Calmodulin as a modulator of NaCl transport in the posterior salt-transporting gills of the Chinese crab Eriocheir sinensis

The possible involvement of calmodulin-dependent processes in the control of Na⁺ and Cl^- transport pathways has been investigated on isolated, perfused preparations of salt-transporting posterior gills of the euryhaline Chinese crab Eriocheir sinensis (collected near Emden, Germany in autumn 1990). The anti-calmodulin phenothiazine drugs Chlorpromazine and Trifluoperazine induced depolarization of the transepithelial potential only when added to the serosal bathing saline (socalled in). This effect is best interpreted by assuming a disturbance of the conductive Cl^- pathways located at the baso-lateral side of the epithelium. In agreement with that conclusion is the fact that Trifluoperazine inhibits the Cl^- transepithelial influx. Trifluoperazine also induces inhibition of the Na⁺ influx when added either to the incubation (out) or to the perfusion (in) medium. These results indicate inhibitory effects of the anticalmodulin drug on both the Na⁺/K⁺ pump and leak system located at the serosal side and on the Na⁺/H⁺ exchange located at the apical side of the epithelium.     

A comparison of transport-related gill enzyme activities and tissue-specific free amino acid concentrations of Baltic Sea (brackish water) and freshwater threespine sticklebacks, Gasterosteus aculeatus, after salinity and temperature acclimation

The osmoregulatory abilities of one freshwater and two brackish water (Baltic Sea) populations of the euryhaline teleost fish Gasterosteus aculeatus were studied with respect to evolutionary physiology. Plasma osmolality, activities of Na⁺K⁺-ATPase, citrate synthase, creatine kinase in the gill and free amino acids in liver, axial muscle and pectoral fin muscle were measured. After transfer from 10 to 35 ppt at 15 °C, time-course changes of plasma osmolality and gill Na⁺K⁺-ATPase showed no significant fundamental differences between the freshwater and one of the Baltic Sea populations. In a multi-factorial experiment, each population was exposed to four different abiotic regimes. Both brackish water populations had high mortality in freshwater at 4 °C, which is discussed as a failure of osmotic regulation (reduced taurine concentrations). Freshwater specimens had higher levels of glycine in the axial and pectoral fin muscles compared to the brackish water populations. This is interpreted as a genetically based effect. In brackish (20 ppt) water of 15 °C, the freshwater population had high activities of Na⁺K⁺-ATPase, but low activities of creatine kinase, whereas both brackish water populations behaved in the opposite way. A fundamental difference between the freshwater and brackish water populations on the level of the osmoregulatory machinery was not observed. Received: 10 December 1998 / Accepted: 22 September 1999     

Inhibition of Na+/K+-ATPase and of active ion-transport functions in the gills of the shore crab Carcinus maenas induced by cadmium

