Cation flux in the ehrlich ascites tumor cell. Evidence for Na+-for-Na+ and K+-for-K+ exchange diffusion.

In a previous study, evidence was presented for an external Na+-dependent, ouabain-insensitive component of Na+ efflux and an external K+-dependent component of K+ efflux in the Ehrlich ascites tumor cell. Evidence is now presented that these components are inhibited by the diuretic furosemide and that under conditions of normal extracellular Na+ and K+ they represent Na+-for-Na+ and K-+for-K+ exchange mechanisms. Using 86Rb to monitor K+ movements, furosemide is shown to inhibit an ouabain-insensitive component of Rb+ influx and a component of Rb+ efflux, both representing approx. 30 percent of the total flux. Inhibition of Rb+ efflux is greatly reduced by removal of extracellular K+. Furosemide does not alter steady-state levels of intracellular K+ and it does not prevent cells depleted of K+ by incubation in the cold from regaining K+ upon warming. Using 22Na to monitor Na+ movements, furosemide is shown to inhibit an ouabain-insensitive component of unidirectional Na+ efflux which represents approx. 22 percent of total Na+ efflux. Furosemide does not alter steady-state levels of intracellular Na+ and does not prevent removal of intracellular Na+ upon warming from cells loaded with Na+ by preincubation in the cold. The ability of furosemide to affect unidirectional Na+ and K+ fluxes but not net fluxes is consistent with the conclusion that these components of cation movement across the cell membrane represent one-for-one exchange mechanisms. Data are also presented which demonstrate that the uptake of alpha-aminoisobutyrate is not affected by furosemide. This indicates that these components of cation flux are not directly involved in the Na+-dependent amino acid transport system A.     

Thiol-Dependent passive K/Cl transport in sheep red cells: IV. Furosemide inhibition as a function of external Rb+, Na+, and Cl−

Summary The effect of the loop diuretic furosemide (4-chloro-N-furfuryl-5-sulfamoyl-anthranilic acid) on the thiol-dependent, ouabain-insensitive K(Rb)/Cl transport in low K⁺ sheep red cells was studied at various concentrations of extracellular Rb⁺, Na⁺ and Cl^–. In Rb⁺-free NaCl media, 2×10^–3 m furosemide inhibited only one-half of thiol-dependent K⁺ efflux. In the presence of 23mm RbCl, however, the concentration of furosemide to produce 50% K⁺ efflux inhibition (IC₅₀) was 5×10^–5 m. In Rb⁺ containing NaCl media, the inhibitory effect of 10^–3 m furosemide was equal to that caused by NO ₃ ^– replacement of Cl^– in the medium. The apparent synergistic action of furosemide and external Rb⁺ on K⁺ efflux was also seen in the ouabain-insensitive Rb⁺ influx. A preliminary kinetic analysis suggests that furosemide binding alters both maximal K⁺(Rb⁺) transport and apparent external Rb⁺ affinity. In the presence of external Rb⁺, Na⁺ (as compared to choline) exerted a small but significant augmentation of the furosemide inhibition of K⁺(Rb⁺) fluxes. There was no effect of Cl^– on the IC₅₀ value of furosemide. As there is no evidence for coupled Na⁺K⁺ cotransport in low K⁺ sheep red cells, furosemide may modify thiol-dependent K⁺(Rb⁺/Cl flux or Rb⁺ (and to a slight degree Na⁺) modulate the effect of furosemide.     

Ba2+-inhibitable86Rb+ fluxes across membranes of vesicles from toad urinary bladder

Summary ⁸⁶Rb⁺ fluxes have been measured in suspensions of vesicles prepared from the epithelium of toad urinary bladder. A readily measurable barium-sensitive, ouabain-insensitive component has been identified; the concentration of external Ba²⁺ required for half-maximal inhibition was 0.6mm. The effects of externally added cations on⁸⁶Rb⁺ influx and efflux have established that this pathway is conductive, with a selectivity for K⁺, Rb⁺ and Cs⁺ over Na⁺ and Li⁺. the Rb⁺ uptake is inversely dependent on external pH, but not significantly affected by internal Ca²⁺ or external amiloride, quinine, quinidine or lidocaine. It is likely, albeit not yet certain, that the conductive Rb⁺ pathway is incorporated in basolateral vesicles oriented right-side-out. It is also not yet clear whether this pathway comprises the principle basolateral K⁺ channel in vivo, and that its properties have been unchanged during the preparative procedures. Subject to these caveats, the data suggest that the inhibition by quinidine of Na⁺ transport across toad bladder does not arise primarily from membrane depolarization produced by a direct blockage of the basolateral channels. It now seems more likely that the quinidine-induced elevation of intracellular Ca²⁺ activity directly blocks apical Na⁺ entry.     

