The transport of sodium into human erythrocytes in vivo

The relative Na²⁴ specific activity of red cells and plasma was measured at periods up to 30 hours following a single intravenous injection of Na²⁴ in normal healthy young adults. The average specific activity of the red cells relative to that of the plasma at 24 hours and beyond was found to average 0.83 ± 0.05 in a series of five normal individuals, significantly different from 1.0. This indicates that all the intracellular Na is not exchangeable in 24 hours, and confirms earlier in vitro results. The red cell Na concentration in man was shown to be 12.1 ± 1.1 m.eq. Na/liter red cell, as measured in a series of nineteen normal healthy young adults. A theoretical analysis of the data on exchangeable cell Na suggests that the red cell Na (5.3 m.eq. Na/liter blood) is divided into a fast compartment comprising 4.25 m.eq. Na/liter blood, and a slow compartment comprising 1.07 m.eq. Na/liter blood. If these compartments are arranged in parallel, the flux between plasma and fast compartment is 1.32 m.eq. Na/liter blood hour, and that between plasma and slow compartment is 0.016 m.eq. Na/liter blood hour. Results of experiments on two patients with congenital hemolytic jaundice suggest that the fraction of slowly exchanging Na may increase with the age of the red cell.     

Heterogeneity in dog red blood cells: sodium and potassium transport

After incubation in isotonic KCl, dog red blood cells can be separated by centrifugation into subgroups which assume different cell volumes and possess different transport characteristics. Those red cells which swell in isotonic KCl exhibit a higher permeability to K and possess a greater volume dependence for transport of K than those red cells which shrink. A high Na permeability characterizes cells which shrink in isotonic KCl and these cells exhibit a larger volume-dependent Na flux than those red cells which swell. These two subgroups of red cells do not seem to represent two cell populations of different age. The results indicate that the population of normal cells is evidently heterogeneous in that the volume-dependent changes in Na and K permeability are distributed between differnt cell types rather than representing a single cell type which reciprocally changes its selectivity to Na and K.     

Influences of cell volume and adrenalectomy on cation flux in dog red blood cells

Rates of ²⁴Na and ⁴²K entry into dog red blood cells were found to be strongly influenced by cell volume. The kinetics of isotope movement were complex, and the cells were not in a steady state. By applying a simple, two-compartment equation to the early times points, values for flux were calculated and corrected for the changes in surface/volume ratio which occur when cells are shrunken or swollen. Curves were thus generated showing Na and K influx as functions of cell water content. A reinvestigation of the effects of adrenalectomy showed that all the observd changes in Na flux could be explained on the basis of alterations in red cell volume.     

Volume-activated Na/H exchange activity in fetal and adult pig red cells: Inhibition by cyclic AMP

Summary Hyposmotic swelling of pig red cells leads to a selective increase in K permeability, whereas hyperosmotic cell shrinkage augments the Na permeability. In this regard, the ouabain-resistant (OR) Na flux of red cells of newborn and adult pigs is characterized in detail. A reduction in cell volume by approximately 18% leads to an increase in the OR Na efflux of fetal and adult cells by 15-and fourfold, respectively. The OR Na influx in both cell types is equally influenced by cell shrinkage. Depletion of cellular K does not influence the volume-activated OR Na efflux. Nor does OR Na influx require external K. Both OR Na efflux and influx activated by shrinkage are inhibited by the diuretics furosemide and amiloride. The rank order of decreasing anion sensitivity for diuretic-sensitive Na efflux was acetate > chloride > gluconate > nitrate. Cell shrinkage induced by the addition of hypertonic salts results in an acidification of the unbuffered and CO₂-free media, provided that both Na and DIDS are present. The qcidification process can be reversed by either of the diuretic agents. These findings suggest that the shrinkageactivated OR Na flux is primarily mediated by a Na/H exchanger rather than by a Na/K/Cl cotransporter. Once loaded with either cAMP or cGMP, cell swelling can no longer activate the Na/H exchanger. The Na/H exchanger activity is detectable in the fetal cells of normal volume but quiescent in adult cells, indicating that the exchanger undergoes a developmental change during the transition from the fetal to adult stage.     

