rapid and persisting increase in Na+-K+-ATPase activity. This increase was seen in all gills but is most pronounced in the posterior gills. The question arises how this rapid rise in enzyme activity is realized. Considering the fact that these crabs encounter in their natural habitat frequent changes in ambient salinity, it would seem most likely from an energetic point of view that this rise is realized through upregulation of existing enzyme or recruitment of silent enzyme rather than by de novo synthesis. Indeed, the largest increase in Na+-K+-ATPase activity (67%) occurred in the first 4 h after transfer to DSW, when the expression of the alpha -subunit had not changed. Thus the increase of the Na+-K+-ATPase activity immediately after hyposmotic stress must result from a stimulation of existing enzyme, and, in the long term, as the need for such an increase persists, synthesis of new enzyme may be enhanced, degradation rate may be reduced, or both.

This rapid response to DSW is of wider occurrence in crabs. Towle and colleagues (44) were the first to show that Na+-K+-ATPase activity in a microsomal fraction isolated from Callinectes gills increases 2-3 h after transfer of the crabs from SW to DSW. More evidence for rapid regulation of Na+-K+-ATPase comes from a study in which hemolymph of a DSW crab (Callinectes sapidus) was injected into a SW crab, causing a rapid, 20-min-lasting increase in the gill Na+-K+-ATPase activity (33). These studies further indicate that a hemolymph-borne, probably endocrine factor is involved in such regulation. The hemolymph-borne factor would convey its message to the sodium pump in the gills as it stimulates sodium influx (19). However, such data are not always corroborated by others: Piller and coworkers (29) found no differences in specific activity of the Na+-K+-ATPase after low-salinity acclimation of C. sapidus and C. similis. Yet, there are indications for a multiple endocrine control of ion transport in gills of crabs. During the first 4 h after hyposmotic stress, dopamine and norepinephrine levels in shore crab increase about twofold above levels seen in steady-state conditions (51). Consensus exists that cAMP is a messenger for dopamine signals and thus a study into the correlation between branchial Na+-K+-ATPase activity and cAMP content was indicated.

In posterior gills of shore crab acclimated to DSW, branchial Na+-K+-ATPase activity had increased and the cAMP concentration decreased (14.6 ± 2.7 pmol/mg protein) compared with the value in SW-acclimated crabs (27.0 ± 7.5 pmol/mg protein). Moreover, the correlation between Na+-K+-ATPase activity and cAMP content became more negative in the first 1 h after transfer, and this, taken with the increase in Na+-K+-ATPase directly after transfer, strongly suggests that activation of the enzyme in the short term requires downregulation of the branchial cAMP content. One could wonder what the impetus is for the rapid decrease in cellular cAMP levels. On a hyposmotic stress, as imposed by transfer to DSW, the hemolymph volume decreases temporarily (8) and causes cells to swell through hydration. For rat cardiac myocytes it was shown that hyposmotic swelling inhibits adenylyl cyclase activity and decreases cAMP content via a pertussis toxin-sensitive pathway (16). It is tempting to assume that in the crab cellular volume regulation triggers the cascade that leads to decreasing cAMP content that eventually governs the Na+-K+-ATPase activity.

The rapid, strong, and negative correlation between Na+-K+-ATPase activity and cAMP content in the posterior gills of crabs acclimating to DSW provides the first compelling evidence for an involvement of cAMP in Na+-K+-ATPase regulation in crustaceans based on in vivo observations. This strongly points to a role for regulation of the sodium pump through a cAMP-dependent PKA pathway (2, 6, 9) as described for vertebrates: inhibition of Na+-K+-ATPase activity mediated by cAMP, in a concentration-dependent way, has been documented in vitro in a variety of vertebrate cells, i.e., liver cells (47), brain (18), pancreatic acinar cells (48), and in adrenal chromaffin cells (23). Na+-K+-ATPase activity in the rat cortical collecting duct is regulated by dopamine that increases cellular cAMP content and inhibits Na+-K+-ATPase through stimulation of a cAMP-dependent PKA pathway (32). Accordingly, we observed a 20% reduction of the Isc in the SW Carcinus hemilamellar preparation when 1 mmol/l 8-bromoadenosine-cAMP was added to the serosal bathing medium (Lucu, unpublished observations). We speculate that in DSW animals the PKA pathway has become desensitized to exogenous cAMP.

In a freshwater crab, Eriocheir sinensis, cAMP increases the transbranchial potential and enhances Na+-K+-ATPase activity (3). Detaille and colleagues (7) demonstrated that dopamine and dibutyryl cAMP when perfused through the isolated gills of Eriocheir hyperpolarize the transbranchial potential, presumably through kinase-mediated phosphorylation of the sodium pump; moreover, cAMP stimulates electrogenic uptake of Na+ and Cl- in a hemilamellar preparation mounted in a Ussing chamber (31). The negative correlation between Na+-K+-ATPase activity and cAMP content reported here for shore crabs thus suggests that cAMP is involved in Na+-K+-ATPase regulation, yet in a diametrically opposite mode in seawater crabs compared with freshwater crabs.

Perspectives

Studies on the gill of the shore crab may contribute new information to the polemic around protein kinase regulation of Na+-K+-ATPase. It is well documented and consensus exists (e.g., 11, 12) that Na+-K+-ATPase activity may be phosphorylated in vitro by cAMP-dependent PKA on a COOH-terminal serine (1) as well as by protein kinase C (PKC) on NH2-terminal sites (11).

Stimulation of cAMP-dependent PKA activity leads to inhibition of Na+-K+-ATPase in distal nephron (14, 40) and pancreatic acinar cells (48) and to activation of the enzyme in shark rectal gland (39). Interestingly, the degree of PKA-mediated phosphorylation of reconstituted Na+-K+-ATPase from shark rectal gland and pig kidney does not predict the enzyme response: phosphorylation of shark enzyme (0.2 mol Pi/mol alpha -subunit) increased hydrolytic activity, but phosphorylation of the pig enzyme (0.9 mol Pi/mol alpha -subunit) left the activity of the enzyme unaltered (6). The notion that an apparently similar signaling pathway provides a stimulus in one case, an inhibitor in the other, or has no apparent effect is not paradoxical considering that enzymes fulfill specific roles in different tissues and in different species. The abundance of Na+-K+-ATPase in posterior gills, the strict correlation of Na+-K+-ATPase and cAMP content in vivo, and the rapid and physiologically clear response of the animal to DSW transfer provide the researcher with a powerful tool to address questions on the consequences of phosphorylations of the enzyme for the in vivo situation. The crab model further provides versatile and easy experimental conditions (SW, DSW, rapid transfer). Activation of the hyperregulatory activity in this animal may prove to be an important tool in the study of membrane trafficking and recruitment of plasma membrane enzymes in sodium-transporting epithelia.


    ACKNOWLEDGEMENTS

Tom Spanings is thanked for excellent animal husbandry, Wim Atsma for computer assistance. Profs. S. E. Wendelaar Bonga, J. Fenwick-Ghikas, and J. C. Fenwick are thanked for critically reading the manuscript.


    FOOTNOTES

C. Lucu gratefully acknowledges the Ministery of Science and Technology of the Republic of Croatia for travel support. C. Lucu was financially supported by the Research School "M&T," Wageningen, The Netherlands.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: G. Flik, Dept. of Animal Physiology, Faculty of Science, Univ. of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands.

Received 28 July 1998; accepted in final form 27 October 1998.


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Top
Abstract
Introduction
Material and methods
Results
Discussion
References

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