DDT inhibits Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>, Mg<Superscript>2+</Superscript>-ATPase in the Intestinal Mucosae and Gills of Marine Teleosts

SODIUM-potassium-activated, magnesium-dependent, adenosine triphosphatase (Na⁺, K⁺, Mg²⁺-ATPase) is widely accepted as an essential factor in sodium transport¹ and observations on fish substantiate this view. There are concurrent increases, for example, of both Na⁺, K⁺, Mg²⁺-ATPase activity and osmoregulatory sodium transport², in the intestinal mucosae^3,4 and the gills^3,5 of euryhaline teleosts during adaptation to seawater. Furthermore, the gills of stenohaline seawater teleosts, which actively secrete sodium, exhibit higher Na⁺, K⁺, Mg²⁺-ATPase activity than the gills of stenohaline freshwater teleosts, which do not actively secrete sodium^3,5. Na⁺, K⁺, Mg²⁺-ATPase therefore seems to be important in maintaining tissue osmolarity well below that of seawater. It is disquieting to report therefore that Na⁺, K⁺, Mg²⁺-ATPase activity in the intestinal mucosae and gills of marine teleosts is inhibited by the organochlorine insecticide DDT. This observation may help to clarify the unexplained sensitivity of teleosts to DDT⁶.     

New isoform HvNHX3 of vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript>-antiporter in barley: Expression and immunolocalization

The gene HvNHX3 encoding a new isoform of vacuolar Na⁺/H⁺-antiporter was identified in barley. This gene is expressed in roots and leaves of barley seedlings, and it encodes a protein consisting of 541 amino acid residues with pre-dicted molecular weight 59.7 kDa. It was found that by its amino acid sequence HvNHX3 is closest to the Na⁺/H⁺-antiporter HbNHX1 of wild type from Hordeum brevisibulatum that grows on salt-marsh (solonchak) soils (95% homology). The expression of HvNHX3 during salt stress is increased several-fold in roots and leaves of barley seedlings. At the same time, the amount of HvNHX3 protein in roots does not change, but in leaves it increases significantly. It was shown using HvNHX3 immunolocalization in roots that this protein is present in all tissues, but in control plants it was clustered and in experimental plants after salt stress it was visualized as small granules. It has been proposed that HvNHX3 is converted into active form during declusterization. The conversion of HvNHX3 into its active form along with its quantitative increase in leaves during salt stress activates Na⁺/H⁺-exchange across the vacuolar membrane and Na⁺ release from cytoplasm, and, as a consequence, an increase of salt stress tolerance.     

Changes in Carboxypeptidase A,Dipeptidase and Na<Superscript>+</Superscript>/K<Superscript>+</Superscript> ATPase Activities in the Intestine of Rats Orally Exposed to Different Doses of Cadmium

The purpose of the present study was to evaluate the effect of cadmium on some protein digestive and absorption enzymes in rats. Thirty-six rats were grouped into three groups of 12 animals each; one group received deionised water and acted as control. One group received 445 μM Cd and the last group received 890 μM Cd in their drinking water for a period of one month. The results obtained indicate that increasing the level of cadmium from 445 μM to 890 μM in the drinking water of the rats led to 29 and 23 increase in accumulated cadmium in the proximal and distal small intestine respectively. The body weight gain of rats exposed to 445 μM and 890 μMCd was decreased by about 24 and 43 respectively when compared with the control. The activities of carboxypeptidase A, dipeptidase and Na+/K+ ATPase were reduced in the mucosa of the proximal end of the small intestine of cadmium exposed rats. The reduction was dose dependent; with the 890 μM Cd exposed rats displaying the least activities. In the distal small intestine, the activities of these enzymes were restored in the 445 μM Cd exposed rats to levels that were not statistically different (P>0.05) from those observed in the controls. In the 890 μMCd exposed rats, dipeptidase activity improved by about 80 compared with the activity of the enzyme in the proximal small intestine. Likewise, Na+/K+ ATPase activity increased by about 125 compared with the observed level in the proximal small intestine. The study suggests that cadmium given to rats in drinking water compromise protein digestion and absorption of nutrients particularly in the proximal region of small intestine and could account for weight reduction associated with cadmium toxicity. Published online December 2004     

