Combined allosteric and competitive interaction between extracellular Na(+) and K(+) during ion transport by the alpha(1), alpha(2), and alpha(3) isoforms of the Na, K-ATPase

A combined allosteric and competitive model describes the interaction between extracellular Na(+) and Rb(+) during ion transport mediated by the Na, K-ATPase. The model was developed from experiments based on (86)Rb uptake by whole cells transfected with rat isoforms of the enzyme. In the absence of Na(+), only a single transport site for extracellular Rb(+) exists. After the occupation of the Na(+)-specific allosteric site, the Rb(+) transport pocket opens to allow occupation by an additional Rb(+) and the subsequent transport of the two Rb(+) ions into the cells. Na(+) can also directly compete with Rb(+) for binding to at least one of the transport sites. While the model derived here applies to each of the three rat isoforms of the Na, K-ATPase expressed in HeLa cells, subtle differences exist among the isoforms. The alpha(3)* isoform has an increased intrinsic affinity for Rb(+) and a lower affinity for the allosteric Na(+) site than alpha(1) or alpha(2)*. The stimulation of uptake observed according to the best-fit model is due to the displacement by Rb(+) of inhibitory Na(+) bound to the transport site.     

A phorbol ester-nonproliferative variant of Swiss 3T3 cells is deficient in Na+K+Cl- cotransport activity

The identity of the genetic defect(s) in Swiss 3T3 TNR-2 and TNR-9 that confers nonresponsiveness to the proliferative effect of 12-0-tetradecanoylphorbol-13-acetate (TPA) is not known. In BALB/c 3T3 cells, loss (via mutation) of a specific membrane ion transport system, the furosemide-sensitive Na+K+Cl- cotransporter, is associated with decreased responsiveness to TPA. In this study, the transport properties of parental Swiss 3T3 cells and the TPA-nonresponsive lines TNR-2 and TNR-9 were determined in the presence and absence of TPA. When the rate of 86Rb+ efflux (as a tracer for K+) was measured from each of the three cell lines, a furosemide- and TPA-inhibitable component of efflux was clearly evident in parental and TNR-9 cells but was virtually absent in TNR-2 cells. 86Rb+ influx measurements indicated the presence in parental 3T3 cells and the TNR-9 line of a substantial furosemide-sensitive flux that could be inhibited by TPA. In contrast, much less furosemide-sensitive influx was present in 3T3-TNR-2 cells and it was relatively unaffected by TPA. In both parental 3T3 and 3T3-TNR-2 cells, most of the furosemide-sensitive 86Rb+ influx is dependent on extracellular Na+ and Cl-. The apparent affinities of the transporter for these two ions, as well as for K+, were similar in both cell lines. In parental cells, the inhibition of furosemide-sensitive 86Rb+ influx was quite sensitive to TPA (K1/2 approximately equal to 1 nM) and occurred very rapidly after phorbol ester exposure. As expected because of its volume-regulatory role, inhibition of Na+K+Cl- cotransport by TPA in parental cells caused a substantial reduction in cell volume (25%). In contrast, because of the reduced level of cotransport activity in TNR-2 cells, TPA had only a slight effect on cell volume. These results suggest that the genetic defect in 3T3-TNR-2 cells (but not TNR-9 cells) responsible for nonresponsiveness to phorbol esters may be the reduction of Na+K+Cl- cotransport activity. Thus this membrane transport system may be an important component of the signal transduction pathway used by phorbol esters in 3T3 cells.     

The mechanism of insulin stimulation of (Na+,K+)-ATPase transport activity in muscle

