Changes in the content of potassium, sodium and potassium influx in human peripheral blood lymphocytes during long-term cultivation with phytohemagglutinin

Intracellular potassium, sodium and potassium influx were examined in PHA-activated human lymphocytes within 6 days of cultivation. DNA flow cytometry was used to estimate the percentage of cells in G1, S and G2 + M phases. Potassium influx and content per g protein were found to be increased, whereas sodium content decreased with the progression of cells from G1 to S phases, being maximum on the 3rd day. Later on the percentage of cells in S phase was seen diminished, and the potassium content decreased just as sodium content increased. It is concluded that ionic changes may correlate with the entering of cells into S phase.     

Dependence of ion transport across the plasma membrane on the density of the cell culture. I. Ion flows and the potassium and sodium content in 3 Chinese hamster cell lines (CHO)

Cation transport has been investigated in three lines of Chinese ovary cells CHO-K1 during the cell culture growth. With the increase in the cell density potassium and sodium contents decreased from 1.2 to 0.8-0.5 and from 0.5 to 0.15-0.1 mmole/g protein, respectively. The time courses of potassium and sodium changes were different, and the increase in intracellular K/Na ratio from 1.5-2.0 to 5-10 with the increase in cell density was revealed. The rubidium influx was found to decrease during the culture growth mainly due to the decrease in ouabain-inhibitable and (ouabain + furosemide)- non-inhibitable influxes. The changes in cation fluxes and cation contents were observed in transformed cells without contact inhibition of division and were considered as a manifestation of density-dependent alterations of plasma membrane.     

Dependence of ion transport across the plasma membrane on cell culture density. II. Active and passive cation transport during the growth of L cell cultures

Rubidium and lithium influxes as well as intracellular potassium and sodium contents were investigated in L cells during the culture growth. In sparse culture over the cell densities 0.5-3 X 10(4) cells/cm2 ouabain-sensitive rubidium influx is small and ouabain-resistant lithium influx in high. With the increase in culture density up to 4-5 X 10(4) cells/cm2 the active rubidium influx, mediated by ouabain-sensitive component, is enhanced, and ion "leakage" tested by lithium influx is diminished. Simultaneously with the exponential growth of culture the intracellular potassium content is increased and the intracellular sodium content is decreased resulting in the higher K/Na ratio in cell. During the further transition to dense culture and in stationary state (10-17 X 10(4) cells/cm2) the sodium content and lithium influx do not change significantly, but the potassium content is decreased. The decrease in intracellular potassium is correlated with that in the portion of cells in S-phase from 27-30 to 12%. Thus, in transformed cells the density-dependent alterations in membrane cation transport are observed.     

Water and ion balance in rat thymocytes under apoptosis induced with dexamethasone or etoposide. Ion-osmotic model of cell volume decrease

Cell ion and water balance was studied with respect to analysis of the osmotic model of apoptotic volume decrease (AVD) in rat thymocytes under dexamethasone (1 microM, 4-6 h) or etoposide (50 microM, 5 h) treatment. Intracellular water content was determined by measurement of cell buoyant density in continuous Percoll gradient, while intracellular potassium and sodium contents were determined by flame emission analysis. Apoptosis was verified by an increase in cell buoyant density, fluorescence of cells stained with Acridine orange and Ethidium bromide (flow cytometry), by changes in the cell cycle and the appearance of sub-diploid peak in the DNA histogram (flow cytometry), and by a decrease in cell size examined with light microscope. A separate fraction of dense cells with reduced size was found to appear after dexamethasone or etoposide treatment. This fraction was considered as apoptotic. An increase in buoyant density of apoptotic cells corresponded to a decrease in cell water content. In apoptotic cells vs. cells with normal buoyant density, the intracellular potassium content was lower, but sodium content was higher. The sum of potassium and sodium contents was lower in apoptotic cells. Taken into account the loss of anions, associated with the loss of cations, the bulk decrease in ions content has been sufficient to be accounted for cell volume decrease on the basis of the ion-osmotic model.     

