Concentration Gradients Of Na Research Articles

Isoform-specific functions of Na,K-ATPase in skeletal muscle

The published data and the results of the author’s own research in the field of the molecular and functional diversity of Na,K-ATPases are reviewed. Na,K-ATPase is an integral membrane protein that maintains the concentration gradients of Na+ and K+ that are essential for electrogenesis, excitability, and several other processes of cellular transport. Most of the Na,K-ATPase of vertebrates is found in the skeletal muscle tissue, which co-expresses the α1 and α2 isoforms of the catalytic and transport α-subunit of Na,KATPase. The activity of Na,K-ATPase is crucial for the contractile function and prolonged activity of skeletal muscle. The data that have accumulated indicate that the α1 isoform of Na,K-ATPase fulfills the major pumping function. The α2 isoform fulfills additional functions related to the specific membrane localization of the protein, the functional interactions with the proteins and lipids of the environment, and fine-tuned regulation by a variety of factors, including motor activity.

Dopamine Transporter Inhibition by Organic Ions & their Effects on GBR12909 Inhibition

Dopamine transporters (DATs) in the presynaptic neuron uptake the remaining dopamine in the synaptic cleft, thereby terminating signal transmission and maintaining presynaptic dopamine storage. DATs co-transport two Na+ and one Cl- with one dopamine; the driving force for dopamine uptake is the concentration gradients of Na+ and Cl-. Consequently, dopamine uptake does not occur when either Na+ or Cl- is not present. Interestingly, however, other inorganic ions act like DAT inhibitors, suggesting they would compete with Na+ or Cl- binding, thus reducing the driving force for dopamine transport. Some organic ions are also known to inhibit dopamine uptake while the underlying mechanism is not yet clear. Here, human DAT (hDAT) inhibition by organic cations (NMDG and choline) and anions (gluconate and isethionate) was studied using fluorescence-based dopamine uptake assays. All tested organic ions inhibited hDAT in a concentration-dependent manner, exhibiting complex inhibitory effects associated with their size and charge (IC50 values ranging from 1 to 37 mM). In particular, NMDG showed a stiff sigmoidal curve with an apparent Hill coefficient (h) of 11 while other ions have h values of 1, suggesting hDAT may have multiple binding sites for NMDG. To investigate further the inhibition mechanism by organic ions, their effects on hDAT inhibition by GBR12909, a DAT specific inhibitor were tested. Interestingly, all tested ions reduced GBR12909 potency by ∼50% in a similar fashion. Among them, isethionate seemed to alter GBR12909 inhibition the most, increasing the IC50 value 3-fold and the h value 2-fold. Since isethionate contains a chemical moiety observed in some atypical DAT inhibitors, it might directly compete with GBR12909 binding. Based on the experimental data and the crystal structure of leucine transporter with ligands, putative organic ion binding sites on hDAT were speculated.

Metabolic Pathways for Ion Homeostasis and Persistent Na+ Current

ATP supply in heart cells is preserved by a number of different mechanisms to maintain a constant level of ATP concentration. ADP, phosphocreatine, inorganic phosphate, and cAMP-activated kinases are effectively involved in ATP supply in abnormal conditions such as ischemia. Intracellular Na(+) and Ca(2+) concentrations in the heart cells are maintained at low levels through the operation of ion transport across the plasma membrane, such as Na(+)-K(+) pumps, as well as Na(+)-Ca(2+), and Na(+)-H(+) exchangers. The activity of the latter two exchanger mechanisms depends on their expression levels and the concentration gradients of Na(+) and Ca(2+) across the membrane. These exchangers may interact, and both are strongly regulated by intracellular Na(+), which is maintained by the Na(+)-K(+) pump, utilizing ATP as an energy source. The mitochondria and the sarcoplasmic reticulum are the organelles responsible for intracellular Ca(2+) stores and release sites for maintaining very low cytoplasmic concentration of Ca(2+). Ischemia disrupts the delicate interactions of these transport mechanisms. This may cause intracellular Na(+) and Ca(2+) accumulation, which can cause decreased contractility and electrical instability. Further, a persistent (noninactivating) Na(+) current may be enhanced during ischemia, and this can contribute to action potential prolongation, and the development of early afterdepolarizations. The Na(+) influx via the persistent Na(+) current may induce further Na(+) and Ca(2+) accumulation in the cells.

