Resolution of Pump and Leak Components of Sodium and Potassium Ion Transport in Human Erythrocytes

Further support for the pump-leak concept was obtained. Net transport was resolved into pump and leak components with the cardiac glycoside, ouabain. The specificity of ouabain as a pump inhibitor was demonstrated by its ineffectiveness when the pump was already inhibited by lack of one of the three pump substrates, sodium ion, potassium ion, or adenosine triphosphate. In the presence of ouabain the rates of passive transport of sodium and potassium ions changed almost in proportion to changes in their extracellular concentrations when one ion was exchanged for the other. In the presence of ouabain and at the extracellular concentrations which produced zero net transport, the ratio of potassium ions to sodium ions was 1.2-fold higher inside the cells than outside. This finding was attributed to a residual pump activity of less than 2% of capacity. The permeability to potassium ions was 10% greater than the permeability to sodium ions. A test was made of the independence of pump and leak. Conditions were chosen to change the rate through each pathway separately or in combination. When both pathways were active, net transport was the sum of the rates observed when each acted separately. A ratio of three sodium ions pumped outward per two potassium ions pumped inward was confirmed.     

Changes in the cation composition and active K+ transport in the red cells of fetal sheep prepartum

The red blood cells of lambs, genotypically low potassium type, undergo a transition from high potassium to low potassium cell type from parturition onwards. This involves gradual changes in cell ion content, sodium pump activity, and ouabain binding. In the present study we investigated the properties of fetal red blood cells from 30 days prepartum using the chronically cannulated pregnant ewe preparation. We demonstrate that intracellular sodium increases and potassium decreases from -30 days onwards. Sodium pump activity monitored either by tracer potassium influx or ouabain binding is markedly higher in the early fetal samples examined and declines fourfold during the final month in utero. Unlike the maternal low potassium cells the early fetal red cells are refractory in terms of sodium pump stimulation by anti-L, the antibody in fact consistently inhibiting the pump. Finally, we have investigated the volume sensitivity and development of the ouabain-insensitive potassium fluxes in these cells and found that both fetal and maternal cells show a marked chloride-dependent, volume-sensitive passive potassium flux. We conclude that the decrease in active sodium transport between fetal red cells and adult low potassium cells is achieved partly by a reduction in the density of sodium pumps per cell, and then later by the introduction into the circulation of cells with Lp-antigen-modified sodium pumps.     

Transmembrane potential of isolated rat alveolar type II cells

Type II cells were isolated from rat lungs by elastase digestion and purified by centrifugal elutriation. The fluorescent dye, Di-S-C3(5), was used as a probe to monitor transmembrane potential (Em) of cells suspended in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered medium. With this technique, the Em of type II cells was estimated to be -27 +/- 2 mV. This resting Em is very close to the equilibrium potential for chloride (-21 mV), which suggests that chloride is passively distributed in type II cells. The resting Em of type II cells is more dependent on the extracellular concentration of potassium (K+) than on external sodium (Na+); i.e., the membrane depolarizes as external sodium is replaced by potassium, suggesting that in unstimulated type II cells the membrane is more permeable to potassium than to sodium. In addition, the resting potential appears to be due, in part, to the activity of a ouabain-sensitive, Na-K pump, which acts to hyperpolarize type II cells. Addition of a membrane perturbant, phorbol myristate acetate (PMA, 10 micrograms/ml), to a type II cell suspension results in an increase in oxygen consumption and membrane depolarization. Both of these responses are sodium dependent and thus appear to be linked to a PMA-induced increase in sodium permeability.     

Differential effect of extracellular calcium on the Na(+)-K+ pump activity in intact polymorphonuclear leucocytes and erythrocytes

The effect of extracellular calcium on the Na(+)-K+ pump activity in human polymorphonuclear leucocytes and erythrocytes was studied and compared with the activity in mixed peritoneal leucocytes from rats. While there was maximal decrease in the pump activity (25-30%) of leucocytes from both rat and human by calcium 0.6 mM, a concentration of 0.1 mM caused a substantial decrease indicating a high sensitivity for extracellular calcium. In contrast, calcium had no effect on the pump activity in erythrocytes. The effect of calcium on the pump activity in leucocytes may be due to regulation of the influx of sodium across the plasma membrane, since in human leucocytes calcium had no effect on the pump activity if the cells were loaded with sodium.     

