Čedomil Lucu

Na++K+-ATPase in gills of aquatic crustacea

The sodium pump, or Na(+)+K(+)-ATPase, provides at least part of the driving force for transepithelial movement of monovalent ions across the gills and other transporting tissues in many aquatic animals including the Crustacea. The crustacean Na(+)+K(+)-ATPase, like that in all animal cells, is composed of a catalytic alpha-subunit and an accompanying beta-subunit. The amino acid sequence of the crustacean alpha-subunit is 71-74% identical to vertebrate alpha-subunit sequences. In brachyuran Crustacea, the Na(+)+K(+)-ATPase is more highly expressed in posterior gills compared with anterior and is found predominantly in mitochondria-rich cells that are morphologically and biochemically specialized to mediate NaCl uptake from the medium. When the external salinity is lowered from that of normal seawater, producing conditions in which many euryhaline Crustacea hyperosmo regulate their hemolymph, both the enzymatic activity of the Na(+)+K(+)-ATPase and the gene expression of the alpha-subunit are increased in these tissues. Although the precise regulatory mechanism is not known, evidence suggests that crustacean hyperglycemic hormone may be responsible for the induction of Na(+)+K(+)-ATPase activity. Whether it also plays a role in activation of gene transcription is not known. A comparison of a range of aquatic Crustacea suggests that the level of Na(+)+K(+)-ATPase function in transporting tissues may be correlated with their ability to invade estuarine habitats.

Na-K-ATPase generates an active transport potential in the gills of the hyperregulating shore crab Carcinus maenas

Perfusing and bathing isolated gills of shore crabs Carcinus maenas with artificial saline or sea (brackish) water enabled us to determine potential differences (PDs) between ambient bathing medium and perfusion solution. Establishment of diffusional PDs was avoided by employment of the same solution on the internal and external side. The PDs measured were therefore of an active nature. We compared the properties of the PDs with the well-known properties of the Na-K-ATPase: dependence on biological energy (ATP), on salinity and sodium concentration, susceptibility of PD to depletion of internal K and to the addition of 5 mM internal ouabain. Considering also the magnitude of the PDs measured, the results obtained indicate that it is the Na-K-ATPase that generates an active transport potential for Na in the gills of shore crabs. This PD represents the driving force for the active uptake of Na in crabs that hyperregulate their body fluids when they inhabit environments of reduced salinity regimes or fluctuating salinities in tidal estuaries. This process counteracts diffusional losses of Na in crabs exposed to dilute media.

Osmoregulation and branchial Na+, K+-ATPase in the lobster Homarus gammarus acclimated to dilute seawater