Inhibition of Na⁺/K⁺-ATPase from gill plasma membranes of the shore crab Carcinus maenas by cadmium was investigated and compared with inhibitory effects by known antagonists (ouabain and Ca²⁺). For comparative considerations the Cd²⁺-inhibition of the enzyme from dog kidney was also tested. Na⁺/K⁺-ATPase from dog kidney and from crab gill differed greatly in sensitivity against ouabain. The inhibition constant K _i of the dog enzyme amounted to 9.1 × 10⁻⁷ mol l⁻¹, i.e. more than 300-fold smaller than the K _i of 2.9 × 10⁻⁴ mol l⁻¹ determined for the crab enzyme. Ca²⁺ inhibited the activity of Na⁺/K⁺-ATPase from crab gill plasma membranes with a K _i of 4.3 × 10⁻⁴ mol l⁻¹. The Na⁺/K⁺-ATPase from crab gill was inhibited by Cd²⁺ with a K _i of 9.1 × 10⁻⁵ mol l⁻¹. Cd²⁺ inhibited the Na⁺/K⁺-ATPase from dog kidney with a K _i (6.4 × 10⁻⁵ mol l⁻¹) comparable to that observed in the crab gill enzyme. Under experimental conditions Cd²⁺-inhibition of Na⁺/K⁺-ATPase was irreversible. Repeated washing, centrifugation and homogenization of the plasma membranes (four times) with Cd²⁺-free buffer did not restore any activity lost in the presence of 1 × 10⁻³ mol l⁻¹ Cd²⁺. Since ouabain-insensitive (nonspecific) ATPases in the plasma membrane fraction of crab gills were inhibited by Cd²⁺ in the same way as Na⁺/K⁺-ATPase, the heavy metal is considered as an unspecific ATPase inhibitor. Comparing these results with literature data on Cd²⁺-binding to electrophoretically separated proteins suggests that Na⁺/K⁺-ATPase is a Cd²⁺-binding enzyme. The results obtained on Na⁺/K⁺-ATPase were reflected by Cd²⁺-inhibition of the branchial ion-transport functions depending on this enzyme. The transepithelial short-circuit current of isolated gill half lamellae, a direct measure of area-specific active ion uptake, and the transepithelial potential difference of isolated, perfused whole gills, also indicative of active ion uptake, were inhibited by the heavy metal in a time- and dose-dependent mode. Remarkably these inhibitions were also irreversible. These findings are ecologically and biomedically significant: even when the actual environmental or tissue concentrations measured are low, biological microstructures such as Na⁺/K⁺-ATPase may accumulate the heavy metal by tight binding over prolonged periods until the first inhibitory effects occur. Received: 25 June 1997 / Accepted: 25 August 1997     

Branchial and intestinal osmoregulatory acclimation in the four-eyed sleeper, <Emphasis Type="Italic">Bostrychus sinensis</Emphasis> (Lacepède), exposed to seawater

Bostrychus sinensis is a facultative air breather that inhabits waters of a wide range of salinities. This study aimed to elucidate whether branchial and intestinal osmoregulatory acclimation occurred in B. sinensis transferred from 5‰ water through a progressive increase in salinities to seawater. Our results indicate that B. sinensis acted as a hyperosmotic regulator in 5‰ water, but exhibited hypoosmotic hypoionic regulation in seawater. During short- (1 day) and medium- (10 days) term acclimation to seawater, there were only minor perturbations in plasma osmolality and [Na⁺], which returned to control levels after 45 days of exposure to seawater. Branchial Na⁺/K⁺-ATPase activity was unaffected by 1, 10 or 45 days of exposure to seawater. However, prolonged (45 days) acclimation to seawater led to a significant increase in Na⁺/K⁺-ATPase α-subunit protein abundance. Taken together, these results indicate that there could be changes in the expression of Na⁺/K⁺-ATPase isoforms and/or post-translational modification of Na⁺/K⁺-ATPase in the gills of fish exposed to seawater. Immunofluorescence microscopy revealed that acclimation to seawater for 10 days only resulted in no change in branchial Na⁺/K⁺-ATPase protein expression, but there were increases in protein expression of cystic fibrosis transmembrane regulator (CFTR)-like chloride channel and Na⁺:K⁺:2Cl⁻ cotransporter (NKCC; probably NKCC1). Indeed, NKCC was undetectable in gills of fish kept in 5‰ water by Western blotting, but it became weakly detectable in fish exposed to seawater for 10 days and prominently expressed in fish exposed to seawater for 45 days. Therefore, our results indicate that branchial CFTR-like chloride channel and NKCC1 were the determining factors in the transition between hyperosmotic regulation and hypoosmotic hypoionic regulation in B. sinensis. Furthermore, the intestine of B. sinensis also served as an important osmoregulatory organ, since there were significant increases in both the activity and protein abundance of intestinal Na⁺/K⁺-ATPase in fish acclimated to seawater for 45 days. The effectiveness of branchial and intestinal osmoregulatory acclimation in B. sinensis during seawater acclimation led to only a minor increase in plasma osmolality, and thus resulted in relatively unchanged free amino acid contents in muscle and liver.     

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.     