Since the mechanism underlying the insulin stimulation of (Na+,K+)-ATPase transport activity observed in multiple tissues has remained undetermined, we have examined (Na+,K+)-ATPase transport activity (ouabain-sensitive 86Rb+ uptake) and Na+/H+ exchange transport (amiloride-sensitive 22Na+ influx) in differentiated BC3H-1 cultured myocytes as a model of insulin action in muscle. The active uptake of 86Rb+ was sensitive to physiological insulin concentrations (1 nM), yielding a maximum increase of 60% without any change in 86Rb+ permeability. In order to determine the mechanism of insulin stimulation of (Na+,K+)-ATPase activity, we demonstrated that insulin also stimulates passive 22Na+ influx by Na+/H+ exchange transport (maximal 200% increase) and an 80% increase in intracellular Na+ concentration with an identical time course and dose-response curve as insulin-stimulated (Na+,K+)-ATPase transport activity. Incubation of the cells with high [Na+] (195 mM) significantly potentiated insulin stimulation of ouabain-inhibitable 86Rb+ uptake. The ionophore monensin, which also promotes passive Na+ entry into BC3H-1 cells, mimics the insulin stimulation of ouabain-inhibitable 86Rb+ uptake. In contrast, incubation with amiloride or low [Na+] (10 mM), both of which inhibit Na+/H+ exchange transport, abolished the insulin stimulation of (Na+,K+)-ATPase transport activity. Furthermore, each of these insulin-stimulated transport activities displayed a similar sensitivity to amiloride. These results indicate that insulin stimulates a large increase in Na+/H+ exchange transport and that the resulting Na+ influx increases the intracellular Na+ concentration, thus activating the internal Na+ transport sites of the (Na+,K+)-ATPase. This Na+ influx is, therefore, the mediator of the insulin-induced stimulation of membrane (Na+,K+)-ATPase transport activity classically observed in muscle.     

Erythroid differentiation and the Na+,K+-pump in ouabain-sensitive and ouabain-resistant Friend erythroleukemia cell lines

The selection and biochemical characterization of ouabain-resistant erythroleukemia cell lines are described. Treatment of ouabain-resistant Friend erythroleukemia cell (FLC) lines with 1 mM ouabain demonstrated a reduced ouabain-sensitive 86Rb+-uptake after Na+-preloading in comparison with ouabain-sensitive cells. The ouabain- and diuretic (piretanide)-insensitive component of the 86Rb+-uptake (residual influx) was significantly enhanced in the ouabain-resistant FLC clones. Measurements of the Na+,K+-ATPase activity (E.C. 3.6.1.3) in plasma membrane preparations of the ouabain-resistant FLC clone B6/2 indicated that a ouabain-resistant Na+,K+-ATPase activity of about 20% of the total enzyme activity existed in the presence of 1 mM ouabain. Further experiments showed that the Na+,K+-ion-gradient in ouabain-resistant B6/2 cells was unaffected by ouabain exposure whereas the gradient collapsed in wild type 12 N cells. Another property of the ouabain-resistant cell lines was a decrease of the 86Rb+-uptake due to the Na+,K+, 2Cl(-)-cotransport system measured as piretanide-sensitive 86Rb+-uptake. The data on ion transport mechanisms in QuaR and QuaS FLC are discussed with respect to mutagen-induced and spontaneous cellular ouabain resistance. In addition, the role of altered ion transport mechanisms is considered for induced erythroid differentiation.     

Insulin and glucagon stimulation of (Na+-K+)-ATPase transport activity in isolated rat hepatocytes

The effects of insulin and glucagon on the (Na+-K+)-ATPase transport activity in freshly isolated rat hepatocytes were investigated by measuring the ouabain-sensitive, active uptake of 86Rb+. The active uptake of 86Rb+ was increased by 18% (p less than 0.05) in the presence of 100 nM insulin, and by 28% (p less than 0.005) in the presence of nM glucagon. These effects were detected as early as 2 min after hepatocyte exposure to either hormone. Half-maximal stimulation was observed with about 0.5 nm insulin and 0.3 nM glucagon. The stimulation of 86Rb+ uptake by insulin occurred in direct proportion to the steady state occupancy of a high affinity receptor by the hormone (the predominant insulin-binding species in hepatocytes at 37 degrees C. For glucagon, half-maximal response was obtained with about 5% of the total receptors occupied by the hormone. Amiloride (a specific inhibitor of Na+ influx) abolished the insulin stimulation of 86Rb+ uptake while inhibiting that of glucagon only partially. Accordingly, insulin was found to rapidly enhance the initial rate of 22Na+ uptake, whereas glucagon had no detectable effect on 22Na+ influx. These results indicate that monovalent cation transport is influenced by insulin and glucagon in isolated rat hepatocytes. In contrast to glucagon, which appears to enhance 86Rb+ influx through the (Na+-K+)-ATPase without affecting Na+ influx, insulin stimulates Na+ entry which in turn may increase the pump activity by increasing the availability of Na+ ions to internal Na+ transport sites of the (Na+-K+)-ATPase.     