Activity of Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase and the content of phospholipids in the blue mussel <Emphasis Type="Italic">Mytilus edulis</Emphasis> L. during environmental temperature changes

Changes in the activity of Na⁺/K⁺-ATPase and content of membrane lipids (phospholipids) in the gills and hepatopancreas of the blue mussel Mytilus edulis L. have been studied during a sharp temperature increase under aquarian managed conditions. The most pronounced changes were recorded in mollusk gills. A correlation of changes in the activity of membrane-bound Na⁺/K⁺-ATPase and phospholipid content (mainly phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and lysophosphatidylcholine) was revealed; this correlation evidences their mutual involvement in compensation for the temperature effect to help mussels adapt to sharp temperature changes.     

K<Superscript>+</Superscript>-induced Ca<Superscript>2+</Superscript> Conductance Responsible for the Prolonged Backward Swimming in K<Superscript>+</Superscript>-agitated Mutant of <Emphasis Type="Italic">Paramecium caudatum</Emphasis>

The K⁺-agitated (Kag) mutant of Paramecium caudatum shows prolonged backward swimming in K⁺-rich solution. To understand the regulation mechanisms of the ciliary motility in P. caudatum, we examined the membrane electrical properties of the Kag mutant. The duration of the backward swimming of the Kag in K⁺-rich solution was about 10 times longer than that of the wild type. In response to an injection of the outward current, the wild type produced an initial action potential and a subsequent membrane depolarization due to I-R potential drop, while the Kag exhibited repetitive action potentials during the depolarization. Under voltage-clamp conditions, the depolarization-activated transient inward current exhibited by the Kag was slightly smaller than that exhibited by the wild type. In response to an application of K⁺-rich solution, both the wild type and the Kag exhibited a depolarizing afterpotential representing the activation of the K⁺-induced Ca²⁺ conductance. The inactivation time course of the K⁺-induced Ca²⁺ conductance of Kag was about 10 times longer than that of the wild type. This difference corresponds well with the difference in behavioral responses between Kag and wild type to K⁺-rich solution. We conclude that the overreaction of the Kag mutant to the K⁺-rich solution is caused by slowing down of the inactivation of the K⁺-induced Ca²⁺ conductance.     

The Expression of NMDA Receptor Subunits in Cerebral Cortex and Hippocampus is Differentially Increased by Administration of Endobain E,a Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Inhibitor

Previous studies showed that endobain E, an endogenous Na⁺, K⁺-ATPase inhibitor, decreases dizocilpine binding to NMDA receptor in isolated membranes. The effect of endobain E on expression of NMDA receptor subunits in membranes of rat cerebral cortex and hippocampus was analyzed by Western blot. Two days after administration of 10 μl endobain E (1 μl = 29 mg fresh tissue) NR1 subunit expression enhanced 5-fold and 2.5-fold in cerebral cortex and hippocampus, respectively. NR2A subunit expression increased 2-fold in cerebral cortex and 1.5-fold in hippocampus. The level of NR2B subunit raised 3-fold in cerebral cortex but remained unaltered in hippocampus. NR2C subunit expression was unaffected in either area. NR2D subunit enhanced 1.6 and 2.1-fold for cerebral cortex and hippocampus, respectively. Results indicate that endogenous Na⁺, K⁺-ATPase inhibitor endobain E differentially modifies the expression of NMDA receptor subunits.     