Cation transport and content in serum-stimulated CHO-773 cells. II. Late changes in rubidium influx and intracellular potassium content

Serum stimulation of stationary cultures of Chinese hamster ovary cells CHO-K1 (clone 773) is accompanied by sustained increase in ouabain-sensitive rubidium (potassium) influx which results in the elevation of intracellular potassium content from 0.5-0.6 to 0.7-0.8 mmole per gram of protein. Cytofluorometric studies of serum-stimulated CHO-773 cultures have shown that the intracellular potassium increase is necessary for successful G1----S progression. The elevation of intracellular potassium was found to occur simultaneously with the cellular protein growth. Cycloheximide (10 micrograms/ml) does not influence the early Na,K-ATPase activation induced by serum; however, it abolishes the sustained increase of both rubidium influx and intracellular potassium content. In serum stimulated cells ouabain increases the potassium efflux; this ouabain effect is not observed after S phase, when rubidium (potassium) influx decreases and intracellular potassium content stops growing.     

Serum stimulation of potassium fluxes,ouabain binding,and sodium fluxes in quiescent chicken embryo fibroblasts

Growth-contingent alterations in potassium and sodium fluxes, ouabain binding, and potassium ion content were examined following serum stimulation of quiescent, density-inhibited chicken embryo fibroblasts. Serum stimulation resulted in very rapid 1.5- to 1.8-fold increases in ouabain-sensitive potassium influx and lesser 1.4- to 1.5-fold increases in potassium efflux and sodium influx. Potassium influx stimulation was maximal after addition of 5–20% calf serum and was unaffected by cycloheximide inhibition of protein synthesis. Reflecting the slightly greater stimulation of potassium influx versus potassium efflux, potassium ion levels were 10–15% higher in serum-stimulated compared to unstimulated cells. Specific ouabain binding levels in stimulated and unstimulated control cells were initially similar, however, by four hours after stimulation a 40–50% increase in specific ouabain binding was observed. Incubation with ouabain was found also to inhibit later serum-stimulated hexose uptake and thymidine incorporation; this blockage may be a consequence of subnormal potassium levels rather than ouabain inhibition of the serum-stimulated potassium influx.     

Early and late changes in potassium transport during the start of proliferation in CHO cell cultures. The action of serum, isoproterenol, propranolol and theophylline

The stimulation of DNA synthesis by serum is accompanied by early (30 minutes) and late (2-8 hours) increase in ouabain-sensitive rubidium (potassium) influx and the elevation of intracellular potassium content from 0.5-0.6 to 0.7-0.8 mmole per gram protein in CHO-K1 cells. Isoproterenol alone induces the transient increase both in potassium influx via Na,K-ATPase and in potassium efflux without any effect on intracellular potassium content and cell proliferation. Isoproterenol acts synergistically with serum in eliciting the early and late changes in potassium transport and in stimulating G1----S transition. The combination of serum and theophylline produces a rapid increase in potassium influx, however, it does not stimulate DNA synthesis and does not induce any later increase in intracellular potassium content. It is concluded that early and late activation of Na,K-ATPase by mitogens can be dissociated; the Na,K-ATPase activation is involved in mitogenic response when producing the sustained potassium influx and the elevation of intracellular potassium content during G1----S transition.     

Evidence for chloride dependent potassium and water transport induced by hyposmotic stress in erythrocytes of the marine teleost,Opsanus tau

Summary Red blood cells of the marine teleost,Opsanus tau (oyster toadfish), were characterized as to their normal hemoglobin, ion and water contents. Cells were exposed to ouabain containing, hyposmotic salt solutions (osmolarity reduced to 2/3 of normal) in which the cation or anion composition was varied. It was found that the initial cell volume expansion due to water influx was independent of the anion present. However, a secondary volume reduction was dependent on the presence of chloride or bromide anions. During volume reduction, cellular potassium and chloride ion contents fell by about equal amounts. Potassium loss was commensurate to the total amount of potassium ions detected extracellularly about 1.5h after the initial osmotic shock. No major changes were seen in the cellular sodium ion contents. When chloride ions within the cells and in the suspending medium were replaced by nitrate, iodide or thiocyanate, the cells failed to return to volumes close to those of isosmotically suspended controls, and the cellular potassium content also remained constant. In hypotonic potassium chloride the cells failed to extrude potassium chloride and water, and hence retained their expanded volume. Neither potassium loss nor volume decrease occurred in cells swollen in hypotonic sodium chloride media containing furosemide or 4,4 diisothiocyano-2,2-stilbene-disulfonic acid (DIDS). These two compounds are known inhibitors of monovalent cation cotransport and anion self exchange, respectively, in mammalian red cells. Hence toadfish red cells respond to osmotic swelling primarily by activation of an ouabain-insensitive, chloride dependent potassium transport system which is sensitive to inhibition by furosemide and DIDS.     