Potassium transport in human erythrocytes: evidence for a three compartment system

Whole human blood is incubated for periods of ½ to 3 hours with K⁴² at 37°C. At the close of this period, called pre-incubation, the plasma is removed from the cells and the cells, now become radioactive, are again incubated in a mixture of plasma and buffer for periods of up to 10 additional hours. The time course of the K⁴² activity of the incubating medium is followed. Characteristically, after 2 hours of pre-incubation, the activity in the medium rises to a peak about 1 and ½ hours after resuspension, and then falls slowly until at 10 hours it is very close to its initial value at the beginning of the resuspension interval. This transient rise in K⁴² activity in the medium is taken to indicate that the red cell does not consist of a single uniform K compartment, but contains at least two compartments. Thus one cellular compartment contains a reservoir of high specific activity K which provides the specific activity gradient necessary to drive the K⁴² content of the medium to its transient peak. Experiments with Na indicate that its behavior in this respect is unlike that of K. The experimental data are matched to a simple model system which is capable of theoretical analysis with the aid of an analogue computer. The model system, whose characteristics agree fairly well with those observed experimentally on red cell suspensions, comprises two intracellular compartments, one containing 2.35 m.eq. K/liter blood, and the other 44.1 m.eq. K/liter blood. The plasma K content is 2.64 m.eq./liter blood. The flux between plasma and the smaller intracellular compartment is 0.65 m.eq. K/liter blood hour; that between the smaller and the larger intracellular compartment, 1.77 m.eq. K/liter blood hour; and that between the larger intracellular compartment and the plasma is 0.34 m.eq. K/liter blood hour.     

Some Kinetic and Metabolic Characteristics of Calcium-Induced Potassium Transport in Human Red Cells

When fresh human erythrocytes or their ghosts are incubated with Ca + IAA (iodoacetic acid) + adenosine, K permeability increases; K permeability also increases when energy-depleted cells or their ghosts are incubated with Ca alone. Na transport decreases or remains unaltered in both situations. The Ca-induced increase in K permeability in the depleted cell system is qualitatively similar to that seen in the fresh cell system and furnishes a means for studying the metabolic dependence of calcium's action. Studies with the depleted system suggest that the normal refractiveness of the cell to calcium is provided by a metabolically dependent substrate. Removal of this substrate allows Ca to enter the cell and exert its effect. By using ⁴⁷Ca, a maximum value was obtained (3–7 x 10^-6 moles/liter of red blood cells) for the quantity of calcium that is taken up by the cell and responsible for the change in K permeability. Measurements of the unidirectional fluxes of K, obtained during the time Ca increases K permeability, appear to satisfy the flux ratio equation for passive diffusion through a 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.     

The Entry of Sodium into Human Red Blood Cells in Vivo

The kinetics of sodium, movement into human red blood cells has been studied in vivo with ²⁴Na. When human serum albumin^-131I is used to measure the percentage of plasma trapped in the packed red blood cells after centrifugation, approximately 30 % of red blood cell sodium is found to equilibrate immediately with plasma. It is concluded that this immediately exchangeable compartment of red blood cell sodium is an experimental artefact, associated with the use of labeled albumin for measuring plasma trapping. This immediately exchangeable fraction disappears when sucrose-¹⁴C is used to measure plasma trapping. The experimental results were examined by compartmental analysis, using an analogue computer. The results obtained, when plasma trapping was measured with sucrose-¹⁴C could be simulated by the use of models containing two compartments, arranged in series or in parallel. The errors of the techniques used and the possible physical basis for the results are discussed.     

Red Cell Membrane Permeability Deduced from Bulk Diffusion Coefficients

The permeability coefficients of dog red cell membrane to tritiated water and to a series of[¹⁴C]amides have been deduced from bulk diffusion measurements through a "tissue" composed of packed red cells. Red cells were packed by centrifugation inside polyethylene tubing. The red cell column was pulsed at one end with radiolabeled solute and diffusion was allowed to proceed for several hours. The distribution of radioactivity along the red cell column was measured by sequential slicing and counting, and the diffusion coefficient was determined by a simple plotting technique, assuming a one-dimensional diffusional model. In order to derive the red cell membrane permeability coefficient from the bulk diffusion coefficient, the red cells were assumed to be packed in a regular manner approximating closely spaced parallelopipeds. The local steady-state diffusional flux was idealized as a one-dimensional intracellular pathway in parallel with a one-dimensional extracellular pathway with solute exchange occurring within the series pathway and between the pathways. The diffusion coefficients in the intracellular and extracellular pathways were estimated from bulk diffusion measurements through concentrated hemoglobin solutions and plasma, respectively; while the volume of the extracellular pathway was determined using radiolabeled sucrose. The membrane permeability coefficients were in satisfactory agreement with the data of Sha'afi, R. I., C. M. Gary-Bobo, and A. K. Solomon (1971. J. Gen. Physiol. 58:238) obtained by a rapid-reaction technique. The method is simple and particularly well suited for rapidly permeating solutes.     

