Inhibition of Na+/K+-ATPase may be one mechanism contributing to potassium efflux and cell shrinkage in CD95-induced apoptosis

To investigate the involvement of K⁺ efflux in apoptotic cell shrinkage, we monitored efflux of the K⁺ congener,⁸⁶ Rb⁺, and cell volume during CD95-mediated apoptosis in Jurkat cells. An anti-CD95 antibody caused apoptosis associated with intracellular GSH depletion, a significant increase in ⁸⁶Rb⁺ efflux, and a decrease in cell volume compared with control cells. Preincubating Jurkat cells with Val-Ala-Asp-chloromethylketone (VAD-cmk), an inhibitor of caspase proteases, prevented the observed ⁸⁶Rb⁺ efflux and cell shrinkage induced by the anti- CD95 antibody. A wide range of inhibitors against most types of K⁺ channels could not inhibit CD95-mediated efflux of⁸⁶ Rb⁺, however, the uptake of⁸⁶ Rb⁺ by Jurkat cells was severely compromised when treated with anti-CD95 antibody. Uptake of⁸⁶ Rb⁺ in Jurkat cells was sensitive to ouabain (a specific Na⁺/K⁺-ATPase inhibitor), demonstrating Na⁺/K⁺-ATPase dependent K⁺ uptake. Ouabain induced significant⁸⁶ Rb⁺ efflux in untreated cells, as well as it seemed to compete with⁸⁶ Rb⁺ efflux induced by the anti-CD95 antibody, supporting a role for Na⁺/K⁺-ATPase in the CD95-mediated⁸⁶ Rb⁺ efflux. Ouabain treatment of Jurkat cells did not cause a reduction in cell volume, although together with the anti-CD95 antibody, ouabain potentiated CD95-mediated cell shrinkage. This suggests that the observed inhibition of Na⁺+/K⁺-ATPase during apoptosis may also facilitate apoptotic cell shrinkage.     

Association of BCL-2 with membrane hyperpolarization and radioresistance

The resting membrane potential of parental, neomycin control, and Bcl-2 transfected cells was measured, and the effect of membrane hyperpolarization or depolarization on radiosensitivity was studied. Bcl-2 transfected cells were significantly more radioresistant than control cells and were significantly hyperpolarized compared to parental and neomycin control transfected PW and HL60 cells. Hyperpolarization of the parental and neomycin control transfected cells by valinomycin significantly increased the radioresistance of these cells to such an extent that there was no longer a significant difference in the survival of the valinomycin treated and irradiated control cells compared to similarly irradiated Bcl-2 transfected cells. In contrast, depolarization of the Bcl-2 transfected PW and HL60 cells decreased the radioresistance of the Bcl-2 transfectants to a level similar to that of the control cells. The data presented here suggest that overexpression of Bcl-2 affects membrane potential and that this hyperpolarization is associated with increased radioresistance of cells that overexpress Bcl-2. Furthermore, Bcl-2 transfected cells were also less susceptible to the specific Na⁺/K⁺-ATPase inhibitor ouabain, suggesting that Bcl-2 may act at the level of the Na⁺/K⁺-ATPase pump. © 1996 Wiley-Liss, Inc.     

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/I) 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.     

Reversible stimulation of the Na+/K+ pump by monensin in cultures of vascular smooth muscle

Two ionophores, monensin and salinomycin, increased total cell Na⁺ and ouabain-sensitive ⁸⁶Rb⁺ uptake in cultures of smooth muscle cells from rat aorta. Monensin was used to produced graded increases in cell Na⁺ in order to assess the Na⁺ dependence of the Na⁺/K⁺ pump in the intact cell. The relationship between internal Na⁺ and ouabain-sensitive ⁸⁶Rb⁺ uptake was hyperbolic (K¹_{Na = 3 mM)}. Monensin did not stimulate ⁸⁶Rb⁺ uptake in the absence of external Na⁺. Loading the cells with Na⁺ by exposing cultures to a K⁺-free medium for 3 hr maximally increased cell Na⁺ and ouabain-sensitive ⁸⁶Rb⁺ uptake to the same extent as monensin. Total cell Na⁺ and pump activity in monensin-treated cells returned to the initial values after removing the ionophore. Monensin was then able to increase total cell Na⁺ and ouabain-sensitive ⁸⁶Rb⁺ uptake to the same extent as the initial treatment with the ionophore.     