Electrogenic effect during active lithium transport through somatic membrane of molluscan neurons

The effect of replacement of sodium and (or) potassium by lithium on the electrogenic effect of active ion transport through the somatic membrane of isolated neurons was studied in the snailPlanorbarius corneus. Changes observed in the electrogenic effect are evidence that intracellular lithium can be actively exchanged for extracellular potassium; lithium can play the role of extracellular potassium during activation of the pump, and intracellular lithium is actively exchanged for extracellular.     

Cell potential and the sodium-potassium pump in vascular smooth muscle.

An electrogenic sodium-potassium pump appears to contribute materially to the steady-state potential and to certain of the transient potential responses of vascular smooth muscle. Since changes in cell potential in turn can lead to changes in contractile state, the pump is implicated in some of the constriction-dilation responses of blood vessels. The vasodilator action of potassium is explainable, for instance, through an effect on cell potential if (and only if) an electrogenic pump is assumed to be extruding sodium at a faster rate than it takes up potassium. This is supported by the observation that ouabain, an inhibitor of Na,K-ATPase activity, will eliminate or reverse the vascular effect of potassium. Furthermore, when the in vivo and in vitro effects on vascular smooth muscle of altered extracellular potassium concentration are compared to calculated cell potentials based on a model that includes an electrogenic pump, the experimental findings are shown to be logical and predictable.     

Potassium transport in the rabbit renal proximal tubule: Effects of barium,ouabain, valinomycin,and other ionophores

Summary Potassium fluxes in a suspension of rabbit proximal tubules were monitored using a potassium-sensitive extracellular electrode. Ouabain (10^–4 m) and barium (5mm) were used to selectively quantitate the potassium efflux pathway (105±5 nmol K⁺·mg protein^–1·min^–1) and the sodium pump-related potassium influx (108±7), respectively. These equal and opposite fluxes suggest that potassium accumulation in the cell occurs mainly through the sodium pump and that potassium efflux occurs mainly through barium-sensitive potassium channels. Thus the activity of the sodium pump (Na, K-ATPase) in the basolateral membrane of the proximal tubule is balanced by the efflux of potassium, presumably across the basolateral membrane, which has a high potassium permeability. In addition, the effect of valinomycin and other ionophores was examined on potassium fluxes and several metabolic parameters [oxygen consumption (QO₂), ATP content]. The addition of valinomycin to the tubules produced a net efflux of potassium which was quantitatively equivalent to the efflux produced by the addition of ouabain. The valinomycin-induced efflux was mainly due to the activity of valinomycin as a mitochondrial uncoupler, which indirectly inhibited the sodium pump by allowing a rapid reduction of the intracellular ATP. Amphotericin, nystatin, and monensin all produced large net releases of intracellular potassium. The action of the ionophores could be localized to the plasma or mitochondrial membrane and classified into three groups, as follows: (a) those which demonstrated full mitochondrial uncoupler activity (FCCP, valinomycin), (b) those which had no uncoupler activity (amphotericin B, nystatin); and (c) those which displayed partial uncoupler activity (monensin, nigericin).     

Extracellular protons regulate the extracellular cation selectivity of the sodium pump

