The roles of the Na,K-ATPase beta 1 subunit in pump sorting and epithelial integrity

In epithelial MDCK cells, the Na,K-ATPase is co-localized with adherens junctions in all stages of monolayer formation starting from initiation of cell–cell contact. The Na,K-ATPase and adherens junction proteins stay partially co-localized even after internalization due to disruption of intercellular contacts by Ca²⁺ deprivation. Similar to adherens junction proteins, the Na,K-ATPase is resistant to extraction with non-ionic detergent, suggesting pump association with the cytoskeleton. In contrast, the heterodimer formed by expressed unglycosylated Na,K-ATPase β₁ subunit and the endogenous α₁ subunit is easily dissociated from the adherens junctions and cytoskeleton by detergent extraction. The MDCK cells in which half of the endogenous β₁ subunits in the lateral membrane are substituted by unglycosylated β₁ subunits display a slower rate of cell-to-cell contact formation and decreased ability to both spread over the surface and migrate. The lack of N-glycans in the Na,K-ATPase β₁ subunit results in an impairment of mature cell–cell junctions as detected by an increase in the paracellular permeability of the MDCK cell monolayers and by a decrease in resistance of adherens junction proteins to extraction by a non-ionic detergent. Therefore the N-glycans of the Na,K-ATPase β₁ subunit are important for retention of the pump at the sites of cell–cell contact. Moreover, they are important for the integrity and stability of cell–cell junctions in mature epithelia. In addition, N-glycans contribute to the formation of cell–cell contacts between surface-attached dispersed cells by mediating lamellipodia formation and stabilizing the newly formed adherens junctions.     

Regulation of Na,K-ATPase during acute lung injury

A hallmark of acute lung injury is the accumulation of a protein rich edema which impairs gas exchange and leads to hypoxemia. The resolution of lung edema is effected by active sodium transport, mostly contributed by apical Na⁺ channels and the basolateral located Na,K-ATPase. It has been reported that the decrease of Na,K-ATPase function seen during lung injury is due to its endocytosis from the cell plasma membrane into intracellular pools. In alveolar epithelial cells exposed to severe hypoxia, we have reported that increased production of mitochondrial reactive oxygen species leads to Na,K-ATPase endocytosis and degradation. We found that this regulated process follows what is referred as the Phosphorylation–Ubiquitination–Recognition–Endocytosis–Degradation (PURED) pathway. Cells exposed to hypoxia generate reactive oxygen species which activate PKCζ which in turn phosphorylates the Na,K-ATPase at the Ser18 residue in the N-terminus of the α1-subunit leading the ubiquitination of any of the four lysines (K16, K17, K19, K20) adjacent to the Ser18 residue. This process promotes the α1-subunit recognition by the μ2 subunit of the adaptor protein-2 and its endocytosis trough a clathrin dependent mechanism. Finally, the ubiquitinated Na,K-ATPase undergoes degradation via a lysosome/proteasome dependent mechanism.     

Polarized membrane distribution of potassium-dependent ion pumps in epithelial cells: Different roles of the <Emphasis Type="Italic">N</Emphasis>-glycans of their <Emphasis Type="Italic">β</Emphasis> subunits

