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.     

Ouabain-insensitive salt and water movements in duck red cells. II. Norepinephrine stimulation of sodium plus potassium cotransport

Catecholamines induce net salt and water movements in duck red cells incubated in isotonic solutions. The rate of this response is approximately three times greater than a comparable effect observed in 400 mosmol hypertonic solutions in the absence of hormone (W.F. Schmidt and T. J. McManus. 1977 a.J. Gen. Physiol. 70:59-79. Otherwise, these two systems share a great many similarities. In both cases, net water and salt movements have a marked dependence on external cation concentrations, are sensitive to furosemide and insensitive to ouabain, and allow the substitution of rubidium for external potassium. In the presence of ouabain, but the absence of external potassium (or rubidium), a furosemide-sensitive net extrusion of sodium against a large electrochemical gradient can be demonstrated. When norepinephrine-treated cells are incubated with ouabain and sufficient external sodium, the furosemide-sensitive, unidirectional influxes of both sodium and rubidium are half- maximally saturated at similar rubidium concentrations; with saturating external rubidium, the same fluxes are half-maximal at comparable levels of external sodium. In the absence of sodium, a catecholamine-stimulated, furosemide-sensitive influx of rubidium persists. In the absence of rubidium, a similar but smaller component of sodium influx can be seen. We interpret these results in terms of a cotransport model for sodium plus potassium which is activated by hypertonicity or norepinephrine. When either ion is absent from the incubation medium, the system promotes an exchange-diffusion type of movement of the co-ion into the cells. In the absence of external potassium, net movement of potassium out of the cell leads to a coupled extrusion of sodium against its electrochemical gradient.     

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.     

Evaluation of ouabain-insensitive red blood cell cation transport in uremic patients

The authors present the results of a simultaneous assay of: intracellular Na+ and K+ concentrations, Na+ and K+ outward bumetanide-sensitive effluxes (Na+, K+ cotransport), Na+ efflux stimulated by extracellular Li+ (Na+, Li+ countertransport), and ouabain- and bumetanide-resistant Na+ and K+ effluxes (passive membrane permeability) performed in red blood cells from 15 uremic patients an regular hemodialysis and from 12 normal subjects, with an established flux assay. Na+ and K+ effluxes by the Na+, K+ cotransport system were significantly (p less than 0.01) lower in uremic patients then in normals (219 +/- 37 vs 82 +/- 17 mumol/l RBC/h and 251 +/- 29 vs 139 +/- 14 mumol/l RBC/h respectively). In normal subjects the bumetanide sensitive Na+ and K+ effluxes were strongly (r = 0.89; p less than 0.01) intercorrelated; and the intracellular Na+ concentration was related to the outward Na+ cotransport flux (r = 0.53; p approximately 0.05). Among uremic patients these correlations were not found. Na+ and K+ intracellular concentrations, passive Na+ and K+ permeability, and Na+, Li+ countertransport activity were not different among uremic patients and normal controls. In conclusion, in uremic dialyzed patients, red blood cell Na+, K+ cotransport activity is quite uniformly suppressed. The possible pathogenesis of this disfunction is still speculative and deserves further studies.     

Racial differences in bumetanide-sensitive cotransport and N-ethylmaleimide-stimulated potassium efflux

Racial differences in erythrocyte potassium effluxes mediated by two loop-diuretic sensitive modes of cotransport were compared. In red cells loaded to contain approximately equimolar amounts of sodium and potassium, black subjects had lower bumetanide-sensitive sodium-dependent net potassium effluxes as compared to whites. In fresh, washed erythrocytes pretreated with N-ethylmaleimide (NEM), maximal net potassium efflux was greater in blacks than in whites. NEM-stimulated potassium efflux was partially inhibited by bumetanide but only at very high concentrations. The quantitative differences in these two modes of potassium efflux suggest that NEM-stimulated potassium efflux is not an altered mode of sodium-dependent potassium efflux.     