The effects of 0.3-10 nM extracellular protons (pH 9.5-8.0) on ouabain-sensitive rubidium influx were determined in 4,4'-diisocyanostilbene-2, 2'-disulfonate (DIDS)-treated human and rat erythrocytes. This treatment clamps the intracellular H. We found that rubidium binds much better to the protonated pump than the unprotonated pump; 13-fold better in rat and 34-fold better in human erythrocytes. This clearly shows that protons are not competing with rubidium in this proton concentration range. Bretylium and tetrapropylammonium also bind much better to the protonated pump than the unprotonated pump in human erythrocytes and in this sense they are potassium-like ions. In contrast, guanidinium and sodium bind about equally well to protonated and unprotonated pump in human red cells. In rat red cells, protons actually make sodium bind less well (about sevenfold). Thus, protons have substantially different effects on the binding of rubidium and sodium. The effect of protons on ouabain binding in rat red cells was intermediate between the effects of protons on rubidium binding and on sodium binding. Remarkably, all four cationic inhibitors (bretylium, guanidinium, sodium, and tetrapropylammonium) had similar apparent inhibitory constants for the unprotonated pump ( approximately 5-10 mM). The K(d) for proton binding to the human pump, with the empty transport site facing extracellularly is 13 nM, whereas the extracellular transport site loaded with sodium is 9.5 nM, and with rubidium is 0.38 nM. In rat red cells there is also a substantial difference in the K(d) for proton binding to the sodium-loaded pump (14.5 nM) and the rubidium-loaded pump (0.158 nM). These data suggest that important rearrangements occur at the extracellular pump surface as the pump moves between conformations in which the outward facing transport site has sodium bound, is empty, or has rubidium bound and that guanidinium is sodium-like and bretylium and tetrapropylammonium are rubidium-like.     

Dog Red Blood Cells : Adjustment of salt and water content in vitro

Dog red blood cells (RBC) lack a ouabain-sensitive sodium pump, and yet they are capable of volume regulation in vivo. The present study was designed to find in vitro conditions under which dog RBC could transport sodium outward, against an electrochemical gradient. Cells were first loaded with sodium chloride and water by preincubation in hypertonic saline. They were then incubated at 37°C in media containing physiologic concentrations of sodium, potassium, chloride, bicarbonate, glucose, and calcium. The cells returned to a normal salt and water content in 16–20 h. Without calcium in the medium the cells continued slowly to accumulate sodium. Removal of glucose caused rapid swelling and lysis, whether or not calcium was present. The net efflux of sodium showed a close relationship to medium calcium over a concentration range from 0 to 5 mM. Extrusion of salt and water was also demonstrated in fresh RBC (no hypertonic preincubation) when calcium levels in the media were sufficiently raised. The ion and water movements in these experiments were not influenced by ouabain or by removal of extracellular potassium. Magnesium could not substitute for calcium. It is concluded that dog RBC have an energy-dependent mechanism for extruding sodium chloride which requires external calcium and is quite distinct from the sodium-potassium exchange pump.     

High and low dietary potassium effects on rat vascular sodium pump activity

Studies of renal and other tissues suggest that chronic elevation or reduction of dietary potassium intake could affect vascular smooth muscle sodium pump (Na-pump) activity. To examine this possibility, the effects of 3 weeks of low (LK: 4 mmole KCl/kg chow), normal (NK; 162 mmole/kg), and high (HK; 1350 mmole/kg) dietary potassium intake on Na-pump activity, the Na-pump activity response to changes in extracellular potassium concentration, and Na-pump site density were determined in tail arteries of rats. Plasma potassium concentration was elevated by 21% in HK rats and reduced by 45% in LK rats. When incubated in autologous plasma, compared to arteries from NK rats, Na-pump activity was decreased in the tail arteries from LK rats but not altered in those from HK rats. When arteries from NK and LK rats were incubated in autologous plasma with the potassium concentration increased to equal that of the HK rats, Na-pump activity exceeded that of HK rat arteries: Na-pump activity of arteries incubated in autologous plasma did not differ from that of arteries incubated in Krebs-Henseleit buffer with the potassium concentration adjusted to equal that of the plasma. Tail artery Na-pump activity for all three dietary potassium groups increased as potassium concentration of the incubation medium was increased from 1 to 12 mM; Na-pump activity was similar for the NK and LK rats at all potassium concentrations, but Na-pump activity of HK rat arteries was less than that of NK arteries at high extracellular potassium concentrations. Na-pump site density was not altered by either HK or LK diet. It is concluded that in tail arteries of rats fed the LK diet, chronically decreased extracellular potassium results in chronically decreased Na-pump activity. In contrast, an adaptive change occurs in tail arteries of rats fed HK diet, such that Na-pump activity remains at normal levels despite elevated extracellular potassium; this adaptive response to chronically increased dietary potassium does not appear to be the result of decreased Na-pump site density.     