Regulation and interconversion of the potassium transport systems of Saccharomyces cerevisiae as revealed by rubidium transport

The kinetics of Rb+ transport in Saccharomyces cerevisiae depended on the K+ content of the cells and on K+ starvation, as follows. In cells with normal K+ (grown at millimolar K+), Rb+ transport was regulated by internal K+. The loss of K+ first decreased the Km and later increased the Vmax of Rb+ transport. K+ starvation of normal-K+ cells for 4-5 h decreased the Km of Rb+ transport below the minimum observed after K+ loss. During this time Eadie-Hofstee plots of Rb+ transport suggest that the existing system was converted into a new one with a higher affinity. Growth at 10 microM K+ only required the system triggered by K+ loss, and the system expressed in K+-starved cells was not expressed under these conditions.     

Die energieabhängige Aufnahme von Thallium durch Chlorella

Summary The uptake of labelled thallous ion into the interior of Chlorella fusca is enhanced by light. This is ascribed to active transport by way of the K (and Rb) pump. The Michaelis constants differ between the higher and the lower concentration ranges, but they are similar for Rb and Tl.The additional, rapid, light-independent adsorption of Tl, mainly by the cell wall, is stronger than that of the alkali ions, but weaker than that of divalent cations. The adsorbed Tl is subject to elution by complexing agents (probably mainly glycolic acid) secreted by the cells, especially in the light.In spite of the similarity of the Michaelis constants, the inhibition of the light-dependent uptake of labelled Rb by unlabelled Tl is far stronger than the inhibition of Tl uptake by Rb. The efficiency of Tl may be due to double action: namely, competition with Rb for the carrier and negative influence on a transport ATPase.

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Herrn Prof. Dr. H. Nowotny gewidmet.     

Induction of cystine transport activity in human fibroblasts by oxygen

The transport activity for cystine in cultured human fibroblasts decreased after incubation of the cells under a low oxygen concentration. After the incubation for 48 h under 3% oxygen, the Vmax of the transport was decreased to less than one-third of that of the control cells, with little change in Km. The similar transport activity was observed in the cells cultured under 3% oxygen for 10-40 days with several times of passages. When these low oxygen-cultured cells were incubated under room air, the activity was enhanced with a lag of about 4 h and was almost completely restored within 24 h. This restoration required protein synthesis. The cystine transport activity increased by 50% after exposure of the cells to hyperoxia (40% oxygen). From these results it is concluded that the transport activity for cystine is induced by oxygen. In contrast, little change in the transport activities for alanine and leucine occurred in the cells exposed to the corresponding hypoxia or hyperoxia. Since the cystine transported into the cells is reduced to cysteine and the cysteine readily exits to the culture medium where it autoxidizes to cystine, a cystine-cysteine cycle across the plasma membrane has been postulated. Since the autoxidation of cysteine in the culture medium was markedly slowed down under the low oxygen concentration, the change in the cystine transport activity in response to the oxygen concentration was regarded as pertinent. Induction of the cystine transport activity may constitute a protective mechanism against the oxidative stress, to which the culture cells are exposed, by providing the cells with cysteine which is mainly incorporated into glutathione.     

Insulin action on cardiac glucose transport. Studies on the role of the Na+/K+ pump

Isolated muscle cells from adult rat heart have been used to study the relationship between myocardial glucose transport and the activity of the Na+/K+ pump. 86Rb+-uptake by cardiac cells was found to be linear up to 2 min with a steady-state reached by 40-60 min, and was used to monitor the activity of the Na+/K+ pump. Ouabain (10(-3) mol/l) inhibited the steady-state uptake of 86Rb+ by more than 90%. Both, the ouabain-sensitive and ouabain-insensitive 86Rb+-uptake by cardiac cells were found to be unaffected by insulin treatment under conditions where a significant stimulation of 3-O-methylglucose transport occurred. 86Rb+-uptake was markedly reduced by the presence of calcium and/or magnesium, but remained unresponsive towards insulin treatment. Inhibition of the Na+/K+ pump activity by ouabain and a concomitant shift in the intracellular Na+ :K+ ratio did not affect basal or insulin stimulated rates of 3-O-methylglucose transport in cardiac myocytes. The data argue against a functional relationship between the myocardial Na+/K+ pump and the glucose transport system.     