Toxic effect of the β-amyloid precursor protein C-terminus fragment and Na+/Ca2+ gradients

There is evidence to suggest that certain metabolic fragments of the beta-amyloid precursor protein (betaAPP) containing the whole of the beta-amyloid (Abeta) sequence are toxic to cells. We showed previously that the 105-amino acid C-terminal peptide (CT105) fragment, incorporating Abeta, is particularly toxic to Xenopus oocytes as well as to mammalian neurons. Here, we investigated the contributions of Na+ and Ca2+ gradients to intracellular CT105-induced toxicity in oocytes, monitored by measuring the membrane resting potential. The concentration gradients of Na+ and Ca2+ were manipulated to determine the involvement of the trans-membrane concentration gradients of these ions in the mode of action of CT105. The results suggested that Na+ influx and intracellular events are mainly responsible for the observed CT105-induced toxicity.

Could cytoplasmic concentration gradients for sodium and ATP exist in intact renal cells?

In renal cells, the Na+ pump maintains a transmembrane concentration gradient for sodium ensuring the net reabsorption of sodium with or without cotransported species. This process requires a significant fraction of the ATP turnover of proximal tubules and thick ascending limbs. To understand the potential regulatory influences of Na+ and ATP on the activity of the Na+ pump in these nephron segments, the apparent kinetics of the membrane-bound Na+-K+ ATPase and of the cellular Na+ pump were studied in different preparations of dog proximal tubules and thick ascending limbs (tubular suspensions, tissue homogenates, and basolateral membrane vesicles) obtained from dog kidney cortex and red medulla. Two determinant kinetic parameters, i.e., the apparent Michaelis constant (Km) and the saturating concentrations for sodium and ATP, were compared with the intracellular concentrations of Na+ and ATP measured under physiological conditions. In both types of tubules, the apparent Km value for Na+ (5-15 mM) is set well below the measured mean intracellular concentration of sodium (50-60 mM), suggesting that the Na+ pump should be saturated by sodium ions under normal conditions. Nevertheless, a modest increment of the Na concentration in the vicinity of the pump, obtained by equilibrating the intra- and extra-cellular sodium concentrations at various extracellular [Na+] with nystatin, increases the activity of the Na+ pump in intact cortical tubules and thick ascending limbs, even when the extracellular [Na+] is set at the estimated intracellular [Na+], demonstrating that the pump is not saturated by sodium in situ. Similarly, the kinetics of the renal Na+ pump as a function of the ATP concentration suggested that the pump should be saturated by ATP in physiological conditions, since in both tissues the cellular ATP level (3-6 mM) is higher than the concentration required to achieve saturation of this activity (< 2.5 mM). However, in renal cortical tubules, the steady-state intracellular [Na+] is affected by modest changes of ATP concentration, suggesting that the Na+ pump is not functionally saturated by ATP. Our data suggest that concentration gradients for Na+ and ATP may exist in the cytosol of renal cells. These gradients would be related to the polarity of sodium transport and of the ATP-consuming and ATP-regenerating processes in intact cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Na+-dependent transport of taurine by membrane vesicles of neuroblastoma x glioma hybrid cells.

The transport of taurine into membrane vesicles prepared from neuroblastoma x glioma hybrid cells 108CC5 was studied. A great part of the taurine uptake by the membrane preparation is due to the transport into an osmotically sensitive space of membrane vesicles. Taurine uptake by membrane vesicles is an active transport driven by the concentration gradient of Na+ across the membrane (outside concentration greater than inside). The Km value of 36 microM for Na+-dependent taurine uptake indicates a high-affinity transport system. The rate of taurine transport by the membrane vesicles is enhanced by the K+ gradient (inside concentration greater than outside) and the K+ ionophore valinomycin. Taurine transport is inhibited by several structural analogs of taurine: hypotaurine, beta-alanine, and taurocyamine. All these results indicate that the taurine transport system of the membrane vesicles displays properties almost identical to those of intact neuroblastoma X glioma hybrid cells.