Evidence that the rotifer<Emphasis Type="Italic"> Brachionus plicatilis</Emphasis> is not an osmoconformer

The rotifer Brachionus plicatilis is euryhaline (growing between 2 and 97 ppt) and has previously been considered an osmoconformer. We suggest that B. plicatilis is an osmoregulator, exhibiting a pattern of Na⁺/K⁺ ATPase activity in response to salinity consistent with that of other osmoregulating euryhaline invertebrates. To examine salinity tolerance, growth rates between 5 and 60 ppt were determined. The activity of Na⁺/K⁺ ATPase was examined, over the same range of salinities, by measuring ATPase activity in rotifer homogenates in the presence and absence of a Na⁺/K⁺ ATPase inhibitor. Maximum specific growth rate (0.95 day^–1) occurred at 16 ppt, highest mean amictic eggs per female (1.41) occurred at 20 ppt, and both parameters decreased rapidly as salinity increased. Egg development time was constant with salinity at 0.92 days. The activity of Na⁺/K⁺ ATPase per milligram protein increased from 3.9 µmol h^–1 at 5 ppt to 6.8 µmol h^–1 at 50 ppt and accounted for 15 and 30% of total ATPase activity, respectively. We suggest that these observations are consistent with increasing stress at high salinities and the occurrence of a hypo-osmoregulatory response. Given the high ATP consumption of Na⁺/K⁺ ATPase at high salinities, it is possible that a proportion of the corresponding decreases in growth rate and egg production are a direct cost of regulation.Communicated by J.P. Thorpe, Port Erin     

Modulation of branchial ion transport protein expression by salinity in glass eels (<Emphasis Type="Italic">Anguilla anguilla</Emphasis> L.)

The Anguillid juvenile glass eel must deal with the osmoregulatory consequences of highly variable environmental salinities on its recruitment migration from coastal to fresh waters. Changes in ionoregulatory parameters and branchial ion transport protein [Na⁺/K⁺-ATPase, Na⁺:K⁺:2Cl⁻ cotransporter (NKCC), cystic fibrosis transmembrane regulator (CFTR) anion channel, V-type proton ATPase] expression (activities, protein and/or mRNA level expression and/or cellular localization) in response to acclimation to a broad range of ionic strengths [distilled water (DW) to hypersaline water (HSW; 150%) sea water (SW 32‰)] was studied. The estuarine glass eels were very euryhaline and successfully acclimated to acute changes in environmental ionic strength from 50% SW, with high mortality only observed in HSW (51%) and sublethal osmoregulatory indicators (whole body water content and sodium levels) disturbed at the extremes (DW and HSW). Central to a high salinity acclimation were elevated branchial Na⁺/K⁺-ATPase, NKCC and CFTR expression. At lower salinity, Na⁺/K⁺-ATPase expression was maintained and NKCC and CFTR expressions were reduced. Branchial chloride cells increased in size up to SW but decreased in HSW. During hypotonic disturbance (DW), no compensatory elevation in V-ATPase or Na⁺/K⁺-ATPase expression was observed.     

Transbranchial potentials and ion fluxes across isolated,perfused gills of Uca rapax

Transbranchial potentials (TP) and sodium or chloride fluxes were measured in an apparatus designed for the simultaneous perfusion of eight isolated gills of Uca rapax. In anterior gills perfused with U. rapax–saline (US) the TP varied almost linearly from-7.5 to +10 mV inside, and in posterior gills from +2 to-8.5 mV (inside), on exposure to salinities ranging from 8.7 through 52, i.e. 25 to 150% seawater (100%=34.6 S). Sodium influx and efflux in anterior gills exposed to US, 8.7 or 43.3 S (0.7 to 4.0 mmol h^–1 g^–1 dry wt) were always greater than in posterior gills (0.5 mmol h^–1). The chloride fluxes were slightly smaller than sodium fluxes in anterior gills, while in the posterior gills the chloride influx (2.8 to 4.6 mmol h^–1) was always larger than chloride efflux (0.6 to 1.1 mmol h^–1) or the sodium fluxes. At least three ion-transport mechanisms may be present in these gills: (1) an internal ( = basolateral), ouabain-sensitive Na⁺, K⁺ pump, restricted to anterior gills; (2) a furosemide-sensitive Na⁺, K⁺, 2Cl^– (plus water) transporter, apparently restricted to posterior gills, and (3) a Na⁺ exchanger (and possibly other as yet unidentified ion transporters, as suggested by large increases of the chloride influxes caused by amiloride), probably located on the apical membranes of the epithelial cells of both gill types. The differential selectivity of the gills of U. rapax for sodium or chloride may limit the transbranchial movements of either ion, without a reduction of the overall permeability of these crabs.Communicated by N.H. Marcus, Tallahassee     

