Increase In Cell Na Research Articles

Role of Na+ conductance, Na(+)-H+ exchange, and Na(+)-K(+)-2Cl- symport in the regulatory volume increase of rat hepatocytes.

1. In rat hepatocytes under hypertonic stress, the entry of Na+ (which is thereafter exchanged for K+ via Na(+)-K(+)-ATPase) plays the key role in regulatory volume increase (RVI). 2. In the present study, the contributions of Na+ conductance, Na(+)-H+ exchange and Na(+)-K(+)-2Cl- symport to this process were quantified in confluent primary cultures by means of intracellular microelectrodes and cable analysis, microfluorometric determinations of cell pH and buffer capacity, and measurements of frusemide (furosemide)/bumetanide-sensitive 86Rb+ uptake, respectively. Osmolarity was increased from 300 to 400 mosmol l-1 by addition of sucrose. 3. The experiments indicate a relative contribution of approximately 4:1:1 to hypertonicity-induced Na+ entry for the above-mentioned transporters and the overall Na+ yield equalled 51 mmol l-1 (10 min)-1. 4. This Na+ gain is in good agreement with the stimulation of Na+ extrusion via Na(+)-K(+)-ATPase plus the actual increase in cell Na+, namely 55 mmol l-1 (10 min)-1, as we determined on the basis of ouabain-sensitive 86Rb+ uptake and by means of Na(+)-sensitive microelectrodes, respectively. 5. The overall increase in Na+ and K+ activity plus the expected concomitant increase in cell Cl- equalled 68 mmol l-1, which fits well with the increase in osmotic activity expected to occur from an initial cell shrinkage to 87.5% and a RVI to 92.6% of control, namely 53 mosmol l-1. 6. The prominent role of Na+ conductance in the RVI of rat hepatocytes could be confirmed on the basis of the pharmacological profile of this process, which was characterized by means of confocal laser-scanning microscopy.

Hypertonicity-induced alkalinization of rat hepatocytes is not involved in activation of Na + conductance or Na +,K +-ATPase

We investigated whether cell alkalinization via activation of Na +/H + exchange is involved in the stimulation of Na + conductance and Na +,K +-ATPase in rat hepatocytes under hypertonic stress. Osmolarity was increased from 300 to 400 mOsm/l at constant extracellular pH (7.4), whereas osmotically induced cell alkalinization (0.3 pH units in HCO 3 −-free solutions) was mimicked by increasing extracellular pH from 7.4 to 7.8 in normosmotic solutions. In intracellular recordings with conventional and ion-sensitive microelectrodes, hypertonic stress led to a transient shift in the voltage response to low Na + solutions (95% in exchange for choline) by −4.3±0.8 mV and a continuous increase in cell Na + from 13.7±1.8 to 18.6±3.0 mmol/l within 8 min. In the presence of 10 −5 mol/l amiloride, these effects were reduced by 80 and 90%, respectively. In contrast, increasing pH did not change the voltage responses to low Na + or cell Na + concentrations significantly. In addition, application of 2 mmol/l Ba 2+ pulses revealed that a sustained membrane hyperpolarization of 15.6±1.4 mV following intracellular alkalinization exclusively reflects an increase in K + conductance. Increasing osmolarity at pH 7.4 augmented ouabain-sensitive 86Rb + uptake from 5.5±1.1 to 8.5±1.6 nmol mg protein −1 min −1. In normosmotic solution at pH 7.8, 86Rb + uptake equalled 4.9±1.6 nmol mg protein −1 min −1, which is not significantly different from control. We conclude that, in rat hepatocytes, cell alkalinization under hypertonic stress is not responsible for the activation of Na + conductance and probably does not participate in the stimulation of Na +,K +-ATPase.

Effects of haemoglobin O2 saturation on volume regulation in adrenergically stimulated red blood cells from the trout, Oncorhynchus mykiss.