CALCIUM CONTENT AND EXCHANGE IN NEOCORTICAL TISSUES DURING THE CATION MOVEMENTS INDUCED BY GLUTAMATES

Abstract—

• 1 Guinea pig neocortical tissues incubated in glucose-bicarbonate media reached stable calcium contents of 2 μat./g of which 0-35 μat.Ca/g was in a space accessible to inulin.
• 2 Addition of l -glutamate salts caused a prompt increase in intracellular calcium at rates up to 7 μat./g tissue/hr.
• 3 Using ⁴⁵Ca, this increase was found to be due to an accelerated influx of Ca and a diminished efflux. The rate of influx could be doubled by 1 mm-l -glutamate.
• 4 Tetrodotoxin at concentrations of 66-330 nM diminished ⁴⁵Ca entry, both in the absence and presence of added glutamate.
• 5 Tissue sodium and potassium contents are also reported under some conditions, and the extent to which calcium may condition sodium and potassium movements is discussed.

     

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.     

Cation transport in murine L fibroblasts cultured long term in a serum-free medium

Cation fluxes and intracellular content in mouse fibroblasts L, growing for more than three years in the Dulbecco modified Eagle's serum-free medium (clone L625sf) were measured as a function of culture density. The cells show no density-dependent inhibition of growth and in continuously growing cultures of L625sf cells internal potassium, and rubidium influx was found to remain high within a wide range of densities (5.10(4)-20.10(4) cells/cm2). A close correlation was revealed between the potassium transport and the proliferative state of L625sf cultures: the addition of 5% calf serum to logarithmically growing cultures leads to the increase in culture growth rate as well as to the increase in ouabain-inhibited rubidium influx and intracellular potassium content; a delay in culture growth rate due to medium depletion is accompanied by decreasing both the rubidium influx and the intracellular potassium content. It is concluded that L625sf cells being capable of multiplicating in serum-free medium remain sensitive to growth factors of serum and may be used for study of growth factor induction of cell proliferation and for identification of autocrine factors of cell growth.     

Increases in microtubule assembly and in tubulin content in mitogenically stimulated mouse splenic T lymphocytes

We have examined the changes in the microtubule and tubulin contents in populations of mouse splenic T lymphocytes stimulated by the mitogen concanavalin A. Indirect immunofluorescence staining with antiserum to tubulin indicated that a more extensive microtubule network was assembled from the centrosome in those cells which had increased in size in response to the mitogen. Direct counts of microtubules from electron micrographs of the centrosome regions of cells showed approximately a 2-fold increase in microtubule number in 48 h stimulated populations and up to a 5-fold increase in the large, fully stimulated, blast cells. Determinations of tubulin and actin contents were made by the measurement of peptides specific to those proteins. As a percentage of total cell protein both of these cytoskeletal proteins increased during the first 24 h of stimulation. Tubulin increased 50% by 24 h and remained high in populations stimulated for 48 h. The tubulin content per cell increased 2.5-fold, from 0.20 to 0.51 μg/10⁶ cells, in the 48 h stimulated population. An increase in tubulin content was also seen following the stimulation of nude mouse B lymphocyte populations and of total splenic lymphocyte populations. Our results show that during lymphocyte stimulation there is a large increase in the numbers of microtubules assembled which is correlated with, and appears dependent on, a similar large increase in the cellular tubulin content.     