Role of passive potassium fluxes in cell volume regulation in cultured HeLa cells

Summary Cultured HeLa cells behave as ideal osmometers when subjected to hyperosmolar media, and show no volume regulatory behavior. In hypoosmolar solutions, cell swelling is not as great as predicted, and this is due largely to a loss of intracellular KCl. In hyperosmolar solutions there is a stimulation of the ouabain-insensitive but loop diuretic-sensitive⁸⁶Rb⁺ (K⁺) pathway. Analysis of the K⁺, Na⁺ and Cl^– dependency of this K⁺ flux pathway demonstrates that the increase is principally due to an increase in its maximal velocity (V _max). The sensitivity of this pathway to diuretic inhibition is unchanged in hyperosmolar media. Diuretic-sensitive⁸⁶Rb⁺ (K⁺) efflux stimulated by hypertonicity shows no marked dependence on external K⁺. The K⁺ loss observed in hypoosmolar media is distinct from the K⁺ transport pathway stimulated by hyperosmolar media on the basis of its sensitivity to furosemide and anion dependence.     

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

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

K+ transport in ‘Tight’ epithelial monolayers of MDCK cells

Summary Bidirectional transepithelial K⁺ flux measurements across high-resistance epithelial monolayers of MDCK cells grown upon millipore filters show no significant net K⁺ flux.Measurements of influx and efflux across the basal-lateral and apical cell membranes demonstrate that the apical membranes are effectively impermeable to K⁺.K⁺ influx across the basal-lateral cell membranes consists of an ouabain-sensitive component, an ouabain-insensitive component, an ouabain-insensitive but furosemide-sensitive component, and an ouabain-and furosemide-insensitive component.The action of furosemide upon K⁺ influx is independent of (Na⁺–K⁺)-pump inhibition. The furosemide-sensitive component is markedly dependent upon the medium K⁺, Na⁺ and Cl^– content. Acetate and nitrate are ineffective substitutes for Cl^–, whereas Br^– is partially effective. Partial Cl^– replacement by NO₃ gives a roughly linear increase in the furosemide-sensitive component. Na⁺ replacement by choline abolishes the furosemide-sensitive component, whereas Li⁺ is a partially effective replacement. Partial Na⁺ replacement with choline gives an apparent affinity of 7mm Na, whereas variation of the external K⁺ content gives an affinity of the furosemide-sensitive component of 1.0mm.Furosemide inhibition is of high affinity (K _1/2=3 m). Piretanide, ethacrynic acid, and phloretin inhibit the same component of passive K⁺ influx as furosemide; amiloride, 4,-aminopyridine, and 2,4,6-triaminopyrimidine partially so. SITS was ineffective.Externally applied furosemide and Cl^– replacement by NO ₃ ^– inhibit K⁺ efflux across the basal-lateral membranes indicating that the furosemide-sensitive component consists primarily of KK exchange.     

Activation of K-Cl Cotransport by Mild Warming in Guinea Pig Red Cells

Unidirectional, ouabain-insensitive K⁺ influx rose steeply with warming at temperatures above 37°C in guinea pig erythrocytes incubated in isotonic medium. The only component of ouabain-insensitive K⁺ influx to show the same steep rise was K-Cl cotransport (Q₁₀ of 10 between 37 and 41°C); Na-K-Cl cotransport remained constant or declined and residual K⁺ influx in hypertonic medium with ouabain and bumetanide rose only gradually. Similar results were obtained for unidirectional K⁺ efflux. Thermal activation of K-Cl cotransport-mediated K⁺ influx was fully dependent on the presence of chloride in the medium; none occurred with nitrate replacing chloride. The increase of K⁺ influx through K-Cl cotransport from 37 to 41°C was blocked by calyculin A, a phosphatase inhibitor. The Q₁₀ of K-Cl cotransport fully activated by hydroxylamine and hypotonicity was about 2. The time course of K⁺ entry showed an immediate transition to a higher rate when cells were instantly warmed from 37 to 41°C, but there was a 7-min time lag in returning to a lower rate when cells were cooled from 41 to 37°C. These results indicate that the steepness of the response of K-Cl cotransport to mild warming is due to altered regulation of the transporter. Total unidirectional K⁺ influx was equal to total unidirectional K⁺ efflux at 37–45°C, but K⁺ influx exceeded K⁺ efflux at 41°C when K-Cl cotransport was inhibited by calyculin or prevented by hypertonic incubation. The net loss of K⁺ that results from the thermal activation of isosomotic K-Cl cotransport reported here would offset a tendency for cell swelling that could arise with warming through an imbalance of pump and leak for Na⁺ or for K⁺. Received: 1 November 1997/Revised: 5 March 1998     