Regulation of Na+, K+-ATPase activity in MDCK kidney epithelial cell cultures: Role of growth state,cyclic AMP,and chemical inducers of dome formation and differentiation

Na⁺,K⁺-ATPase activity was monitored in MDCK kidney epithelial cell monolayers and in cell extracts as a function of cell density, cAMP elevation, and exposure to hexamethylene bisacetamide (HMBA) and dimethylsulfoxide (Me₂SO). Ouabain-sensitive Na⁺,K⁺-ATPase and ⁸⁶Rb⁺ uptake activities, and the number of [³H]-ouabain binding sites were maximal in subconfluent cultures and decreased accompanying the development of a confluent monolayer. A sodium pump density of 8 × 10⁷ pumps/cell was estimated for subconfluent cultures, declining to 9 × 10⁵ pumps/cell at confluence. Previous studies have shown that dibutyryl cyclic AMP (Bt₂cAMP), 1-methyl-3-isobutylxanthine (IBMX), or the differentiation inducers HMBA and Me₂SO, which also caused cAMP elevation, all stimulated dome formation, a visible manifestation of active transepithelial Na⁺ and water transport (Lever, 1979). In the present study, all of these inducers were found to elevate intracellular Na⁺ content, implicating this variable in control of induction of dome formation. Operationally, inducers could be divided into two classes. HMBA and Me₂SO partially inhibited ouabain-sensitive ⁸⁶Rb⁺ influx. Ouabain, at a concentration that caused partial sodium pump inhibition and increased intracellular Na⁺ content, was also effective as an inducer. The second class, exemplified by IBMX and Bt₂cAMP caused a furosemide-sensitive increase in intracellular Na⁺ content. This class of inducers stimulated ouabain-sensitive ⁸⁶Rb⁺ uptake, presumably by substrate effects due to increased Na⁺ levels. The Na⁺ or ATP activation of Na⁺,K⁺-ATPase activity assayed in cell-free extracts, the affinity of the transport system for Rb⁺ in intact cells and intracellular ATP levels were unchanged by inducer treatment. Elevation of intracellular Na⁺ concentration, either by cAMP-stimulated, furosemide-sensitive mechanisms or by partial inhibition of the sodium pump may stimulate the induction of dome formation in MDCK cells.     

Modulation of β-Cell Ouabain-Sensitive 86Rb+ Influx (Na+/K+ Pump) by D-Glucose,Glibenclamide or Diazoxide

The activity of the β-cell Na⁺/K⁺ pump was studied by using ouabain-sensitive (lmM ouabain) ⁸⁶Rb⁺ influx in β-cell-rich islets of Umeå-ob/ob mice as an indicator of the pump function. The present results show that the stimulatory effect of glucose on ouabain-sensitive ⁸⁶Rb⁺ influx reached its approximate maximum at 5mM glucose. Pre-treatment of the islets with 20mM glucose for 60 min strongly reduced the glucose-induced stimulation of the Na⁺/K⁺ pump. Pre-treatment (60 or 180 min) of islets at 0mM glucose, on the other hand, did not affect the magnitude of the glucose-induced stimulation of ⁸⁶Rb⁺ influx dunng the subsequent 5-min incubation. Glibenclamide stimulated the ouabain-sensitive ⁸⁶Rb⁺ uptake in the same manner as glucose. The stimulatory effect, showed its apparent maximum at 0.5μM. Pre-treatment (60 min) of islets with 1μM glibenclamide did not reduce the subsequent stimulation of the ouabain-sensitive ⁸⁶Rb⁺ influx. The stimulatory effect of glibenclamide and D-glucose were not .additive, suggesting that they may have the same mechanism of action. No direct effect of glibenclamide (0.01-1μM) was observed on the Na⁺/K⁺ ATPase activity in homogenates of islets. Diazoxide (0.4mM) inhibited the Na⁺/K⁺ pump. This effect was sustained even after 60 min of pre-treatment of islets with 0.4mM diazoxide. The stimulatory effect of glibenclamide and D-glucose were abolished by diazoxide. It is concluded that nutrient as well as non-nutrient insulin secretagogues activate the Na⁺/K⁺ pump, probably as part of the membrane repolarisation process.     