Abstract Osmoregulatory ability and Na + ,K + -ATPase activity in gills and epipodites were examined following transfer of the lobster Homarus gammarus from sea water (SW; 1292 mOsmol l −1 ; 38 ppt salinity) to dilute seawater (DSW; 680±5 mOsmol l −1 ; 20 ppt salinity). The lobster H. gammarus behaves as an osmoconformer in sea water and a poor hyperosmoregulator when equilibrated in dilute seawater, where haemolymph was maintained 154 mOsmol l −1 above that of the medium. Lobsters could withstand direct transfer from SW to DSW and, after about 12 h, the initial drop in blood osmoconcentration and principal inorganic osmolytes ceased and a gradual increase followed during the next 15 days. Chloride and sodium were the main osmotic effectors responsible for the slow gradual adjustment of the blood hyperosmoticity. In DSW-acclimated lobsters, the Na + ,K + -ATPase specific activities of gills (arthrobranchia and podobranchia) homogenates and partially purified membrane vesicle fractions were, respectively, 2.0 and 3.5 to 3.9-fold higher that those of the SW-acclimated animals. Enzyme specific activities of homogenate and membrane vesicle fractions isolated from DSW pleurobranchia were 1.4-fold higher than those of the SW-acclimated animals. In trichobranchiate gills and epipodites, saponin treatment increased the Na + ,K + -ATPase activity over the native enzyme activity of the respective tissues. The plasma membrane vesicles from trichobranchiate gills and epipodites were better purified when isolated from DSW- than from SW-acclimated lobsters. Homogenates and membrane vesicles (enrichment factors ranging from seven to eight) from epipodites allowed a several-fold increase in enzyme specific activity over trichobranchiate gills. When lobsters were acclimated to DSW, the Na + ,K + -ATPase specific activity measured in saponin-treated homogenates of epipodites was positively correlated with the sodium concentration gradient between haemolymph and DSW. The results suggest that epipodites Na + ,K + -ATPase activation in DSW should be correlated with the blood sodium gradient, i.e. osmoregulatory ability. Short-circuit current of the hemiepipodite isolated from lobsters acclimated to DSW and mounted in a micro-Ussing chamber was −232 μA cm −2 (negative charge flow driven from apical to basolateral side of preparation) and conductance was 65 mS cm −2 , a value characteristic of a leaky epithelium. Half-maximal inhibition of current by the specific Na + ,K + -ATPase inhibitor ouabain occurs at 0.78 mM. At 1 mM ouabain, more then 70% of the inhibited current is Na + ,K + -ATPase-related ion transport in epipodites. These results provide, for the first time, physiological evidence that, besides trichobranchiate gills, epipodites have an osmoregulatory role in the lobster Homarus gammarus .

Active uptake of sodium in the gills of the hyperregulating shore crab Carcinus maenas

Isolated posterior gills of shore crabs,Carcinus maenas, previously acclimated for at least 1 month to brackish water of 10 ‰ S, were connected with an artificial hemolymph circulation by means of thin polyethylene tubings. Gills were symmetrically perfused and bathed with 50 % sea water. Transepithelial potential differences (PDs) and fluxes of sodium between medium and blood were measured under control conditions and following reductions of PDs by means of 5 mM internal (blood side) ouabain, 0.5 mM internal and external (bathing medium) NaCN or by exhaustion of energy reserves along with a prolonged perfusion period of more than 9 h. In these experiments22Na was used as tracer. Each of the three modes of reducing transepithelial potential differences resulted in a decrease in sodium influxes from 500–1000 µmoles g−1 h−1 to 250–400 µmoles g−1 h−1. The findings suggest that sodium influx, which normally greatly exceeds efflux, was diminished by its active component. The remaining non-inhibitable influx equals efflux values. Our findings thus indicate that efflux is completely passive, while influx has — beside a passive component of efflux magnitudes — an additional active portion which is much larger than the passive component. Since ouabain is a specific inhibitor of the Na-K-ATPase, our results confirm previous findings (Siebers et al., 1985) that the basolaterally located Na-K-ATPase generates the transepithelial potential difference in the gills, which is inside negative by about 6–12 mV. Inhibition of the active portion of sodium influx by internal ouabain along with reduced PDs suggests that transepithelial PDs generated by the branchial sodium pump are the driving force for active sodium uptake in hyperregulating brackish water crabs.

Linkage of Cl− fluxes with ouabain sensitive Na/K exchange through Carcinus gill epithelia

Abstract 1. 1. Mechanisms of the sodium and chloride transport and TBP (transbranchial potentials) were investigated in isolated Carcinus gill filaments perfused with diluted sea-water identical to the external bathing solutions. 2. 2. TBP were observed in the range from −4 to −8 mV and Na+ and Cl− influxes exceed that of effluxes. 3. 3. Ouabain (5 mmol/1) perfused internally resulted in markedly reduced sodium and chloride influxes. K-free solutions (basolateral side) resulted in substantially decreased Cl− influxes and changed TBP close to zero. 4. 4. Since the effects of ouabain and NaCN on Na+ and Cl− influxes (Ja→b) occur after basolaterally but not after apically applied drugs, it can be assumed that at least one portion of the chloride influxes are secondary active processes linked with an active ATPase sensitive Na, K pump.