Ion Absorption and Retention by Chlorella Pyrenoidosa. II. Permeability of the Cell to Sodium and Rubidium

The Na and Rb permeability of Chlorella pyrenoidosa were estimated from the rates of radioisotope self-diffusion.

The isotopic exchange in absence of net ionic movements followed first order kinetics. This suggested that for sodium, which reached isotopic equilibrium in approximately 90 minutes, the cell behaved as 1 compartment with respect to isotopic exchange. Rubidium in 180 minutes approached isotopic equilibrium by 67%; thus, the existence of a single compartment for Rb has not been demonstrated. Net fluxes, calculated from the isotope exchange data, and expressed on a dry weight and surface area base showed that Na fluxes were approximately 7 times larger than Rb fluxes. Net Na fluxes of 90 milli-equivalents per 100 g dry weight per hour were far in excess of the observed maximum net accumulation of Na. However, Rb fluxes of 13 milliequivalents per 100 g dry weight per hour were of similar magnitude as the rate of Rb accumulation. Thus, permeability could be a limiting factor for Rb but not for Na accumulation. Sodium and Rb fluxes in absence of net ionic movements were inhibited by low temperature, dark air and dark N₂ conditions. This change in flux rates was explained mainly on the basis of metabolically dependent changes in the cell surface layers.

Isotope fluxes of Rb were drastically reduced in dark air and dark N₂ in the absence or presence of net cation movements. Dark N₂ essentially eliminated net cation accumulation, whereas dark air had relatively little effect on the net K and Rb accumulation by Chlorella. Thus the 2 major factors involved in net cation accumulation in the Chlorella cell, permeability and processes leading to cation retention, respond differently to metabolic inhibition permitting a separation of these 2 important aspects of cation accumulation.

     

Diffusion and permeation of cations in human and dog erythrocytes

The kinetics of movement of tracer Na into human and dog red cells have been studied. The time courses of these processes and of K transfer were compared with the theoretical time course for saturation of a flat sheet having a resistive surface. The theoretical and the experimental curves when plotted against t^½ have a considerable portion which is linear; on the basis of this resemblance the results are interpreted in terms of a permeability constant and an internal diffusion constant. It is supposed that selective adsorption acts to bring about concentration of K in the human cell and that the bulk of the Na of that cell is present in a thin outer region, while the K is in the interior. The action of strophanthin is to remove the usual limit to the Na capacity of the cell and it is proposed that the Na region increases in thickness at the expense of the K region. Omission of K from the medium has a similar result. Na uptake into poisoned cells measured either with tracer or as a net gain has a linear dependence upon t^½ after a delay. The permeability of the dog cell to Na is reduced when K is added to the medium; this may be due to the formation of an outer K-rich region which imposes a resistance to Na movement.     

Cell volume regulation by Amphiuma red blood cells. The role of Ca+2 as a modulator of alkali metal/H+ exchange