Early transport changes during erythroid differentiation of Friend leukemic cells

Early transport changes occurring during Friend erythroleukemic cell differentiation are reported. A decrease in the rate of 86Rb transport was observed beginning approximately five hours after stimulation with 1.5% dimethylsulfoxide (DMSO), a potent inducer of Friend cell differentiation. By 12 to 14 hours after DMSO addition, the transport rate had stabilized at close to 60% of control level. This decrease in the rate of 86Rb transport preceded a previously reported decrease in cell volume. Other chemical inducers of Friend cells, such as hypoxanthine and ouabain, also caused early decreases in 86Rb influx. In contrast, xanthine, which does not induce Friend cell differentiation, also did not affect 86Rb influx. The transport of two amino acid analogues, alpha-aminoisobutyric acid and 2-aminobicyclo [2,2,1]-heptane-2-carboxylic acid, which differ in their mode of uptake, was also measured following induction by DMSO. The transport rates of both compounds decreased after a 12-hour exposure to DMSO. In contrast, the uptake of 3H-colchicine, a drug which diffuses passively across the cell membrane, was not significantly affected. Studies with several variant cell lines which do not synthesize hemoglobin in response to DMSO indicate that these non-inducible cells can be divided into two classes--those that demonstrate early changes in transport very similar to the changes observed in inducible cell lines and those which exhibit only small changes in transport. Results obtained using a revertant clone have helped to distinguish between those transport changes which are associated with the induction of hemoglobin synthesis and those which are not. In addition, these early transport changes may be useful in defining the stage in the differentiation process at which a particular variant line is blocked.     

Methanogenesis and the K+ transport system are activated by divalent cations in ammonia-treated cells of Methanospirillum hungatei

We describe a K+ transport system in Methanospirillum hungatei cells depleted of cytoplasmic K+ via an ammonia/K+ exchange reaction (Sprott, G. D., Shaw, K. M., and Jarrell, K. F. (1984) J. Biol. Chem. 259, 12602-12608). Ammonia-treated cells contained low concentrations of ATP and were unable to make CH4 or to transport 86Rb+. All of these properties were restored by CaCl2, MgCl2, or MnCl2, and not by CoCl2 or NiCl2. The Rb+ transport system had a Km of 0.42 and Vmax of 29 nmol/min X mg; K+ inhibited competitively. Both H2 and CO2 were required for appreciable transport, whereas air, valinomycin, or nigericin were potent inhibitors. The influx of Rb+ was electrogenic and associated with proton efflux, producing a delta pH (alkaline inside) in acidic media. In the absence of K+ (or Rb+), the activation of CH4 synthesis by Mg2+ produced little change in the cytoplasmic pH, showing that methanogenesis did not elicit a net efflux of protons. The pH optimum for transport was in the range 6.0-7.3 where the transmembrane pH gradient would contribute minimally to the proton motive force. Protonophores at pH 6.3 caused a partial decline in CH4 synthesis and the ATP content and dramatically collapsed Rb+ transport. These and other inhibitor experiments, coupled with the fact that the Rb+ gradient was too large to be in equilibrium with the proton motive force alone, suggest a role for both ATP and the proton motive force in Rb+ transport. Also, a role for K+ in osmoregulation is indicated.     

Regulatory interaction of ATP Na+ and Cl- in the turnover cycle of the NaK2Cl cotransporter

