K-Cl cotransporter expression in the human kidney.

The K-Cl cotransporter protein KCC1 is a membrane transport protein that mediates the coupled, electroneutral transport of K and Cl across plasma membranes. The precise cell type(s) in the kidney that express the K-Cl cotransporter have remained unknown. The aim of the present investigation was to define the distribution of KCC1 mRNA in the human kidney. We used in situ hybridization with a nonradioactive digoxigenin-labeled riboprobe. We identified abundant KCC1 mRNA expression in the epithelial cells throughout the distal and proximal renal tubular epithelium. The transporter was also expressed in glomerular mesangial cells and endothelial cells of the renal vessels. These findings suggest that the K-Cl cotransporter may have an important role in transepithelial K and Cl reabsorption.

Urea inhibits the Na-K pump in human erythrocytes.

Recent studies have established that urea alters the activity of several volume-sensitive cation transport pathways. However, it has remained unclear whether urea has any effect on transport pathways that are not volume-sensitive. We examined the effect of urea on Na-K pump in the human erythrocytes. In cells from nine subjects, 500 mM urea inhibited 52 +/- 10% of the pump activity measured as the ouabain-sensitive (OS) K influx. Urea inhibited the OS K influx reversibly, in a concentration-dependent manner. [3H] oubain binding, a measure of the number of Na-K pump sites remained unchanged with urea. Urea decreased the Vmax for ouabain-sensitive K influx, but did not alter the apparent K(m) for external K. Furthermore, urea did not alter the apparent K(m) for intracellular Na. The ion turnover per pump site was decreased in the presence of urea. Thus, physiologically relevant urea concentration inhibit the Na-K pump in human erythrocyte. The inhibition of the Na-K pump by urea suggests that the effects of urea may not be limited to volume-sensitive transporters, but may be more widespread.

Urea inhibits Na-K-2Cl cotransport in medullary thick ascending limb cells.

We examined the effect of physiological concentrations of urea (100-500 mM) on Na-K-2Cl cotransport in cultured cells from mouse medullary thick ascending limb (mTAL). Urea acutely inhibited bumetanide-sensitive K influx in mTAL cells in a concentration-dependent fashion, with a statistically significant inhibition (19%) at 100 mM and 86% inhibition at 500 mM. The effect of urea was entirely reversible and was blocked by prior treatment with okadaic acid, a phosphatase inhibitor, suggesting that urea exerts its action upstream of the phosphorylation-dephosphorylation step. Cell volume was unchanged in the presence of 500 mM urea. The number of [3H]bumetanide binding sites, a measure of the number of functioning cotransporter sites, was decreased in the presence of urea, and the decrease in bumetanide binding was proportional to the decrease in bumetanide-sensitive K influx. Urea also stimulated the Ba-sensitive swelling-activated K efflux from mTAL cells. Thus urea, in concentrations that prevail in the renal medulla, alters ion transport in mTAL cells.

Prostaglandin E2 inhibits Na-K-2Cl cotransport in medullary thick ascending limb cells.

Prostaglandin E2 (PGE2) is known to inhibit transepithelial Cl transport in medullary thick ascending limb (mTAL), but the mechanism of inhibition or the transport pathway affected has not been identified. We undertook this study to examine the effect of PGE2 on Na-K-2Cl cotransport in mouse mTAL cells in culture. In nanomolar concentrations, PGE2 inhibited the Na- and Cl-dependent, bumetanide-sensitive K influx by 45%, and this inhibition was also observed in the presence of 3 mM ouabain. Although PGE2 also inhibited ouabain-sensitive K flux, that inhibition was abolished in the presence of apical nystatin, suggesting that the pump inhibition was secondary to diminished Na entry into the cells. The effect of PGE2 was concentration dependent. Inhibition was observed at a concentration of < 1 nM, and half-maximal effect was observed at 2.5 nM. The effect of PGE2 was not mediated by an action on cytosolic Ca because cytosolic Ca was unchanged after the addition of PGE2. PGE2 reduced the maximal velocity for the cotransporter but had no effect on the affinity of the cotransporter for external Na, K, or Cl. Specific [3H]bumetanide binding was reduced in the presence of PGE2, suggesting that PGE2 affected bumetanide-sensitive K influx by downregulating the number of functioning Na-K-2Cl cotransporters. These results suggest that Na-K-2Cl cotransport in the mTAL cells may be under tonic inhibitory control of PGE2.