Acclimation of Solea senegalensis to different ambient temperatures: implications for thyroidal status and osmoregulation

We have investigated the regulation of thyroidal status and osmoregulatory capacities in juveniles from the teleost Solea senegalensis acclimated to different ambient temperatures. Juveniles, raised in seawater at 19°C, were acclimated for 3 weeks to temperatures of 12, 19 and 26°C. Since our preliminary observations showed that at 12°C feed intake was suppressed, our experimental design controlled for this factor. The concentration of branchial Na⁺,K⁺-ATPase, estimated by measurements of enzyme activity at the optimum temperature of this enzyme (37°C), did not change. In contrast, an increase in Na⁺,K⁺-ATPase activity (measured at 37°C), was observed in the kidney of 12°C-acclimated fish. In fish acclimated to 12°C, the hepatosomatic index had increased, which correlated with increased plasma levels of triglycerides and non-esterified fatty acids. Plasma cortisol levels did not differ significantly between the experimental groups. In liver and gills, the amount of iodothyronine deiodinases that exhibit thyroid hormone outer ring deiodination was up-regulated only when fish did not feed. When assayed at the acclimation temperature, kidney deiodinase activities were similar, indicating a temperature-compensation strategy. 3,5,3′-triiodothyronine (T3) tissue concentrations in gills and kidney did not differ significantly between experimental groups. However, at 12°C, lower T3 tissue levels were measured in plasma and liver. We conclude that S. senegalensis adjusts its osmoregulatory system to compensate for the effects of temperature on electrolyte transport capacity. The organ-specific changes in thyroid hormone metabolism at different temperatures indicate the involvement of thyroid hormones in temperature acclimation.     

Reduced hypoosmoregulatory ability and alteration in gill chloride cell distribution in mature chum salmon (Oncorhynchus keta) migrating upstream for spawning

Osmoregulatory ability of mature chum salmon (Oncorhynchus keta) during spawning migration was examined by following the changes in gill Na⁺, K⁺-ATPase activity and in the distribution and morphology of chloride cells. Mature chum salmon caught in Otsuchi Bay, northern Honshu Island, Japan, died within 5 d in seawater (SW) in association with a marked increase in plasma osmolality, whereas the fish transferred to fresh water (FW) maintained plasma osmolality efficiently. Gill Na⁺, K⁺-ATPase activity decreased in both SW-maintained and FW-transferred fish. Well-developed chloride cells, identified by immunocytochemical staining specific for Na⁺, K⁺-ATPase, were present mainly in the filament epithelium of immature fish caught in the ocean. In mature fish caught in the bay, however, additional chloride cells were also found in the lamellar epithelium. The number of filament chloride cells decreased markedly in the mature fish both in SW and in FW, whereas the number of lamellar chloride cells was maintained. These results suggest that the loss of hypoosmoregulatory ability in mature chum salmon may be attributable to the decrease in filament chloride cells and associated decrease in gill Na⁺, K⁺-ATPase activity, and also that appearance of lamellar chloride cells may be preparatory to the forthcoming upstream migration. Received: 14 April 1997 / Accepted: 5 May 1997     

Biomarkers as tools to assess the chronic toxicity of ammonia in the juvenile Mugil cephalus

With juvenile fish as the subject, the effects of low concentration ammonia on antioxidant system were studied using Mugil cephalus. Samples of gill and liver tissue were obtained from 0.35, 0.70, 1.5 and 3?mg/L ammonia groups at 0, 5, 10, 15 and 20 days of exposure, at which times the biomarkers were measured. Results showed that gill malondialdehyde (MDA) content exhibited an initial significant increase (p?≤?0.05) at unionised ammonia concentrations of 0.70, 1.5 and 3.0?mg/L on day 5, followed by subsequent declines, while liver MDA levels exhibited significant increases (p?≤?0.05) at unionised ammonia concentrations of 1.5?mg/L starting on day 10 and at 3.0?mg/L starting on day 5. With exposure to ammonia at different concentrations, Na⁺-K⁺-ATPase activity in liver and gill decreased over time. The Na⁺-K⁺-ATPase activity was negatively related to ammonia concentration from 0.70 to 3.0?mg/L. Overall, our results show that MDA and Na⁺-K⁺-ATPase, evaluated here as potential biomarkers of ammonia exposure, exhibited responses to sublethal concentrations of ammonia that were concentration dependent.     