Involvement of the antiplatelet activity of magnesium sulfate in suppression of protein kinase C and the Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger

Magnesium sulfate is widely used to prevent seizures in pregnant women with hypertension. The aim of this study was to examine the inhibitory mechanisms of magnesium sulfate in platelet aggregation in vitro. In this study, magnesium sulfate concentration-dependently (0.6–3.0 mM) inhibited platelet aggregation in human platelets stimulated by agonists. Magnesium sulfate (1.5 and 3.0 mM) also concentration-dependently inhibited phosphoinositide breakdown and intracellular Ca⁺² mobilization in human platelets stimulated by thrombin. Rapid phosphorylation of a platelet protein of M_r 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12-13-dibutyrate (PDBu, 50 nM). This phosphorylation was markedly inhibited by magnesium sulfate (3.0 mM). Magnesium sulfate (1.5 and 3.0 mM) further inhibited PDBu-stimulated platelet aggregation in human platelets. The thrombin-evoked increase in pHi was markedly inhibited in the presence of magnesium sulfate (3.0 mM). In conclusion, these results indicate that the antiplatelet activity of magnesium sulfate may be involved in the following two pathways: (1) Magnesium sulfate may inhibit the activation of protein kinase C, followed by inhibition of phosphoinositide breakdown and intracellular Ca⁺² mobilization, thereby leading to inhibition of the phosphorylation of P47. (2) On the other hand, magnesium sulfate inhibits the Na⁺/H⁺ exchanger, leading to reduced intracellular Ca⁺² mobilization, and ultimately to inhibition of platelet aggregation and the ATP-release reaction.     

Pivotal Role of Reduced Glutathione in Oxygen-induced Regulation of the Na<Superscript><Emphasis Type="Bold">+</Emphasis></Superscript>/K<Superscript><Emphasis Type="Bold">+</Emphasis></Superscript> Pump in Mouse Erythrocyte Membranes

This study addresses the mechanisms of oxygen-induced regulation of ion transport pathways in mouse erythrocyte, specifically focusing on the role of cellular redox state and ATP levels. Mouse erythrocytes possess Na⁺/K⁺ pump, K⁺-Cl^– and Na⁺-K⁺-2Cl^– cotransporters that have been shown to be potential targets of oxygen. The activity of neither cotransporter changed in response to hypoxia-reoxygenation. In contrast, the Na⁺/K⁺ pump responded to hypoxic treatment with reversible inhibition. Hypoxia-induced inhibition was abolished in Na⁺-loaded cells, revealing no effect of O₂ on the maximal operation rate of the pump. Notably, the inhibitory effect of hypoxia was not followed by changes in cellular ATP levels. Hypoxic exposure did, however, lead to a rapid increase in cellular glutathione (GSH) levels. Decreasing GSH to normoxic levels under hypoxic conditions abolished hypoxia-induced inhibition of the pump. Furthermore, GSH added to the incubation medium was able to mimic hypoxia-induced inhibition. Taken together these data suggest a pivotal role of intracellular GSH in oxygen-induced modulation of the Na⁺/K⁺ pump activity.     

Sulfatide and Na<Superscript>+</Superscript>-K<Superscript>+</Superscript>-ATPase: A Salinity-sensitive Relationship in the Gill Basolateral Membrane of Rainbow Trout

We investigated the effect of salinity on the relationship between Na⁺-K⁺-ATPase and sulfogalactosyl ceramide (SGC) in the basolateral membrane of rainbow trout (Oncorhynchus mykiss) gill epithelium. SGC has been implicated as a cofactor in Na⁺-K⁺-ATPase activity, especially in Na⁺-K⁺-ATPase rich tissues. However, whole-tissue studies have questioned this role in the fish gill. We re-examined SGC cofactor function from a gill basolateral membrane perspective. Nine SGC fatty acid species were quantified by tandem mass spectrometry (MS/MS) and related to Na⁺-K⁺-ATPase activity in trout acclimated to freshwater or brackish water (20 ppt). While Na⁺-K⁺-ATPase activity increased, the total concentration and relative proportion of SGC isoforms remained constant between salinities. However, we noted a negative correlation between SGC concentration and Na⁺-K⁺-ATPase activity in fish exposed to brackish water, whereas no correlation existed in fish acclimated to freshwater. Differential Na⁺-K⁺-ATPase/SGC sensitivity is discussed in relation to enzyme isoform switching, the SGC cofactor site model and saltwater adaptation.This revised version was published online in June 2005 with a corrected cover date.     