Selective inhibition by bis(2-chloroethyl)methylamine (nitrogen mustard) of the Na/K/Cl cotransporter of murine L1210 leukemia cells

Incubation of L1210 murine leukemia cells in vitro with 10 μM of the bifunctional alkylating agent bis(2-chloroethyl)methylamine (nitrogen mustard, HN2) for 10 min brought about a fall of more than 99.9% in their ability to form colonies when the cells were suspended in 0.5% nutrient agar. Incubation with HN2 also inhibited the influx of the potassium congener ⁸⁶Rb⁺ to exponentially proliferating L1210 cells in a concentration-dependent manner. This inhibition was specific and was accounted for by a reduction of a diuretic-sensitive component of ⁸⁶Rb⁺ influx, identified in the preceding paper (Wilcock, C. and Hickman, J.A. (1988) Biochim. Biophys. Acta 946, 359–367) as being mediated by a Na⁺/K⁺/Cl⁻ cotransporter. Inhibition by 10 μM HN2 was complete after a 3-h incubation. There was no inhibition at this time of the ouabain-sensitive component of ⁸⁶Rb⁺ influx, mediated by Na⁺/K⁺-ATPase. After 3 h of incubation with 10 μM HN2 there was also no change in the membrane potential of the treated cells as measured by the distribution of the [³H]TPMP⁺, no decrease in cellular ATP concentration and no change in intracellular pH, and the ability of the cells to exclude the vital dye Trypan blue was not significantly different from control values. These effects of HN2, therefore, appeared to follow lethal damage, but precede cell death. In the stationary phase of L1210 cell growth, the component of HN2 and diuretic-sensitive K⁺ influx to L1210 cells was reduced, whilst the component constituting the HN2-insensitive ouabain-sensitive sodium pump was increased. The monofunctional alkylating agent MeHN1 (2-chloroethyldimethylamine) which cannot cross-link cellular targets and has no antitumour activity, did not inhibit ⁸⁶Rb⁺ influx to L1210 cells when incubated at equimolar or equitoxic concentrations to HN2. Intracellular potassium concentration was maintained close to control values of 138 ± 10 mM in HN2-treated cells because of an approx. 35% fall in cell volume. The results suggest that the Na⁺/K⁺/Cl⁻ cotransporter is a selectively inhibitable target for HN2, and the lesion is discussed with reference to the cytotoxic effects of this agent.     

Regulation of Na,K-ATPase Transport Activity by Protein Kinase C

Considerable evidence indicates that the renal Na⁺,K⁺-ATPase is regulated through phosphorylation/dephosphorylation reactions by kinases and phosphatases stimulated by hormones and second messengers. Recently, it has been reported that amino acids close to the NH₂-terminal end of the Na⁺,K⁺-ATPase α-subunit are phosphorylated by protein kinase C (PKC) without apparent effect of this phosphorylation on Na⁺,K⁺-ATPase activity. To determine whether the α-subunit NH₂-terminus is involved in the regulation of Na⁺,K⁺-ATPase activity by PKC, we have expressed the wild-type rodent Na⁺,K⁺-ATPase α-subunit and a mutant of this protein that lacks the first thirty-one amino acids at the NH₂-terminal end in opossum kidney (OK) cells. Transfected cells expressed the ouabain-resistant phenotype characteristic of rodent kidney cells. The presence of the α-subunit NH₂-terminal segment was not necessary to express the maximal Na⁺,K⁺-ATPase activity in cell membranes, and the sensitivity to ouabain and level of ouabain-sensitive Rb⁺-transport in intact cells were the same in cells transfected with the wild-type rodent α1 and the NH₂-deletion mutant cDNAs. Activation of PKC by phorbol 12-myristate 13-acetate increased the Na⁺,K⁺-ATPase mediated Rb⁺-uptake and reduced the intracellular Na⁺ concentration of cells transfected with wild-type α1 cDNA. In contrast, these effects were not observed in cells expressing the NH₂-deletion mutant of the α-subunit. Treatment with phorbol ester appears to affect specifically the Na⁺,K⁺-ATPase activity and no evidence was observed that other proteins involved in Na⁺-transport were affected. These results indicate that amino acid(s) located at the α-subunit NH₂-terminus participate in the regulation of the Na⁺,K⁺-ATPase activity by PKC. Received: 10 July 1996/Revised: 19 September 1996     