In response to osmotic perturbation, the Amphiuma red blood cell regulates volume back to "normal" levels. After osmotic swelling, the cells lose K, Cl, and osmotically obliged H2O (regulatory volume decrease [RVD] ). After osmotic shrinkage, cell volume is regulated as a result of Na, Cl, and H2O uptake (regulatory volume increase [RVI] ). As previously shown (Cala, 1980 alpha), ion fluxes responsible for volume regulation are electroneutral, with alkali metal ions obligatorily counter-coupled to H, whereas net Cl flux is in exchange for HCO3. When they were exposed to the Ca ionophore A23187, Amphiuma red blood cells lost K, Cl, and H2O with kinetics (time course) similar to those observed during RVD. In contrast, when cells were osmotically swollen in Ca-free media, net K loss during RVD was inhibited by approximately 60%. A role for Ca in the activation of K/H exchange during RVD was suggested from these experiments, but interpretation was complicated by the fact that an increase in cellular Ca resulted in an increase in the membrane conductance to K (GK). To determine the relative contributions of conductive K flux and K/H exchange to total K flux, electrical studies were performed and the correspondence of net K flux to thermodynamic models for conductive vs. K/H exchange was evaluated. These studies led to the conclusion that although Ca activates both conductive and electroneutral K flux pathways, only the latter pathways contribute significantly to net K flux. On the basis of observations that A23187 did not activate K loss from cells during RVI (when the Na/H exchange was functioning) and that amiloride inhibited K/H exchange by swollen cells only when cells had previously been shrunk in the presence of amiloride, I concluded that Na/H and K/H exchange are mediated by the same membrane transport moiety.     

Ion transport and permeability in the mouse egg

Sodium, potassium, and chloride unidirectional fluxes have been studied in the mature mouse egg. Their relationship to cell membrane potential and conductance has been investigated. Unidirectional Na efflux is composed of a ouabain sensitive component, presumably representing an active Na efflux, an external Na-dependent component and a diffusional component. The data indicate that the external Na-dependent component represents a Na:Na exchange mechanism. There also exists an ouabain-sensitive component of K influx. The stoichiometry of the ouabain-sensitive fluxes is approx. 2.7:1 (Na to K). From the diffusional components of Na and K flux, the membrane permeability to these cations has been estimated. P_Na and P_K are 1.2 × 10⁻⁷ cm sec⁻¹ and 0.8 × 10⁻⁷ cm sec⁻¹ respectively. These permeabilities, in conjunction with the internal exchangeable fractions of Na and K and the external concentrations, predict an egg membrane potential of −11 mV (inside negative). Microelectrode measurements yield an egg membrane potential of −14 ± 0.4 mV, indicating that the cell membrane potential is predominantly a result of the Na and K permeabilities and distributions. Internal exchangeable Cl is 67 ± 3 mM in standard medium, as determined from ³⁶Cl distribution. The chloride equilibrium potential is therefore −15 mV, which is not significantly different from the egg membrane potential. This suggests that Cl distributes passively across the egg membrane, reflecting the egg membrane potential. Hyperpolarization of the egg membrane potential to −27 ± 1.5 mV by reduction of external Na results in an exchangeable internal Cl of 49 ± 8 mM. This yields a Cl equilibrium potential of −24 mV, indicating that the Cl distribution shifts in the predicted manner upon a change in cell membrane potential. Tracer flux data indicate that Cl conductance comprises the bulk of the total membrane conductance with Na and K sharing the remainder in approximately equal amounts.     

Temperature sensitivity of potassium flux into red blood cells in the familial pseudohyperkalaemia syndrome

The temperature dependence of potassium flux into the red cells of normal and pseudohyperkalaemic individuals over the range 4-40 degrees C was measured using 86RbCl as tracer. Flux through the pump was measured as the ouabain-sensitive component (0.2 mM ouabain) and flux via Na+,K+-cotransport was measured as the decrease in the rate of K+ influx in the presence of 1 mM furosemide. The residual passive permeability of the red cell plasma membranes to K+ was that influx which was unaffected by either inhibitor. When Na+ influxes were measured, the ratio of Na+ to K+ transported via the furosemide-sensitive component was 1 over the full temperature range studied. The temperature sensitivity of K+ influx via the pump was normal as was the enzymic activity of the Na+,K+-ATPase. In contrast, the activity of the Na+,K+-cotransport system in pseudohyperkalaemics was more temperature sensitive than that of controls and affected individuals also showed a greater passive permeability to K+ at low temperatures. Red cell membranes from affected individuals have significantly increased amounts of phosphatidylcholine which are balanced, to a degree, by a decreased content of phosphatidylethanolamiane. It is proposed that in this example of familial pseudohyperkalaemia there is an alteration in the structure of the red cell plasma membrane which influences the temperature sensitivity of both its cotransport and passive permeability properties.     

Delivery of ion pumps from exogenous membrane-rich sources into mammalian red blood cells.