Cardiotonic steroids: potential endogenous sodium pump ligands with diverse function

The highly conserved cardiotonic steroid (CS) binding site present on the ubiquitous membrane sodium pump, sodium, potassium-ATPase, appears to have been conserved by no force other than its capacity to bind CS: a family that includes plant-derived cardiac glycosides and putative endogenous vertebrate counterparts. Binding of ligand is inhibited by increased extracellular potassium. This implies functional coordination because inhibition of the sodium pump would be counterproductive when extracellular potassium is elevated. The interesting biology of the CS binding site continues to stimulate investigations into the identity of endogenous ligands, their role as pump regulators at the cellular level, and as mediators of body fluid balance and blood pressure regulation. In addition to inhibition of sodium and potassium transport, there is considerable recent evidence suggesting that the sodium pump may act as a cell signaling receptor activated by CS binding and responding by coordination of intracellular signaling pathways that can be dependent on and also independent of the reduction in transmembrane ion flux resulting directly from pump inhibition. This signaling may influence cell survival, growth, and differentiation. Recent insight into the biology of pump regulation by CS is reviewed.     

An electrogenic NA+/K+ pump in the choroid plexus.

Intracellular electrical potential and potassium activity was measured by means of microelectrodes in the epithelial cells of choroid plexus from bullfrogs (Rana catesbeiana). Ouabain applied from the ventricular side caused an abrupt depolarisation of 10 mV but only a gradual loss of potassium from the cells. Readministration of potassium to the ventricular solution of plexuses which were previously depleted of potassium, caused a hyperpolarisation of about 4 mV. These two experiments are consistent with the notion of an electrogenic Na+/K+ pump situated at the ventricular membrane and which pumps potassium into the cell and sodium into the ventricle. The numerical values obtained suggest that 3 sodium ions are pumped for 2 potassium ions. The permeability coefficient for potassium exit from the cell is calculated to be 1.24 . 10(-5) cm-1 . s-1 expressed per cm2 of flat epithelium.     

The ionic basis of electrical activity in embryonic cardiac muscle

The intracellular sodium concentration reported for young, embryonic chick hearts is extremely high and decreases progressively throughout the embryonic period, reaching a value of 43 mM immediately before hatching. This observation suggested that the ionic basis for excitation in embryonic chick heart may differ from that responsible for electrical activity of the adult organ. This hypothesis was tested by recording transmembrane resting and action potentials on hearts isolated from 6-day and 19-day chick embryos and varying the extracellular sodium and potassium concentrations. The results show that for both young and old embryonic cardiac cells the resting potential depends primarily on the extracellular potassium concentration and the amplitude and rate of rise of the action potential depend primarily on the extracellular sodium concentration.     

Increases in Na/K pump numbers in isolated human lymphocytes exposed to lithium in vitro. Reversal by myo-inositol and by inhibitors of protein kinase C and the Na/H antiport

Lithium (1-8 mM) caused a dose-dependent increase in the number of [3H]ouabain binding sites and in sodium/potassium (Na/K) pump activity in normal lymphocytes after incubation for 72 h. The increase in Na/K pump activity was due to an increase in the Vmax of the pump, with no change in the apparent affinity (Km) for potassium (rubidium). There was no change in the turnover number of the pump and the intracellular sodium concentration fell. The increase in [3H]ouabain binding sites was prevented by the addition of myo-inositol (10 mM), by inhibition of the protein kinase C with staurosporine (100 nM) and by inhibition of the Na/H antiport with dimethylamiloride (50 microM). These results suggest that the increase in Na/K pump activity caused by lithium is due to an increase in pump numbers and not due to increased activity of individual pumps or to an alteration in the affinity of the pumps for potassium. The increase in Na/K pump numbers and activity in lymphocytes exposed to lithium for 72 h may be related to altered Na/H antiport activity secondary to inhibition of phosphoinositol breakdown by lithium.     