Adrenergic stimulation of trout red blood cells activates a Na+/H(+)-exchange. If unopposed, the ensuing increase in cell Na+ leads to an isosmotic cell swelling. In this study the effect of the level of haemoglobin O2 saturation on volume regulation has been investigated in adrenergically stimulated red blood cells from trout: at full haemoglobin O2 saturation, net influx of Na+ through the Na+/H(+)-exchanger was balanced by net efflux of K+ and no increases in cell volume took place. In contrast, at low O2 saturation (8-14%) adrenergic stimulation led to a substantial increase in cell Na+, K+ and volume. Moreover, cell volume recovery after adrenergic swelling was incomplete at low O2 saturation, whereas cells at high O2 saturation exhibited a fast and complete cell volume recovery. In cells exposed to alternating high and low O2 saturation, volume regulation was similar to the regulation found in cells maintained at high O2 saturation. In cells at high O2 saturation, extrusion of cellular Na+ by the Na+/K(+)-pump significantly contributed to the volume decrease. It is concluded that trout red blood cells at high or alternating O2 saturations possess a powerful regulatory volume decrease response that is shut off at low O2 saturation. The physiological implications of this regulation is discussed.

Hydroxyurea affects cell morphology, cation transport, and red blood cell adhesion in cultured vascular endothelial cells

Hydroxyurea (HU) significantly increases fetal hemoglobin (Hb) production and concomitantly affects passive erythrocyte K transport and cell volume in patients homozygous for Hb S, thus decreasing disease severity. Red blood cells (RBCs) with Hb S display a greater adherence to vascular endothelial cells (VECs) than do Hb A cells, thus increasing the probability of vaso-occlusive crisis. The effect of HU on the structure and function of VECs is still unknown. In the present study, HU significantly changed, in a dose-dependent manner, the morphology and monovalent cation composition of cultured VECs after incubation in normal culture medium for up to 10 days in the absence and presence of 0.3 (therapeutic dose) and 3.0 (toxic dose) mmol/L HU. Treated cells showed significant morphologic changes such as an increase in apparent cell size and the formation of multinucleated giant cells. The protein content per dish decreased by 50% and 80% at 0.3 and 3.0 mmol/L HU, respectively, accompanied by an increase in cell Na (maximum, approximately 200%) and cell K (maximum, approximately 50%) contents at about days 4 to 6 and 8 to 10, respectively. In addition, HU decreased RBC adherence to VECs in experiments with 51Cr- loaded Hb A or Hb S RBCs. The HU-induced changes in VEC morphology, cation composition, and RBC adherence may be caused or accompanied by alterations in cell membrane permeability, transformation of endothelial cells, or decreased number/density of VEC adhesion molecules. Precise mechanisms of the HU effects warrant further investigation in light of the reported beneficial effects of HU in the treatment of sickle cell anemia.

Hydroxyurea Affects Cell Morphology, Cation Transport, and Red Blood Cell Adhesion in Cultured Vascular Endothelial Cells

AbstractHydroxyurea (HU) significantly increases fetal hemoglobin (Hb) production and concomitantly affects passive erythrocyte K transport and cell volume in patients homozygous for Hb S, thus decreasing disease severity. Red blood cells (RBCs) with Hb S display a greater adherence to vascular endothelial cells (VECs) than do Hb A cells, thus increasing the probability of vaso-occlusive crisis. The effect of HU on the structure and function of VECs is still unknown. In the present study, HU significantly changed, in a dose-dependent manner, the morphology and monovalent cation composition of cultured VECs after incubation in normal culture medium for up to 10 days in the absence and presence of 0.3 (therapeutic dose) and 3.0 (toxic dose) mmol/L HU. Treated cells showed significant morphologic changes such as an increase in apparent cell size and the formation of multinucleated giant cells. The protein content per dish decreased by 50% and 80% at 0.3 and 3.0 mmol/L HU, respectively, accompanied by an increase in cell Na (maximum, ~200%) and cell K (maximum, ~50%) contents at about days 4 to 6 and 8 to 10, respectively. In addition, HU decreased RBC adherence to VECs in experiments with 51Cr-loaded Hb A or Hb S RBCs. The HU-induced changes in VEC morphology, cation composition, and RBC adherence may be caused or accompanied by alterations in cell membrane permeability, transformation of endothelial cells, or decreased number/density of VEC adhesion molecules. Precise mechanisms of the HU effects warrant further investigation in light of the reported beneficial effects of HU in the treatment of sickle cell anemia.