Sodium and potassium content and membrane transport properties in red blood cells from newborn puppies

The intracellular sodium and potassium concentrations and membrane transport properties for these ions were investigated in red blood cells from newborn puppies and adult dogs. At birth the intracellular concentrations of sodium and potassium are much higher than those found in adult dog red cells. During the first few weeks of life the intracellular concentrations of these ions gradually decrease until the adult level is reached. Changes in the membrane transport properties develop concurrently. The rate of active potassium influx, as measured by ouabain-sensitivity, and the pump to leak ratio are greater in red cells from newborn puppies than in those from adult animals. No ouabain-sensitive sodium efflux could be demonstrated in red cells from older puppies or adult dogs. When either puppy or adult dog red cells are depleted of ATP (by incubation at 37°C with no substrate), potassium permeability increases, and the permeability of the membrane to sodium decreases. The addition of adenosine reverses the effect of depletion.     

Effect of bradykinin on Na-K-2Cl cotransport and bumetanide binding in aortic endothelial cells

Simultaneous measurements of potassium influx and binding of [3H]bumetanide were performed in endothelial cells cultured from bovine aortas to determine how bradykinin regulates Na-K-2Cl cotransport. [3H]Bumetanide displayed saturable binding and was displaced by low concentrations of unlabeled bumetanide. All three transported ions were required for binding and high concentrations of chloride inhibited binding, consistent with binding of bumetanide to the second chloride site of the transporter. Scatchard analysis of binding under maximal conditions (100 mM sodium, 30 mM potassium, 30 mM chloride) revealed a single class of binding sites with a binding constant of 112 nM and a density of 22 fmol/cm2 or approximately 122,000 sites/cells. Na-K-2Cl cotransport, measured as bumetanide-sensitive potassium influx, was stimulated 118 +/- 30% by bradykinin (p less than 0.01) at physiologic ion concentrations. Stimulation was inhibited by increased potassium or decreased external chloride concentrations and was not seen in conditions required for maximal binding of bumetanide. Simultaneous measurement of the binding of tracer [3H]bumetanide and its inhibition of potassium influx in medium containing 10 mM potassium and 130 mM chloride revealed a turnover number for the cotransporter of 293 +/- 68 s-1 which increased to 687 +/- 105 s-1 with bradykinin (p less than 0.001). There was no change in cell volume and only a 5.6 mM increase in intracellular sodium concentration associated with this stimulation. Bradykinin also increased the affinity of the cotransporter for bumetanide as indicated by a decrease in the Ki for potassium influx from 464 +/- 46 nM to 219 +/- 19 nM (p less than 0.005). Our results show that [3H]bumetanide can be used to quantitate Na-K-2Cl cotransporter sites in aortic endothelial cells and to determine the mechanism by which cotransport is regulated. The stimulation of cotransport in aortic endothelial cells by bradykinin is due to an increase in the activity of existing transporters rather than to an increase in the number of transporters. This, together with the increased affinity for bumetanide, strongly suggests that a change in cotransporter structure is occurring in response to bradykinin.     

Relationships between membrane lipids and ion transport in red blood cells of Dahl rats

Distinct changes of membrane lipid content could contribute to the abnormalities of ion transport that take part in the development of salt hypertension in Dahl rats. The relationships between lipid content and particular ion transport systems were studied in red blood cells (RBC) of Dahl rats kept on low- and high-salt diets for 5 weeks since weaning. Dahl salt-sensitive (SS/Jr) rats on high-salt diet had increased blood pressure, levels of plasma triacylglycerols and total plasma cholesterol compared to salt-resistant (SR/Jr) rats. Furthermore, RBC of SS/Jr rats differed from SR/Jr ones by increased content of total membrane phospholipids, but membrane cholesterol was not changed significantly. SS/Jr rats had higher RBC intracellular Na+ (Na(i)+) content and enhanced bumetanide-sensitive Rb+ uptake. RBC membrane content of cholesterol and phospholipids correlated positively with RBC Na(i)+ content, with the activity of Na+-K+ pump and Na+-K+-2Cl- cotransport and also with Rb+ leak. The content of phosphatidylserines plus phosphatidylinositols was positively associated with RBC Na(i)+ content, with the activity of Na+-K+ pump and Na+-K+-2Cl- cotransport and with Rb+ leak. The content of sphingomyelins was positively related to Na+-K+-2Cl- cotransport activity and negatively to ouabain-sensitive Rb+-K+ exchange. We can conclude that observed relationships between ion transport and the membrane content of cholesterol and/or sphingomyelins, which are known to regulate membrane fluidity, might participate in the pathogenesis of salt hypertension in Dahl rats.     

