Saturable ammonium ion transport in myeloma and hybridoma cells is mediated by the Na+K+2Cl--cotransporter

The total entry of ammonium ions into Sp2/0-Ag14 myeloma cells and hybridoma cells consists of a saturable and a non-saturable component. The plasma membrane Na+K+2Cl--cotransporter was identified as the saturable ammonium ion transporter in both cell lines, and the non-saturable entry was due to simple diffusion of ammonium ions. The theoretical maximum transport rate via the Na+K+2Cl--cotransporter was identical in the two cell lines, but the ammonium ion diffusion rate was considerably higher in the hybridoma cells. We speculate that this is an effect of different membrane properties caused by dissimilar expression of tumour characteristics.     

Role of Ca2+, Mg2+ and K+ ions in determining apoptosis and extent of suppression afforded by bcl-2 during hybridoma cell culture

We have addressed the possibility that Ca²⁺, Mg²⁺ and K⁺ ions play a central role in governing the morphological and biochemical changes attributed to apoptotic cell death. By removing Ca²⁺, Mg²⁺ or K⁺ ions from the cell culture medium we were able to assess the contribution of each ion to hybridoma cell growth and viability. The differences were explained in terms of a possible reduction in their respective intracellular levels. From several lines of evidence, the deprivation of K⁺ ions was the most detrimental to cellular growth and viability and induced significant levels of early apoptotic cells. Another effect of this deprivation was to weaken the plasma membranes without causing membrane breakdown; exposure to high agitation rates confirmed fragility of the cell membranes. Removal of Mg²⁺ caused a reduction in the levels of early apoptotic cells and predisposed cells to high levels of primary necrotic death. The lower levels of apoptotic cells failed to demonstrate the classic nuclear morphology associated with apoptosis, while retaining other apoptotic features. These results highlighted the importance of utilizing several assays for the determination of apoptosis. The absence of Ca²⁺ appeared to be the mildest insult, but its deprivation did accelerate a significant decline in culture by increasing apoptotic death. Hybridoma cells overexpressing the apoptotic suppresser gene bcl-2 were protected from the predominantly necrosis inducing effects of Mg²⁺ ion deprivation and apoptosis inducing effects of Ca²⁺ ion deprivation. However, apoptosis was not as effectively suppressed in bcl-2 cells responding to incubation in K⁺ free medium. The inclusion of bcl-2 activity in the mechanisms of Ca²⁺ Mg²⁺ or K⁺ deprivation induced cell death emphasizes a close relationship between ionic dissipation and the apoptotic process.     

Potassium and sodium balance in U937 cells during apoptosis with and without cell shrinkage.

Staurosporine (STS) and etoposide (Eto) induced apoptosis of the human histiocytic lymphoma cells U937 were studied to determine the role of monovalent ions in apoptotic cell shrinkage. Cell shrinkage, defined as cell dehydration, was assayed by measurement of buoyant density of cells in continuous Percoll gradient. The K+ and Na+ content in cells of different density fractions was estimated by flame emission analysis. Apoptosis was evaluated by confocal microscopy and flow cytometry of acridine orange stained cells, by flow DNA cytometry and by effector caspase activity. Apoptosis of U937 cells induced by 1 muM STS for 4 h was found to be paralleled by an increase in buoyant density indicating cell shrinkage. An increase in density was accompanied by a decrease in K+ content (from 1.1 to 0.78 mmol/g protein), which exceeded the increase in Na+ content (from 0.30 to 0.34 mmol/g) and resulted in a significant decrease of the total K+ and Na+ content (from 1.4 to 1.1 mmol/g). In contrast to STS, 50 microM Eto for 4 h or 0.8-8 microM Eto for 18-24 h induced apoptosis without triggering cell shrinkage. During apoptosis of U937 cells induced by Eto the intracellular K(+)/Na+ ratio decreased like in the cells treated with STS, but the total K+ and Na+ content remained virtually the same due to a decrease in K+ content being nearly the same as an increase in Na+ content. Apoptotic cell dehydration correlated with the shift of the total cellular K+ and Na+ content. There was no statistically significant decrease in K+ concentration per cell water during apoptosis induced by either Eto (by 13.5%) or STS (by 8%), whereas increase in Na+ concentration per cell water was statistically significant (by 27% and 47%, respectively). The data show that apoptosis can occur without cell shrinkage-dehydration, that apoptosis with shrinkage is mostly due to a decrease in cellular K+ content, and that this decrease is not accompanied by a significant decrease of K+ concentration in cell water.     