Posidonia australis Growing in Altered Salinities: Leaf Growth, Regulation of Turgor and the Development of Osmotic Gradients

The marine angiosperm Posidonia australis Hook f. is always submerged and the leaf cells are able to exchange ions, gases and water with the surrounding seawater. The base of the youngest lamina is surrounded by sheaths from older leaves and a gradient in cell osmotic pressure existed from the base of the lamina enclosed within the sheath to the emerged zone at the top of the sheath. For plants grown in seawater, the cells at the base of the lamina had an osmotic pressure of 1.34 MPa (seawater = 2.54 MPa); the osmotic pressure increased with distance along the lamina to the emerged lamina value of 3.09 MPa. The osmotic gradient was accounted for by cell concentration gradients of Na+ (73 mol m-3 increasing to 412 mol m-3), K+ (91 mol m-3 increasing to 279 mol m-3) and Cl- (62 mol m-3 increasing to 578 mol m-3). Gradients also existed in the cell concentrations of sucrose and amino acids. It is proposed that, within the solution enclosed by the sheath, a standing osmotic gradient is created by ion uptake from the sheath solution. Leaf growth was unaffected by salinities from 13‰ to 57‰ and net photosynthesis was unaffected by reduction in salinity from 34‰ to 19‰. The cells of the leaves and rhizome adjusted their osmotic pressure by changes in Na+, K+ and Cl- concentrations such that turgor varied only between 0.67 and 1.52 MPa over a range in external osmotic pressures from 0.83 to 3.89 MPa. The tolerance of P. australis to changes in salinity in the absence of severe physical disturbance is due, largely, to the sheath and to the osmotic pressure gradient.

Distribution of chemical elements in the cellular and extracellular components of the stomach and duodenum of rats

Electron-probe x-ray microanalysis was performed on freeze-dried sections, 4 μm in thickness, of the walls of the stomach and duodenum of Sprague-Dawley rats by using a scanning electron microscope equipped with an energy dispersive x-ray detector and a pulse-height analysis system. Comparable cellular and extracellular components, i.e., the luminal epithelium, lamina propria, muscularis mucosae, submucosa, tunica muscularis, and serosa of the stomach and duodenum were probed with an electron beam using an acceleration voltage of 15 kV. This results in the emission of x-rays that are characteristic of the elements present in the volume probed. Six elements of biologic interest were detected, specifically Na, Mg, P, S, Cl, and K. Based on the peak /continuum ratios for these elements, relative concentrations were measured for each of the probe sites. The findings indicate significant differences exist between the relative amounts of Na (P < 0.01) and Cl (P < 0.001) in the submucosae of the stomach and duodenum. Also, between successive layers of the walls of the stomach and duodenum there are apparent differences in the relative concentrations of certain elements. Concentration gradients for Na, Cl, K, and P are apparent between the mucosal and serosal surfaces of these organs. Other elemental concentration and gradient differences are seen between and within the walls of the stomach and duodenum. Furthermore, these studies indicate that different cellular and extracellular probe sites in the stomach and duodenum show different spectral profiles due to subtle differences in peak intensities of the various elements. The findings of the present study suggest that electron-probe x-ray microanalysis might provide useful information on the relative distribution and concentration of chemical elements concerned with the physiologic processes of the gastrointestinal tract.

Concentration gradients of Na+ and Cl? ions between the external medium and gall bladder wall tissues in rabbits

Concentration gradients of Na+ and Cl? ions between the external medium and gall bladder wall tissues in rabbits

The Na + gradient hypothesis in cytoplasts derived from Ehrlich ascites tumor cells

The concentration gradients of Na + and the non-metabolizable amino acid, α-aminoisobutyric acid, and the membrane potential were measured in cytoplasts derived from Ehrlich ascites tumor cells in order to test the Na + gradient hypothesis for the active transport of neutral amino acids in animal cells. According to this hypothesis, the Na + electrochemical gradient and the amino acid activity gradient should be equal at the steady state. It has been difficult to measure the Na + electrochemical gradient in intact Ehrlich cells because Na + may be sequestered in the nuclei of these cells. This problem is avoided with cytoplasts derived from Ehrlich cells because they do not contain internal compartments where Na + could be sequestered. Since these cytoplasts also maintain steady state concentrations of Na +, K +, and α-aminoisobutyric acid similar to those found in whole Ehrlich cells, they are uniquely suited for testing the Na + gradient hypothesis. Assuming the activity coefficients of external and cytoplasmic Na + are equal, the energy in the Na + electrochemical gradient of cytoplasts was 90% of that in the α-aminoisobutyric acid concentration gradient at the steady state. If the Na + gradient hypothesis is correct, the 10% difference between these two gradients cannot be explained in terms of the sequestration of Na + in the nucleus because cytoplasts do not contain internal compartments.