To probe the mechanism by which intracellular ATP, Na+, and Cl- influence the activity of the NaK2Cl cotransporter, we measured bumetanide-sensitive (BS) 86Rb fluxes in the osteosarcoma cell line UMR- 106-01. Under physiological gradients of Na+, K+, and Cl-, depleting cellular ATP by incubation with deoxyglucose and antimycin A (DOG/AA) for 20 min at 37 degrees C reduced BS 86Rb uptake from 6 to 1 nmol/mg protein per min. Similar incubation with 0.5 mM ouabain to inhibit the Na+ pump had no effect on the uptake, excluding the possibility that DOG/AA inhibited the uptake by modifying the cellular Na+ and K+ gradients. Loading the cells with Na+ and depleting them of K+ by a 2-3- h incubation with ouabain or DOG/AA increased the rate of BS 86Rb uptake to approximately 12 nmol/mg protein per min. The unidirectional BS 86Rb influx into control cells was approximately 10 times faster than the unidirectional BS 86Rb efflux. On the other hand, at steady state the unidirectional BS 86Rb influx and efflux in ouabain-treated cells were similar, suggesting that most of the BS 86Rb uptake into the ouabain-treated cells is due to K+/K+ exchange. The entire BS 86Rb uptake into ouabain-treated cells was insensitive to depletion of cellular ATP. However, the influx could be converted to ATP-sensitive influx by reducing cellular Cl- and/or Na+ in ouabain-treated cells to impose conditions for net uptake of the ions. The BS 86Rb uptake in ouabain-treated cells required the presence of Na+, K+, and Cl- in the extracellular medium. Thus, loading the cells with Na+ induced rapid 86Rb (K+) influx and efflux which, unlike net uptake, were insensitive to cellular ATP. Therefore, we suggest that ATP regulates a step in the turnover cycle of the cotransporter that is required for net but not K+/K+ exchange fluxes. Depleting control cells of Cl- increased BS 86Rb uptake from medium-containing physiological Na+ and K+ concentrations from 6 to approximately 15 nmol/mg protein per min. The uptake was blocked by depletion of cellular ATP with DOG/AA and required the presence of all three ions in the external medium. Thus, intracellular Cl- appears to influence net uptake by the cotransporter. Depletion of intracellular Na+ was as effective as depletion of Cl- in stimulating BS 86Rb uptake.(ABSTRACT TRUNCATED AT 400 WORDS)     

Coupling of a loop diuretic-sensitive Na+ influx with the net loop diuretic-sensitive K+ efflux in mouse NIH 3T3 cells

Mouse 3T3 fibroblasts have a loop diuretic sensitive Na+ transport system, responsible for more than 50% of the total Na+ influx. This transport system is dependent on the simultaneous presence of all three ions; Na+, K+, (Rb+) and Cl- in the extracellular medium. The same requirement for these three ions was also found for the loop diuretic-sensitive K+ efflux. In addition, the sensitivities of Na+ influx and Rb+ efflux for the two loop diuretics, furosemide and bumetanide were found to be similar. The similar ionic requirement and sensitivity towards loop diuretics of the two fluxes, support the hypothesis, that this loop diuretic-sensitive Na+ influx in mouse 3T3 cells, is accompanied by the net loop diuretic-sensitive K+ efflux.     

Correlation between Ion Fluxes and Ion-stimulated Adenosine Triphosphatase Activity of Plant Roots

The energy-dependent influx of Rb(+) into excised roots of corn, wheat, and barley has been determined and compared to the Rb(+)-stimulated ATPase activity of membrane fractions obtained from root homogenates of these species. The external Rb(+) concentrations studied were in the range of 1 to 50 mm. The ratio of Rb(+) influx/Rb(+)-stimulated ATPase was approximately 0.85 and was nearly constant for all the species and Rb(+) concentrations studied. The correlation coefficient for Rb(+) influx versus Rb(+)-activated ATPase was 0.94. The results support the concept that ATP is the energy source for ion transport in roots and that an ATPase participates in the energy transduction process involved in energy-dependent ion transport.     

Characterization of Rb uptake into Sf9 cells using cation chromatography: evidence for a K-Cl cotransporter

To assess cation-chloride cotransporter activity in Sf9 cells, cation chromatography was used to measure initial uptake rates of Rb. Rb exchanged with cellular K, with 30% of cellular K replaced after a 40 min exposure to Rb. Rb uptake into Sf9 cells was not inhibited by 50 μmol l(-1) ouabain. Rb uptake was approximately 65% inhibited by 250 μmol l(-1) bumetanide added to the assay solution, and was more than 95% inhibited when cells were pre-incubated for 20 min with bumetanide (100 and 1000 μmol l(-1)). Uptake of Rb and Cl followed simple Michaelis-Menten kinetics, with a K(m) for Rb of 17.1+/-2.2 mmol l(-1) and a K(m) for Cl of 93.7+/-5.6 mmol l(-1). Rb uptake was not dependent upon extracellular Na. Two min exposures to solutions with reduced [Na] or [Cl] produced small but significant changes in cellular Na content. We conclude that the primary Rb uptake pathway in Sf9 cells is a K-Cl cotransporter and that cation chromatography can be used to effectively study kinetic parameters of cotransporter function in tissue culture cells. Characterization of baseline cation-chloride cotransporter activity in Sf9 cells strengthens their utility as a tool for expression and characterization of exogenous proteins.     