Evidence for Cl− exchangers in perfused Carcinus gills

Abstract 1. 1. The effects of inhibitors and ion substitution on TBP (transbranchial potentials) and unidirectional 22 Na and 36 Cl fluxes from the bathing medium across the apical and basolateral sides into the perfusion solution J a→b of isolated Carcinus gill epithelia were studied. Identical, diluted sea-water (DSW) and artificial solutions were used in the bathing solution and perfusate (202 ± 5 mM Na). 2. 2. Externally applied bumetanide (10 −3 M) did not affect 36 Cl and 22 Na fluxes. In addition, 36 Cl and 22 Na unidirectional J a→b fluxes obtained by isosmotic substitution of co-ions did not provide evidence for the presence of a Na-K-2Cl co-transporter on the apical membrane. 3. 3. 10 −3 M 4-acetamido-4 isothiocyanostilbene 2,2 disulphonic acid (SITS) in the bathing solution and on both sides (apical and basolateral surfaces) effected a rapid, reversible and statistically significant inhibition of 36 Cl J a→b , fluxes of 27.6 and 39.4%, respectively. In addition, 10 mM acetazolamide reduced Cl − (external and both sides) and Na + J a→b , fluxes. Furthermore, Cl − influxes were stimulated by addition of NaHCO 3 (15.5mM) on both epithelial sides by 64%. 4. 4. On the basis of the experimental evidence described, it is suggested that there is a Cl − /HCO 3 (or OH − ) antiporter located on the apical side of the gill surface.

Changes in Na+/K+-ATPase activity, unsaturated fatty acids and metallothioneins in gills of the shore crab Carcinus aestuarii after dilute seawater acclimation.

We have assessed the activity of Na(+)/K(+)-ATPase, cAMP, free fatty acids (FFA) and metallothionein (MT) in the posterior gills of the brackish water shore crab Carcinus aestuarii during acclimation to 10 ppt dilute seawater (DSW). Following 3-18 days acclimation in DSW specific activity of Na(+)/K(+)-ATPase in native gill homogenates and partially purified membrane vesicles was progressively increased, from 1.7- to 3.9-fold. After short-term acclimation of crabs in DSW with added sucrose to make media isosmotic with the haemolymph the specific Na(+)/K(+)-ATPase activity in homogenates was not increased, relative to SW enzyme activity. Moreover, hyposmotic conditions led to depletion of cAMP in gills. In partially purified membrane vesicles isolated from posterior gills, fatty acids with compositions 16:0, 18:0, 18:1, 20:4 and 20:5 dominated in both SW- and DSW-acclimated Carcinus. During a year in which the metabolic activity of crabs was increased, the arachidonic/linoleic acids ratio (ARA/LA) for DSW-acclimated crabs was markedly increased relative to that in SW. Increased Na(+)+K(+)-ATPase activity under hyposmotic stress may be modulated at least partially by the changed proportion of fatty acids in the purified membranes of posterior gills. Long-term acclimation of shore crabs to DSW resulted in a 2.6-fold increase in cytosolic metallothionein (MT) content in posterior gills over those in SW crabs. Assuming an antioxidant role of MT associated with intracellular zinc partitioning, the observed MT induction in posterior gills may be considered an adaptive response of C. aestuarii to hyposmotic stress.

Effects of amiloride on sodium chloride transport across isolated perfused gills of shore crabs Carcinus maenas acclimated to brackish water

Abstract 1. 1. Bathing and perfusing isolated posterior gills of the shore crab Carcinus maenas with 50% sea-water (200 mM Na + , symmetrical exposure) resulted in a spontaneously generated transepithelial potential difference (PD) of some millivolts (blood side negative). 2. 2. Externally applied amiloride decreased the PDs rapidly, reversibly and in a concentration-dependent mode by inhibition of sodium influxes and non-inhibition of chloride influxes. The two influxes are thus independent of each other. 3. 3. Symmetry PDs and amiloride effects were abolished by cyanide ions. Asymmetrically ( 400 200 mM Na + ) exposed gills exhibited passive PDs which were sensitive to amiloride even after cyanide poisoning. 4. 4. The results communicated are consistent with the presence of two amiloride-sensitive transport sites for sodium: an apical Na + /H + exchanger, which depends on Na-K-ATPase and carboanhydrase activity and a passive, preferably sodium-selective paracellular pathway.