Na+/K+/Cl− cotransport in cultured vascular smooth muscle cells: Stimulation by angiotensin II and calcium ionophores,inhibition by cyclic AMP and calmodulin antagonists

Summary The specific activity of the Na⁺/K⁺/Cl^– cotransporter was assayed by measuring the initial rates of furosemide-inhibitable⁸⁶Rb⁺ influx and efflux. The presence of all three ions in the external medium was essential for cotransport activity. In cultured smooth muscle cells furosemide and bumetanide inhibited influx by 50% at 5 and 0.2 m, respectively. The dependence of furosemide-inhibitable⁸⁶Rb⁺ influx on external Na⁺ and K⁺ was hyperbolic with apparentK _m values of 46 and 4mm, respectively. The dependence on Cl^– was sigmoidal. Assuming a stoichiometry of 112 for Na⁺/K⁺/Cl^–, aK _m of 78mm was obtained for Cl^–. In quiescent smooth muscle cells cotransport activity was approximately equal to Na⁺ pump activity with each pathway accounting for 30% of total⁸⁶Rb⁺ influx. Growing muscle cells had approximately 3 times higher cotransport activity than quiescent ones. Na⁺ pump activity was not significantly different in the gorwing and quiescent cultures. Angiotensin II (ANG) stimulated cotransport activity as did two calcium-transporting ionophores, A23187 and ionomycin. The removal of external Ca²⁺ prevented A23187, but not ANG, from stimulating the cotransporter. Calmodulin antagonists selectively inhibited⁸⁶Rb⁺ influx via the cotransporter. Beta-adrenoreceptor stimulation with isoproterenol, like other treatments which increase cAMP, inhibited cotransport activity. Cultured porcine endothelial cells had 3 times higher cotransport activity than growing muscle cells. Calmodulin antagonists inhibited cotransport activity, but agents which increase cAMP or calcium had no effect on cotransport activity in the endothelial cells.     

Furosemide-sensitive K+ channel in glioma cells but not neuroblastoma cells in culture

A furosemide-sensitive, ouabain-insensitive [⁸⁶Rb⁺] uptake is described in glioma cells in culture which is dependent upon external Na⁺, K⁺, and Cl^? concentrations. This transport activity was also inhibited by bumetanide at 100-fold lower concentrations than furosemide. Furosemide-sensitive swelling of glioma cells is demonstrated and this activity is dependent upon external Na⁺ and K⁺ in a manner similar to [⁸⁶Rb⁺] uptake. This transport activity was not detected in neuroblastoma cells and the possible relevance of these findings to extracellular K⁺ buffering by glia is discussed.     

Effect of Hypoxia on Na+-K+-Cl− Cotransport in Cultured Brain Capillary Endothelial Cells of the Rat

Abstract: The effect of hypoxia on Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport activity in cultured rat brain capillary endothelial cells (RBECs) was investigated by measuring ⁸⁶Rb⁺ uptake as a tracer for K⁺. RBECs expressed both Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport activity (4.6 and 5.5 nmol/mg of protein/min, respectively). Hypoxia (24 h) decreased cellular ATP content by 43.5% and reduced Na⁺,K⁺-ATPase activity by 38.9%, whereas it significantly increased Na⁺-K⁺-Cl^? cotransport activity by 49.1% in RBECs. To clarify further the mechanism responsible for these observations, the effect of oligomycin-induced ATP depletion on these ion transport systems was examined. Exposure of RBECs to oligomycin led to a time-dependent decrease of cellular ATP content (by ～65%) along with a complete inhibition of Na⁺,K⁺-ATPase and a coordinated increase of Na⁺-K⁺-Cl^? cotransport activity (up to 100% above control values). Oligomycin augmentation of Na⁺-K⁺-Cl^? cotransport activity was not observed in the presence of 2-deoxy-d -glucose (a competitive inhibitor of glucose transport and glycolysis) or in the absence of glucose. These results strongly suggest that under hypoxic conditions when Na⁺,K⁺-ATPase activity is reduced, RBECs have the ability to increase K⁺ uptake through Na⁺-K⁺-Cl^? cotransport.     