Proton-activated rubidium transport catalyzed by the sodium pump

Although the sodium pump normally exchanges three sodium for two potassium ions, experiments with inside-out red cell membrane vesicles show that the stoichiometry is reduced when the cytoplasmic sodium concentration is decreased to less than 1 mM. The present study was designed to gain insight into the question whether other monovalent cations, particularly protons, can act as sodium congeners in effecting pump-mediated potassium transport (ATP-dependent rubidium efflux from inside-out vesicles). The results show that at low cytoplasmic sodium concentration, an increase in proton concentration effects a further reduction in sodium:rubidium stoichiometry, to a value less than the minimal expected (1Na+:3Rb+). Furthermore, when vesicles containing 86RbCl are incubated in nominally sodium-free medium. ATP-dependent net rubidium efflux (normal influx) occurs when the pH is reduced from approximately 7.0 to 6.2 or less. This efflux is inhibited by strophanthidin and vanadate. These experiments support the notion that the sodium pump can operate as an ATP-dependent proton-activated rubidium (potassium) pump without obligatory countertransport of sodium ions.     

Bumetanide-sensitive potassium transport and volume regulation in turkey erythrocytes

The bumetanide-sensitive (K+ + Na+ + 2Cl-)-cotransport system in turkey erythrocytes is activated by either of two treatments: addition of epinephrine or an increase in osmolarity. At elevated (20 mM) K+ concentration, cotransport activity induced by epinephrine slowly (within 90 min) declines to background level again. This time-dependent inactivation has been linked to bumetanide-sensitive cell swelling. We have compared both the initial rate of cotransport activity and its time dependence after induction by either epinephrine, increased osmolarity or a combination of the two treatments. As a measure of cotransport activity we took the bumetanide-sensitive fraction of 86Rb+ influx. Immediately after activation, several kinetic characteristics of this flux (Vmax; Km towards K+; Ki towards bumetanide; pH profile) were identical in cells activated by either treatment. By contrast, cotransport activated by hypertonicity was significantly more resistant towards subsequent inactivation. We show this to be due to the increase in intracellular ion concentrations brought about by hypertonic cell shrinkage. This tended to reverse the driving force for cotransport, and thereby prevented the bumetanide-sensitive swelling associated with inactivation. Our data support the notion that cell volume plays a key role both in the activation and in the time-dependent inactivation of bumetanide-sensitive transport.     

A thermodynamic study of electroneutral K-Cl cotransport in pH- and volume-clamped low K sheep erythrocytes with normal and low internal magnesium

Swelling-induced human erythrocyte K-Cl cotransport is membrane potential independent and capable of uphill transport. However, a complete thermodynamic analysis of basal and stimulated K-Cl cotransport, at constant cell volume, is missing. This study was performed in low K sheep red blood cells before and after reducing cellular free Mg into the nanomolar range with the divalent cation ionophore A23187 and a chelator, an intervention known to stimulate K- Cl cotransport. The anion exchange inhibitor 4,4''diisothiocyanato- 2,2''disulfonic stilbene was used to clamp intracellular pH and Cl or NO3 concentrations. Cell volume was maintained constant as external and internal pH differed by more than two units. K-Cl cotransport was calculated from the K effluxes and Rb (as K congener) influxes measured in Cl and NO3, at constant internal K and external anions, and variable concentrations of extracellular Rb and internal anions, respectively. The external Rb concentration at which net K-Cl cotransport is zero was defined as flux reversal point which changed with internal pH and hence Cl. Plots of the ratio of external Rb concentrations corresponding to the flux reversal points and the internal K concentration versus the ratio of the internal and external Cl concentrations (i.e., the Donnan ratio of the transported ions) yielded slopes near unity for both control and low internal Mg cells. Thus, basal as well as low internal Mg-stimulated net K-Cl cotransport depends on the electrochemical potential gradient of KCl.     