Transfection alters ion transport in MDCK cells.

In the course of an investigation into the effect of Tamm-Horsfall protein (THP) on ion transport, we performed stable transfection of THP into MDCK cells using the SV40 or the cytomegalovirus (CMV) promoter. As controls, we transfected MDCK cells with an "empty" plasmid containing SV40 or CMV promoter but without THP cDNA. In another set of controls, we subjected cells to transfection procedures without DNA (mock transfection). K influx was not altered in cells subjected to mock transfection procedures without DNA, but both ouabain sensitive (OS) and ouabain resistant (OR) components of K influx were diminished in cells transfected with THP cDNA using either SV40 or CMV promoter. However, K influx was also reduced in cells transfected with a control plasmid containing either the SV40 promoter alone, or the CMV promoter alone, without the THP cDNA. Thus, the transport alterations were caused by transfection and not by THP. The reduction in ouabain-sensitive K influx was accompanied by a proportional reduction in the abundance of Na-K pump units as assessed by [3H] ouabain binding. [3H] bumetanide binding, a measure of the number of functioning NaK2Cl cotransporter sites, was reduced pari passu with the reduction in bumetanide-sensitive K influx. These results highlight the possibility that alterations in properties of transfected cells may not be solely due to the presence of transfected protein, but the result of some process associated with transfection itself. Without appropriate controls to evaluate this possibility, results of transfection studies are subject to potentially faulty and misleading interpretation.

Urea inhibits NaK2Cl cotransport in human erythrocytes.

We examined the effect of urea on NaK2Cl cotransport in human erythrocytes. In erythrocytes from nine normal subjects, the addition of 45 mM urea, a concentration commonly encountered in uremic subjects, inhibited NaK2Cl cotransport by 33 +/- 7%. Urea inhibited NaK2Cl cotransport reversibly, and in a concentration-dependent fashion with half-maximal inhibition at 63 +/- 10 mM. Acute cell shrinkage increased, and acute cell swelling decreased NaK2Cl cotransport in human erythrocytes. Okadaic acid (OA), a specific inhibitor of protein phosphatase 1 and 2A, increased NaK2Cl cotransport by nearly 80%, suggesting an important role for these phosphatases in the regulation of NaK2Cl cotransport. Urea inhibited bumetanide-sensitive K influx even when protein phosphatases were inhibited with OA, suggesting that urea acted by inhibiting a kinase. In cells subjected to shrinking and OA pretreatment, maneuvers expected to increase the net phosphorylation, urea inhibited cotransport only minimally, suggesting that urea acted by causing a net dephosphorylation of the cotransport protein, or some key regulatory protein. The finding that concentrations of urea found in uremic subjects inhibited NaK2Cl cotransport, a widespread transport pathway with important physiological functions, suggests that urea is not only a marker for accumulation of other uremic toxins, but may be a significant uremic toxin itself.

The Malone antegrade continence enema for neurogenic and structural fecal incontinence and constipation.

Problems of fecal elimination are commonly encountered by the pediatric urologist and surgeon. The Malone antegrade continence enema has been described as a means to administer a large volume enema via a continent catheterizable appendicocecostomy, resulting in reliable fecal elimination. Of 22 patients undergoing this procedure 16 reported total continence 4 months or longer after surgery. Complications are relatively minor and tap water appears to be a safe solution for the antegrade continence enema. A nonrefluxing, imbricated appendicocecostomy is preferable to prevent cutaneous fecal or gas leaks.

Urea activation of K-Cl transport in human erythrocytes.

This report prompted us to examine the effect of urea on K-Cl cotransport in human erythrocytes. In human erythrocytes, urea activated K-Cl cotransport reversibly and in a concentration-dependent manner. Pretreatment with okadaic acid abolished the urea activation of transport, suggesting that exposure to urea resulted in net dephosphorylation of the transporter or a key regulator and that the action of urea was exerted proximal to the phosphorylation-dephosphorylation step. At a concentration of 200 mM, urea activated K-Cl cotransport without any delay, even in the absence of cell swelling. However, with increasing urea concentrations, an appreciable increase in lag time was observed before the final steady-state flux was reached, suggesting that urea inhibits a regulatory kinase. The latter conclusion was also supported by the finding that, at any given urea concentration, the lag time for activation was greater than the lag time for deactivation. Mg depletion activated cotransport, and urea had no additional stimulatory effect in Mg-depleted cells. In urea-pretreated cells, swelling further activated cotransport, but without any measurable delay, in contrast to a time lag of 8 min when control cells (not exposed to urea) were swollen. The latter finding suggests that urea promotes the conversion of transporters from the resting to the partially activated state. These findings raise the possibility that high concentrations of urea in the renal medulla may play a role in the decrease in cell volume that occurs during the maturation of reticulocytes and young erythrocytes, both in normal subjects and in subjects with hemoglobinopathies.