The Na+,K+,2Cl−-cotransport system in HeLa cells: Aspects of its physiological regulation

We have previously reported on the biochemical properties of a Na⁺,K⁺,2Cl^?-cotransport in HeLa cells and here we deal with aspects of its physiological regulation. Na⁺,K⁺,2Cl^?-cotransport in HeLa cells was studied by ⁸⁶Rb⁺ influx and ⁸⁶Rb⁺/²²Na⁺ efflux measurements. The effects of rat atrial natriuretic peptide (ANP), isoproterenol, and amino acids on ⁸⁶Rb⁺ flux, mediated by the bumet-anide-sensitive Na⁺, K⁺, 2Cl^?-cotransport system and the ouabain-sensitive Na⁺/K⁺-pump, were investigated. ANP reduced bumetanide-sensitive ⁸⁶Rb⁺ influx under isotonic as well as under hypertonic conditions. Similar decrease of bumetanide-sensitive ⁸⁶Rb⁺ influx was observed in the presence of 8-bromo-cGMP, while neither isoproterenol as a β-receptor agonist nor 8-bromo-cAMP-could alter bumetanide-sensitive ⁸⁶Rb⁺ influx. Furthermore, efflux of ⁸⁶Rb⁺ and ²²Na⁺ was greatly reduced in the presence of bumetanide and ANP. Together with our recent findings, showing functionally active, high affinity receptors for ANP on HeLa cells (Kort and Koch, Biochim. Biophys. Res. Commun. 168:148–154, 1990), this study indicates that ANP participates in the regulation of the Na⁺, K⁺, 2Cl^?-cotransport system in HeLa cells. Further measurements revealed that amino acids as present in the growth medium (Joklik's minimal essential medium) and the amino acid derivative α-methyl-aminoisobutyric acid (metAlB, 1 and 5 mM, respectively) also reduced Na⁺, K⁺, 2Cl^?-cotransport-mediated ⁸⁶Rb⁺ uptake and diminished the stimulatory effect of hypertonicity on the cotransporter. In addition, the Na⁺/K⁺-pump was markedly stimulated in the presence of amino acids, while neither ANP and 8-Br-cGMP nor isoproterenol and 8-Br-cAMP had a significant effect on the activity of the Na⁺/K⁺-pump.     

Enhancement of ornithine decarboxylase and Na+, K+ ATPase in osteoblastoma cells by intermittent compression

Rat osteoblatoma cells (ROS $\text{2}\text{3}$) were subjected in culture to a physiologic, intermittent, compressive force. The mechanical perturbation enhanced the activity of ornithine decarboxylase by 60%. Investigation of the mechanism of enzyme activation revealed an increase in ouabain inhibitable ⁸⁶Rb⁺ uptake, indicating an elevated Na⁺, K⁺ ATPase activity. Ouabain (1 μM) reduced ornithine decarboxylase activity by 75% in control cultures. This inhibition was partially overcome by intermittent compression. It appears that a functioning Na⁺, K⁺ ATPase is essential for the maintenance of ornithine decarboxylase activity and that activation of Na⁺, K⁺ ATPase may be associated with the trophic effects of mechanical stimuli in these cells.     

Effects of ouabain on proliferation of human endothelial cells correlate with Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase activity and intracellular ratio of Na<Superscript>+</Superscript> and K<Superscript>+</Superscript>