Alterations in Ca2+-binding on plasma membrane after adaptation to salt stress of tobacco cells in suspension

Electrophoresis was used to study effects of salinity on the characteristics of Ca2+ binding to the outer surface of plasma membrane (PM) of protoplasts isolated from two types of tobacco (Nicotiana tabacum L., cv. Bright Yellow) cultured cells that were adapted (tolerant) and unadapted (sensitive) to 50 mM NaCl stress. Electrophoretic analysis of salt-sensitive NaCl-unadapted cells shows that Na+ induced an appreciably higher degree of reduction in the amount of Ca2+ bound to PM compared with K+ with increasing concentration from 0.1 to 30 mM. In salt-tolerant NaCl-adapted cells, however, both Na+ and K+ ions induced almost the same degree of reduction in the amount of Ca2+ bound to PM in the physiological concentration range of Ca2+ in the medium between 2 and 4 mM. These results suggest that, under the physiological conditions, PM of salt-sensitive NaCl-unadapted cells has an appreciable amount of PM-bound Ca2+ that is desorbed much easier by Na+ than K+, whereas PM of salt-tolerant NaCl-adapted cells has the PM-bound Ca2+ that can be equally desorbed by Na+ and K+.     

Analysis of the monovalent ion fluxes in U937 cells under the balanced ion distribution: recognition of ion transporters responsible for changes in cell ion and water balance during apoptosis

Unidirectional (22)Na, Li(+) and Rb(+) fluxes and net fluxes of Na(+) and K(+) were measured in U937 human leukemic cells before and after induction of apoptosis by staurosporine (1 microM, 4 h) to answer the question which ion transporter(s) are responsible for changes in cell ion and water balance at apoptosis. The original version of the mathematical model of cell ion and water balance was used for analysis of the unidirectional ion fluxes under the balanced distribution of major monovalent ions across the cell membrane. The values of all major components of the Na(+) and K(+) efflux and influx, i.e. fluxes via the Na(+),K(+)-ATPase pump, Na(+) channels, K(+) channels, Na/Na exchanger and Na-Cl symport were determined. It is concluded that apoptotic cell shrinkage and changes in Na(+) and K(+) fluxes typical of apoptosis in U937 cells induced by staurosporine are caused by a complex decrease in the pump activity, Na-Cl symport and integral Na(+) channel permeability.     

Secondary structure of heart sarcolemmal proteins during interaction with metallic cofactors of (Na+ + K+)-ATPase

he secondary structure of membrane proteins was studied in rat heart sarcolemma by circular dichroism under conditions of interaction with metallic cofactors of (Na+ + K+)-ATPase at their optimal concentrations and under metal free conditions. Approximately 80 per cent of polypeptide chains in the membrane were organized in alpha-helical structure. Upon stabilizing the E1. Na conformation state of (Na+ + K+)-ATPase by Mg2+ and Na+ ions, only a slight increase in the protein alpha-helix content (to 83 per cent) was observed. On the other hand, simultaneous addition of Mg2+ and K+ ions resulting in the establishment of the E2 . K conformational state of the enzyme, was followed by a significant decrease in the membrane protein helicity (to 72 per cent). The presence of all three metallic cofactors of (Na+ + K+)-ATPase did not induce any further conformational change in sarcolemmal proteins as compared to the state induced by the interaction with Mg2+ and Na+ ions. In contrast to results obtained with Mg2+ ions, the interaction of Na+ with the sarcolemmal membranes led to a considerable decrease and that of K+ to a significant increase in alpha-helicity of the membrane polypeptides. These findings have confirmed the regulatory role of magnesium in transition of the conformational state from E1 to E2 in the reaction sequence of (Na+ + K+)-ATPase. Specific modulation by Na+ and K+ of the helicity of sarcolemmal proteins in the presence of Mg2+ and in the absence of ATP might be considered as a preprint of conformational changes which will occur in the presence of ATP.     