Effect of salinity fluctuation on the osmotic pressure and Na+, Ca2+ and Mg2+ ion concentrations in the hemolymph of bivalve molluscs

Specimens of Chlamys opercularis, Modiolus modiolus, Mytilus edulis, Crassostrea gigas, Scrobicularia plana and Mya arenaria were exposed to both gradual (sinusoidal) and abrupt (square-wave) salinity fluctuations and measurements made of osmotic, Na⁺, Mg²⁺ and Ca²⁺ concentrations in the hemolymph and where applicable in the mantle fluid. In both sinusoidal and square-wave regimes fluctuating between 100 and 50% seawater (100%=ca. 32 S), the hemolymph Na⁺, Mg²⁺, Ca²⁺ and osmotic concentrations followed the concentrations of the external medium in Chlamys opercularis. The hemolymph and mantle fluid osmotic Na⁺, Mg²⁺ and Ca²⁺ concentrations of Modiolus modiolus, Mytilus edulis, Crassostrea gigas and S. plana followed those of the external medium as long as the molluscs' shell valves remained open. There were no changes in the ionic or osmotic concentrations of the hemolymph or mantle fluid of any of these species during periods of shell-valve closure. The hemolymph osmotic, Na⁺ and Mg²⁺ concentrations of wedged-open Modiolus modiolus, Mytilus edulis, C. gigas and S. plana followed those of the external medium. Hemolymph Ca²⁺ concentrations showed a damped response in C. gigas and Mytilus edulis. The hemolymph osmotic, Na⁺, Ca²⁺ and Mg²⁺ concentrations of Mya arenaria fluctuated in a similar manner to the external medium, but were damped. Wedged-open Mytilus edulis exposed to fluctuating salinity and supplied with a constant supply of 10 mM Ca²⁺ showed greater changes in hemolymph ionic and osmotic concentrations than M. edulis exposed to the same salinity fluctuation without a constant Ca²⁺ supply. Chlamys opercularis and Modiolus modiolus survived in a 50% seawater minimum sinusoidal salinity fluctuation for 10 days; wedged-open M. modiolus survived only 3 days. Burrowing had no effect on the osmotic, Na⁺, Mg²⁺ or Ca²⁺ concentrations of the hemolymph of Mya arenaria or S. plana exposed to fluctuating salinities. All of the species studied were shown to be osmoconformers.     

Respiratory quotient and ammonia excretion in Tilapia mossambica

Tilapia mossambica (Peters), acclimated to and tested in fresh water at 30°C, maintained a routine respiratory quotient (R Q) of about unity and an ammonia quotient (A Q) (Vol. NH₂/Vol. O₂) of about 0.2 at high ambient oxygen concentrations. At low oxygen concentrations (below 2 ppm) R Q and A Q increased sharply to values of 8 and 1, respectively (at 0.6 ppm), indicating a close relationship of increase in anaerobic energy utilization and increase in protein metabolism at inadequate oxygen concentrations. T. mossambica (8 cm), exercised continuously with intervening sampling and flushing stops for 6 h, at a swimming speed of about 2 body lengths/sec, derived some anaerobic energy throughout the exercise (R Q: 1.2), utilizing more protein the longer the exercise. The coupling of the increased protein metabolism and anaerobic energy utilization may be of advantage in preventing acidosis and also in conserving sodium (Na⁺) in fish.Part of this work was included in a paper presented at the Centennial Meeting of The American Fisheries Society, September 13–16, 1970, New York.     