Palytoxin down-modulates the epidermal growth factor receptor through a sodium-dependent pathway

Palytoxin, a non-12-O-tetradecanoylphorbol-13-acetate type tumor promoter, has been shown to inhibit epidermal growth factor (EGF) binding to both high and low affinity receptors through a protein kinase C-independent pathway. In the present paper, we have investigated the mechanism of palytoxin action in Swiss 3T3 cells. Two lines of evidence indicate that calcium is not required for palytoxin activity. First, palytoxin can induce the loss of EGF binding sites in the absence of external calcium. Second, studies with the photosensitive protein aequorin indicate that palytoxin does not cause the influx of external calcium or the release of calcium from internal stores under the conditions used in these studies. However, palytoxin action does appear to be dependent upon the presence of sodium. When extracellular sodium is replaced by either choline, Tris, or sucrose, palytoxin is unable to decrease EGF binding to either high or low affinity receptors. Studies of sodium influx indicate that palytoxin induces rapid sodium uptake and that the rate of sodium uptake is dose-dependent. Furthermore, there appears to be a direct correspondence between the extent of inhibition of EGF binding by palytoxin and the rate of sodium uptake. Finally, the palytoxin-induced inhibition of EGF binding can be mimicked by monensin, a sodium ionophore. The specificity of this sodium dependence was tested by substituting lithium, potassium, or cesium for sodium. Although lithium is an effective substitute for sodium, palytoxin can no longer inhibit EGF binding when sodium is replaced by either potassium or cesium. Marked inhibition of palytoxin action is also obtained when 5.4 mM potassium or 5.4 mM cesium are added to the sodium-containing medium. These studies suggest that palytoxin is able to down-modulate the EGF receptor through a novel mechanism involving the activation or formation of a sodium pump or channel.     

X-ray microanalysis of elemental changes in human parathyroid glands in primary and secondary hyperparathyroidism

The elemental composition of chief cells of parathyroid glands from patients with adenomatous primary hyperparathyroidism (HPT) and uremic secondary HPT was studied by X-ray microanalysis. Glands histologically deemed normal were used as controls. The analyses were also carried out on tissue specimens incubated in hypo-, normo- and hypercalcemic media (0.5, 1.25, and 3.0 mM calcium concentration). Analysis of chief cells from normal glands did not reveal any significant differences in ionic composition after exposure to the different calcium concentrations. In chief cells from adenomatous and uremic hyperplastic glands, elemental changes were noted. In comparison with specimens incubated in 1.25 mM calcium medium, cells in 0.5 mM calcium medium had a lower content of potassium and phosphorus. After stimulation with increasing extracellular concentration, an increase in the K/Na ratio was observed, due to a marked decrease of sodium and an increase of potassium: the calcium concentration was almost unchanged. Our findings indicate that in HPT an increase in serum calcium concentration might exert a stimulatory effect on the Na/K pump (sodium pump) and on the calcium-activated potassium channels. Either of these mechanisms might contribute to a lowering of cytoplasmic calcium. Our observations suggest that changes in ionic content of the parathyroid cells may be of importance for the stimulus secretion process in the cells.     

Potassium modulation of taurine transport across the frog retinal pigment epithelium