Immunolocalization of Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase in the branchial cavity during the early development of the crayfish <Emphasis Type="Italic">Astacus leptodactylus</Emphasis> (Crustacea,Decapoda)

The ontogeny of osmoregulation was examined in the branchial cavity of embryonic and early post-embryonic stages of the crayfish Astacus leptodactylus maintained in freshwater, at the sub-cellular level through the detection of the sodium–potassium adenosine triphosphatase (Na⁺,K⁺-ATPase). The embryonic rate of development was calculated according to the eye index (EI) which was 430–450 m at hatching. The distribution of the enzyme was identified by immunofluorescence microscopy using a monoclonal antibody IgG5 raised against the avian -subunit of the Na⁺,K⁺-ATPase. Immunoreactivity staining, indicating the presence of Na⁺, K⁺-ATPase appeared in the gills of late embryos (EI400 m), i.e. a few days before hatching time, and steadily increased throughout the late embryonic and early post-embryonic development. The appearance of the enzyme correlates with the ability to osmoregulate which also occurs late in the embryonic development at EI 410–420 m and with tissue differentiation within the gill filaments. These observations indicate that the physiological shift from osmoconforming embryos to hyper-regulating late embryos and post-hatching stages in freshwater must originate partly from the differentiation in the gill epithelia of ionocytes which are the site of ion pumping, as suggested by the location of Na⁺,K⁺-ATPase. Only the gills were immunostained and a lack of specific staining was noted in the lamina and the branchiostegites. Therefore, osmoregulation through Na⁺active uptake is likely achieved in embryos at the gill level; all the newly formed gills in embryos function in ion regulation; other parts of the branchial chamber such as the branchiostegites and lamina do not appear to be involved in osmoregulation.     

Transport of Na<Superscript>+</Superscript> and K<Superscript>+</Superscript> by an antiporter-related subunit from the <Emphasis Type="Italic">Escherichia coli </Emphasis>NADH dehydrogenase I produced in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The NADH dehydrogenase I from Escherichia coli is a bacterial homolog of the mitochondrial complex I which translocates Na⁺ rather than H⁺. To elucidate the mechanism of Na⁺ transport, the C-terminally truncated NuoL subunit (NuoL_N) which is related to Na⁺/H⁺ antiporters was expressed as a protein A fusion protein (ProtA–NuoL_N) in the yeast Saccharomyces cerevisiae which lacks an endogenous complex I. The fusion protein inserted into membranes from the endoplasmatic reticulum (ER), as confirmed by differential centrifugation and Western analysis. Membrane vesicles containing ProtA–NuoL_N catalyzed the uptake of Na⁺ and K⁺ at rates which were significantly higher than uptake by the control vesicles under identical conditions, demonstrating that ProtA–NuoL_N translocated Na⁺ and K⁺ independently from other complex I subunits. Na⁺ transport by ProtA–NuoL_N was inhibited by EIPA (5-(N-ethyl-N-isopropyl)-amiloride) which specifically reacts with Na⁺/H⁺ antiporters. The cation selectivity and function of the NuoL subunit as a transporter module of the NADH dehydrogenase complex is discussed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Occurrence of ghrelin-producing cells,the ghrelin receptor and Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase in tissues of Atlantic halibut (<Emphasis Type="Italic">Hippoglossus hippoglossus</Emphasis>) during early development