ATP-driven exchange of Na+ and K+ ions by Streptococcus faecalis

We describe the characterization of KtrII, a novel potassium transport system of Streptococcus faecalis, first discovered by H. Kobayashi [1982) J. Bacteriol. 150, 506-511). KtrII requires sodium ions and mediates the stoichiometric exchange of internal Na+ for external K+. Potassium accumulation is not energized by the electrochemical potentials of either H+ or Na+; the energy source is probably ATP. Two lines of evidence indicate that KtrII is a manifestation of the sodium-stimulated ATPase reported earlier (Heefner, D. L., and Harold, F. M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2798-2802). (i) Mutants that lack the ATPase also lack KtrII, and revertants recover both in parallel. (ii) KtrII and the Na+-ATPase are induced in parallel when cells are grown on media rich in sodium, particularly under conditions that limit the generation of a proton potential. KtrII is not induced in response to K+ deprivation. We propose that the Na+-ATPase exchanges Na+ for K+ ions.     

Arachidonic acid induces both Na+ and Ca2+ entry resulting in apoptosis

Marked accumulation of arachidonic acid (AA) and intracellular Ca2+ and Na+ overloads are seen during brain ischemia. In this study, we show that, in neurons, AA induces cytosolic Na+ ([Na+](cyt)) and Ca2+ ([Ca2+](cyt)) overload via a non-selective cation conductance (NSCC), resulting in mitochondrial [Na+](m) and [Ca2+](m) overload. Another two types of free fatty acids, including oleic acid and eicosapentaenoic acid, induced a smaller increase in the [Ca2+](i) and [Na+](i). RU360, a selective inhibitor of the mitochondrial Ca2+ uniporter, inhibited the AA-induced [Ca2+](m) and [Na+](m) overload, but not the [Ca2+](cyt) and [Na+](cyt) overload. The [Na+](m) overload was also markedly inhibited by either Ca2+-free medium or CGP3715, a selective inhibitor of the mitochondrial Na+(cyt)-Ca2+(m) exchanger. Moreover, RU360, Ca2+-free medium, Na+-free medium, or cyclosporin A (CsA) largely prevented AA-induced opening of the mitochondrial permeability transition pore, cytochrome c release, and caspase 3-dependent neuronal apoptosis. Importantly, Na+-ionophore/Ca2+-free medium, which induced [Na+](m) overload, but not [Ca2+](m) overload, also caused cyclosporin A-sensitive mitochondrial permeability transition pore opening, resulting in caspase 3-dependent apoptosis, indicating that [Na+](m) overload per se induced apoptosis. Our results therefore suggest that AA-induced [Na+](m) overload, acting via activation of the NSCC, is an important upstream signal in the mitochondrial-mediated apoptotic pathway. The NSCC may therefore act as a potential neuronal death pore which is activated by AA accumulation under pathological conditions.     

Toxin-induced K+ efflux through the Na+ channel of neuroblastoma cells

Neurotoxins which modify the gating system of the Na+ channel in neuroblastoma cells and increase the initial rate of 22Na+ influx through this channel also give rise to the efflux of 86Rb+ and 42K+. These effluxes are inhibited by tetrodotoxin and are dependent on the presence in the extracellular medium of cations permeable to the Na+ channel. These stimulated effluxes are not due to membrane depolarization or increases in the intracellular content of Na+ and Ca2+ which occur subsequent to the action of neurotoxins. The relationships of 22Na+ influx and 42K+ (or 86Rb+) effluxes to both the concentration of neurotoxins and the concentration of external permeant cations strongly suggest that the open form of the Na+ channel stabilized by neurotoxins permits an efflux of K+ ions. Our results indicate that for the efflux of each K+ ion there is a corresponding influx of two Na+ ions into the Na+ channel.     

