How does reduced external K+ concentration affect the rate of Na+ efflux? Evidence against the K-Na coupled pump but in support of the association-induction hypothesis.

Recent frog-muscle studies produced the following findings: 1. Contrary to the theory of K+--Na+ coupled pump, reduction of external K+ concentration to near zero did not significantly reduce the rate of efflus of the fraction of cell Na+ conventionally regarded as rate-limited by membrane permeability. 2. Reduction of external K+ concentration profoundly reduced the rate of the efflux of this fraction only if the muscles were exposed to the low K+ while being loaded with radioactive Na+. 3. The data indicate that the fraction of Na+ efflux which in normal cells at room temperature has a half-time exchange (t1/2) of 20-40 min is not rate-limited by membrane permeability but by desorption from cellular adsorption sites. Surface-limited Na+ exchange between free Na+ in the cell and the external environment is represented by a faster fraction with a t1/2 of 2 to 4 min. 4. The data further indicate that the slow-down of the rate of efflux of the (slow) fraction arises from a cooperative shift of those beta- and gamma-carboxyl groups from adsorbing K+ to adsorbing Na+ when external K+ concentration is reduced below a critical level. The enhanced adsorption energy of the newly adsorbed Na+ raises the activation energy, hence a slower rate of exchange is seen as a slow-down in the "efflux curves." It is therefore only when free labeled Na+ is present in the cell water and thus available to the newly emerging Na+ adsorption sites that the effect of low external K+ can be visualized in a labeled-Na+ efflux study. Application of low K+ Ringer's solution after free labeled Na+ in and out of the cells has been washed away only causes enhanced adsorption of non-labeled Na+, which is not detected in isotope efflux study.     

Fast and slow fractions of K+ flux in human lymphocytes.

Potassium influx and efflux were studied in human peripheral blood lymphocytes equilibrated over a wide range of external K+ levels. The absence of a net ion movement throughout the flux study was established, trapped space was measured with polyethylene glycol, and cells were separated from incubation media without exposure to any washing solution. There are both rapid and slow cellular fractions of 42K influx and efflux, and half-times of exchange of around 2 minutes, and 400 minutes, respectively. The rapid component is identical in magnitude to the smaller non-saturable component of cell K+, while the slow component is identified with the larger, sigmoidal, saturable component of cell K+ that was previously shown to follow a cooperative adsorption isotherm. These results support the association-induction hypothesis, which predicts (a) a rapid fraction of K+ flux due to equilibration of ion within cell water existing in a state of polarized multilayers, and (b) a slower component of K+ flux limited by adsorption onto, or desorption from, fixed anionic sites existing throughout the cell. K+ influx, as a function of external K+, showed a triphasic relation with a peak around 1 mM K+ex, then a trough around 4mM K+ex, and then a gradual rise. This relation was readily explained, in terms of the association-induction hypothesis, by the cooperative interaction between, and ion occupancy of, fixed anionic sites that adsorb K+ or Na+.     

Experimental verification of an expected relation between time of incubation and magnitude of the fast and slow fractions of the sodium efflux from amphibian eggs.

The Na+ efflux curves of single ovarian eggs are separable into two fractions. The magnitude of the slow fraction increases slowly with time of exposure of the eggs to labeled Na+, long after the fast fraction has reached equilibrium. The data agree with the theory that the fast fraction is rate-limited by surface permeation and that the slow fraction is rate-limited by desorption from intracellular adsorption sites.     

Kinetic investigation of K+ efflux during glycerol treatment of muscle

The kinetics of K+ efflux was investigated in the membranes of frog sartorius muscle after disintegration by glycerol treatment. Data of the K+ concentration in the muscle as a function of time of the glycerol treatment fitted well the sum of two exponential fractions (with the correlation coefficient of more than 0.98). The half-lives of the two fractions of the K+ efflux were 1 and 75 hours respectively. On the basis of the value of its half life the efflux of the faster fraction was suggested to correspond to the free diffusion. At low temperature the magnitude of the faster fraction increased in a Na+-containing milieu. This could be due to K+-Na+ ion exchange. From the rate of loss of the slower fraction of K+ one finds that movement of K+ in cells without membranes is significantly slower than free diffusion. Presumably, part of the bound potassium exists in intra- or intermolecular "ion-bridges" of muscle proteins.     