Characterization of low potassium-resistant mutants of the Madin-Darby canine kidney cell line with defects in NaCl/KCl symport

Three independent mutants of the Madin-Darby canine kidney cell line (MDCK) have been isolated which were capable of growth in media containing low concentrations of potassium. All three mutants were deficient to varying extents in furosemide- and bumetanide-sensitive 22Na+, 86+b+, and 36Cl- uptake. The two mutants most resistant to low K+ media had lost essentially all of the 22Na+, 86Rb+, and 36Cl- uptake activities of this system. The third mutant was partially resistant to low K+ media and had reduced levels of bumetanide-sensitive uptake for all three ions. Extrapolated initial uptake rates for 22Na+, 86Rb+, and 36Cl- revealed that the partial mutant exhibited approximately 50% of the parental uptake rates for all three ions. The stoichiometries of bumetanide-sensitive uptake in both the parental cell line and the partial mutant approximated 1 Rb+:1 Na+:2 Cl-. The results of this study provide genetic evidence for a single tightly-coupled NaCl/KCl symporter in MDCK cells. The correlation between the ability to grow in low K+ media and decreased activity of the bumetanide-sensitive co-transport system suggests that the bumetanide-sensitive transport system catalyzes net K+ efflux from cells in low K+ media. The results of 86Rb+ efflux studies conducted on ouabain-pretreated mutant and parental cells are consistent with this interpretation. Cell volume measurements made on cells at different densities in media containing normal K+ concentrations showed that none of the mutants differed significantly in volume from the parental strain at a similar cell density. Furthermore, all three mutants were able to readjust their volume after suspension in hypotonic media. These results suggest that in the MDCK cell line, the bumetanide-sensitive NaCl/KCl symport system does not function in the regulation of cell volume under the conditions employed.     

Copper ion redox state is critical for its effects on ion transport pathways and methaemoglobin formation in trout erythrocytes.

We have studied the mechanism of copper uptake by the cells, its oxidative action and effects on ion transport systems using rainbow trout erythrocytes. Cupric ions enter trout erythrocytes as negatively charged complexes with chloride and hydroxyl anions via the band 3-mediated Cl-/HCO3- exchanger. Replacement of Cl- by gluconate, and complexation of cupric ions with histidine abolish rapid Cu2+ uptake. Within the cell cupric ions interact with haemoglobin, causing methaemoglobin formation by direct electron transfer from heme Fe2+ to Cu2+, and consecutive proton release. Ascorbate-mediated reduction of cupric ions to cuprous decreases copper-induced metHb formation and proton release. Moreover, cuprous ions stimulate Na+H+ exchange and residual Na+ transport causing net Na+ accumulation in the cells. The effect requires copper binding to an externally facing thiol group. Copper-induced Na+ accumulation is accompanied by K+ loss occurring mainly via K+-Cl- cotransporter. Taurine efflux is also stimulated by copper exposure. However, net loss of osmolytes is not as pronounced as Na+ uptake and modest swelling of the cells occurs after 5 min of copper exposure. Taken together the results indicate that copper toxicity, including copper transport into the cells and its interactions with ion transport processes, depend on the valency and complex formation of copper ions.     