Cell shrinkage and apoptosis: a role for potassium and sodium ion efflux

In this study we have shown that redistribution of the lipid composition of the external and internal leaflets of the PM during apoptosis results in two main alterations in the cell surface, externalisation of PS, and a looser packing of the lipid hydrophobic head groups in the external leaflet. Significantly, neither of these alterations can be directly implicated in the mechanism of apoptotic cell shrinkage, however they do have functions in other phases of the apoptotic process. Progressional studies involving morphological and flow cytometric evaluation, and DNA gel electrophoresis revealed that apoptotic cell shrinkage is associated with a decrease in [Na+]i and [K+]i which occurs after visualisation of chromatin condensation and internucleosomal DNA fragmentation, and prior to apoptotic body formation. When apoptotic cultures were supplemented with inhibitors of the Na+, K+-ATPase pump or the Ca2+-dependent K+ channel, essentially all of the respective Na+ or K+ efflux during apoptosis can be inhibited, suggesting that essentially all of the Na+ and K+ efflux can be ascribed to active pumping via the Na+, K+-ATPase pump and the Ca2+-dependent K+ channel.     

Regulatory volume increase (RVI) and apoptotic volume decrease (AVD) in U937 cells placed into hyperosmotic medium

Time course of changes in intracellular water, K+ and Na+ of U937 cells incubated in hyperosmolar medium with addition of 200 mM sucrose was studied. Ouabain-sensitive and ouabain-resistant Rb+ (K+) influxes were measured during regulatory cell volume increase (RVI) and apoptotic volume decrease (AVD). Microscopy of cells stained by Acrydine orange, Ethydium bromide, APOPercenrage Dye and polycaspase marker FLICA was performed. We found that initial osmotic cell shrinkage induced both RVI and AVD responses. RVI dominated at the early stage whereas AVD prevailed at the later stage. In view of the data obtained in U937 cells the current opinion that RVI "dysfunction" is a prerequisite for apoptosis and AVD (Subramanyam et al., 2010) should be revised. U937 cells are capable to trigger of apoptosis and AVD in spite of the unimpaired RVI response. It is concluded that AVD plays a significant role in preventing osmotic lysis of apoptotic cells rather than in the initiation of apoptosis.     

Induction of apoptosis by bufalin in human tumor cells is associated with a change of intracellular concentration of Na+ ions.

In an attempt to characterize the mechanisms that are operative at the early stages of the induction of apoptosis by bufalin, a component of the traditional Chinese medicine chan'su, we examined the effects of bufalin on plasma membrane potential, as determined by monitoring the uptake by cells of rhodamine 123. Bufalin induced apoptosis in human monocytic leukemia THP-1 cells, in human lymphoblastic leukemia MOLT-3 cells, and in human colon adenocarcinoma COLO320DM cells but not in normal human leukocytes, for example, polymorphonuclear cells and lymphocytes, and not in murine leukemia P388D1 and M1 cells. Treatment for 3 h with bufalin at 10(-6) M caused a decrease in the plasma membrane potential in several lines of human tumor cells but not in murine leukemia cells. No changes in mitochondrial membrane potential, as monitored with the fluorescent dye JC-1, and no release of cytochrome c were observed within at least 6 h after the start of treatment with bufalin. Moreover, overexpression of bcl-2 in human leukemia HL60 cells that had been transfected with cDNA for bcl-2 prevented bufalin-induced apoptosis but had no significant effect on the change in plasma membrane potential induced by bufalin. Since bufalin specifically inhibits the Na+,K(+)-ATPase of human but not murine tumor cells, and since this inhibition leads to a change in intracellular concentration of Na+ ions, our findings suggest that bufalin induces apoptosis in human tumor cells selectively via inhibition of the Na+,K(+)-ATPase, which acts upstream of the bcl-2 protein.     

Role of Na+ in transport of Hg2+ and induction of the Tn21 mer operon.

The effects of sodium ions on the uptake of Hg2+ and induction of the Tn21 mer operon were studied by using Escherichia coli HMS174 harboring the reporter plasmids pRB28 and pOS14. Plasmid pRB28 carries merRT', and pOS14 carries merRTPC of the mer operon, both cloned upstream of a promoterless luciferase gene cassette in pUCD615. The bioluminescent response to 1 microM Hg2+ was significantly inhibited in E. coli HMS174(pRB28) in minimal medium supplemented with sodium ions at 10 to 140 mM. After initial acceleration, light emission declined at 50 nM Hg2+ in the presence of Na+. The mer-lux assay with resting cells carrying pRB28 and 203Hg2+ uptake experiments showed increased induction and enhanced mercury uptake, respectively, in media supplemented with sodium ions. The presence of Na+ facilitated maintenance of bioluminescence in resting HMS174(pRB28) cells induced with 50 nM Hg2+. External K+ stimulated bioluminescent response in HMS174(pRB28) and HMS174(pOS14) grown in sodium phosphate minimal medium devoid of potassium ions. Sodium ions appear to facilitate mercury transport. We propose that sodium-coupled transport of mercuric ions can be one of the mechanisms for mercury uptake by E. coli and that the Na+ gradient may energize the transport of Hg2+.     