Na+/Na+ exchange and Na+/H+ antiport in rabbit erythrocytes: Two distinct transport systems

Rabbit erythrocytes are well known for possessing highly active Na+/Na- and Na+/H+ countertransport systems. Since these two transport systems share many similar properties, the possibility exists that they represent different transport modes of a single transport molecule. Therefore, we evaluated this hypothesis by measuring Na+ transport through these exchangers in acid-loaded cells. In addition, selective inhibitors of these transport systems such as ethylisopropyl-amiloride (EIPA) and N-ethymaleimide (NEM) were used. Na+/Na+ exchange activity, determined as the Na+o-dependent 22Na efflux or Na+i-induced 22Na entry was completely abolished by NEM. This inhibitor, however, did not affect the H+i-induced Na+ entry sensitive to amiloride (Na+/H+ exchange activity). Similarly, EIPA, a strong inhibitor of the Na+/H+ exchanger, did not inhibit Na+/Na- countertransport, suggesting the independent nature of both transport systems. The possibility that the NEM-sensitive Na+/Na+ exchanger could be involved in Na+/H+ countertransport was suggested by studies in which the net Na+ transport sensitive to NEM was determined. As expected, net Na+ transport through this transport system was zero at different [Na+]i/[Na+]o ratios when intracellular pH was 7.2. However, at pHi = 6.1, net Na+ influx occurred when [Na+]i was lower than 39 mM. Valinomycin, which at low [K+]o clamps the membrane potential close to the K+ equilibrium potential, did not affect the net NEM-sensitive Na+ entry but markedly stimulated the EIPA- and NEM-resistant Na+ uptake. This suggest that the net Na+ entry through the NEM-sensitive pathway at low pHi, is mediated by electroneutral process possibly involving Na+/H+ exchange. In contrast, the EIPA-sensitive Na+/H+ exchanger is not involved in Na+/Na+ countertransport, because Na+ transport through this mechanism is not affected by an increase in cell Na+ from 0.4 to 39 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of pH on growth of mouse renal cortical tubule cells in primary culture.

We examined the effect of the medium pH on growth of primary cultures of mouse cortical tubule cells grown in defined medium. A significantly higher DNA content was observed within 24 h of lowering medium pH from 7.4 to 6.8 or 7.1 and persisted for the duration of the study. Further studies revealed that either medium acidification or insulin plus prostaglandin E1 nearly doubled uptake of [3H]thymidine in cells deprived of other growth factors for the previous 72-110 h. Moreover, the effects of insulin, prostaglandin E1, and medium acidification on [3H]thymidine uptake of quiescent cells were additive. An alkaline medium pH appeared to have a small but significant effect on cell hypertrophy, since cells exposed to pH 7.4 and 7.7 had a higher protein-to-DNA ratio than cells incubated at a lower pH. Cell pH of monolayers grown on glass slides determined from fluorescence of the carboxyfluorescein analogue 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) was linearly correlated with medium pH, and changes in medium pH resulted in changes in steady-state cell pH of a similar magnitude. Four hours after medium acidification, relative increases in cell Na+ and water content occurred, whereas medium alkalinization led to decreases in cell Na+ and water content. The increases in cell Na+ and cell water content at pH 6.8 could be inhibited by amiloride. We conclude that decreasing the cell pH can be a mitogenic stimulus for renal tubule cells. Medium acidification is accompanied by changes in cell Na+ transport, which may be mediated in part by altered Na+-H+ antiporter activity.

Blood pressure and erythrocyte Na+ transport systems in a French urban male population.