Radioactive 86rubidium influx into red blood cells in essential hypertension in relation to the plasma renin activity

Changes in several mechanisms of sodium transport across the cell membranes are described in essential hypertension. We studied ouabain-sensitive and insensitive 86Rb+ influx into the red blood cells (RBC) of 16 healthy controls and 51 patients with essential hypertension (EH) divided according to their plasma renin activity (PRA) in 3 groups: 11 patients with high PRA (HREH), 18 patients with normal PRA (NREH) and 22 patients with low PRA (LREH). In addition to studying 86RB+ uptake by patients RBC, we tested also the effect of the patients' sera on 86Rb+ influx into the RBC of healthy subjects. Red blood cells of patients with HREH and NREH had lower ouabain-sensitive 86Rb+ influx in comparison with controls. No significant differences were found between these hypertensive groups. In contrast 86Rb+ uptake by the RBC of LREH patients was always higher than in controls or HREH and NREH. It was chiefly the ouabain-sensitive component that was raised, but some increase in ouabain-insensitive 86Rb+ influx also could be seen. The serum of patients with HREH and NREH, when incubated with RBC of healthy controls, lowered their ouabain-sensitive 86Rb+ influx. The decrease was more pronounced in NREH than in HREH group. Plasma from LREH patients increased both ouabain-sensitive and ouabain-insensitive 86Rb+ influx into the control RBC. These findings indicate that there may be differences in the sodium/potassium transport mechanisms across the cell membrane in various kinds of EH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Arsenate substitutes for phosphate in the human red cell sodium pump and anion exchanger

The sodium pump of human red blood cells mediates a Rb:Rb exchange that is dependent for maximal rates upon the simultaneous presence of intracellular ATP (or ADP) and phosphate. We have measured ouabain-sensitive 86Rb uptake into resealed ghosts of human red cells containing ADP and show that arsenate will substitute for phosphate in supporting the Rb:Rb exchange transport mode. The concentration dependence of arsenate-supported Rb:Rb exchange in ghosts containing 2 mM ADP shows both activating and inhibiting phases; the dependence upon phosphate shows similar characteristics. Elevation of the external [Rb] lowers the apparent affinity for arsenate since there is a shift to higher concentrations of arsenate in the activating and inhibiting phases of the arsenate concentration dependence curve. Similarly, elevation of [ADP] substantially reduces the inhibition of Rb:Rb exchange observed at higher [arsenate]. These effects are also observed in phosphate-supported Rb:Rb exchange. The phosphate requirement for Rb:Rb exchange involves phosphorylation of the sodium pump protein; the close agreement between the effects of arsenate and phosphate in supporting Rb:Rb exchange makes it likely that arsenylation of the sodium pump occurs during Rb:Rb exchange. Arsenate efflux from red blood cell ghosts into arsenate-free chloride medium is partially inhibited (77-80%) by DNDS (4,4'-dinitro-2,2'-stilbenedisulfonic acid), this compares with 82-87% inhibition by DNDS of phosphate efflux under the same conditions. It appears that Band III, the red cell anion transport system, accepts arsenate in a similar fashion to phosphate and that a fraction of the flux of both anions may occur through pathways other than Band III. Thus, in human red blood cells, both the sodium pump and the anion exchange transport system will accept arsenate as a phosphate congener and the protein-arsenate interactions are very similar to those with phosphate.     

Stimulation of K+ transport systems by Ha-ras

The expression of Ha-ras in quiescent NIH3T3 cells carrying a glucocorticoid-inducible human Ha-ras gene (Val-Gly mutation at codon 12) stimulates total 86Rb+ influx. This effect is predominantly due to an elevated 86Rb+ uptake through an ouabain-resistant, furosemide-sensitive system. The ouabain-sensitive Na+/K(+)-ATPase is less affected. The transport which is resistant to both inhibitors is not altered by Ha-ras. Overexpression of the Ha-ras proto-oncogene causes only a marginal increase in total 86Rb+ uptake. The stimulation of the furosemide-sensitive influx by Ha-ras is paralleled by an increase in mean cell volume which can be inhibited by furosemide. A rapid stimulation of the furosemide-sensitive Rb+ influx is also observed after addition of bombesin to growth-arrested cells. Furosemide inhibits the mitogenic response after expression of Ha-ras or addition of bombesin. Both the Ha-ras and the bombesin-induced stimulation of the furosemide-sensitive Rb+ transport can be blocked by protein kinase C depletion or the protein kinase C inhibitor staurosporine. In contrast to bombesin-induced phosphatidylinositol-4,5-bisphosphate hydrolysis which is down-modulated by Ha-ras, the stimulation of the furosemide-sensitive Rb+ influx by bombesin is elevated in Ha-ras-expressing cells. This is in accordance with the increased mitogenic activity of bombesin in Ha-ras-expressing cells.