Side-by-side with inhibition of the Na⁺,K⁺-ATPase ouabain and other cardiotonic steroids (CTS) can affect cell functions by mechanisms other than regulation of the intracellular Na⁺ and K⁺ ratio ([Na⁺]_i/[K⁺]_i). Thus, we compared the doseand time-dependences of the effect of ouabain on intracellular [Na⁺]_i/[K⁺]_i ratio, Na⁺,K⁺-ATPase activity, and proliferation of human umbilical vein endothelial cells (HUVEC). Treatment of the cells with 1-3 nM ouabain for 24-72 h decreased the [Na⁺]_i/[K⁺]_i ratio and increased cell proliferation by 20-50%. We discovered that the same ouabain concentrations increased Na⁺,K⁺-ATPase activity by 25-30%, as measured by the rate of ⁸⁶Rb⁺ influx. Higher ouabain concentrations inhibited Na⁺,K⁺-ATPase, increased [Na⁺]_i/[K⁺]_i ratio, suppressed cell growth, and caused cell death. When cells were treated with low ouabain concentrations for 48 or 72 h, a negative correlation between [Na⁺]_i/[K⁺]_i ratio and cell growth activation was observed. In cells treated with high ouabain concentrations for 24 h, the [Na⁺]_i/[K⁺]_i ratio correlated positively with proliferation inhibition. These data demonstrate that inhibition of HUVEC proliferation at high CTS concentrations correlates with dissipation of the Na⁺ and K⁺ concentration gradients, whereas cell growth stimulation by low CTS doses results from activation of Na⁺,K⁺-ATPase and decrease in the [Na⁺]_i/[K⁺]_i ratio.     

Generation of plasma membrane potential by the Na+-pump coupled to proton extrusion

Lettré cells maintain a plasma membrane potential near — 60mV, yet are scarcely depolarized by 80 mM Rb⁺ and are relatively impermeable to ⁸⁶Rb⁺. They are depolarized by ouabain without a concomitant change in intracellular cation content. Addition of K⁺ to cells suspended in a K⁺ free medium, or of Na⁺ to cells in a Na⁺ free medium, hyperpolarizes the cells. They contain electroneutral transport mechanisms for Na⁺, K⁺ and H⁺ which can function as Na⁺:K⁺ and Na⁺:H⁺ exchanges. It is concluded that plasma membrane potential of Lettré cells, in steady-state for Na⁺ and K⁺, is produced by an electrogenic Na⁺ pump sustained by electroneutral exchanges, and restricted by anion leakage.     

Identification of a third isoform of Na+, K+-ATPase activity in rat brain synaptosomes

³H-ouabain binding and ouabain-inhibitable ⁸⁶Rb⁺ (K⁺) uptake were investigated as a means to identify a third isoform of Na⁺, K⁺-ATPase in crude synaptosome preparations. The specific binding of low concentrations (10 nM and 1 uM) of ³H-ouabain, in crude synaptosome preparations, was markedly inhibited by K⁺ (0.5–5 mM). Accordingly, ⁸⁶Rb⁺ (K⁺) uptake, in the presence of 5 mM K⁺ was not sensitive to inhibition by low concentrations (10⁻¹¹–10⁻⁷ M) of ouabain. Higher concentrations (10⁻⁶–10^−2.6 M) of ouabain resulted in a biphasic inhibition of K⁺ uptake, which distinguished the activities of the presumed alpha 2 and alpha 1 isozymes of Na⁺, K⁺-ATPase. Reduction of K⁺ (1.25 mM and 0.5 mM) in the incubation, resulted in the observation of a third component of ouabain- sensitive K⁺ uptake. This Na⁺, K⁺-ATPase activity, which was defined, pharmacologically, as very sensitive (VS) to ouabain, exhibited IC₅₀s of 3.6 nM and 92 nM at 1.25 mM K⁺ and 0.5 mM K⁺, respectively. Inhibition of ouabain binding and VS-dependent K⁺ uptake, at a high, physiological cocentration (5 mM) of K⁺, suggests that VS may be an inactive isoform of brain Na⁺, K⁺-ATPase under resting conditions.     

Cell growth and ouabain-sensitive Rb uptake and (Na + K)-ATPase activity in 3T3 and SV40 transformed 3T3 fibroblasts

The uptake of ouabain-sensitive ⁸⁶Rb⁺ uptake measured at 5 min and the uptake measured at 60 min was 4.5- and 2.7-fold greater respectively for SV40 transformed 3T3 cells compared to 3T3 cells during the late log phase of growth. This uptake, however, varied markedly with cell growth. Ouabain-sensitive ⁸⁶Rb⁺ uptake was found to be a sensitive indicator of protein synthesis as measured by total protein content. Cessation of cell growth as measured by total protein content was associated with a decline in ouabain-sensitive ⁸⁶Rb⁺ uptake in both cell types. This increased ouabain-sensitive cation transport was reflected in increased levels of (Na⁺ + K⁺)-ATPase activity for SV40 3T3 cells, which showed a 2.5-fold increase V but the same K_rmm as 3T3 cells.These results are compared with the results of related work. Possible mechanisms for these effects are discussed and how changes in cation transport might be related to alterations in cell growth.     