Motional characteristics of K+ and Na+ in intact and sucrose-permeabilized rat lymphocytes.

Most, if not all, cells maintain an unequal distribution of Na+ and K+ against their environment. These two monovalent ions are in constant exchange between the cell and the extracellular space since both ions have proved to be permeable through the cell membrane. The distribution of Na+ and K+ in intact and "sucrose-permeabilized" rat lymphocytes were studied ("sucrose-permeabilization" means homogenization in isotonic sucrose solution). Both the intact and the permeabilized lymphocytes were incubated in Hanks' solution and then transferred into K+, Na(+)-free isotonic sucrose solution. Alternatively, the cells were incubated only in the sucrose solution or in Hanks' solution. The Na+ and K+ content of the cells were determined at the conclusion of each period of incubation in the same or different medium. We found that K+ did not equilibrate under any conditions in intact lymphocytes but Na+ responded to changes of the incubation media. In the permeabilized cells Na+ freely equilibrated with the extracellular medium while K+ did not, although its concentration decreased compared to that of intact cells.     

Inversion of the intracellular Na+/K+ ratio blocks apoptosis in vascular smooth muscle at a site upstream of caspase-3.

Long term elevation of the intracellular Na+/K+ ratio inhibits macromolecule synthesis and proliferation in the majority of cell types studied so far, including vascular smooth muscle cells (VSMC). We report here that inhibition of the Na+,K+ pump in VSMC by ouabain or a 1-h preincubation in K+-depleted medium attenuated apoptosis triggered by serum withdrawal, staurosporine, or okadaic acid. In the absence of ouabain, both DNA degradation and Caspase-3 activation in VSMC undergoing apoptosis were insensitive to modification of the extracellular Na+/K+ ratio as well as to hyperosmotic cell shrinkage. In contrast, protection of VSMC from apoptosis by ouabain was abolished under equimolar substitution of Na+o with K+o, showing that the antiapoptotic action of Na+,K+ pump inhibition was caused by inversion of the intracellular Na+/K+ ratio. Unlike VSMC, the same level of increment of the [Na+]i/[K+]i ratio caused by a 2-h preincubation of Jurkat cells with ouabain did not affect chromatin cleavage and Caspase-3 activity triggered by treatment with Fas ligand, staurosporine, or hyperosmotic shrinkage. Thus, our results show for the first time that similar to cell proliferation, maintenance of a physiologically low intracellular Na+/K+ ratio is required for progression of VSMC apoptosis.     

Effects of NH4+ and K+ on the energy metabolism in Sp2/0-Ag14 myeloma cells

Potassium ions decrease the transport rate of ammonium ions into myeloma and hybridoma cells, one effect of the involved transport processes being an increased energy demand (Martinelle and Häggström, 1993; Martinelle et al., 1998b). Therefore, the effects of K+ and NH4+ on the energy metabolism of the murine myeloma cell line, Sp2/0-Ag14, were investigated. Addition of NH4Cl (10 mM) increased the metabolism via the alanine transaminase (alaTA) pathway, without increasing the consumption of glutamine. As judged by the alanine production, the energy formation from glutamine increased by 155%. The presence of elevated concentrations of KCl (10 mM) was positive, resulting in a decreased uptake of glutamine (45%), and an even larger suppression of ammonium ion formation (70%), while the same throughput via the alaTA pathway (and energy production from glutamine) was retained as in the control culture. However, the simultaneous presence of 10 mM K+ and 10 mM NH4+ was more inhibitory than NH4Cl alone; an effect that could not be ascribed to increased osmolarity. Although the culture with both K+ and NH4+ consumed 60% more glutamine than the culture with NH4+ alone, the energy generation from glutamine could not be increased further, due to the suppression of the glutamate dehydrogenase pathway. Furthermore, the data highlighted the importance of evaluating the metabolism via different energy yielding pathways, rather than solely considering the glutamine consumption for estimating energy formation from glutamine.     