The Na,K-ATPases and the H,K-ATPases are two potassium-dependent homologous heterodimeric P₂-type pumps that catalyze active transport of Na⁺ in exchange for K⁺ (Na,K-ATPase) or H⁺ in exchange for K⁺ (H,K-ATPase). The ubiquitous Na,K-ATPase maintains intracellular ion balance and membrane potential. The gastric H,K-ATPase is responsible for acid secretion by the parietal cell of the stomach. Both pumps consist of a catalytic α-subunit and a glycosylated β-subunit that is obligatory for normal pump maturation and trafficking. Individual N-glycans linked to the β-subunits of the Na,K-ATPase and H,K-ATPase are important for stable membrane integration of their respective α subunits, folding, stability, subunit assembly, and enzymatic activity of the pumps. They are also essential for the quality control of unassembled β-subunits that results in either the exit of the subunits from the ER or their ER retention and subsequent degradation. Overall, the importance of N-glycans for the␣maturation and quality control of the H,K-ATPase is greater than that of the Na,K-ATPase. The roles of individual N-glycans of the β-subunits in the post-ER trafficking, membrane targeting and plasma membrane retention of the Na,K-ATPase and H,K-ATPase are different. The Na,K-ATPase β ₁-subunit is the major β-subunit isoform in cells with lateral location of the pump. All three N-glycans of the Na,K-ATPase β ₁-subunit are important for the lateral membrane retention of the pump due to glycan-mediated interaction between the β ₁-subunits of the two neighboring cells in the cell monolayer and cytosolic linkage of the α-subunit to the cytoskeleton. This intercellular β ₁–β ₁ interaction is also important for formation of cell–cell contacts. In contrast, the N-glycans unique to the Na,K-ATPase β ₂-subunit,which has up to eight N-glycosylation sites, contain apical sorting information. This is consistent with the apical location of the Na,K-ATPase in normal and malignant epithelial cells with high abundance of the β ₂-subunit. Similarly, all seven N-glycans of the gastric H,K-ATPase β-subunit determine apical sorting of this subunit. Supported in part by NIH grants DK46917, DK58333, D53642, and USVA     

Expression of the Avian Na,K-ATPase Subunits in Dictyostelium discoideum

This study explored whether Dictyostelium discoideum can be used to express the avian Na,K-ATPase, a heterodimeric membrane protein. Dictyostelium was able to express mRNAs encoding the avian Na,K-ATPase subunits. However, Dictyostelium expressed avian Na,K-ATPase protein when only when a Dictyostelium consensus ribosomal binding sequence, AAAATAAA, was inserted in front of the open reading frames of the α₁- and β₁-subunit cDNAs and the first eight codons following the start-translation codons were changed to Dictyostelium preferred codons. These modified mRNAs appeared to be much less stable than the forms that were not readily translated. Dictyostelium could express the avian β-subunit alone but only expressed the α₁-subunit when the β₁-subunit was co-expressed. Subunit assembly occurred in cells expressing both α₁- and β₁-subunits. The bulk of the exogenously expressed sodium pump subunits remained in an intracellular compartment, presumed to be the endoplasmic reticulum. Dictyostelium exported little or no Na,K-ATPase or free β-subunit to the plasma membrane. Received: 7 July 1998/Revised: 8 October 1998     

Development of the salivary glands in embryos of Ixodes ricinus (Acari: Ixodidae)

We examined Ixodes ricinus embryos between 18 and 28 days of development with light, scanning and transmission electron microscopy. The differences in inner structure attested to establish three successive developmental stages: days 18–20, day 23, and days 26–28. Between 18 and 20 days the embryos are at early stages of organogenesis. Salivary glands cannot be identified at that stage. In 23-day-old embryos salivary glands are already outlined but the structure of alveoles is still different from that in larvae in which the embryonic development has been completed. Gland cells start to form alveoles and become active between 26 and 28 days of the development. This revised version was published online in July 2006 with corrections to the Cover Date.     

Immunolocalization of the Na,K-ATPase in photoreceptor cells of the moth Manduca sexta-evidence for Na,K-ATPase on both the nonmicrovillar and the microvillar domains of the plasma membrane

Immunohistochemical and physiological studies on various insect photoreceptors have demonstrated that the Na,K-ATPase (sodium pump) is restricted to the nonreceptive nonmicrovillar area of the plasma membrane. Here, we examined the distribution of the Na,K-ATPase in photoreceptor cells of the superposition-type compound eye in the moth Manduca sexta. Using immunofluorescent and immunogold cytochemistry, we show that the Na,K-ATPase is localized to both the nonmicrovillar and the microvillar parts of the plasma membrane. Manduca photoreceptors thus deviate from the common concept that the sodium pump and the molecular components of the photoreceptive machinery reside on different domains of the plasma membrane.     