This paper reports an investigation of blood pressure (taken as a continuous variable) as a function of: erythrocyte Na+ content; Na+,K+ pump; Na+,K+ cotransport and Na+,Li+ countertransport fluxes, and passive cation permeabilities in fresh erythrocytes from 129 French males who were living in an urban area and were not under treatment for any medical condition (after allowing for the effects of age, body mass index, alcohol and tobacco consumption). In contrast with previous findings in a North American population, we were unable to confirm that blood pressure was correlated with erythrocyte Na+ content and Na+,K+-AT-Pase activity. Conversely, the only transport parameter correlated (negatively) with blood pressure was outward Na+,K+ cotransport [r = -0.20, P less than 0.05 and r = -0.19, P less than 0.05, for systolic (SAP) and diastolic arterial pressure (DAP), respectively; n = 114]. When allowing for age, body mass index and alcohol consumption, the correlation coefficient between the Na+,K+ cotransport system and blood pressure increased from -0.20 to -0.28 (P less than 0.01) for SAP and from -0.19 to -0.28 (P less than 0.01) for DAP (n = 105). We conclude that the correlations between blood pressure and erythrocyte Na+ transport function could differ between North American and French (or Mediterranean) populations. In any case, a decreased pump or outward Na+,K+ cotransport activity may lead hypertensive subjects to a similar increase in cell Na+ (and Ca2+) content in the vascular wall.

Na+, K+, Cl-]-cotransport function and dysfunction in different forms of primary hypertension.

We investigated the effect of an increase in cell Na+ content on outward and inward unidirectional fluxes catalyzed by the [Na+, K+, Cl-]-cotransport system in human erythrocytes (incubated in Li-Rb media). Erythrocytes with low Na+ content exhibited an uncoupled K+ efflux. The increase in cell Na+ content resulted in a more marked stimulation of outward Na+, K+ than of inward Li+, Rb+ cotransport fluxes (with stoichiometries not very different from one-to-one). These results suggest that in human erythrocytes and in nonepithelial cells with small but outwardly directed electrochemical Cl- gradients, the [Na+, K+, Cl-]-cotransport system may behave as a "second pump" by using the extra energy supplied by an additional net [K+, Cl-] efflux. The [Na+, K+, Cl-]-cotransport system (of vascular cells and/or noradrenergic endings) may play two different roles in primary hypertension: (a) "defective second pump" in some essential hypertensive patients with decreased cotransport affinity for internal Na+ and (b) "compensatory second pump" in other forms of primary hypertension where abnormalities in the Na+, K+ pump or in other ion transport systems may predispose the cell to a defective extrusion of excess cell Na+ content.

A Study of the Interaction between the Na+, K+ Pump and Na+ : Ca 2+ Exchange in Macrophages and Vascular Smooth Muscle Cells

Blaustein (Am J Physiol 1977;232:C165-C173) postulated that Na+:Ca2+ exchange in vascular smooth muscle plays a key role in the link between sodium and hypertension. Investigation of this hypothesis was facilitated by the use of: a) Sr2+, a slowly transported Ca2+ analogue, and b) new quasispecific inhibitors of Na+:Ca2+ exchange such as 2',4'-dimethylbenzamil. Preliminary experiments in mouse macrophages showed that the initial rate of Sr2+ uptake lasted for at least 15 minutes and was therefore easier to measure than the initial rate of unidirectional isotopic Ca2+ influx (which lasted less than 30 seconds). In cells with normal Na+ content, basal Sr2+ influx (432 +/- 77 mumol [L cells X h]-1; mean +/- SEM of seven experiments) exhibited properties compatible with a ground membrane leak for divalent cations (quasilinear dependence on the external Sr2+ concentration, partial or full resistance to external Ca2+, Ba2+, verapamil, and 2',4'-dimethylbenzamil). Membrane depolarization by external K+ was unable to modify basal Sr2+ uptake. Conversely, a 100% increase in cell Na+ content by preincubation with ouabain increased the rate of Sr2+ uptake by 233 +/- 48 mumol (L cells X h)-1 (mean +/- SEM of seven experiments). 2',4'-Dimethylbenzamil, but not the Ca2+ antagonists diltiazem or methoxyverapamil, inhibited ouabain-stimulated Sr2+ influx (IC50 of about 3 X 10(-5) M). 2',4'-Dimethylbenzamil was also able to inhibit Na+ efflux (by 3.05 +/- 0.98 mmol (L cells X h)-1; mean +/- SEM of three experiments) suggesting the existence of Na+:Sr2+ exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant murine interferon-gamma-induced differentiation of pre-B lymphocytes is associated with Na+/H+ exchange-dependent and -independent cytoplasmic alkalinization.