Blunt renal trauma in the pediatric population: indications for radiographic evaluation.

OBJECTIVES: The purpose of this study was to define more clearly the clinical indications for radiographic evaluation of blunt renal injury in the pediatric population. METHODS: Children evaluated for blunt abdominal trauma at the Children's Hospital of Los Angeles and Los Angeles County/University of Southern California Medical Center undergo routine physical examination, laboratory analysis, and computed tomography (CT) scan of the abdomen and pelvis regardless of urinalysis results. We retrospectively evaluated the abdominal and pelvic CT scans of 412 children sustaining blunt abdominal trauma between June 1985 and June 1990. A total of 48 children, ages 6 months to 14 years (mean 5.6 years), with CT-documented renal injuries secondary to blunt trauma were identified. The radiographic findings were correlated with clinical presentation in this group of patients. RESULTS: Of the 48 children sustaining renal injuries (12% of the group), 23 (48%) had renal contusions and 25 children (52%) sustained more serious (significant) renal injuries. Of the children with significant renal injuries, 17 (68%) had minor renal lacerations and 8 (32%) had major renal lacerations. No child sustained a renal pedicle injury. All 25 children sustaining significant renal injuries presented with hematuria: 17 (68%) had microscopic (more than 3 red blood cells per high-power field) and 8 (32%) had gross hematuria. In the 23 children with renal contusions, 4 (17%) had no hematuria, 13 (57%) had microscopic hematuria, and 6 (26%) presented with gross hematuria. Hypotension occurred in 2 of the 25 children with significant renal injuries and in 2 of 23 children with renal contusions. Fifteen of the 25 patients (60%) with significant renal injuries had associated organ injuries, and 17 of the 23 children (74%) with renal contusions had associated organ injuries. CONCLUSIONS: In adults, gross hematuria and microscopic hematuria with hypertension following blunt trauma have been correlated with significant renal injuries requiring radiographic investigation. We conclude that these clinical criteria proposed to guide the radiographic evaluation of the adult population with blunt trauma do not apply to children. In our study, the degree of hematuria did not correlate with the degree of renal injury, and significant renal injury did occur with microhematuria in the absence of hypotension. We suggest that any child with a history of blunt abdominal trauma and any evidence of hematuria should undergo abdominal and pelvic CT scanning for the proper diagnosis and staging of renal and other associated intra-abdominal injuries.

Na+/K+/2Cl- cotransport in medullary thick ascending limb cells: kinetics and bumetanide binding.

We examined the properties of Na+/K+/2Cl- cotransport in cultured mouse mTAL cells with respect to its kinetics, the contribution of K/K exchange to K fluxes mediated by the cotransporter, and [3H]bumetanide binding and turnover numbers in media with varying osmolality. The addition of bumetanide, the replacement of external Na+ or the replacement of external Cl- resulted in an almost identical (approx. 50%) decrease in K+ influx, suggesting that Na(+)-dependent, Cl(-)-dependent, BS K+ influx was a measure of Na+/K+/2Cl- cotransport. The kinetics of the BS K+ influx revealed a high affinity for external Na+ (apparent Km 7 mM) and external K+ (apparent Km 1.3 mM), but a very low affinity for external Cl- (apparent Km 67 mM with a two-site model). Of interest was the finding that none of the K+ (86Rb+) efflux was sensitive to bumetanide, suggesting the absence of cotransport mediated K/K exchange in this cell type. Specific [3H]bumetanide binding was a saturable function of free bumetanide concentration with a Kd of 0.20 microM and maximum binding (Bmax) of 0.63 pmol/mg, or about 53,000 sites per cell. Simultaneous transport and bumetanide binding assays yielded a turnover number of 255 min-1. The omission of external Na+, K+ or Cl- reduced specific [3H]bumetanide binding to values indistinguishable from zero. Changing medium osmolarity resulted in a co-ordinate change in BS K+ influx and bumetanide binding, with a monotonic increase in both transport and bumetanide binding with increase in osmolality from 200 to 400 mosmol/kg. About 85% of the cotransporter sites were located on the apical side, as in the intact mTAL tubule. The simultaneous measurement of BS ion transport and [3H]bumetanide binding in the mTAL cell may provide valuable insights into the regulation of Na+/K+/2Cl- cotransport in this nephron segment.