Some characteristics of Na/K-ATPase from rat intestinal basal lateral membranes

Summary Basal lateral membrane vesicles were isolated from rat intestinal epithelial cells. The sodium potassium triphosphatase (Na/K-ATPase) of these plasma membranes has been characterized by (1) the molecular weight of the phosphorylated intermediate, (2) the sensitivity of the phosphorylated intermediate to hydroxylamine, (3) its ouabain binding constants, and (4) its susceptibility to digestion by pronase. The phosphorylated intermediate was shown by SDS polyacrylamide gel electrophoresis to be a protein of 100,000 Daltons apparent mol wt. Its extensive hydrolysis in hydroxylamine demonstrated that it was an acyl phosphate. The isolated basal lateral membranes bound ouabain with a dissociation constant,K _m (1.5×10^–5 m), similar to the inhibitory constantK _I (3×10^–5 m), measured for ouabain inhibition of the Na/K-ATPase activity. The association rate constant measured for ouabain binding at 22°C was 1.3×10³ m ^–1 sec^–1 and is similar to the association rate constants reported for other tissues and species. The high dissociation rate constant, 3.6×10^–2 sec^–1, is consistent with the insensitivity of the rat to ouabain. Digestion of the intact cells by pronase yielded basal lateral membranes in which the Na/K-ATPase had been unaffected. The phosphorylated intermediate ran as a sharp band at 100,000 Daltons on electrophoresis, and the ouabain dissociation constant appeared to be unchanged. In these membranes, protein stains of polyacrylamide gels revealed digestion of the major high mol wt proteins including the major protein at 100,000 Daltons. This suggests that the Na/K-ATPase represent a minor component, less than 1%, of the basal lateral membrane protein. From these characteristics of the phosphorylated intermediate and the ouabain binding constants, we conclude that the Na/K-ATPase of the basal lateral membranes of rat intestinal epithelial cells is similar to that found in other tissues and species. Estimates of the number of pump sites and the turnover number predict rates of Na transport that are consistent with observed values.This paper is dedicated to the memory of Professor David H. Smyth, FRS, who died on September 10, 1979.     

Palytoxin Acts on Na+,K+-ATPase but not Nongastric H+,K+-ATPase

Palytoxin (PTX) opens a pathway for ions to pass through Na,K-ATPase. We investigate here whether PTX also acts on nongastric H,K-ATPases. The following combinations of cRNA were expressed in Xenopus laevis oocytes: Bufo marinus bladder H,K-ATPase α₂- and Na,K-ATPase β₂-subunits; Bufo Na,K-ATPase α₁- and Na,K-ATPase β₂-subunits; and Bufo Na,K-ATPase β₂-subunit alone. The response to PTX was measured after blocking endogenous Xenopus Na,K-ATPase with 10 μm ouabain. Functional expression was confirmed by measuring ⁸⁶Rb uptake. PTX (5 nm) produced a large increase of membrane conductance in oocytes expressing Bufo Na,K-ATPase, but no significant increase occurred in oocytes expressing Bufo H,K-ATPase or in those injected with Bufo β₂-subunit alone. Expression of the following combinations of cDNA was investigated in HeLa cells: rat colonic H,K-ATPase α₁-subunit and Na,K-ATPase β₁-subunit; rat Na,K-ATPase α₂-subunit and Na,K-ATPase β₂-subunit; and rat Na,K-ATPase β₁- or Na,K-ATPase β₂-subunit alone. Measurement of increases in ⁸⁶Rb uptake confirmed that both rat Na,K and H,K pumps were functional in HeLa cells expressing rat colonic HKα₁/NKβ₁ and NKα₂/NKβ₂. Whole-cell patch-clamp measurements in HeLa cells expressing rat colonic HKα₁/NKβ₁ exposed to 100 nm PTX showed no significant increase of membrane current, and there was no membrane conductance increase in HeLa cells transfected with rat NKβ₁- or rat NKβ₂-subunit alone. However, in HeLa cells expressing rat NKα₂/NKβ₂, outward current was observed after pump activation by 20 mm K⁺ and a large membrane conductance increase occurred after 100 nm PTX. We conclude that nongastric H,K-ATPases are not sensitive to PTX when expressed in these cells, whereas PTX does act on Na,K-ATPase.     