Self-exchange of sodium in human lymphocytes.

Self-exchanges of Na and K in human lymphocytes were measured by isotopic efflux techniques. In washed cells, K exchanged in a single slow exponential fraction, but the Na exchange had a marked curvature. It was shown that the curvature was not caused by simple bulk-phase diffusion, and it was resolved into three major fractions: fast (F) (half-time, t1/2 = 2-4 min), intermediate (I) (t1/2 = 12 min), and slow (S) (t1/2 = 125 min). Each of these appeared to follow an exponential function. The I fraction contained approximately 10 mmol Na/kg cells (25-30% of normal cellular Na), was not affected by manipulations that cause lymphocytes to gain Na, and had little or no temperature dependence. The S fraction of Na in normal cells (S1) contained approximately 10 mmol Na/kg cells, had only a slight temperature dependence, and the amount and rate of S1 were independent of external K concentration (Kex). Another slow fraction (S2) appeared when the cells underwent a net gain of Na in exchange for K, and was characterized by a steep temperature dependence and a peak rate around the transition point (the point at which half of cellular K is replaced by Na) at 0.4 mM Kex. The results are discussed within context of a theory that assigns the exchange of the major part of K in its slow exponential fraction and the Na exchange in S2 to interactions of these ions with fixed anionic sites, on intracellular macromolecules, which have been shown previously to interact cooperatively in their association with K and Na.     

The Effect of Ouabain and External Potassium on the Ion Transport of Rabbit Red Cells

Na efflux of rabbit RBC is approximately 10 mmoles/kg wet weight. hr. One-half of this consists of a ouabain-insensitive exchange diffusion component. Ouabain inhibits 2.5 mmoles/kg.hr of Na efflux. K influx is 3.0 mmoles/kg.hr; 2.2 mmoles/kg.hr are inhibited by ouabain. In contrast with human RBC, ouabain inhibition of Na efflux and K influx of rabbit RBC is easily reversible. After 2 hr, ouabain inhibition of Na efflux is completely compensated for by increased internal Na concentration and Na efflux returns to initial levels. Removal of ouabain at this stage results in stimulation of the efflux by 4.3 mmoles/kg.hr. Na influx is initially not affected by ouabain but is increased by 2.4 mmoles/kg.hr after 2 hr incubation with the drug. Removal of K from normal Ringer does not affect Na efflux and increases Na influx by 1.6 mmoles/kg.hr. Addition of ouabain to K-free Ringer inhibits Na efflux and influx to the same extent (1.6 mmoles/kg.hr). Removal of Na from K-free Ringer has an inhibitory effect on efflux similar to that of ouabain. These findings suggest that the fraction of Na efflux inhibited by removal of external K is completely replaced by a new, ouabain-sensitive exchange diffusion of Na ions.     

The effects of temperature and ouabain on steady-state Na and K exchanges in human lymphocytes

The temperature-dependence of steady-state exchanges of K and Na were determined under conditions in which cell viability, ATP, water, Ca, and Mg were not confounding variables. Steady-state ion contents are near-normal between 37° and 10° C, Below 10° C K is replaced by Na in a mole-for-mole fashion with significant net K retention and Na exclusion occurring even below 3°C. The rates of steady-state Kand Na exchanges have markedly different temperature-dependences; between 37° and 10° C, for example, that of K decreases markedly while that of Na remains near-normal, and there is no consistent correlation between the steady-state exchanges and the ionic contents. Ouabain increases steady-state Na flux at 37° C and induces a more marked temperature-dependence over the entire temperature range. This effect is not due simply to inhibition of some processes and unmasking others; it mirrors a pronounced effect of ouabain on the intrinsic properties of Na self-exchange. These results are compatible with a model based on two simple concepts: (1) partial ionic exclusion from cellular water that is ordered by interaction with proteins; and (2) ionic accumulation mediated by adsorption onto and desorption from fixed macromolecular anionic sites, the majority of which interact with one another in a cooperative fashion. In this view, the sharp temperature transition in the net replacement of K by Na below 10° C is due to a critical transition in the selectivity of the cooperatively interacting adsorption sites. The rates of steady-state self-exchanges of K and Na are determined by parameters of ion-site interaction, and the major set of ion-adsorbing sites that interact cooperatively have a steep thermal activation energy of ionic self-exchange. When they are in the K-preferring state above 10° C, exchange of K has a steep temperature-dependence. When they are in the Na-preferring state below 10° C, exchange of Na has a steep temperature-dependence. When these sites are forced into a Na-preferring state at all temperatures by treatment with ouabain, exchange of Na acquires a steep temperature-dependence over the entire temperature range 37° to 0° C.     