Sodium and potassium fluxes and membrane potential of human neutrophils: evidence for an electrogenic sodium pump

Sodium and potassium ion contents and fluxes of isolated resting human peripheral polymorphonuclear leukocytes were measured. In cells kept at 37 degrees C, [Na]i was 25 mM and [K]i was 120 mM; both ions were completely exchangeable with extracellular isotopes. One-way Na and K fluxes, measured with 22Na and 42K, were all approximately 0.9 meq/liter cell water . min. Ouabain had no effect on Na influx or K efflux, but inhibited 95 +/- 7% of Na efflux and 63% of K influx. Cells kept at 0 degree C gained sodium in exchange for potassium ([Na]i nearly tripled in 3 h); upon rewarming, ouabain-sensitive K influx into such cells was strongly enhanced. External K stimulated Na efflux (Km approximately 1.5 mM in 140-mM Na medium). The PNa/PK permeability ratio, estimated from ouabain insensitive fluxes, was 0.10. Valinomycin (1 microM) approximately doubled PK. Membrane potential (Vm) was estimated using the potentiometric indicator diS-C3(5); calibration was based on the assumption of constant-field behavior. External K, but not Cl, affected Vm. Ouabain caused a depolarization whose magnitude dependent on [Na]i. Sodium-depleted cells became hyperpolarized when exposed to the neutral exchange carrier monensin; this hyperpolarization was abolished by ouabain. We conclude that the sodium pump of human peripheral neutrophils is electrogenic, and that the size of the pump-induced hyperpolarization is consistent with the membrane conductance (3.7-4.0 microseconds/cm2) computed from the individual K and Na conductances.     

Regulation of hepatic synthesis of proteins by the chronology of protein ingestion

Circadian variations in liver protein synthesis were were assessed in control rats fed a mixed 10% protein diet and in rats fed proteins as a separate meal either at 09:00 (SF 09) or at 21:00 (SF 21) and provided with a protein-free diet ad libitum. Protein synthesis was measured by incorporation of labelled leucine over a short period of time (15 min) at time-points regularly spaced over 24 h. In controls, the circadian variations observed were of moderate amplitude (from 2.75 mg/h per g at 09:00 to 5.77 mg/h per g at 06:00) correlated with increased protein and RNA contents of the liver. In separately fed animals ingestion of the protein meal triggered a 300% increase in protein synthesis within 1 h while the feeding pattern was unaltered. In the SF 09 group, high synthetic activity was not followed by an increase of hepatic protein content while hepatic urea concentrations were sharply increased and glucogenic amino acid pools were greatly depleted. It is suggested that the high influx of amino acids consecutive to the absorption of the dietary proteins is the key factor stimulating protein synthesis, while synchronisation with the energetic metabolism controls the degree of degradation. The possible involvement of variations in the insulin to glucagon ratio is discussed.     

Further studies on a novel class of genetic variants of the L1210 cell with increased folate analogue transport inward. Transport properties of a new variant, evidence for increased levels of a specific transport protein, and its partial characterization following affinity labeling

Studies are reported on the characterization of a new isolate within a novel class of variants of the L1210 cell exhibiting markedly increased transport inward of folate analogues. This variant (L1210/R83), which was selected in the presence of the antifolate metoprine, exhibited a 40-fold increase in [3H]aminopterin influx compared to parental cells and a modest (4-5-fold) increase in [3H]aminopterin efflux. The increase in influx was associated with a comparable increase in influx Vmax for the one-carbon, reduced folate transport system and the same increase in the amount of specific binding of [3H]aminopterin on the cell surface. Values for influx Km for [3H]aminopterin and specificity for various folate structures were unchanged. The alteration in influx Vmax and more rapid efflux accounted for the different level of intracellular exchangeable level of drug at steady state in this variant compared with parental L1210 cells. Otherwise, membrane potential was unchanged. The N-hydroxysuccinimide ester of [3H]aminopterin was used to covalently label the specific binding protein for folate compounds in the plasma membrane of variant and parental L1210 cells. Incorporation of label into this protein was stable under a variety of conditions and accounted for 97 and 52% of total cellular labeling, respectively, for membrane derived from R83 and parental L1210 cells at a reagent concentration of 20 nM. Specific affinity labeling on the surface of parental and variant cells was decreased in the presence of aminopterin, methotrexate, or 5-formyltetrahydrofolate, but not in the presence of folic acid. Also, [3H]aminopterin influx in these cells was inhibited by the N-hydroxysuccinimide ester of aminopterin or methotrexate, but not the N-hydroxysuccinimide ester of folic acid. These findings, in addition to the increased affinity labeling of this variant, which corresponds to the increase in influx of [3H] aminopterin also seen, appears to identify the affinity labeled protein as a component of the "classical" one-carbon, reduced folate transport system in these cells. The affinity labeled protein from each cell type was solubilized in sodium dodecyl sulfate or extracted in detergent in the presence of proteinase inhibitors and was found to elute from Sephacryl S-300 and migrate during sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single peak of Mr = 45,000-48,000. Recovery of labeled binding protein in these fractions from R83 variant cells was approximately 40 times greater than that from parental cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Decreased membrane permeability to potassium is responsible for the cell volume increase that drives lens fiber cell elongation