Ecotoxicity of silver nanoparticles on earthworm Eisenia fetida: responses of the antioxidant system,acid phosphatase and ATPase

Ecotoxicity of nanoparticles has received growing attention in recent years. This study investigated the influence of silver nanoparticles (Ag-NP) on earthworm Eisenia fetida. The experiment was performed with five test groups: control (without Ag-NP), 10?nm Ag-NP groups (20, 100 or 500?mg?kg^?1) and positive control (787?mg?kg^?1 AgNO₃). After 14-day acute exposure, activities of various enzymes, including glutathione S-transferase (GST), glutathione reductase (GR), acid phosphatase (AP), and Na⁺, K⁺-ATPase were determined. Effects of Ag-NP with different sizes (10 and 80?nm) were also tested. Data showed that the activity of GR was significantly lower at 500?mg?kg^?1. The activities of AP and Na⁺, K⁺-ATPase were inhibited following the increase of Ag-NP concentration. When treated with Ag-NP with different sizes, activities of AP and Na⁺, K⁺-ATPase of the 10?nm group were significantly lower than the control group, but those of the 80?nm group were similar to the control group. Data indicate that Ag-NP may be harmful to the earthworm E. fetida at 500?mg?kg^?1, and the toxicity of Ag-NP with 10?nm size is greater than 80?nm. In addition, AP and Na⁺, K⁺-ATPase are sensitive biomakers to the effects of Ag-NP.     

Activity and inhibitor sensitivity of ATPases in the hydrothermal vent tubeworm Riftia pachyptila : a comparative approach

Phosphorylated ATPases may be involved in the effective pH regulation seen in the hydrothermal vent tubeworm Riftia pachyptila. R. pachyptila appears not only to have a large concentration of ATPases, but the main function of these ATPases seems to have shifted from other types of transport, such as Na⁺ and K⁺ movement, to the facilitation of H⁺ elimination. Plume and trophosome ATPase activity for R. pachyptila measured 646.2 ± 29.5 and 481.4 ± 32.0 μmol Pi (inorganic phosphate) g⁻¹ wet wth⁻¹, respectively. Plume tissue ATPase activity (both mass-specific and protein-specific) in R. pachyptila was higher (between 7% and 55%) than the activity measured in any tissue for 7 other shallow- and deep-living species, in this study. This supports the hypothesis that R. pachyptila regulates acid/base balance via high concentrations of H⁺-ATPases, including Na⁺/H⁺ and K⁺/H⁺ exchangers and possibly electrogenic H⁺-ATPases, as evidenced by a higher total ATPase concentration (646 μmol Pi g⁻¹ wet wt h⁻¹), lesser Na⁺/K⁺-ATPase activity (13% of the total, as compared to 20−40% found in other animals), and higher H⁺-ATPase activity (226–264 μmol Pi g⁻¹wet wt h⁻¹). Overall, R. pachyptila appears to demonstrate elevated ATPase activity, with a greater fraction of the enzymes devoted to proton elimination, in order to effectively control its extracellular pH in the face of processes acting to acidify the internal environment. Received: 9 May 2000 / Accepted: 4 October 2000     

Effects of lead and selected pyrethroids/piperonyl butoxide alone and in combination on Na,K‐ATPase

In vitro effects of Pb²⁺, the pyrethroid insecticides cypermethrin, fenvalerate and the syner‐gist piperonyl butoxide on sodium‐potassium‐activated adenosine triphosphatase (Na,K‐ATPase) from dog kidney were determined. Pb²⁺ with an estimated IC50 value of 5.2 μM was found to be a potent inhibitor of Na,K‐ATPase activity, whereas Na,K‐ATPase was less sensitive to the pyrethroids tested and piperonyl butoxide. Investigation with circular dichroism (CD) spec‐troscopy showed that inhibition occurs through conformational changes of the α‐subunit of the enzyme. The kinetic characteristics of inhibition of Na,K‐ATPase with varying substrate (ATP) concentrations as well as with varying Na⁺ concentrations exhibited a competitive type of inhibition with Pb²⁺ in the μM range. With Pb²⁺ alone in the enzyme assay no conformational changes of the protein could be observed which confirmed the assumption that Pb²⁺ can bind to the Na⁺ binding site of the α‐subunit. Uncompetitive type of inhibition occurred with varied K⁺ concentrations demonstrating that this cation binding site is not affected directly by Pb²⁺.