Multiple fractions of sodium exchange in human lymphocytes

Human lymphocytes contain a large, saturable fraction of K⁺ that exchanges slowly with K⁺ in the external medium, and a small non-saturable fraction that exchanges rapidly. We determined whether or not Na⁺ exchanges in a similar manner with external Na⁺. Cells were pre-equilibrated to ensure absence of net ion movements. Efflux was studied by loading with ²²Na and transferring without washing to a non-labeled medium. Influx was studied by transferring to labeled medium and separating large samples of cells at 6,000g. There are fast, intermediate, and slow fractions of Na⁺ exchange, with half-times of 2, 14, and 120 minutes. At normal external K⁺, most cell Na⁺ exchanges rapidly, while at lower external K⁺ the Na⁺ that replaces cell K⁺ exchanges slowly. Parallel sources of fast and slow fractions, such as extracellular ones and subpopulations of cells, were ruled out by simultaneous ⁴²K and ²²Na fluxes and by a quantitative analysis of the combined K⁺ and Na⁺ content and flux data over a range of external K⁺ and Na⁺ levels. Five possible models of ion fluxes occurring in series were considered. Surface matrix, surface binding sites, and cytoplasmic channels with rapid nuclea exchange were eliminated as sources of the fast fractions. Therefore, the fast fractions of K⁺ and Na⁺ must reflect the permeability of the surface membrane. This left only two possible sources of the slow fractions. One, a subcellular compartment (e.g., nucleus), was eliminated by the combined content and flux data. We conclude that the slow fractions of ion flux are rate-limited by adsorption onto and desorption from cellular macromolecules. The data support the association-induction hypothesis and are understood by reference to two fundamental concepts: that of rapid solute exclusion from cell water existing in a polarized state; and that of solute accumulation limited by adsorption onto fixed anionic sites within the cell.     

Na+ and K+ levels in living cells: do they depend on the rate of outward transport of Na+?

At 25 degrees C, frog sartorius muslces rapidly gained Na+ and lost K+ in iodoacetamide and pure nitrogen. Beginning at normal levels, the concentrations of these ions in the cells reached those in the surrounding Ringer solution in 140 min. Yet during that time the Na+ efflux rate showed no sign of the slowing down demanded by Na-pump theory. The data support the view that maintenance and alterations of N1+ levels in frog muslce cells reflect adsorption on protein sites and the solubility property of bulk phase water and are independent of the rate at which Na+ leaves the cell surface.     

Sodium and proton transport in Mycoplasma gallisepticum.

When washed cells of Mycoplasma gallisepticum were incubated at 37 degrees C in 250 mM 22NaCl, the intracellular Na+ increased, and the K+ decreased. The addition of glucose to these Na+-loaded cells caused Na+ efflux and K+ uptake (both ions moving against concentration gradients). This effect of glucose was blocked by the ATPase inhibitor dicyclohexylcarbodiimide, which prevents the generation of a proton motive force in these cells. In additional experiments, Na+ extrusion was studied by diluting the 22Na+-loaded cells into Na+-free media and following the loss of 22Na+ from the cells. Glucose stimulated 22Na+ extrusion in such cells by a dicyclohexylcarbodiimide-sensitive mechanism. Proton movement was studied by measuring the pH gradient across the cell membrane with the 9-aminoacridine fluorescence technique. Glucose addition to cells preincubated with cations other than Na+ resulted in cell alkalinization (which was prevented by dicyclohexylcarbodiimide). This observation is consistent with the operation of a proton-extruding ATPase. When glucose was added to Na+-loaded cells and diluted into Na+-free media, intracellular acidification was observed, followed several minutes later by a dicyclohexylcarbodiimide-sensitive alkalinization process. The initial acidification was probably due to the operation of an Na+-H+ antiport, since Na+ exit was occurring simultaneously with H+ entry. When Na+-loaded cells were diluted into Na+-containing media, the subsequent addition of glucose resulted in a weak acidification, presumably due to H+ entry in exchange for Na+ (driven by the ATPase) plus a continuous passive influx of Na+. All of the data presented are consistent with the combined operation of an ATP-driven proton pump and an Na+ -H+ exchange reaction.     