Furosemide-sensitive cation transport in frog skeletal muscle fibers

Furosemide-inhibitable components in unidirectional cation fluxes have been identified in frog skeletal muscle. In sodium loaded muscles, placed in sodium-free rubidium lithium media, furosemide (1 mM) inhibits partially rubidium and lithium influxes as well as potassium and sodium outfluxes. The furosemide-inhibitable components were found to depend on the presence of ouabain. They were greatly diminished in sodium-free magnesium media and were present in chloride-free nitrate containing media. The dependence of furosemide-inhibitable sodium efflux on internal sodium content was also described.     

Cation and ATP content of ferret red cells

Ferret red cells were shown to have the following properties: 1. They have a high sodium (96 mmol/l cell) and low potassium (3.9 mmol/l cell) content. 2. The majority do not appear to have an active sodium pump in their membranes. 3. Their membranes are highly permeable to rubidium indicating that they are probably also highly permeable to potassium. 4. Their magnesium (3.01 mmol/l cell) and calcium (0.01 mmol/l cell) contents are similar to those of red cells from other species. 5. Their ATP content (0.6 mmol/l cell) is similar to that of cat and dog red cells and is sufficiently high to activate known ion transport systems.     

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.     

Changes in sodium pump transport activity and Na+, K+-ATPase expression level following lymphocytes activation in humans

Ouabain-inhibitable rubidium influxes, intracellular sodium content (Nai), and alpha 1-subunit abundance have been studied in human blood lymphocytes, stimulated by phytohemagglutinin (PHA) or by the phorbol 12,13-dibutyrate (PDBu), and calcium ionophore--ionomycin. It is shown that at early stages of PHA-induced activation, the Na/K pump expression (as determined by Wesrn blots of alpha 1 protein in membrane fractions of total cell lysates) does not change, and the increase in Rb influx is due to the increase in Nai and results from the enhanced transport activity of Na/K pumps present in plasma membrane. During the late stages of G0-->G1-->S transit (16-48 h), the increase in Rb influx occurs without changes in Nai, and monensin increases both Nai, and the Rb influx via the Na/K pump. To the end of the first day of mitogen activation, the alpha 1 protein content was found to increase by 5-7 times. A correlation was revealed between changes in ouabain-inhibitable Rb influxes, alpha 1 protein abundance, and the proliferation rate. It is concluded that blasttransformathion of normal human lymphocytes is accompanied by the increase in membrane-associated pool of alpha 1-subunit of Na+,K(+)-ATPase, and the enhanced activity of sodium pump during the G0-->G1-->S progression is provided by increased number of Na+,K(+)-ATPase pumps in plasma membrane.     

Rubidium uptake in single cells

Summary Rubidium uptake was measured in single erythroid and myeloid cells of rabbit by means of X-ray microanalysis. It was found in the nucleated bone marrow cells that after incubation in rubidium the sums of potassium and rubidium concentrations were similar to the original potassium concentrations, indicating that there was one-to-one replacement of potassium by rubidium. Although the nuclear potassium and rubidium concentrations were higher than those in the cytoplasm, the nuclear and cytoplasmic ratios of K/Rb were similar. This implies that the potassium in both compartments exchanged freely with rubidium. In the erythroid line of cells there was a continuous reduction of potassium transport activity during the maturation process as indicated by the decrease in rubidium uptake rates. The uptake was measured in seven groups of cell types that could be distinguished on the basis of morphology and chemical composition. The order of the groups from high to low rubidium uptake were: esosinophilic myelocyte > early erythroblast and thinrimmed erythroblast > late erythroblast > early bone marrow red cell > late bone marrow red cell > peripheral blood red cell. Thus, there is a continuous decrease in rubidium transport as the erythroid cells mature.     