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

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

Ouabain-resistant Na+, K+ transport system in mouse NIH 3T3 cells

Summary It is shown that the ouabain-resistant (OR) furosemide-sensitive K⁺(Rb⁺) transport system performs a net efflux of K⁺ in growing mouse 3T3 cells. This conclusion is based on the finding that under the same assay conditions the furosemidesensitive K⁺(Rb⁺) efflux was found to be two- to threefold higher than the ouabain-resistant furosemide-sensitive K⁺(Rb⁺) influx. The oubain-resistant furosemide-sensitive influxes of both²²Na and⁸⁶Rb appear to be Cl^– dependent, and the data are consistent with coupled unidirectional furosemide-sensitive influxes of Na⁺, K⁺ and Cl^– with a ratio of 1 1 2. However, the net efflux of K⁺ performed by this transport system cannot be coupled to a ouabain-resistant net efflux of Na⁺ since the unidirectional ouabain-resistant efflux of Na⁺ was found to be negligible under physiological conditions. This latter conclusion was based on the fact that practically all the Na⁺ efflux appears to be ouabainsensitive and sufficient to balance the Na⁺ influx under such steady-state conditions. Therefore, it is suggested that the ouabain-resistant furosemide-sensitive transport system in growing cells performs a facilitated diffusion of K⁺ and Na⁺, driven by their respective concentration gradients: a net K⁺ efflux and a net Na⁺ influx.     

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

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

The ionic relations of Porphyra purpurea(Roth) C.Ag. (Rhodophyta, Bangiales)

Abstract Radioisotope equilibration techniques have been used to determine the intracellular concentration of K⁺, Na⁺ and Cl_?, together with the unidirectional ion fluxes across the plasmalemma of Porphyra purpurea. Influx and efflux of ⁴²K⁺, ²⁴Na⁺ and ³⁶C1^? are biphasic, the rapid, initial uptake and loss of tracer from individual thalli being attributable to desorption from extracellular regions. Cellular fluxes are slower and monophasic, cells discriminating in favour of K⁺ and Cl^? and against Na⁺. A comparison between the equilibrium potential of individual ion species and the measured membrane potential demonstrates that there is an active component of K⁺ and Cl^? influx and Na⁺ efflux. ‘Active’ uptake and ‘passive’ loss of K⁺ and Cl^? are reduced when plants are kept in darkness, suggesting that a fraction of the transport of K⁺ and Cl^? may be due to ‘exchange diffusion’ (K⁺/K⁺ and Cl^?/Cl^?antiport).     

Effects of acetylcholine and caerulein on 86Rb+ efflux in the mouse pancreas. Evidence for a sodium-potassium-chloride cotransport system

The effect of acetylcholine and the cholecystokinin-like peptide, caerulein on the fractional efflux of ⁸⁶Rb⁺ from preloaded isolated segments of mouse pancreas were studied. Both secretagogues evoked a marked transient increase in ⁸⁶Rb⁺ efflux. The removal of Ca²⁺ from the superfusing medium and addition of 10^?4 M EGTA, markedly reduced, but did not abolish the responses to either acetylcholine or caerulein. Furosemide ($\text{10}{}^{\mathrm{?5}}\text{?10}{}^{\mathrm{?3}}\text{M}$) or piretanide (10^?4 M) reduced the basal efflux and inhibited the secretagogue-elicited responses. Stimulus-induced ⁸⁶Rb⁺ outflow was abolished when the Cl^? component of the superfusing solution was replaced by either NO₃^?, SO₄^2? or I^? but not in case of replacement by Br^?, When Na⁺ was replaced with either Li⁺ or choline⁺ both acetylcholine and caerulein failed to elicit any detectable increase in ⁸⁶Rb⁺ outflow. However, when Tris⁺ was substituted for Na⁺, acetylcholine caused a moderate increase in ⁸⁶Rb⁺ efflux which was abolished by either furosemide (10^?4 M) or chloride depletion (nitrate substitution). The removal of extracellular K⁺ or pretreatment with 10^?3 M ouabain had little effect on secretagogue-evoked ⁸⁶Rb⁺ efflux. These results indicate the presence of a diuretic-sensitive Na⁺-K⁺-Cl^? cotransport system in the mouse pancreatic acinar cell membrane.     