A cooperative transition theory applied to the kinetics of ionic exchanges in cells

Manifestations of a cooperative interaction between ion-adsorbing sites in cells include steep, sigmoidal equilibrium adsorption isotherms of K+ and Na+, critical temperature transitions of net exchanges of Na+ for K+, and the allosteric nature of the effects of ligands on cellular K+ and Na+. Cooperative ionic adsorption is described by a one-dimensional Ising model. The experimentally-determined equilibrium parameters permit prediction of the kinetics of exchange of K+ for Na+ (the approach to equilibrium) by stochastic or hydrodynamic solutions of a time-dependent Ising model. Studies of the rates of self-exchange of adsorbed ions reveal properties of the cooperatively interacting adsorption sites and their dependence on temperature and chemical potential. High rates of isotopic exchanges of K+ and Na+ occur near the transition point. This is explained by the hypothesis of an increase in susceptibility of the ensemble to slight variations of microK or microNA near the phase transition, which leads to an increase in microscopic fluctuations within the ensemble. It is suggested that the isotopic ion exchange experiment may be a means to explore the microscopic states of the ensemble and their transition probabilities.     

Sodium extrusion by the sea-water-acclimated fiddler crab Uca pugilator: comparison with other marine crustacea and marine teleost fish.

1. Measurements of the blood Na concentration and transepithelial electrical potential (T.E.P.) across Uca pugilator acclimated to sea water indicate that Na is maintained out of electrochemical equilibrium with sea water. The resulting net Na influx as well as the sodium gain due to ingestion of the medium must be balanced by extrarenal Na extrusion. 2. The small T.E.P. (-0.7 mV) and the 'transport numbers' of Na and Cl indicate that the permeability to these ions is equivalent. 3. Removal of external K results in a significant stimulation of unidirectional Na efflux that is dependent upon external Na but is not inhibited by ouabain. 4. Transfer of Uca to K and Na-free sea water results in a 54% decline in unidirectional efflux, which is not due to T.E.P. changes. Readdition of 25mM-K stimulates Na efflux much more than can be accounted for by changes in the T.E.P. Readdition of 25mM-Na to potassium-free sea water does not change the Na efflux. 5. The results indicate that Na extrusion by Uca is via a Na/K exchange mechanism which partially inhibits Na/Na exchange. Cessation of Na/K exchange (in K-free sea water) removes this inhibition and allows rapid Na/Na exchange. It is not known whether Na/K and Na/Na exchange are via the same or parallel carrier systems.     

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.     

Volume-sensitive K transport in human erythrocytes

Studies have been carried out on human erythrocytes to examine the alterations of K transport induced by swelling or shrinking the cells by osmotic and isosmotic methods. Hypotonic swelling of erythrocytes (relative cell volume, 1.20) resulted in a striking, four- to fivefold augmentation in the ouabain-resistant K influx over the value obtained at a normal cell volume. Shrinking the cells in hypertonic media resulted in a small but statistically significant reduction in K influx. Three different methods of varying cell volume gave similar results. These include the addition of sucrose and of NaCl to hypotonic media and the isosmotic (nystatin) method. The major fraction of the K influx in swollen cells is specific in its requirement for Cl or Br and is not supported by thiocyanate, iodide, nitrate, methylsulfate, or acetate. Bumetanide (0.1 mM), MK-196 (0.2 mM), and piretanide (1 mM) are poorly effective in suppressing K uptake in swollen cells, but at higher concentrations, bumetanide (1 mM) inhibits 80% of the Cl-dependent K influx in swollen cells. The bumetanide concentration required to inhibit 50% of the Cl-dependent K influx is 0.17 mM. The volume-sensitive K influx is independent of both extracellular and intracellular Na, so that the (Na + K + 2Cl) cotransport pathway is not a likely mediator of the volume-sensitive K transport. A variety of inhibitors of the Ca-activated K channel are ineffective in suppressing swelling-induced K influx. Like K uptake, the efflux of K is also enhanced by cell swelling. Swelling-activated K efflux is Cl dependent, is independent of extracellular and intracellular Na, and is observed with both hypotonic and isosmotic methods of cell swelling. The activation of K efflux by cell swelling is observed in K-free media, which suggests that the volume-sensitive K transport pathway is capable of net K efflux. The addition of external K to hypotonic media resulted in an increase in K efflux compared with the efflux in K-free media, and this increase was probably due to K/K exchange. Thus, hypotonic or isosmotic swelling of human erythrocytes results in the activation of a ouabain-resistant, Cl-dependent, Na-independent transport pathway that is capable of mediating both net K efflux and K/K exchange.     