Pig reticulocytes. V. Development of Rb+ influx during in vitro maturation

Influx of the K⁺ analogue Rb⁺ was measured through the ouabain-sensitive Na⁺/K⁺ pump and the ouabain-insensitive “leak” pathways in Cl^? or NO in mature red cells from adult pigs and in reticulocytes naturally occurring in 7-day-old piglets. In reticulocytes, Rb⁺ influxes by the two pathways were of about equal magnitude in Cl^? (13 and 10 mmoles/liter cells × hr) and at least 25-fold larger than in mature red cells (0.5 and 0.4 mmoles/liter cells × hr). In Na ⁺ media, a portion of the ouabain-insensitive “leak” flux of Rb⁺ was Cl^? dependent (Rb⁺Cl^? transport) as NO replacement reduced Rb⁺ influx by 90% in reticulocytes and by 40% in mature red cells. The sulfhydryl reagent N-ethylmaleimide (NEM) stimulated Rb⁺Cl^? transport about twofold in reticulocytes and up to 13-fold in mature red cells. When reticulocytes matured to erythrocytes during in vitro incubation, about 90% of both ouabain-sensitive Rb⁺ pump and ouabain-insensitive Rb⁺Cl^? influx were lost. In contrast, the NEM-stimulated Rb⁺Cl^? transport changed much less throughout this period, suggesting an entity operationally but not necessarily structrually distinct from the basal Rb⁺Cl^? transport. Although the experimental variability precluded a full assessment of significant changes in the small Na⁺/K⁺(Rb⁺) pump and Rb⁺Cl^? fluxes in mature pig red cells kept for the same time period in vitro, Rb⁺ flux changes in reticulocytes appear to be maturational in nature, reflecting parallel activity transitions of Na⁺/K⁺ pump and Cl^?-dependent K⁺ fluxes in vivo.     

Lyn regulates creatine uptake in an imatinib-resistant CML cell line

BackgroundImatinib mesylate (imatinib) is the first-line treatment for newly diagnosed chronic myeloid leukemia (CML) due to its remarkable hematologic and cytogenetic responses. We previously demonstrated that the imatinib-resistant CML cells (Myl-R) contained elevated Lyn activity and intracellular creatine pools compared to imatinib-sensitive Myl cells.MethodsStable isotope metabolic labeling, media creatine depletion, and Na⁺/K⁺-ATPase inhibitor experiments were performed to investigate the origin of creatine pools in Myl-R cells. Inhibition and shRNA knockdown were performed to investigate the specific role of Lyn in regulating the Na⁺/K⁺-ATPase and creatine uptake.ResultsInhibition of the Na⁺/K⁺-ATPase pump (ouabain, digitoxin), depletion of extracellular creatine or inhibition of Lyn kinase (ponatinib, dasatinib), demonstrated that enhanced creatine accumulation in Myl-R cells was dependent on uptake from the growth media. Creatine uptake was independent of the Na⁺/creatine symporter (SLC6A8) expression or de novo synthesis. Western blot analyses showed that phosphorylation of the Na⁺/K⁺-ATPase on Tyr 10 (Y10), a known regulatory phosphorylation site, correlated with Lyn activity. Overexpression of Lyn in HEK293 cells increased Y10 phosphorylation (pY10) of the Na⁺/K⁺-ATPase, whereas Lyn inhibition or shRNA knockdown reduced Na⁺/K⁺-ATPase pY10 and decreased creatine accumulation in Myl-R cells. Consistent with enhanced uptake in Myl-R cells, cyclocreatine (Ccr), a cytotoxic creatine analog, caused significant loss of viability in Myl-R compared to Myl cells.ConclusionsThese data suggest that Lyn can affect creatine uptake through Lyn-dependent phosphorylation and regulation of the Na⁺/K⁺-ATPase pump activity.General significanceThese studies identify kinase regulation of the Na⁺/K⁺-ATPase as pivotal in regulating creatine uptake and energy metabolism in cells.