Changes in K<Superscript>+</Superscript>, Na<Superscript>+</Superscript>, Cl<Superscript>−</Superscript> balance and K<Superscript>+</Superscript>, Cl<Superscript>−</Superscript> fluxes in U937 cells induced to apoptosis by staurosporine: On cell dehydration in apoptosis

The K⁺, Na⁺, and Cl⁻ balance and K⁺ (Rb⁺) and ³⁶Cl⁻ fluxes in U937 cells induced to apoptosis by 0.2 or 1 μM staurosporine were studied using flame emission and radioisotope techniques. It is found that two-thirds of the total decrease in the amount of intracellular osmolytes in apoptotic cells is accounted for by monovalent ions and one-third consists of other intracellular osmolytes. A decrease in the amount of monovalent ions results from a decrease in the amount of K⁺ and Cl⁻ and an increase in the Na⁺ content. The rate of ³⁶Cl⁻, Rb⁺ (K⁺), and ²²Na⁺ equilibration between cells and the medium was found to significantly exceed the rate of apoptotic change in the cellular ion content, which indicates that unidirectional influxes and effluxes during apoptosis may be considered as being in near balance. The drift of the ion flux balance in apoptosis caused by 0.2 μM staurosporine was found to be associated with the increased ouabain-resistant Rb⁺ (K⁺) channel influx and insignificantly altered the ouabain-sensitive pump influx. Severe apoptosis induced by 1 μM staurosporine is associated with reduced pump fluxes and slightly changed channel Rb⁺ (K⁺) fluxes. In apoptotic cells, the 1.4–1.8-fold decreased Cl⁻ level is accompanied by a 1.2–1.6-fold decreased flux.     

Role of the furosemide-sensitive Na+/K+ transport system in determining the steady-state Na+ and K+ content and volume of human erythrocytesin vitro andin vivo

Summary To study the physiological role of the bidirectionally operating, furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes, the effect of furosemide on red cell cation and hemoglobin content was determined in cells incubated for 24 hr with ouabain in 145mm NaCl media containing 0 to 10mm K⁺ or Rb⁺. In pure Na⁺ media, furosemide accelerated cell Na⁺ gain and retarded cellular K⁺ loss. External K⁺ (5mm) had an effect similar to furosemide and markedly reduced the action of the drug on cellular cation content. External Rb⁺ accelerated the Na⁺ gain like K⁺, but did not affect the K⁺ retention induced by furosemide. The data are interpreted to indicate that the furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes mediates an equimolar extrusion of Na⁺ and K⁺ in Na⁺ media (Na⁺/K⁺ cotransport), a 1:1 K⁺/K⁺ (K⁺/Rb⁺) and Na⁺/Na⁺ exchange progressively appearing upon increasing external K⁺ (Rb⁺) concentrations to 5mm. The effect of furosemide (or external K⁺/Rb⁺) on cation contents was associated with a prevention of the cell shrinkage seen in pure Na⁺ media, or with a cell swelling, indicating that the furosemide-sensitive Na⁺/K⁺ transport system is involved in the control of cell volume of human erythrocytes. The action of furosemide on cellular volume and cation content tended to disappear at 5mm external K⁺ or Rb⁺. Thein vivo red cell K⁺ content was negatively correlated to the rate of furosemide-sensitive K⁺ (Rb⁺) uptake, and a positive correlation was seen between mean cellular hemoglobin content and furosemide-sensitive transport activity. The transport system possibly functions as a K⁺ and waterextruding mechanism under physiological conditiosin vivo. The red cell Na⁺ content showed no correlation to the activity of the furosemide-sensitive transport system.     

K+ Secretion in Rat Distal Jejunum

The Ussing chamber technique was used to measure unidirectional Rb⁺ fluxes under short-circuit conditions across tissue sheets from proximal, central, and distal jejunum of rats. Whereas the proximal and central parts of the jejunum did not show any net transport of Rb⁺, there was a net secretion of around 0.2 μmol hr⁻¹ cm⁻² in the distal segment. This secretion could not be influenced significantly by mucosal application of K⁺ channel blockers such as Ba²⁺ (5 mm), tetraethylammonium (20 mm) or quinine (1 mm). Serosal ouabain (1 mm) blocked net secretion by increasing mucoserosal flux. Blockers of H⁺/K⁺ ATPases could not alter net fluxes of Rb⁺. Stimulation of Cl⁻ secretion by forskolin (10 μm) or of Na⁺ absorption by serine (10 mm) failed to influence the observed secretion of Rb⁺. Adrenaline (10 μm) also had no effect on Rb⁺ fluxes. Blocking Na⁺/H⁺ exchange by 5-(N-Ethyl-N-isopropyl)-amilorid (100 μm) blocked net secretion by increasing mucoserosal flux, as did the addition of Na⁺ acetate (30 mm) to the mucosal solution. We conclude that the distal jejunum of the rat secretes K⁺ under short-circuit conditions. This secretion does not seem to occur via K⁺ channels, but through a pH dependent mechanism. Received: 16 February 1999/Revised: 29 June 1999     