Kinetic Analyses of Calcium Movements in HeLa Cell Cultures : II. Calcium efflux

Calcium efflux was studied in monolayers of HeLa cells. The fast phase of exchange was studied in an open system by continuous washout. Its half-time was 1.58 min which is practically identical to the fast phase of calcium influx previously found to be 1.54 min. This suggests that the fast component of efflux represents calcium exchange from an extracellular compartment probably from calcium bound to the cell membrane surface. Dinitrophenol (DNP) and iodoacetate (IAA) do not inhibit calcium efflux from this compartment. The slow phase of calcium exchange was studied in a closed three compartment system. The half-time of calcium efflux measured under these conditions is almost identical to that obtained previously in studies of calcium influx: 33.0 and 37.0 min, respectively. This slow compartment is likely to be the intracellular exchangeable calcium pool. DNP and IAA inhibit calcium efflux from this compartment, lengthening the half-time from 33 min to 55.0 and 216 min, respectively. This suggests that calcium extrusion from the cell is an active process. Since calcium influx is not affected by metabolic inhibitors, the cellular calcium concentration increases as would be predicted under these conditions. Calcium efflux is also markedly depressed by lowering the temperature.     

Effects of extracellular ATP on ion transport systems and [Ca2+]i in rat parotid acinar cells. Comparison with the muscarinic agonist carbachol

The effects of extracellular ATP on ion fluxes and the intracellular free Ca2+ concentration ([Ca2+]i) were examined using a suspension of rat parotid acinar cells and were contrasted with the effects of the muscarinic agonist carbachol. Although ATP and carbachol both rapidly increased [Ca2+]i about threefold above the resting level (200-250 nM), the effect of ATP was due primarily to an influx of Ca2+ across the plasma membrane, while the initial response to carbachol was due to a release of Ca2+ from intracellular stores. Within 10 s, ATP (1 mM) and carbachol (20 microM) reduced the cellular Cl- content by 39-50% and cell volume by 15-25%. Both stimuli reduced the cytosolic K+ content by 57-65%, but there were marked differences in the rate and pattern of net K+ movement as well as the effects of K+ channel inhibitors on the effluxes initiated by the two stimuli. The maximum rate of the ATP-stimulated K+ efflux (approximately 2,200 nmol K+/mg protein per min) was about two-thirds that of the carbachol-initiated efflux rate, and was reduced by approximately 30% (vs. 60% for the carbachol-stimulated K+ efflux) by TEA (tetraethylammonium), an inhibitor of the large conductance (BK) K+ channel. Charybdotoxin, another K+ channel blocker, was markedly more effective than TEA on the effects of both agonists, and reduced the rate of K+ efflux initiated by both ATP and carbachol by approximately 80%. The removal of extracellular Ca2+ reduced the ATP- and the carbachol-stimulated rates of K+ efflux by 55 and 17%, respectively. The rate of K+ efflux initiated by either agonist was reduced by 78-95% in cells that were loaded with BAPTA to slow the elevation of [Ca2+]i. These results indicated that ATP and carbachol stimulated the efflux of K+ through multiple types of K(+)-permeable channels, and demonstrated that the relative proportion of efflux through the different pathways was different for the two stimuli. ATP and carbachol also stimulated the rapid entry of Na+ into the parotid cell, and elevated the intracellular Na+ content to 4.4 and 2.6 times the normal level, respectively. The rate of Na+ entry through Na(+)-K(+)-2Cl- cotransport and Na(+)-H+ exchange was similar whether stimulated by ATP, carbachol, or ionomycin, and uptake through these two carrier-mediated transporters accounted for 50% of the ATP-promoted Na+ influx. The remainder may be due to a nonselective cation channel and an ATP-gated cation channel that is also permeable to Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