Net taurine transport across the frog retinal pigment epithelium-choroid was measured as a function of extracellular potassium concentration, [K+]o. The net rate of retina-to-choroid transport increased monotonically as [K+]o increased from 0.2 mM to 2 mM on the apical (neural retinal) side of the tissue. No further increase was observed when [k+]o was elevated to 5 mM. The [K+]o changes that modulate taurine transport approximate the light-induced [K+]o changes that occur in the extracellular space separating the photoreceptors and the apical membrane of the pigment epithelium. The taurine-potassium interaction was studied by using rubidium as a substitute for potassium and measuring active rubidium transport as a function of extracellular taurine concentration. An increase in apical taurine concentration, from 0.2 mM to 2 mM, produced a threefold increase in active rubidium transport, retina to choroid. Net taurine transport can also be altered by relatively large, 55 mM, changes in [Na+]o. Apical ouabain, 10(-4) M, inhibited active taurine, rubidium, and potassium transport; in the case of taurine, this inhibition is most likely due to a decrease in the sodium electrochemical gradient. In sum, these results suggest that the apical membrane contains a taurine, sodium co-transport mechanism whose rate is modulated, indirectly, through the sodium pump. This pump has previously been shown to be electrogenic and located on the apical membrane, and its rate is modulated, indirectly, by the taurine co-transport mechanism.     

Characteristics of electrogenic sodium pumping in rat myometrium

Sodium-rich myometrium, obtained from the uteri of pregnant rats, rapidly hyperpolarized when 4.6–120 mM potassium was added to the bathing medium at 37°C. Hyperpolarization was due to sodium pumping since the process was markedly temperature dependent, was abolished by ouabain, and required both intracellular sodium and extracellular potassium. The observed membrane potential exceeded the calculated potassium equilibrium potential during hyperpolarization providing evidence that sodium pumping was electrogenic. Hyperpolarization was reduced in the presence of chloride. The rate of sodium pumping may influence potassium permeability since potassium apparently did not short-circuit the pump during hyperpolarization.     

Molecular characterization and expression of the (Na+ + K+)-ATPase alpha-subunit in Drosophila melanogaster.

The (Na+ + K+)-ATPase (sodium pump) is an ouabain-sensitive, electrogenic ion pump responsible for maintaining the balance of sodium and potassium ions in almost all animal cells. Robust, ouabain-sensitive rubidium uptake, indicative of the sodium pump, was found in tissue-cultured Drosophila cells, and both larvae and adults die when fed a diet containing ouabain. A monoclonal antibody to the avian sodium pump alpha-subunit was found to cross-react with the Drosophila sodium pump alpha-subunit. Immunofluorescence microscopy was used to obtain a semi-quantitative view of the expression of the sodium pump in Drosophila tissues: high levels of the sodium pump were detected in malpighian tubules, indirect flight muscles and tubular muscles, and throughout the nervous system. The cDNA encoding this sodium pump alpha-subunit in Drosophila melanogaster was cloned, sequenced and expressed in mouse L cells. At the amino acid level, its deduced sequence of 1038 residues (the first such sequence for an invertebrate) is approximately 80% similar to alpha-subunit sequences reported for vertebrates. Only one gene was found in Drosophila, located on the third chromosome at position 93B. A restriction site polymorphism has been found, and several mutations exist that may involve the alpha-subunit gene.     

细胞外钠离子浓度对羊浦肯野纤维膜钠泵活动的影响

采用离子选择电极测量羊浦肯野纤维细胞膜内钠离子活度(~(ai)N_a),细胞间钾离子活度(a~ok)及细胞膜电位(v_m),观察不同浓度低钠,无钙液对其影响,在无钙低钠液中,细胞内Na~+逐出,α~iNa 降低,其变化速率,幅值与[Na]_o 相关,同时也受细胞 a~iNa 初始水平(aiNa(o))的影响。aiNa 下降6min 时的稳态水平与[Na]_o 呈直线正相关,这些结果表明,[Na]_o 降低时,细胞膜钠泵活动加强,细胞内 Na~+逐出增加,其最终结果是使 Na+跨膜梯度维持相对稳定,因而可以认为是 Na~+跨膜梯度而不是单纯的细胞内 Na~+控制膜钠泵活动。在低 Na~+液引起细胞内 Na~+主动逐出增加的同时,细胞膜出现超极化,[Na]_o 愈低,膜超极化程度愈高,从低钠液引起的 a~i_(Na),V_m,α~o_k 变化之间的时程关系看,膜超极化主要由加大的外向泵电流引起,同时发生的细胞间 K~+浓度变化对其也有一定影响。