Differentiation of lens epithelial cells into fiber cells involves an increase in cell volume which previously was proposed to be the direct cause of the extensive cell elongation which accompanies fiber formation. In this study we have continued to investigate the mechanism underlying cell elongation by defining the minimum nutrient and ion requirements of elongating cells, measuring potassium and sodium fluxes in stimulated and unstimulated lens epithelia, and determining the effects of several pharmacological agents on elongation and ion transport. We have shown that elongation will occur in a basic salt/glucose solution with Insulin-like growth factor I/somatomedin-C stimulation. Neither sodium nor any metabolite appears to be the osmotically active species which drives the increase in cell volume. However, potassium efflux rate coefficient was 47% lower in differentiating cells than in unstimulated cells, whereas potassium uptake rates and ouabain effects were similar. Cells did not elongate in potassium-free medium nor in the presence of several drugs which prevent the accumulation of intracellular potassium or hinder osmotic water flux. Unstimulated cells elongated, however, with the application of quinidine, a drug known to block potassium channels. We propose that stimulation of lens epithelial cells with an insulin-like growth factor signals the closure of a certain population of potassium channels. As a result, potassium efflux from the differentiating cells slows while active potassium uptake continues at a constant rate. Potassium then accumulates within the cell causing water influx, an increase in cell volume, and cell elongation.     

Ascorbate increases the synthesis of procollagen hydroxyproline by cultured fibroblasts from chick embryo tendons without activation or prolyl hydroxylase

An improved procedure was developed to extract prolyl hydroxylase from tendon cells of chick embryos with detergent, and improved assays were developed for both the activity of the enzyme and the amount of enzyme protein. Freshly isolated tendon cells were found to contain approx. 100 μg of enzyme protein per 10⁸ cells and 40–50% of the enzyme protein was active. When the cells were cultured, they were found to contain the same amount of enzyme protein by only 15–20% of the enzyme protein was active. Gel filtration of cell extracts indicated that the active form of prolyl hydroxylase in freshly isolated tendon cells and in cultured tendon cells had the same apparent size and the same activity per μg of immunoreactive protein as enzyme which was shown to be a tetramer. The inactive form was found to have about the same apparent size as subunits of the enzyme.When freshly isolated cells were incubated for 2 h in the presence of 40 μg per ml of ascorbate, there was a slight increase in the rate of hydroxyproline synthesis. In cultured cells, ascorbate at a concentration of 40 μg per ml caused a 2-fold increase in the rate of hydroxyproline synthesis within 30 min. However, ascorbate did not increase the activity of prolyl hydroxylase in extracts from either cell system. Therefore it appears that the influence of ascorbate on synthesis of procollagen hydroxyproline by the cells studied here must be ascribed to a cofactor effect on the hydroxylation reaction similar to that observed with purified enzyme, and it does not involve “activation” of inactive enzyme protein to active enzyme as has been observed in cultures of L-929 and 3T6 mouse fibroblasts.     