We have previously shown that in a murine pre-B lymphocyte cell line, 70Z/3, interleukin-1-induced IgM expression (differentiation) was associated with Na+/H+-mediated cytoplasmic alkalinization. Because interferon-gamma also induces 70Z/3 differentiation, in this study we examined the effects of recombinant murine interferon-gamma (rIFN-gamma) on cell pH. We found that rIFN-gamma (50 units/ml) induced a sustained increase in cell pH, averaging 0.09 pH units above the base line at 30 min and 0.08 at 4 h. Because rIFN-gamma also induced increases in cell Na+ concentration, the data suggests stimulation of Na+/H+ exchange across the cell membrane. Amiloride inhibited the rIFN-gamma-mediated pH rise by only 31% at 30 min, but at 4 h the inhibition was more complete (89%). In contrast to pHi, amiloride totally blocked the Na+ rise at both 30 min and 4 h. This indicates that at the earlier time point the pH rise had a Na+/H+ exchange-dependent and -independent component while at the later time most of the pH rise was due to the Na+/H+ exchanger. The presence of a Na+ independent amiloride-insensitive component was further confirmed by the 0.05 pH rise induced by rIFN-gamma in Na+-free media. Failure to block the Na+-independent rIFN-gamma-mediated pHi rise by anion exchange inhibitors or removal of Ca2+ indicate that this component was not mediated by Cl-/OH- or Ca2+/H+ exchange. The nature of the Na+-independent cell alkalinization process or its role in rIFN-gamma-mediated 70Z/3 differentiation remains to be determined. Because amiloride did not have an effect on rIFN-gamma-induced surface Ig expression or the rIFN-gamma-mediated increase in new kappa light chain-specific mRNA the results indicate that rIFN-gamma-stimulated Na+/H+ is not required for 70Z/3 differentiation.

Alterations in cation homeostasis in cultured chick ventricular cells during and after recovery from adenosine triphosphate depletion.

Alterations in cation homeostasis during and after recovery from myocardial ischemia may account for some of the reversible and irreversible components of myocardial cell injury. To investigate possible mechanisms involved, we exposed cultured layers of spontaneously contracting chick embryo ventricular cells to media containing 1 mM cyanide (CN) and 20 mM 2-deoxyglucose (2-DG), and zero glucose for up to 6 h, and then allowed cultured cells to recover in serum-free culture medium for 24 h. Changes in Na, K, and Ca contents, 42K uptake and efflux, ATP content, cell water content, and lactate dehydrogenase (LDH) release were measured, and compared with changes produced by exposure to 10(-3) M ouabain and severe hypoxia. Exposure to CN and 2-DG caused marked increase in cell Na (sevenfold) and Ca (fivefold) contents, and a decrease in K content (one-fifth normal), coincident with ATP depletion to one-tenth normal levels. This produced only slight cell injury, evidenced by increased LDH release. Recovery for 24 h resulted in return to near normal values (expressed in nanomoles per milligram of protein) of Na, Ca, and ATP contents. However, there was failure of cell K content to return to normal, associated with a persistent reduced net uptake of 42K, and an increase in the rate of 42K efflux. These abnormalities in K homeostasis were associated with a decrease in cell volume and water content per milligram of protein. More marked ATP depletion (to 1/100 normal values) was produced by hypoxia plus 2-DG and zero glucose, and was associated with much more severe cell injury manifested by LDH loss. Ouabain exposure resulted in a much greater Ca gain (20-30-fold), relative to increase in Na content, than did either CN and 2-DG or hypoxia; and ouabain effects were not reversible (after a 15-fold or greater increase in Ca content was produced) and were associated with significant LDH release. We conclude that these cells are resistant to cell injury caused by moderately severe Ca overload and ATP depletion produced by exposure to CN and 2-DG. However, metabolic inhibition of ATP production produces persistent abnormalities in K homeostasis, associated with functional abnormalities.