Using polyethylene glycol-mediated fusion of ATP-ase-enriched (native) microsomes with red blood cells, we have delivered sarcoplasmic reticulum (SR) Ca-ATPase and kidney Na,K-ATPase into the mammalian erythrocyte membrane. Experiments involving delivery of the SR Ca-ATPase into human red cells were first carried out to assess the feasibility of the fusion protocol. Whereas there was little detectable 45Ca2+ uptake into control cells in either the absence or presence of extracellular ATP, a marked time-dependent uptake of 45Ca2+ was observed in the presence of ATP in cells fused with SR Ca-ATPase. Comparison of the kinetics of uptake into microsome-fused cells versus native SR vesicles supports the conclusion of true delivery of pumps into the red cell membrane. Thus, the time to reach steady state was more than two orders of magnitude longer in the (large) cells versus the native SR vesicles. Na,K-ATPase from dog and rat kidney microsomes were fused with red cells of humans, sheep, and dogs. Using dog kidney microsomes fused with dog red cells which are practically devoid of Na,K-ATPase, functional incorporation of sodium pumps was evidenced in ouabain-sensitive Rb+ uptake and Na+ efflux energized by intracellular ATP, as well as in ATP-stimulated Na+ influx and Rb+ efflux from inside-out membrane vesicles prepared from the fusion-treated cells. From analysis of the biphasic kinetics of ouabain-sensitive Na+ efflux under conditions of limited intracellular Na+ concentration, it is concluded that the kidney pumps are incorporated into a relatively small fraction (approximately 15%) of the red cells. This system provides a uniquely useful system for studying the behavior of native sodium pumps in a compartment (red cell) of small surface/volume ratio. The newly incorporated native kidney pumps, while of the same isoform as the endogenous red cell pump, behave differently from the endogenous red cell sodium pump with respect to their very low "uncoupled" Na+/O flux activity.     

Cation Transport in Dog Red Cells

Studies have been made on the cation transport system of the dog red cell, a system of particular interest because it has been shown that there is a marked dependence of cation fluxes on the cell volume. We have found that a 10% decrease in cell volume causes a large increase in 1 hr uptake of ²⁴Na as well as a considerable inhibition of ⁴²K uptake. This effect cannot be produced by a difference in medium osmolality but rather requires the cell volume to change. Dog red cell uptake of ²⁴Na is not inhibited by iodoacetate. Phloretin inhibits ²⁴Na uptake and lactate production, and virtually abolishes the volume effect on Na uptake. These several observations may be accounted for in terms of a working hypothesis which presupposes a cation carrier complex which pumps K into and Na out of cells of normal volume. When the cells are shrunken the carrier specificity shifts to an external Na-specific mode and there is a large increase in ²⁴Na uptake, driven by the inwardly directed Na electrochemical potential gradient.     

Effects of adenosine receptor agonists on volume-activated ion transport in pig red cells.

Swelling of pig red cells leads to an increase in a chloride-dependent K flux which can be potentiated by cAMP, whereas cell shrinking causes a selective increase in Na movement which is mediated by a Na/H exchanger. We examined the influence of adenosine and adenosine receptor agonists on the volume-sensitive, ouabain-resistant, chloride-dependent K flux, referred to as Rb flux and volume-activated Na/H exchange pathway. It was found that adenosine and adenosine receptor agonists inhibited the Rb flux. N6-cyclohexyl adenosine (CHA) has been found to be the most potent inhibitor with EC50 of approximately 4.5 microM followed by 2-chloroadenosine (Cl-ado) with EC50 of approximately 27 microM and 5'-(N-ethyl)-carboxamido-adenosine (NECA) with EC50 of approximately 185 microM. CHA also inhibits the cAMP-stimulated Rb flux. However, CHA does not alter the basal intracellular cAMP level nor the intracellular cAMP content raised by exogenously added cAMP. In contrast to the adenosine agonist action on the Rb flux, Na/H exchange, which is activated upon cell shrinkage, exhibits a slight stimulation in response to CHA. These findings suggest that the presence of A1 adenosine receptors on the surface of red cells influences the regulation of volume-activated ion transport.     