Complete reversal of Pb²⁺ by DTT confirms that a possible target for interaction of this heavy metal ion with Na, K‐ATPase are specific SH groups.

Synergistic effects could only be determined with higher Pb²⁺ concentrations of 3, 5 and 7 μM plus piperonyl butoxide while all other combinations with this heavy metal plus organic substances where of the additive type. With CD spectroscopy also only additive effects were observed. These results demonstrate that higher concentrations of piperonyl butoxide favor the binding of Pb²⁺ to the Na⁺ binding site by conformational changes of the protein.     

Chemical composition of rivers and streams in the forest and savanna zones of Nigeria

The aim of this study is to investigate the differences in the chemical conditions of lotic waterbodies in the two major ecosystems in Nigeria, the forest and savanna zones. The forest waters were slightly acidic (mean±SD pH = 6.72±0.58) while the savanna waters were slightly alkaline (pH = 7.11±0.33). The cationic order of dominance in the forest waters was Na⁺ > Ca²⁺ > Mg²⁺ > K⁺ in contrast to Ca²⁺ > Mg²⁺ > Na⁺ > K⁺ in savanna waters. The forest waters were chlorided (typical of coastal and/or marine waters) whereas the savanna waters were carbonated in nature, typical of the worldwide freshwater. Organic carbon was significantly higher in forest waters than in the savanna waters (p < 0.05) while nutrient compounds were significantly higher in savanna waters than in forest waters. The seasonal variation of the chemical parameters was generally more evident in savanna than in forest waters. The differences in water quality between the two major vegetation zones reflect the differences in the biogeochemical processes and nutrient cycling that characterise forest and savanna ecosystems.     

Sodium- and potassium-activated adenosine triphosphatase in the excretory organs of Sepia officinalis (Cephalopoda)

Sodium- and potassium-activated ATPase (Na⁺–K⁺-ATPase) has been demonstrated in excretory organs of Sepia officinalis, using a cytochemical procedure. In the renal appendages, both epithelia of the pancreatic appendages, the folded epithelium of the branchial heart appendage and the transport-active epithelium of the gill, the enzyme is localized exclusively in the basolateral cell membranes, i.e., the membranes of the basal labyrinth and the lateral plasma membranes. In addition, Na⁺–K⁺-ATPase is also located in the sarcolemma of the muscle fibres of the branchial heart. Distribution and localization of the enzyme is further substantiated by [³H]-ouabain autoradiography. The possible involvement of Na⁺–K⁺-ATPase in the excretion of ammonia and in ionic regulation in dibranchiate cephalopods is discussed.This study was supported by the Deutsche Forschungsgemeinschaft and is part of a doctoral dissertation     

Molecular studies on the ouabain binding site of the Na+, K+-ATPase in milkweed butterflies

Summary. The Na⁺, K⁺-ATPase of the Monarch butterfly (Danaus plexippus) is insensitive to the inhibition by cardiac glycosides due to an amino acid replacement: histidine instead of asparagine at position 122 of the α-subunit representing the ouabain binding site. By PCR amplification of the DNA sequence of this site, a PCR product of 270 bp was obtained from DNA extracted from Danainae species (Danaus plexippus, D. chrysippus, D. gillipus, D. philene, D. genutia, Tirumala hamata, Euploea spp., Parantica weiskei, P. melusine), Sphingidae (Daphnis nerii) and mimics of milkweed butterflies (Hypolimnas missipus, Limenitis archippus and L. arthemis, Nymphalidae). Analysis of the nucleotide sequences revealed that the single point mutation in the ouabain binding domain (AAC-Asn for CAC-His) was present only in Danaus plexippus, but not in the other species investigated. Since these milkweed butterflies also store cardenolides, other structural modifications of the Na⁺, K⁺-ATPase may have occurred or other strategies of cardenolide tolerance have been developed. Received 15 May 2000; accepted 29 June 2000