The sink action of cerebrospinal fluid in uremia.

The distribution of water, electrolytes and non-electrolytes in various compartments of the central nervous system was analyzed in rats subjected to various stages of uremia. After bilateral nephrectomy for various periods of time ranging from 1 to 18 h, there is a progressive hyperkalemia, hyponatremia and metabolic acidosis. Although the cerebrospinal fluid (CSF) [K] progressively increases to a concentration of 6 mmol/l at 48 h post-nephrectomy, the ratio of CSF [K]/plasma [K] remains relatively constant throughout the duration of the uremic challenge; on the other hand, the concentration gradient for Na (CSF to plasma) markedly increases with progression of uremia. The steady rise in cellular [K] in both the choroid plexus and in at least one compartment of the cerebral cortex is presumably a reflection of stimulattion of the Na-K pump caused by a build-up in [K] in the extracellular fluids of the brain. The blood-brain and blood-CSF barrier systems to both 14C-urea and 131I remain intact 24--48 h after nephrectomy; and there is not any evidence of cerebral edema at any stage of uremia. Thus, several lines of evidence indicate that the CSF-sink effect is maintained even in advanced uremia.

Some effects of the ionophore X-537A on the isolated rat tail artery.

The effects of micromolar concentrations of the ionophore X-537A (RO 2-2985) were studied using isolated preparations of the rat tail artery. The ionophore causes complete release of catecholamines from adrenergic nerves, which is the sole cause of the transient contractile response. The amines are released by a nonexocytotic process which seems to be related to the ability of X-537A to act as an efficient transmembrane carrier of Na+, k+, and H+. The ionophore also causes an almost complete and irreversible loss of the cocaine-sensitive component of metaraminol uptake by the tissue. X-537A dissipates the transmembrane concentration gradients of Na and K in the smooth muscle component of the preparation. This effect is unrelated to the release of endogenous catecholamines, and it can also be observed after the Na pump has been inhibited with ouabain. It is fully reversible, though not readily, and it can be induced repeatedly. In catecholamine-depleted strips, X-537A dissipates the transmembrane Na+ and K+ gradients without causing any change in tension. Stimulation of the rate of O2 consumption by X-537A in catecholamine-depleted tissue is reversible, and it is unaffected by ouabain and (or) removal of external Ca2+.

Microprobe measurement of Na, K and Cl concentration profiles in epithelial cells and intercellular spaces of rabbit ileum.

THE absorption of many solutes and water by the intestinal mucosa is linked passively to an active transport of Na+ to produce an absorbate which is isotonic with the capillary plasmal1,2. Since Curran3 first proposed a double membrane model with a hypertonic compartment within the tissue, it has generally been accepted that the primary sites of Na+ transport4,5 and solute–water coupling are the lateral intercellular spaces (LIS) within the epithelium (for reviews see refs 6 and 7). Several theoretical models have been proposed to explain how the secretion of Na+ into LIS generates flow of water, but the sites within the tissue where the final isotonicity of the absorbate is achieved remain controversial8–15. This is chiefly because it has not been possible directly to measure the profiles of ionic concentrations in the US and surrounding tissue compartments. Indirect evidence has suggested that the extracellular spaces in gall bladder16, feline intestine17 and rabbit ileum18 contain higher concentrations than those found in external bathing solutions. We have now used electron microprobe X-ray analysis19–22 of frozen-hydrated sections to measure the concentration profiles of Na, K, Cl, Ca, S and P in the mucosal tissue of rabbit ileum. The results establish the hypertonicity of the fluid in the LIS and the concentration gradients of Na, K and Cl in the cells and in the LIS.