Osmotic and ionic regulation in shore crabsCarcinus maenas inhabiting a tidal estuary

Shore crabsCarcinus maenas were exposed to salinities fluctuating according to the natural tidal rhythm. To this end they were maintained in net cages positioned in the estuarine waters of the river Elbe. The cages were lifted every hour, and between 8–12 specimens were analyzed for hemolymph concentrations of Na, K, Ca, Mg, and osmolality. The results obtained were compared with the respective data measured in external brackish water. In addition, the specific activity of Na−K-ATPase in a posterior gill was determined. Hemolymph Na and Mg as well as branchial Na−K-ATPase were also determined in crabs collected in the North Sea and the Baltic. The results show that inC. maenas living in salinities fluctuating with the tides by approx. 15‰ S, Na, K and Ca were hyperregulated, and Mg was effectively hyporegulated. The concentrations of all hemolymph ions and the activity of the Na−K-ATPase were kept constant over the whole tidal cycle. In Baltic crabs, Na was effectively hyperregulated and gill Na−K-ATPase was significantly elevated by a factor of ca 2 when compared with North Sea crabs. It is suggested that long-term hyperregulation of Na in constant salinities results from an increased number of Na−K-ATPase molecules which may change by synthesis or degradation following salinity stress. Constant hemolymph levels of hyperregulated Na in crabs inhabiting fluctuating brackish water are accomplished by activation of existing Na−K-ATPase by low Na and inhibition by higher ambient concentrations. This work is part of the first author's doctoral thesis submitted to the Department of Biology at the University of Hamburg.     

Developmental changes in β-subunit composition of Na,K-ATPase in the Drosophila eye

The Drosophila genome contains at least three loci for the Na,K-ATPase β-subunit; however, only the protein products of nrv1 and nrv2 have been characterized hitherto. Here, we provide evidence that nrv3 also encodes for a functional Na,K-ATPase β-subunit, as its protein product co-precipitates with the Na,K-ATPase α-subunit. Nrv3 expression in adult flies is restricted to the nervous system in which Nrv3 is enriched in selective types of sensory cells. Because Nrv3 expression is especially prominent in the compound eye, we have analyzed the subcellular and developmental distribution of Nrv3 within the visual cells and related this distribution to those of the α-subunit and of the β-subunits Nrv1 and Nrv2. Prospective visual cells express Nrv2 in the third larval instar stage and during the first half of pupal development. During the last third of pupal life, Nrv3 gradually replaces Nrv2 as the Na,K-ATPase β-subunit in the photoreceptor cells. Adult photoreceptors express Nrv3 as their major β-subunit; the visual cells R1–R6 co-express Nrv2 at a low level, whereas R7 and R8 co-express Nrv1. Notably, β-subunits do not co-distribute exactly with the α-subunit at some developmental stages, supporting the concept that the α-subunit and β-subunit can exist in the plasma membrane without being engaged in α/β heterodimers. The non-visual cells within the compound eye express almost exclusively Nrv2, which segregates together with the α-subunit to septate junctions throughout development.     

Fatty Acid-Induced Modulation of Ouabain Responsiveness of Rat Na,K-ATPase Isoforms

Membrane phospholipids represent a potential influence on the enzymatic properties of the Na,K-ATPase. Little is known concerning the effects of the fatty acid environment surrounding the enzyme on the kinetic properties of the Na,K-ATPase. We used the most obvious difference among the α isoforms of rat, their affinities for digitalis glycosides, to examine the relationship between the lipid environment and the Na,K-ATPase. Specific membrane environments that differ in their fatty acid composition were produced by drug-induced diabetes, as well as variations in diet. The α1 isoforms in various tissues were then characterized by their resistance to ouabain in Na,K-ATPase-enriched membrane microsomal fractions. The Na,K-ATPase activity in nerves and hearts were altered by diabetes and partially restored in nerves after a fish oil diet. Evaluation of enzyme kinetics (dose-response curves for ouabain) in membrane preparations allowed us to correlate the ouabain affinity of α1 isoform with fatty acid composition. The affinity of the α1 isoform for ouabain was significantly increased with accretions in the total amount of fatty acids of the n-6 series (P < 0.0001). Our observations provide a partial explanation for the observed difference in isoform properties among tissues. Moreover, these results underline the interaction between membrane fatty acids and the glycoside binding site of the Na,K-ATPase α1 subunit. Received: 15 June 1998/Revised: 18 November 1998     