Plasma membrane depolarization without repolarization is an early molecular event in anti-Fas-induced apoptosis

The movement of intracellular monovalent cations has previously been shown to play a critical role in events leading to the characteristics associated with apoptosis. A loss of intracellular potassium and sodium occurs during apoptotic cell shrinkage establishing an intracellular environment favorable for nuclease activity and caspase activation. We have now investigated the potential movement of monovalent ions in Jurkat cells that occur prior to cell shrinkage following the induction of apoptosis. A rapid increase in intracellular sodium occurs early after apoptotic stimuli suggesting that the normal negative plasma membrane potential may change during cell death. We report here that diverse apoptotic stimuli caused a rapid cellular depolarization of Jurkat T-cells that occurs prior to and after cell shrinkage. In addition to the early increase in intracellular Na(+), (86)Rb(+) studies reveal a rapid inhibition of K(+) uptake in response to anti-Fas. These effects on Na(+) and K(+) ions were accounted for by the inactivation of the Na(+)/K(+)-ATPase protein and its activity. Furthermore, ouabain, a cardiac glycoside inhibitor of the Na(+)/K(+)-ATPase, potentiated anti-Fas-induced apoptosis. Finally, activation of an anti-apoptotic signal, i.e. protein kinase C, prevented both cellular depolarization in response to anti-Fas and all downstream characteristics associated with apoptosis. Thus cellular depolarization is an important early event in anti-Fas-induced apoptosis, and the inability of cells to repolarize via inhibition of the Na(+)/K(+)-ATPase is a likely regulatory component of the death process.     

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)     

Retinoic-acid-induced differentiation of HL-60 cells is associated with biphasic activation of the Na+− K+ pump

The human promyelocytic leukemia cell line, HL-60, can be induced to differentiate into granulocyte-like cells when cultured in the presence of 10(-6) M retinoic acid (RA) for several days. Following the addition of RA two kinds of changes occur. First, there are early changes that comprise an increase in the intracellular concentration of sodium ions [Na]i, which reaches its maximum after 6 h, and an increase in the activity of the Na+-pump, which is reflected by an ouabain-sensitive K+ influx that peaks at 8 h (170% of the control value) and that occurs without any change in the number of pump molecules, as measured by the binding of 3H-ouabain. Second, beginning after 12 h of culture with RA, a decrease in the number of ouabain-binding sites occurs, this being accompanied by an increase in the number of K+ ions actively transported by each site. The effect of modulation of Na+-pump activity on the RA-induced differentiation of HL-60 cells was studied using low, noncytotoxic concentrations of ouabain which, although alone having no differentiating effect, accelerated and potentiated the effect of RA on differentiation. When added in combination, these drugs induced rapid stimulation of the Na+-pump, which reached its peak after 2 h. These results indicate that a concomitant increase in the level of [Na+]i and in the activity of the Na+-pump constitute primary events in the interaction between RA and HL-60 cells, and that cation fluxes may play a role in the initiation of the process of differentiation.     

Binding of monovalent cations to Na+,K+-dependent ATPase purified from porcine kidney. III. Marked changes in affinities for monovalent cations induced by formation of an ADP-insensitive but no an ADP-Sensitive phosphoenzyme

We measured the amounts of Rb+ ions (a K+ congener) as well as Na+ and K+ ions bound to the ATPase during the ATPase reaction at pH 7.5 and 0 degrees C. The affinity of the Na+-binding sites for three Na+ ions decreased markedly but that of the K+-binding sites for two K+ or Rb+ ions increased markedly upon formation of an ADP-insensitive phosphorylated intermediate. Furthermore, the present experiment did not give any indication of a change in the Hill coefficient of 2, and showed an increase in the affinity of the K+-binding sites for Rb+ ions of about 28 times upon the formation of an ADP-insensitive EP. The enzyme state with a high affinity for Rb+ was maintained after the disappearance of EP. When the ATPase was treated with N-ethylmaleimide (NEM), almost all the EP formed was ADP-sensitive. The formation of an ADP-sensitive EP with the NEM-treated enzyme induced no change in the affinities of the ATPase for Na+ and Rb+ ions.