Mechanism of alteration of sodium potassium pump of erythrocytes from patients with chronic renal failure.

We examined intracellular electrolytes, K influx, and [3H]ouabain-binding capacity of erythrocytes from 32 normal subjects and 45 patients with end-stage renal failure on dialysis, including 16 with high intracellular Na (mean 17.3 +/- 3.9 mmol/liter cell water). The [3H]ouabain-binding capacity of erythrocytes with high cell Na was markedly reduced as compared with that of erythrocytes from normal subjects (274 +/- 52 vs. 455 +/- 59 sites/cell, P less than 0.001). The mean serum creatinine was higher in the uremic group with high cell Na. There was a significant linear correlation between intracellular Na and [3H]ouabain-binding in both normal and uremic subjects. Cross-incubation of normal cells with uremic plasma for 24 h failed to reduce [3H]ouabain-binding capacity of normal cells. In spite of a substantial increase in cell Na, K pump influx was not higher in uremic erythrocytes with high cell Na. When intracellular Na was altered with nystatin (cell Na equal to 120 mmol/liter cell water in both groups), K pump influx was proportional to the number of Na-K pump sites so that the ion turnover rate per pump site was similar in the two groups. Uremic plasma failed to depress K pump influx of normal erythrocytes. The passive net influx of Na in uremic cells with high intracellular Na was not different from that observed in erythrocytes from normal subjects. When erythrocytes were separated by age on Percoll density gradients, the number of Na-K pump sites of the youngest uremic cells was significantly lower than that of the youngest normal cells, suggesting that decreased synthesis of Na-K pump sites, rather than accelerated loss of Na-K pump sites during aging, was responsible for the decrease in [3H]ouabain-binding capacity of erythrocytes from uremic subjects. Taken together, these findings suggest that a decrease in the number of Na-K pump sites plays a major role in the abnormality of Na-K pump of erythrocytes from patients with chronic renal failure.

Cardiac glycoside receptors in cultured heart cells—II: Characterization of a high affinity and a low affinity binding site in heart muscle cells from neonatal rats

The binding of [ 3H]ouabain has been studied in (Na + + K +)-ATPase enriched cardiac cell membranes, as well as in cardiac muscle and non-muscle cells in culture—all obtained from hearts of neonatal rats. The binding has been correlated with ouabain-induced inhibition of (Na + + K +)-ATPase (cardiac cell membranes) and the inhibition of active ( 86Rb + + K +)-influx (cardiac muscle and non-muscle cells in culture). Furthermore, the effect of ouabain on the amplitude of cell-wall motion and contraction velocity has been studied in electrically driven cardiac muscle cells. In muscle and non-muscle cells, two classes of ouabain binding sites have been identified. In rat heart muscle cells, the high affinity binding site has a dissociation constant ( K D ) of 3.2 × 10 −8 M and a binding capacity ( B) of 0.2 pmole/mg protein (80,000 sites/cell); the values for the low affinity binding site are: K D = 7.1 × 10 −6 M; B = 2.6 pmole/mg protein (10 6 sites/cell). The binding to both types of binding sites is depressed by K + and abolished after heat denaturation of the cells. The kinetics of [ 3H]ouabain binding to rat heart muscle cells (association and dissociation rate constants, K +- and temperature-dependence of association and dissociation processes) have been characterized. In rat heart muscle and non-muscle cells, the binding of [ 3H]ouabain to the low affinity site results in inhibition of the ( 86Rb + + K +)-influx ( EC 50 = 1.3 and 1.5 × 10 −5 M ouabain), a decrease in cell-K + ( EC 50 = 1.9 and 1.4 × 10 −5 M ) and an increase in cell-Na + (10 −5-10 −4 M). The ouabain-induced positive inotropic effect (increase in amplitude of cell-wall motion, increase in contraction velocity) in cardiac muscle cells is observed only at ouabain concentrations ⩾5 × 10 −6 M, and it is therefore probably attributed to occupation of the low affinity binding site. Coupling of occupation of the low affinity site by ouabain with drug-induced inhibition of the sodium pump and with drug-induced positive inotropic action is further substantiated by kinetic measurements. In contrast, occupation of the high affinity binding site does not produce any measurable inhibition of the sodium pump activity or positive inotropy. In agreement with the findings in intact cells, two classes of ouabain binding sites ( K D = 2.2 × 10 −8 and 1.3 × 10 −4 M have been characterized in (Na + + K +)-ATPase enriched cardiac cell membranes from neonatal rats; only the binding of ouabain to the low affinity binding site produces a measurable inhibition of (Na + + K +)-ATPase activity ( EC 50 = 4.8 × 10 −5 M ouabain). Our results support the finding of different types of ouabain binding sites in rat heart. The nature of the high affinity/low capacity ouabain binding site remains to be elucidated.