Light-sensitive membrane potentials in onion guard cells

INTRACELLULAR electrical recordings in onion guard cells show that they maintain a membrane potential difference (MPD), inside negative. The MPD is light-sensitive; cells subjected to short light and dark cycles depolarise in the dark and hyperpolarise in the light. The swiftness of the electrical changes makes them among the fastest known stomatal responses, suggesting a causal relationship between the reception of light, the changes in membrane potential and the ion fluxes known to be associated with stomatal movement. The magnitude and electrical sign of the responses are consistent with the activity of a light-sensitive proton pump which would provide the driving force for ion transport across the cell membrane. Guard cells, the specialised structures that control gas exchange in the leaves of higher plants, use potassium to modulate their intracellular osmotic potential and perform the mechanical work needed to open and close the stomatal pore1. When guard cells accumulate K+ (with intracellular concentrations reaching up to 0.5 M (refs 2 and 3)), water content increases and swelling occurs, causing the pore to open. During the reverse process, K+ is extruded (intracellular concentration diminishes to 0.1 M (refs 2 and 3)), leading to water loss and pore closure. Control of intracellular K+ concentrations is a universal feature of living organisms. Nerve cells sore energy used to generate electrical signals by maintaining concentration gradients of Na+ and K+ (ref. 4); bacterial cells selectively accumulate K+ (ref. 5) and, in plants, cells expand by increasing their turgor through uptake of K+ and its counter-ions6. While the cellular basis of K+ uptake in stomatal guard cells is poorly understood, one might expect common mechanisms for K+ transport even in widely divergent species. Two distinct mechanisms for K+ transport have been defined. In one, common in animal cells, K+ uptake is driven by ATP through a Na+/K+ ATPase7; in the second system, demonstrated in bacteria, ion transport is conducted by a proton-motive force8 generated by pH and electrical potential gradients across the membrane9. In showing that wall-less guard cell protoplasts swell when illuminated, we previously argued that a proton-motive force could drive K+ uptake into the guard cells10. The hypothesis generates the basic prediction of a light-sensitive, inside negative, MPD across the guard cell membrane. We report here the experimental verification of that prediction, through intracellular recordings in guard cells of Allium cepa.

An isolated rabbit kidney preparation for use in organ preservation research

An isolated rabbit kidney preparation has been developed principally for use as a laboratory assay to compare experimental renal preservation techniques. Perfusion was carried out at 37°C and an arterial pressure of 110 mm Hg using bloodless perfusates containing bovine serum albumin, dextran, hydroxyethyl starch, or Pluronic F108. Glomerular filtration rate and protein leakage were determined with each perfusate. It was found that low filtration rates and high degrees of protein leakage were obtained with perfusates containing either albumin, dextran, or a mixture of these colloids, but both pluronic and hydroxyethyl starch gave acceptable glomerular filtration rates and relatively low protein leakage. The pluronic perfusate was selected for further study. Isolated rabbit kidneys had a greatly reduced vascular resistance, and medullary flow was increased to a proportionally greater degree than cortical flow. The concentration gradient of Na+ normally found between cortex and medulla was lost, and this was associated with an inability of perfused kidneys to effect osmolal concentration of urine. Oxygen consumption was within the limits published for the organ in vivo, and during perfusion the total nucleotide content of renal tissue was well maintained, but there was a small decrease in the ATP/ADP ratio. Function improved steadily during the first hour of perfusion to give a GFR during the second hour that was close to the physiological norm. The fractional reabsorption of water, glucose, and sodium reached maxima of 63, 80, and 67%, respectively. Although the performance of the preparation is subphysiological, it is stable during the time required for the comparison of preserved kidneys and is reproducible. The preparation has already yielded valuable data in this field.

Electrical potential profile in rabbit ileum: role of rheogenic Na transport.

The electrical potential profile of rabbit ileum was investigated in vitro with the microelectrode technique. The transmural electrical potential difference (PD), designated psims, was immediately reduced by 60% upon cooling the tissue from 37 to 7 degrees C; the PD across the mucosal membrane (transmucosal PD, psimc) was simultaneously reduced by 37%. These electrical changes could not be attributed to alternations in either transmembrane ion concentration gradients or total tissue conductance. The psimc and psims may have substantial values even after the concentration gradients of Na and K across the cell membane are eliminated, provided that active transport mechanisms are still operative. Conversely, in the presence of approximately normal transmembrane ion concentration gradients, but when active transport mechanisms have been inhibited. psimc is reduced by 45% and psims is zero. These observations are consistent with a model of electrolyte transport in which psims and the normal transmembrane cation concentration gradients are established by rheogenic active transport of Na out of the cell. The psimc is generated both by rheogenic active Na transport and by cation concentration gradients which exist across the cell membrane. The Koefoed-Johnsen and Ussing model (Acta Physiol. Scand., 1958, vol. 42, p. 298) of electrolyte transport by epithelial cells does not adequately describe the electrical properties of ileum.

Platelet membrane function studies on platelets from patients with hereditary thrombopathic thrombocytopenia.