Reproductive biology of the bigeye thresher shark,Alopias superciliosus (Lowe, 1839) (Chondrichthyes: Alopiidae), in the northwestern Pacific

Reproductive aspects ofAlopias superciliosus in the northwestern Pacific were described in detail, on the basis of 629 specimens (429 females and 200 males) collected from January 1984 to October 1984 and from October 1992 to March 1994.Alopias superciliosus embryos are oophagous. Six developmental stages (3 encapsulative and 3 posthatching) based on embryonic morphology and source of nutrition were recognized. The species bears 2 embryos per litter, their size at birth being between 135 and 140 cm TL. The sex ratio of embryos was 1∶1. Total length of females at maturity was 332–341. 1. cm; of males 270.1–287.6 cm. The gestation period could not be determined because most adult females were pregnant throughout the year. The typical reproductive strategy ofA. superciliosus is the production of a few large embryos per litter, with no fixed mating or birthing season.     

Changes of gas exchange parameters and of functional-biochemical properties of erythrocytes in dynamics of experimental anemia in rats

In experiments on male Wistar rats, in a specially constructed computerized installation, O₂ consumption by the animals in comparison with changes of hematological, biochemical, and rheological blood properties is studied after anemization—acute blood loss (12–15% of the total blood mass). An increase of the O₂ consumption by the organism and tissues by 18–28% has been revealed for the first 7 days after the blood loss, in spite of a pronounced decrease of hematocrit and of the amount of erythrocytes and hemoglobin in peripheral blood by 20–25% of the initial level. There was a 5–10-fold increase of the content of immature erythrocyte forms—reticulocytes and a progressive rise of cell acidic resistance, which is characteristic of young erythrocyte forms. An increase of O₂ consumption at a decrease of the blood oxygen capacity (a low hemoglobin level) seems to be due to the more efficient transport and yield of O₂ to tissues. At the 3rd and 7th day after the blood loss, activity of Na,K-ATPase has been found to increase by 60% and 20%, respectively. Analysis of the erythrocyte rheological properties has shown that the maximal firmness of aggregates (U_q) and the aggregation rate (1/T) decrease progressively beginning from 3 days after the blood loss; index of deformability (I_max) turned out to be elevated by 7–11%, probably due to an increase of the cell membrane elasticity. The conclusion is made that changes of erythrocyte rheological properties are interconnected with changes of the Na,K-ATPase activity and are directed at optimization of blood circulation in large vessels and the capillary network.     

Somatic embryogenesis and in vitro rosmarinic acid accumulation in Salvia officinalis and S. fruticosa leaf callus cultures

The effect of explant age, plant growth regulators and culture conditions on somatic embryogenesis and rosmarinic acid production from leaf explants of Salvia officinalis and S. fruticosa plants collected in Greece was investigated. Embryogenic callus with numerous spherical somatic embryos could be induced on explants derived from both species and cultured for 3 weeks on a Murashige and Skoog (MS) medium supplemented with 1.8–18 μm 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (Kin) or 10.5–21 μm 1-naphthalenacetic acid and 6-benzyladenine. Only explants from young plants (with six to eight leaves) responded to the culture treatments and, in general, low light intensities (50 μmol m^–2 s^–1) favoured callus formation and induction of somatic embryos. Somatic embryos were further developed on the same medium. Heart- and torpedo-shaped embryos (1–2 mm long) were subcultured on a growth-regulator-free MS medium for maturation. Maximum rosmarinic acid accumulation in S. officinalis and S. fruticosa callus cultured on 4.5 μm 2,4-D and 4.5 μm Kin was 25.9 and 29.0 g/l, respectively. Received: 17 January 1997 / Revision received: 26 May 1997 / Accepted: 30 June 1997     