Voltage dependence of the Na/Ca exchange in voltage-clamped, dialyzed squid axons. Na-dependent Ca efflux

A combination of the voltage-clamp and the intracellular dialysis techniques has been used to study the membrane potential dependence of the Nao-dependent Ca efflux in squid giant axons. In order to improve axon survival, experiments were carried out using internal solutions prepared with large impermeant organic anions and cations, which did not affect the operation of the Na/Ca exchange mechanism. In axons dialyzed with solutions prepared without internal Na, the Nao-dependent Ca efflux had a small sensitivity to membrane potential changes. For a 25-mV membrane displacement in the hyperpolarizing direction, the basal Ca efflux increased by only 7.4% (n = 13). When the dialysis medium contained Na (from 20 to 55 mM), the efflux increased 32.3% (n = 25) for the same membrane potential change. The K1/2 for this effect is approximately 5 mM Na, and saturation appears to occur at a Na concentration above 20 mM. Adding ATP to the dialysis medium increased the magnitude of the Nao-dependent Ca efflux without changing its voltage sensitivity. Wide changes in the intracellular ionized Ca concentration (from 0.1 to 230 microM) did not modify the voltage sensitivity of the exchange system. Elimination of the reversal of Na/Ca exchange (Nai-dependent Ca influx) by removing Cao did not modify the voltage sensitivity of the Nao-dependent Ca efflux. When the axon membrane potential was submitted to prolonged changes, the corresponding changes in the Ca efflux were not sustained, but declined exponentially to intermediate values. This effect may indicate a slow inactivation process in the Na/Ca exchange mechanism. Voltage-clamp pulse experiments revealed: (a) the absence of a fast inactivation process in the Na/Ca exchange, and (b) that the activation of the carrier for hyperpolarizing pulses occurs as rapidly as 1 ms.     

ADP+orthophosphate (P(i)) stimulates an Na/K pump-mediated coefflux of P(i) and Na in human red blood cell ghosts

The Na/K pump in human red blood cells that normally exchanges 3 Nai for 2 Ko is known to continue to transport Na in a ouabain-sensitive and ATP-dependent manner when the medium is made free of both Nao and Ko. Although this Na efflux is called "uncoupled" because of removal of ions to exchange with, the efflux has been shown to be comprised of a coefflux with cellular anions. The work described in this paper presents a new mode of operation of uncoupled Na efflux. This new mode not only depends upon the combined presence of ADP and intracellular orthophosphate (P(i))i but the Na efflux that is stimulated to occur is coeffluxed with (P(i))i. These studies were carried out with DIDS- treated resealed red cell ghosts, suspended in buffered (NMG)2SO4, that were made to contain, in addition to other constituents, varying concentrations of ADP and P(i) together with Na2 SO4, MgSO4 and hexokinase. While neither ADP nor P(i) was effective alone, ouabain- sensitive uncoupled Na efflux, (measured with 22Na) could be activated by [ADP+P(i)] where the K0.5 for ADP in the presence of 10 mmol (P(i))i/liter ghosts was 100-200 mumol/liter ghosts and the K0.5 for (P(i))i, in the presence of 500 mumol ADP/liter ghosts was 3-4 mmol/liter ghosts. [ADP+P(i)] activation of this Na efflux could be inhibited by as little as 2 mumol ATP/liter ghosts but the inhibition could be relieved by the addition of 50 mM glucose, given entrapped hexokinase. While ouabain-sensitive Na efflux was found to be coeffluxed with P(i) (measured with entrapped [32P]H3PO4), this was not so for SO4 (measured with 35SO4). The stoichiometry of Na to P(i) efflux was found to be approximately 2 to 1. Na efflux as well as (P(i))i efflux were both inhibited by 10 mM Nao (K0.5 approximately equal to 4 mM). But, whereas 20 mM Ko (K0.5 approximately equal to 6 mM) inhibited the efflux of (P(i))i, as would be expected from previous work, Na efflux was actually increased. When Ko influx was measured in this situation there was a 1 for 1 exchange of Nai for Ko, that is, of course, downhill with respect to the gradient of each ion. Surprisingly AsO4 was unable to replace P(i) for activation of Na efflux but Na efflux could be inhibited by vanadate and oligomycin. In terms of mechanism, it is likely that ADP acts to promote the formation of the phosphoenzyme (EP) by (P(i))i that would otherwise be inhibited by Nai.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modes of operation and variable stoichiometry of the furosemide- sensitive Na and K fluxes in human red cells