K+-dependent net Na+ efflux in roots of barley plants

Summary The influence of K⁺ ions on the net Na⁺ fluxes in cells of excised barley roots (Hordeum distichon L.) and roots of whole barley plants was investigated. The fluxes were determined by flame photometry in the external solution. In both cases a transient net Na⁺ efflux against the external Na⁺ concentration was observed upon addition of K⁺. The results stress the effectiveness of the K⁺-dependent Na⁺ efflux mechanism residing at the plasmalemma, and its involvement in K–Na-selectivity in whole barley plants.     

Stimulation of monovalent cation fluxes by electron donors in the human red cell membrane

When human red cells are incubated at 37°C with the artificial electron donor system ascorbate + phenazine methosulphate the fluxes of Rb⁺ (K⁺) through the cell membrane are increased. The effect of this donor system is much stronger in energy-depleted than in normal cells. The same effects are produced by HS-glutathione, NADH or NADPH loaded into resealed ghosts, but these electron donors were ineffective when added to the incubation medium. The Rb⁺ (K⁺) fluxes induced by electron donors resemble closely those induced by an increase of intracellular Ca²⁺ (Gardos effect). The electron donors require the presence of intracellular Ca²⁺ to be effective, but at levels that do not stimulate by themselves the fluxes of K⁺. Flavoenzyme inhibitors (atebrin and chlorpromazine), oligomycin and quinine prevented the effects of both electron donors and Ca²⁺ alone; antimycin, uncouplers and ethacrynic acid inhibited them partially; ouabain, furosemide, and rotenone had no effect.The results could be explained if the effect of electron donors is to bring about a change in the redox state of some membrane component(s) that makes intracellular Ca²⁺ more effective to elicit rapid K⁺ movements. Plasma membrane oxidoreductase activities could be engaged in this change.     

Insulin action on cardiac glucose transport Studies on the role of the Na+/K+ pump

Isolated muscle cells from adult rat heart have been used to study the relationship between myocardial glucose transport and the activity of the Na⁺/K⁺ pump. ⁸⁶Rb⁺-uptake by cardiac cells was found to be linear up to 2 min with a steady-state reached by 40–60 min, and was used to monitor the activity of the Na⁺/K⁺ pump. Ouabain (10^?3 mol/I) inhibited the steady-state uptake of ⁸⁶Rb⁺ by more than 90%. Both, the ouabain-sensitive and ouabain-insensitive ⁸⁶Rb⁺-uptake by cardiac cells were found to be unaffected by insulin treatment under conditions where a significant stimulation of 3-O-methylglucose transport occurred. ⁸⁶Rb⁺-uptake was markedly reduced by the presence of calcium and/or magnesium, but remained unresponsive towards insulin treatment. Inhibition of the Na⁺/K⁺ pump activity by ouabain and a concomitant shift in the intracellular Na⁺:K⁺ ratio did not affect basal or insulin stimulated rates of 3-O-methylglucose transport in cardiac myocytes. The data argue against a functional relationship between the myocardial Na⁺/K⁺ pump and the glucose transport system.     

Effect of ouabain upon diuretic-sensitive K+ transport in cultured cells evidence for separate modes of operation of the transporter

(1) Unidirectional K⁺ (⁸⁶Rb) influx and efflux were measured in subconfluent layers of MDCK renal epithelial cells and HeLa carcinoma cells. (2) In both MDCK and HeLa cells, the furosemide-inhibitable and chloride-dependent component of K⁺ influx/efflux was stimulated 2-fold by a 30 min incubation in 1 · 10^?3 M ouabain. (3) Measurements of net K⁺ loss and Na⁺ gain in ouabain-treated cells at 1 h failed to show any diuretic sensitive component, confirming the exchange character of the diuretic-sensitive fluxes. (4) Prolonged incubations for 2.5 h in ouabain revealed a furosemide- and anion-dependent K⁺ (Cl^?) outward net flux uncoupled from net Na⁺ movement. Net K⁺ (Cl^?) outward flux was half-maximally inhibited by 2 μM furosemide. (5) After 2.5 h ouabain treatment, the anion and cation dependence of the diuretic-sensitive K⁺ influx/efflux were essentially unchanged when compared to untreated controls.