Cell growth and net Na+ flux are inhibited by a protein produced by kidney epithelial cells in culture.

Proliferation of confluent kidney epithelial cell cultures (BSC-1 line) is inhibited by a protein (Mr approximately equal to 24,000) that is secreted by the cells. The mechanism of action of this growth inhibitor was sought by studying its effect on net Na+ flux because increased availability of Na+ in the culture medium had been shown to stimulate cell growth. The increase in cell Na+ content observed during stimulation of the growth after a medium change was attenuated in the presence of the purified inhibitor. Inhibition of both cell Na+ accumulation and growth in the presence of the protein was reversed completely by addition of NaCl to the medium. These results suggest that control of net Na+ flux and growth in kidney epithelial cells could be mediated, at least in part, by a secreted cellular protein.

Calcium elevation in cultured heart cells: its role in cell injury.

Inhibition of the Na+-K+ pump in cultured embryonic chick heart cells promotes the elevation of intracellular Ca2+ and is a useful manipulation to study the relationship between Ca2+ and myocardial cell injury. One hour of Na+-K+ pump inhibition resulted in a fourfold increase in cell Na+, a 50% decline in cell K+, and a 5- to 10-fold increase in cell Ca2+, 45% of which is mitochondrial. The degree of cell injury induced by Ca2+ loading was evaluated by monitoring the content of adenosine 5'-triphosphate (ATP) and the release of the intracellular enzyme lactate dehydrogenase (LDH). Under these conditions ATP content declined by 25-30% and LDH release increased from 1 to 1.4% of the total LDH. Furthermore, cells subjected to 1 h of Na+-K+ pump inhibition and returned to control solution for 5 h showed that Ca2+ decreased to near control levels and ATP content was restored. Although inhibition of Na+-K+ transport caused a large increase in cell Ca2+, neither Na+-K+ pump inhibition nor elevation in total cell Ca2+ per se resulted in irreversible myocardial cell injury.

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 (K 1 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.

Interrelation between membrane transport and the contents of alkali metal cations in HeLa cells.