Ghrelin is a pituitary growth hormone (GH)-secretagogue that also has metabolic, reproductive, proliferative, immunological and brain functions in mammals. Far less is known about its role in fish. We have therefore performed an immunohistochemical determination of its tissue distribution in the developing Atlantic halibut (Hippoglossus hippoglossus) to gain insights into its potential function. Ghrelin immunoreactivity was detected in first-feeding halibut larvae in the skin, urinary bladder, gastrointestinal (GI) tract and olfactory lobe of the brain. In subsequent stages up to metamorphosis, ghrelin immunoreactivity declined in the skin and became evident in the gills. When the stomach developed, ghrelin immunoreactivity declined throughout the GI tract with the exception of the stomach, which exhibited an intense signal. Immunoreactive ghrelin cells were also present in the olfactory lobe, nerve and epithelium and in occasional cells of the buccal cavity and oesophagus. Ghrelin immunoreactivity had an overlapping distribution with that for Na⁺,K⁺-ATPase, colocalisation also being observed in some ionocytes of the gill. The co-expression of ghrelin and the GH-secretagogue receptor in the same tissue indicates that ghrelin can exert both endocrine and paracrine actions in the developing halibut. The presence of immunoreactive ghrelin in several osmoregulatory tissues, the GI tract and sensory tissue provides strong evidence that ghrelin has multiple functions during development and also suggests targets for future investigations.     

Protoplast Formation of the Coccolithophorid <Emphasis Type="Italic">Pleurochrysis haptonemofera</Emphasis> in Hypoosmotic K<Superscript>+</Superscript> Solution: Shedding of the Coccosphere and Regrowth of the Protoplast in Normal Medium

Both coccolith-bearing cells (C-cells) and naked cells (N-cells) of the coccolithophorid Pleurochrysis haptonemofera can grow in salinities of more than 7‰ (about 20% of a “normal” sea water salinity [35‰]), with the highest growth rates in salinities of more than 14‰. Microscopic observations of cells suspended in 100 mM NaCl (7‰) showed that, while N-cells were swelling uniformly all over the cell surface, C-cells were bulging the plasma membrane from the hole of the coccosphere at the apical (flagellar) pole of the cell. Effects of several cations and anions on the morphological change of C-cells under hypoosmotic pressure were investigated. When 100 mM K⁺ was used, protoplasts were released from the coccosphere completely in almost all the cells. This phenomenon was shown with K⁺ most effectively. The protoplasts could grow in the fresh medium and form the first coccolith within 9 h.     

Study of the involvement of osmotic adjustment and H<Superscript>+</Superscript>-ATPase activity in the resistance of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> suspension cells to salt stress

The salt-induced H⁺-ATPase activity and osmotic adjustment responses of Catharanthus roseus (L.) G. Don suspension cultures were studied. Cells were treated with 0, 50 or 100mM NaCl for 7days or were maintained for 8 months with 50 mM NaCl (50T cells). Growth, osmotic potential (), ions content, soluble sugars, proline and total amino acids were determined in the sap of control and salt-treated cells. Salinity reduced cell growth and . The higher decrease in the in salt-treated cells was due to higher accumulation of Na⁺ and Cl^–. The levels of organic solutes, such as soluble sugars, free proline and total amino acids, increased with salt treatment. These results suggest that salt-tolerant cells are able to osmotically adjust. Salinity treatments stimulated H⁺-ATPase activity. Immunodetection of the enzyme showed that the increased activity was due to an increased amount of protein in the plasmalemma. The induction by NaCl, especially at 100 mM NaCl and for 50T cells, could account for the K⁺ and Cl^– uptake but not for higher or lower tolerance.     

Cloning and functional expression in <Emphasis Type="Italic">Saccharomyces cereviae</Emphasis> of a K<Superscript>+</Superscript> transporter,AlHAK, from the graminaceous halophyte, <Emphasis Type="Italic">Aeluropus littoralis</Emphasis>

High-affinity K⁺ transporters play an important role in K⁺ absorption of plants. We isolated a HAK gene from Aeluropus littoralis, a graminaceous halophyte. The amino acid sequence of AlHAK showed high homology with HAK transporters obtained from Oryza sativa (82%) and Hordeum vulgare (82%). When expressed in Saccharomyces cereviae WΔ3, AlHAK performed high-affinity K⁺ uptake with a K_m value of 8 μM, and the growth of transformants was dramatically inhibited by 150 mM Rb⁺ and 150 mM Cs⁺ but less affected by 300 mM Na⁺. AlHAK may thus improve the capacity of plants to maintain a high cytosolic K⁺/Na⁺ ratio at high salinity.