Cation Activation of the Basolateral Sodium-Potassium Pump in Turtle Colon

The current generated by electrogenic sodium-potassium exchange at the basolateral membrane of the turtle colon can be measured directly in tissues that have been treated with serosal barium (to block the basolateral potassium conductance) and mucosal amphotericin B (to reduce the cation selectivity of the apical membrane). We studied the activation of this pump current by mucosal sodium and serosal potassium, rubidium, cesium, and ammonium. The kinetics of sodium activation were consistent with binding to three independent sites on the cytoplasmic side of the pump. The pump was not activated by cellular lithium ions. The kinetics of serosal cation activation were consistent with binding to two independent sites with the selectivity Rb > K > Cs > NH₄. The properties and kinetics of the basolateral Na/K pump in the turtle colon are at least qualitatively similar to those ofthe well-characterized Na/K-ATPase of the human red blood cell .     

Uptake and utilization of14C-glycine by embryos ofSebastes melanops

Synopsis The ability of embryos of the viviparous scorpaenidSebastes melanops to take up nutrients from an exogenous substrate was demonstrated by incubating embryos at various stages of development (18–30 days after fertilization) in¹⁴C-labeled glycine for 24 h. Uptake was highest for embryos at the latest stages (28–30 days) and increased at a linear rate during the incubation period. Nutrient uptake was not time dependent in embryos at the early stages (18–22 days). Nutrient utilization byS. melanops embryos was measured by the oxidation of¹⁴C-labeled glycine to¹⁴CO₂. The amount of respired¹⁴CO₂ by the oldest embryos increased significantly at a linear rate over the 24 h incubation period. There was no evidence of nutrient utilization by the youngest embryos. The developmental changes we observed in the uptake and utilization of exogenous glycine are supported by our previous findings that the oldest embryos have fully developed mouths and guts, and require additional nutrition from intraovarian sources at this stage of development.     

Immunolocalization of Na,K-ATPase in blowfly photoreceptor cells

The Na,K-ATPase (sodium pump) plays a central role in the physiology of arthropod photoreceptors as it re-establishes gradients for Na⁺ and K⁺ after light stimulation. We have mapped the distribution of the Na,K-ATPase in the photoreceptors of the blowfly (Calliphora erythrocephala) by immunofluorescent and immunogold cytochemistry, and demonstrate that the distribution pattern is more complex than previously presumed. High levels of sodium pumps have been detected consistently in all photoreceptors R1-8 on the nonreceptive surface, but no sodium pumps are found on the microvillar rhabdomere. Within the nonreceptive surface of the cells R1-6, however, the sodium pumps are confined to sites juxtaposed to neighboring photoreceptor or glial cells; no sodium pumps have been detected on the parts of the nonreceptive surface exposed to the intra-ommatidial space. In R7 and R8, the sodium pumps are found over the entire nonreceptive surface. The cytoskeletal protein spectrin colocalizes with the sodium pumps suggesting that linkage of the pump molecules to the spectrin-based submembrane cytoskeleton contributes to the maintenance of the complex pattern of pump distribution.     

Survival of the mosquito predator,Notonecta unifasciata[Hemiptera: Notonectidae] embryos at low thermal gradients

Notonecta unifasciata Guerin eggs maintained at different stages of embryonic development in water at variable temperatures (2.2–25.6 °C) and for periods of 4–12 weeks revealed maximum viability (>80 %) at the highest temperature. However, optimum nondevelopmental viability was at 14.4 °C with eight-day-old embryos (>35 %). Short term (4 weeks) storage at 14.4 °C significantly increased egg viability. Survival was poor (<20 %) at the 2 lowest temperatures. Eggs held at 14.4 °C for 12 weeks and sustainingca. 50 % mortality, may be a practical procedure for biological control.