Further photophysical and photochemical characterization of flavins associated with single-shelled vesicles

Summary In order to investigate whether the loop diuretic sensitive, sodium-chloride cotransport system described previously in shark rectal gland is in fact a sodium-potassium chloride cotransport system, plasma membrane vesicles were isolated from rectal glands ofSqualus acanthias and sodium and rubidium uptake were measured by a rapid filtration technique. In addition, the binding of N-methylfurosemide to the membranes was investigated. Sodium uptake into the vesicles in the presence of a 170mm KCl gradient was initially about five-fold higher than in the presence of a 170mm KNO₃ gradient. In the presence of chloride, sodium uptake was inhibited 56% by 0.4mm bumetanide and 40% by 0.8mm N-methylfurosemide. When potassium chloride was replaced by choline chloride or lithium chloride, sodium uptake decreased to the values observed in the presence of potassium nitrate. Replacement of potassium chloride by rubidium chloride, however, did not change sodium uptake. Initial rubidium uptake into the membrane vesicles was about 2.5-fold higher in the presence of a 170mm NaCl gradient than in the presence of a 170mm NaNO₃ gradient. The effect of chloride was completely abolished by 0.4mm bumetanide. Replacement of the sodium chloride gradient by a lithium chloride gradient decreased rubidium uptake by about 40%; replacement by a choline chloride gradient reduced the uptake even further. Rubidium uptake was also strongly inhibited by potassium. Sodium chloride dependence and bumetanide inhibition of rubidium flux were also found in tracer exchange experiments in the absence of salt gradients. The isolated plasma membranes bound³[H]-N-methylfurosemide in a dose-dependent manner. In Scatchard plots, one saturable component could be detected with an apparentK _D of 3.5×10^–6 m and a number of sitesn of 104 pmol/mg protein. At 0.8 m, N-methylfurosemide binding decreased 51% when sodium-free or low-potassium media were used. The same decrease was observed when the chloride concentration was increased from 200 to 600mm or when 1mm bumetanide or furosemide were added to the incubation medium. These studies indicate that the sodium-chloride cotransport system described previously in the rectal gland is in fact a sodium-potassium chloride cotransport system. It is postulated that this transport system plays an essential role in the secondary active chloride secretion of the rectal gland.     

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.     

Heavy water inhibition of alkali cation transport across the muscle membrane. II. A comparison of the action of D20 and ouabain on the sodium efflux and rubidium influx in magnesium media

The action of heavy water and ouabain on sodium effluxes and rubidium influxes has been measured and compared in frog muscles (m. sartorius, R. temporaria). Approximately half of muscle sodium was substituted by lithium by preliminary incubation in mixed sodium-lithium media. The ratio of the ouabain-sensitive parts of rubidium influx and sodium efflux is 7.3:10.5, and that of D2O-sensitive parts of corresponding parameters is 7.5:11.3. A conclusion is made that D2O-effect on the Na, K-ATPase system of muscles under investigation resembles ouabain-effect on sodium effluxes as well as on rubidium influxes.     

Calcium antagonists in hypertension: relation to abnormal sodium transport.

Leucocyte sodium efflux rate constants and intracellular electrolyte contents were estimated in 13 patients with untreated essential hypertension. There was no correlation between intracellular sodium or potassium content or efflux rate constant and blood pressure. The patients were then treated with oral nifedipine and blood pressure controlled. Sodium efflux rate constants and electrolyte contents were estimated one and three months after the start of treatment. There was a significant fall in blood pressure, but mean sodium efflux rate constant and intracellular sodium content were unchanged. There was no correlation between the fall in blood pressure, initial sodium efflux, or intracellular sodium content. These data do not support the hypothesis that the sodium pump and intracellular sodium content have a direct role in generating raised blood pressure, or that treatment of hypertension with calcium antagonists corrects a fundamental alteration of calcium-sodium exchange across the cell membrane.