We report in this paper different modes of Na and K transport in human red cells, which can be inhibited by furosemide in the presence of ouabain. Experimental evidence is provided for inward and outward coupled transport of Na and K, Ki/Ko and Nai/Nao exchange, and uncoupled Na or K efflux. The outward cotransport of Na and K was defined as the furosemide-sensitive (FS) component of Na and K effluxes into choline medium and as the Cl-dependent or cis-stimulated component of the ouabain-resistant (OR) Na and K effluxes. Inward cotransport of Na and K was defined by the stimulation by external Na (Nao) of the K influx and the stimulation by external K (Ko) of the Na influx in the presence of ouabain. Both effects were FS and Cl dependent. Experimental evidence for an FS Ki/Ko exchange pathway of the Na/K cotransport was provided by (a) the stimulation by external K of FS K influx and efflux, and (b) the stimulation by internal Na or K of FS K influx in the absence of external Na. Evidence for an FS Nai/Nao exchange pathway was provided by the stimulation of FS Na influx by internal Na from a K-free medium (130 mM NaCl). This pathway was four to six times smaller than the Ki/Ko exchange. In cells containing only Na or K, incubated in media containing only Na or K, respectively, there was FS efflux of the cation without simultaneous inward transport (FS uncoupled Na and K efflux). The stoichiometric ratio of FS outward cotransport of Na and K into choline medium varied with the ratio of Nai-to-Ki concentrations, and when Nai/Ki was close to 1, the ratio of FS outward Na to K flux was also 1. In choline media, FS Na efflux was inhibited by external K (noncompetitively), whereas FS k efflux was stimulated. The stimulation of FS K efflux was due to the stimulation by Ko of the Ki/Ko exchange pathway. Thus, the stoichiometry of FS Na and K effluxes also varied in the presence of external K. A minimal model for a reaction scheme of FS Na and K transport accounts for cis stimulation, trans inhibition, and trans stimulation, and for variable stoichiometry of the FS cation fluxes.     

Properties of the Na+-dependent Cl-/HCO3- exchange system in U937 human leukemic cells

U937 cell possess two mechanisms that allow them to recover from an intracellular acidification. The first mechanism is the amiloride-sensitive Na+/H+ exchange system. The second system involves bicarbonate ions. Its properties have been defined from intracellular pH (pHi) recovery experiments, 22Na+ uptake experiments, 36Cl- influx and efflux experiments. Bicarbonate induced pHi recovery of the cells after a cellular acidification to pHi = 6.3 provided that Na+ ions were present in the assay medium. Li+ or K+ could not substitute for Na+. The system seemed to be electroneutral. 22Na+ uptake experiments showed the presence of a bicarbonate-stimulated uptake pathway for Na+ which was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate. The bicarbonate-dependent 22Na+ uptake component was reduced by depleting cells of their internal Cl- and increased by removal of external Cl-. 36Cl- efflux experiments showed that the presence of both external Na+ and bicarbonate stimulated the efflux of 36Cl- at a cell pHi of 6.3. Finally a 36Cl- uptake pathway was documented. It was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate (K0.5 = 10 microM) and bicarbonate (K0.5 = 2 mM). These results are consistent with the presence in U937 cells of a coupled exchange of Na+ and bicarbonate against chloride. It operates to raise the intracellular pH. Its pHi and external Na+ dependences were defined. No evidence for a Na+-independent Cl-/HCO3- exchange system could be found. The Na+-dependent Cl-/HCO3- exchange system was relatively insensitive to (aryloxy)alkanoic acids which are potent inhibitors of bicarbonate-induced swelling of astroglia and of the Li(Na)CO3-/Cl- exchange system of human erythrocytes. It is concluded that different anionic exchangers exist in different cell types that can be distinguished both by their biochemical properties and by their pharmacological properties.