Routine clotting tests, general platelet function tests, platelet Na&lt;sup&gt;+&lt;/sup&gt; and K&lt;sup&gt;+&lt;/sup&gt; concentrations, the influx and efflux kinetics of &lt;sup&gt;86&lt;/sup&gt;Rb&lt;sup&gt;+&lt;/sup&gt; (as K&lt;sup&gt;+&lt;/sup&gt; analogue) and the osmotic resistance have been studied in platelets from patients with a dominant hereditary macrocytic thrombopathic thrombocytopenia. Calculated on a cell-volume basis the pathological platelets showed no abnormalities in the concentration gradients for Na&lt;sup&gt;+&lt;/sup&gt; and K&lt;sup&gt;+&lt;/sup&gt;. In all patients a normal active uptake and a strikingly decreased passive efflux rate of &lt;sup&gt;86&lt;/sup&gt;Rb&lt;sup&gt;+&lt;/sup&gt; from the platelets was observed. Ouabain increased the efflux rate of &lt;sup&gt;86&lt;/sup&gt;Rb&lt;sup&gt;+&lt;/sup&gt; from control platelets, whereas no or weak efflux-increasing effect was observed in the pathological platelets. The platelets of all patients studied showed a shift towards a more resistant osmotic fragility curve, possibly caused by an increased surface to volume ratio of the cells. The findings are explained in terms of a pathological platelet membrane which is more rigid in structure and which requires more drastic stimuli in order to function properly.

Concentration gradient of Na + and K + ions in outer segments of retinal rods.

The localization of the NA+ ion in the outer segments of frog retinal rods and the state (free or bound) of NA+ and K+ ions in isolated outer segments of bovine rods were studied. The Na+ ion localized mainly in discs of the outer segments; interdisc spaces, continuations of the cell cytoplasm, were rich in K+. About 80% of Na+ and K+ ions in the outer segments are in a relatively free state. Phospholipids of the segment membranes are bound mainly with Na+. The concentration gradient of the cations between contents of the discs (high Na+, low K+) and interdisc spaces (high K+, low Na+) was calculated approximately from the content, distribution, and state of Na+ and K+ in native outer segments which can be regarded as a battery of extracellular compartments separated by intracellular contents. If isolated segments are incubated in media differing in Na+ and K+ content, the accumulation of these cations is limited although dependent on their concentration in the incubation medium.

INHIBITION OF Mg- AND Mg, Na, K-ACTIVATED ADENOSINE TRIPHOSPHATASE ACTIVITY BY L-ASCORBIC ACID AND L-CYSTEINE

It is now commonly postulated that the electrical excitability of neural cells depends on the concentration gradients of Na+ and K+ across the cell membrane and the gradients are maintained by active cation transport system. Further, a membrane-bound, Na+ and K+-stimulated and ouabain-sensitive adenosine triphosphatase (Na-K-ATPase) has been proved to be involved in the active cation transport system in the brain as well as in many other tissues (1). While cardiac glycosides have been well known to be specific inhibitors of Na-K-ATPase, some biogenic substances were also considered to participate in the transport mechanism. In this respect, two biogenic substances, ascorbic acid and cysteine, came to light. They were described as rather specific inhibitors of Na-K-ATPase in the electric organ of eel (2) and the liver of rat (3). The present report describes how ascorbic acid and cysteine affect Na-K-ATPase and Mg-ATPase of the rabbit brain, histochemically and biochemically. For reference the enzymes of rabbit liver were tested.

The influence of ions on the labelling of adenosine triphosphate in red cell ghosts

1. The ionic composition of human red cell ghosts and suspending Ringer solutions have been varied independently. Measurements were made of the incorporation of [(32)P]o-phosphate ((32)P(i)) into ATP associated with different concentration gradients of Na and K across the membrane.2. Some incorporation of (32)P(i) was always found irrespective of the ionic composition of ghosts or media. However, additional labelling of energy-rich phosphate occurred when low Na, high K ghosts were incubated in a high Na, K-free medium. This did not occur when there was only a gradient of either Na or K. Downhill movements of both Na into and K out of the ghosts were needed for the extra labelling.3. Even in the presence of suitable ionic gradients, the extra incorporation was prevented by ouabain or by adding a small amount of external K sufficient to facilitate normal operation of the Na pump.4. Increase in internal P(i) stimulated the incorporation.5. The results show that the conditions for forward and backward running of the ATPase system associated with the Na pump are such that both reactions cannot proceed optimally at the same time.