Conductive sodium entry in gill cells of the shore crab, Carcinus maenas

Isolated posterior gills of shore crabs, Carcinus maenas, collected from the Baltic Sea, were perfused and bathed with sea water and solutions of alkali chlorides. The preparation was used to measure fluxes of sodium from the external medium across the gills into the hemolymph and to determine transepithelial potential differences (PDs). Internally negative active transport PDs resulted from perfusion and bathing the gills symmetrically with the same medium (50% sea water). Passive (asymmetry) PDs following employment of 100% sea water as internal and 50% sea water as external medium were — in contrast to symmetry PDs — insensitive to cyanide and ouabain. This result indicates that the gill recognized the desired hyperosmotic state and responded by switching off the active transport component observed under symmetry conditions. Diffusional potential differences and fluxes of Na+ were inhibited by the externally applied diuretic amiloride. Gradients of pure alkali chlorides between medium and blood were accompanied by cation specific PDs. These PDs and their amiloride sensitivity were inversely related to the diameter of the unhydrated cation and allowed the calculation of the permeability sequence P: Li+>Na+>K+>Rb+>Cs+>Cl-. The results obtained show that the permeability of the gills to cations greatly exceeds that to anions. In addition, these findings indicate that the initial amiloride-sensitive step in Na+ transport across the gill is not represented by an electroneutral sodium/proton exchange but by a conductive mechanism.

Role of seawater concentration and major ions in oxygen consumption rate of isolated gills of the shore carb Carcinus mediterraneus Csrn

In the posterior gills isolated from the shore crab Carcinus mediterraneus, the effects of seawater concentration, ion substitution and ion transport inhibitors on oxygen consumption was studied. Oxygen consumption rate (VO2) of the excised gills depended on the seawater concentration of the adaptation medium of intact crabs. The VO2 measured in the gills isolated from the crabs acclimated to dilute seawater (DSW) was higher than VO2 in the gills excised from the crabs living in ordinary seawater. In the gills isolated from seawater-acclimated crabs, and transferred to dilute medium the VO2 was not increased but, rather, slightly decreased. A specific inhibitor of the enzyme Na+K+-ATPase ouabain (2.5 × 10−3 mol 1−1)_reduced oxygen consumption rate by 30%. Moreover, in the K+-free saline, the VO2 was decreased by 40% suggesting the key role of the enzyme in oxidative requirements for ionic regulatory purposes. In Ca2+-free saline, containing 0.1 mmol 1−1 EGTA VO2 was markedly decreased. Significant differences between the specific activity of Na+K+-ATPase in homogenates and membrane vesicles isolated from gills acclimated to seawater and DSW were found. In DSW-acclimated crabs, the specific activity of gill homogenate and membrane vesicles Na+K+-ATPase was increased, respectively by 71.6 and 122.9%, compared to the gills isolated from seawater-acclimated crabs. Addition of NaCl salts in solution preparated by isoosmotic replacement of Na+ by N-methyl-D-glucamine (NMDG) steadily increased O2 consumption. The curve was similar to an enzyme catalyzed reaction, Km amounted to 16.0 mmol Na 1−1 and Vmax to 465 μl O2 h−1 per gram gill (w.w.). The inhibitor of H+-ATPase, N-ethylmaleimide (NEM), reduced O2 consumption; I50 (50% reduced oxygen consumption from control) was attained at 5 × 10−5 mol 1−1 NEM. The results are discussed in terms of energetic demands of the gills in ion regulation.