The relation of membrane transport of alkali cations to their external concentrations or to their cellular contents was studied in HeLa cells. Chilling the cells at 0 degrees C reversed cell Na+ and K+ to a mirror image of the normal pattern. Upon rewarming to 37 degrees C the ouabain-sensitive Rb+ uptake became 2-fold faster than the control. A kinetic analysis revealed that the stimulation was due to an increase in the maximal rate of Rb+ uptake, Jmax. The increase in apparent Km was relatively small. The analysis also showed that the ouabain-sensitive cation transport system seemed to have two binding sites for Rb+. The stimulation of Rb+ uptake was related to an increase in cell Na+, and an addition of ouabain abolished such a relation. Net Na+ flux which was in the direction from inside the cells to the medium at hypernormal cell Na+ was iiincreased when cell Na+ ncreased. In contrast, net Na+ flux which was in the opposite direction in the presence of ovabain was reduced and became almost 0 at cell Na+ of 900 nmol/mg of protein. The Na+/Rb+ coupling ratio in the ouabain-sensitive cation transport was apparently less than 1 at nearly physiological cell Na+, but it approached 1.5 when cell Na+ was sufficiently high. The sum of cell K+ plus Rb+ varied inversely with cell Na+, and this relation was unaffected upon treatment with ouabain. When Rb+ uptake declined below 80% of the control, cell K+ plus Rb+ was reduced, however, 40% of the sum of cell cations was still preserved even after complete inhibition of the cation pumps by ouabain treatment of 2 hr. Interrelations of these results are discussed.

Regulation of Sodium and Potassium Transport in Phytohemagglutinin-Stimulated Human Blood Lymphocytes

Phytohemagglutinin (PHA) or concanavalin A treatment of lymphocytes causes an increase in membrane permeability so that the leak rates of Na and K increase 1.5- to 2-fold. Active Na and K transport increase proportionately in response to the increased membrane permeability. We have examined the role of lymphocyte Na concentration in sustaining the increased Na and K transport observed after PHA treatment. Cell Na concentration increases from 14.8 to 20.5 mmol/liter cell water in PHA-treated lymphocytes (P < 0.001). Four lines of evidence suggest that the 5-6 mmol/liter cell water increase in lymphocyte Na accounts for the increase in active Na and K transport in mitogen-treated lymphocytes. First, PHA does not increase directly the maximal Na, K-ATPase activity of isolated lymphocyte membrane vesicles. Second, when the Na concentration is increased by 6 mmol/liter cell water in unstimulated lymphocytes, Na and K transport increase nearly twofold. Third, the cell Na concentration (15 mmol/liter cell water) is near the K(m) for Na activation of the Na, K-ATPase in lymphocyte membranes. The ATPase activity thus, is capable of increasing as the cell Na rises above normal. Fourth, if lymphocytes are incubated in a medium containing a low Na concentration, K transport does not maintain the internal K concentration and the fall in cell K is accentuated in PHA-treated lymphocytes. These studies indicate that the adaptive acceleration of Na and K transport in mitogen-treated lymphocytes is mediated by a small increase in cell Na.

Interaction between cell sodium and the amiloride-sensitive sodium entry step in rabbit colon.

Ouabain abolishes the short-circuit current (Isc) and decreases the transepithelial conductance (Gt) of rabbit colon. In contrast, amphotericin B elicits a maximum Isc and markedly increases Gt. However, in both instances the amiloride-sensitive Na entry step is completely blocked, presumably due to an increase in cell Na. Conversely, when Na-depleted tissues are suddenly exposed to 140 mM Na, the amiloride-sensitive Isc and the amiloride-sensitive component of Gt (alphaGNa) increase abruptly to their maximum values and then decline to steady-state plateaus with a half time of approximately 6 min; throughout the decline (Isc/alphaGNa) = ENa is constant at a value of 95 mV. In the presence of amphotericin B, the Isc abruptly rises to the same maximum but does not decline. These findings indicate that in the presence of 140 mM Na the conductance of the amiloride-sensitive Na entry step can vary from a maximum value of approximately 1.6 mmhos/cm2 when cell Na is depleted, to zero when cell Na is abnormally elevated (e.g., in the presence of ouabain or amphotericin B). Our findings are consistent with a system in which the pathway responsible for transcellular Na transport parallels another cellular compartment with which it communicates. The Na capacity of the active transport pathway appears to be very small so that this compartment fills rapidly after exposure of Na-depleted cells to 140 mM Na, and active transepithelial Na transport is initiated and reaches steady-state levels quickly. The Na capacity of the second compartment is much larger; the Na content of this compartment appears to be responsible for the negative feedback effect on the permeability of the amiloride-sensitive entry step.