Delta endotoxin is a potent inhibitor of the (Na,K)-ATPase

A 68-kDa protein, delta endotoxin, produced by Bacillus thuringiensis ssp. Kurstaki inhibits ion transport, (Na,K)-ATPase, and K+-p-nitrophenylphosphatase activity catalyzed by the Na+ pump. The Ki for inhibition of the K+-p-nitrophenylphosphatase activity of purified dog kidney (Na,K)-ATPase was approximately 0.37 microM. Delta endotoxin had a similar Ki for inhibition of (Na,K)-ATPase activity when assayed at low Na+ concentration (10 mM) but the inhibition was reversed when high concentrations of Na+ (100 mM NaCl) were added to the assay. Phosphorylation of the active site aspartyl residue with 32PO3-4 was also blocked by delta endotoxin. Ouabain-sensitive 86Rb+ uptake into intact human red blood cells was not inhibited by externally added toxin; however, strophanthidin-inhibitable 22Na+ uptake into inside-out vesicles from red blood cells was completely blocked by delta endotoxin (Ki = 0.73 microM). These data suggest that delta endotoxin must enter the cell before it can inhibit the Na+ pump.     

pH regulatory Na/H exchange by Amphiuma red blood cells

In Amphiuma red blood cells, the Na/H exchanger has been shown to play a central role in the regulation of cell volume following cell shrinkage (Cala, P. M. 1980. Journal of General Physiology. 76:683- 708.) The present study was designed to evaluate the existence of pH regulatory Na/H exchange in the Amphiuma red blood cell. The data illustrate that when the intracellular pHi was decreased below the normal value of 7.00, Na/H exchange was activated in proportion to the degree of acidification. Once activated, net Na/H exchange flux persisted until normal intracellular pH (6.9-7.0) was restored, with a half time of approximately 5 min. These observations established a pHi set point of 7.00 for the pH-activated Na/H exchange of Amphiuma red blood cell. This is in contrast to the behavior of osmotically shrunken Amphiuma red blood cells in which no pHi set point could be demonstrated. That is, when activated by cell shrinkage the Na/H exchange mediated net Na flux persisted until normal volume was restored regardless of pHi. In contrast, when activated by cell acidification, the Na/H exchanger functioned until pHi was restored to normal and cell volume appeared to have no effect on pH-activated Na/H exchange. Studies evaluating the kinetic and inferentially, the molecular equivalence of the volume and pHi-induced Amphiuma erythrocyte Na/H exchanger(s), indicated that the apparent Na affinity of the pH activated cells is four times greater than that of shrunken cells. The apparent Vmax is also higher (two times) in the pH activated cells, suggesting the involvement of two distinct populations of the transporter in pH and volume regulation. However, when analyzed in terms of a bisubstrate model, the same data are consistent with the conclusion that both pH and volume regulatory functions are mediated by the same transport protein. Taken together, these data support the conclusion that volume and pH are regulated by the same effector (Na/H exchanger) under the control of as yet unidentified, distinct and cross inhibitory volume and pH sensing mechanisms.     

Active sodium and potassium transport in high potassium and low potassium sheep red cells

The kinetic characteristics of the ouabain-sensitive (Na + K) transport system (pump) of high potassium (HK) and low potassium (LK) sheep red cells have been investigated. In sodium medium, the curve relating pump rate to external K is sigmoid with half maximal stimulation (K_1/2) occurring at 3 mM for both cell types, the maximum pump rate in HK cells being about four times that in LK cells. In sodium-free media, both HK and LK pumps are adequately described by the Michaelis-Menten equation, but the K_1/2 for HK cells is 0.6 ± 0.1 mM K, while that for LK is 0.2 ± 0.05 mM K. When the internal Na and K content of the cells was varied by the PCMBS method, it was found that the pump rate of HK cells showed a gradual increase from zero at very low internal Na to a maximum when internal K was reduced to nearly zero (100% Na). In LK cells, on the other hand, no pump activity was detected if Na constituted less than 70% of the total (Na + K) in the cell. Increasing Na from 70 to nearly 100% of the internal cation composition, however, resulted in an exponential increase in pump rate in these cells to about ⅙ the maximum rate observed in HK cells. While changes in internal composition altered the pump rate at saturating concentrations of external K, it had no effect on the apparent affinity of the pumps for external K. These results lead us to conclude that the individual pump sites in the HK and LK sheep red cell membranes must be different. Moreover, we believe that these data contribute significantly to defining the types of mechanism which can account for the kinetic characteristics of (Na + K) transport in sheep red cells and perhaps in other systems.