Cation transport and content in serum-stimulated CHO-773 cells. I. Rapid changes in rubidium and lithium influxes and intracellular sodium content

The cultures of Chinese hamster ovary cells (CHO-K1 clone 773) can be brought to the stationary state with most of cellular populations in G1 phase by growing continuously for 4 days up to the cultural density (10-12) X 10(4) cells/cm2. Upon introduction of fresh Eagle medium with 10% calf serum the cells progress from G1 to S phase for 7-9 hours. It is shown that within the first minutes of serum addition ouabain-sensitive rubidium influx increases, however, lithium influx, which serves a test for passive sodium pathways in the membrane, increases or does not change. No correlation was found between the rubidium influx and intracellular sodium changes, induced by serum. From comparative studies of ouabain-sensitive rubidium influx, lithium influx and intracellular sodium content it is concluded that the increase in intracellular sodium is not responsible for serum-induced Na,K-ATPase activation.     

Ouabain-insensitive net sodium influx in erythrocytes of normotensive and essential hypertensive humans

The Na+ content of erythrocytes is elevated in people with essential hypertension. There is conflicting evidence about its cause. The present study was designed to investigate whether the increase in content is due to a defect in a ouabain-resistant Na+ flux. Net Na+ influx was determined from the increase in Na+ content of erythrocytes during incubation in the presence of ouabain. Na+ content of erythrocytes from 24 normotensive Caucasian subjects with no known family history of hypertension was 6.9 +/- 1.3 mmol per litre of cells. It was 7.9 +/- 2.0 mmol per litre of cells in 18 subjects with essential hypertension. The difference was less and not significant when the two non-Caucasian subjects of the hypertensive group were excluded. Net Na+ influx was 1.83 mmol/h per litre of cells in the normotensive group. In eight subjects it was measured on a second occasion after an interval of several months. The coefficient of a variation of the duplicate tests was 2.4%. Net Na+ influx was significantly higher in the hypertensive group, the value was 2.18 +/- 0.15 mmol/h per litre of cells. In 11 of these subjects, Na+ influx was measured on a second occasion. The coefficient of variation was 6.2%, significantly greater than in the control group. In some of these subjects Na+ influx was within the normal range on one of the two occasions. When the groups were compared with use of the mean values from the duplicate tests, net Na+ influx was elevated in 17 of the 18 hypertensive subjects. The findings are discussed with reference to previous work and in relation to the established facilitatory effects of an increased intracellular Na+ concentration on excitable cells that influence blood pressure.     

Characteristics of an amiloride-sensitive sodium entry pathway in cultured rodent glial and neuroblastoma cells

We have studied the induction of an amiloride-sensitive sodium influx into C6 glioma, NIE, and NB2A neuroblastoma cell lines. In late log phase, cells grown continuously in the presence of 10% fetal calf serum showed Na⁺ influxes of approximately 25–30 nmol/mg protein min; < 5% of this flux was inhibited by amiloride. Removal of serum for 24 h caused a decrease in the total Na⁺ influx to 15–20 nmol/mg protein/min. Upon readdition of serum to the incubation medium, there was an increase in total Na⁺ influx, depending on the cell type, of 20–400% within 2 min. This increment in Na⁺ influx represented an increase in amiloride-sensitive Na⁺ transport with an apparent K′, of 0.4 mM. By adding serum back at various times after serum deprivation, it was determined that 4 h was required to observe a detectable increase in the amiloride-sensitive Na⁺ flux. Thus, serum removal results in the induction of the amiloride transport system which, however, remains latent until the reintroduction of serum to the medium. Addition of 5 μg/ml of cycloheximide blocked the increase in Na⁺ transport, indicating that de novo protein synthesis mediated this serum deprivation–induced increase in Na⁺ transport. Moreover, inhibition of de novo lipid synthesis by 0.1 mM fenfluramine also blocked the induction of this transport activity, suggesting that a coordinated synthesis of lipid and protein is required for the expression of this sodium transport site. We have also found that this serum stimulated Na⁺ influx did not saturate with Na⁺ concentration, up to 140 mM. Also, among commonly used inhibitors of passive Na⁺ entry into epithelial tissues, only amiloride was capable of inhibiting this transport system in these neural cell lines.