Effect of membrane potential on K-Cl transport in human erythrocytes.

We examined whether swelling-activated K-Cl cotransport is electrogenic in human erythrocytes. Baseline membrane potential, measured by the change in fluorescence of the carbocyanine dye diS-C3-5, was not different in hypotonically swollen (-7.6 mV) or isosmotically swollen cells (-9.5 mV). We used hemisodium, a new highly selective Na ionophore, in varying concentrations, in the presence of a fixed outwardly directed Na gradient (intracellular Na, 75 mM; external Na, 1 mM) to vary membrane potential over a wide range despite identical K and Cl concentrations. The membrane potential varied between -8 and -90 mV. K influx increased slightly with hyperpolarization in swollen and nonswollen cells. However, the difference between the two fluxes, swelling-activated K influx, a measure of K-Cl cotransport, was unaffected by voltage changes, as was swelling-activated K efflux. We conclude that K-Cl cotransport in human erythrocytes is electroneutral and by inference has a 1:1 stoichiometry.

Hemisodium, a novel selective Na ionophore. Effect on normal human erythrocytes.

Hemisodium is a novel Na ionophore that belongs to the class of compounds called cryptands. These compounds possess an electron-rich cavity for binding of cations and are conformationally organized during synthesis to favor the selective binding of one cation over another. In media containing 145 mM NaCl and 5 mM KCl, hemisodium (10(-5) M) increased erythrocyte Na content from 23 to 345 mmol/kg.dry cell solid (dcs) over 4 h and increased water content from 1.8 to 3.5 liter/kg.dcs over the same period. K content decreased somewhat over the same time period, but this fall in K content was prevented entirely by incubation in either low Na media (to prevent net Na entry) or in Cl free media. Thus, the decrease in K content in high NaCl media was due to cell swelling, which activated KCl cotransport, and not due to a direct action of hemisodium on K permeability. Hemisodium-mediated Na transport was conductive, because erythrocyte membrane potential (Vm), determined by diS-C3-5 fluorescence, changed from -9 to +22 mV in high Na media in the presence of hemisodium and DIDS. In cells equilibrated with sulfamate, an anion with low conductive permeability, Vm changed 54 mV per 10-fold change in external Na concentration with the addition of hemisodium. In contrast, a 10-fold change in the external concentration of K, Rb, Cs, or T1 failed to alter Vm in the presence of hemisodium, suggesting a high Na specificity of the ionophore. Na conductance determined from net fluxes increased from 0.04 to 5.2 microS/cm2 with 10 microM hemisodium, and with that concentration the ratio of Na to K conductance was 45:1. Among the Na ionophores available so far, hemisodium appears to have the greatest specificity. Hemisodium may be a valuable tool in membrane transport studies.

The effects of extracorporeal shock wave lithotripsy on renal growth, function and arterial blood pressure in an animal model.

The long-term effects of extracorporeal shock wave lithotripsy (ESWL*) on children treated for renal calculi are unclear. To study the long-term bio-effects of this mode of treatment on the immature animal we evaluated 30 New Zealand white rabbits at 7 weeks of age for weight, serum blood urea nitrogen and creatinine, and arterial blood pressure after which they underwent left nephrectomy. Each group of 5 rabbits received ESWL of varying levels (500 to 3,000 shock waves) to the remaining right kidney using the Northgate SD3 lithotriptor (spark gap mediated). One control group received no shock waves. At maturity (16 weeks) the aforementioned parameters were measured again, and the kidneys and any grossly abnormal adjacent organs were examined. We found no significant change in total animal growth, renal growth, renal function or perirenal organs in the post-ESWL groups versus the control group. All post-ESWL groups had an increase in mean arterial blood pressure versus the control group with 3 of 6 groups showing significant increases (p less than 0.05). Histological renal changes, seen at all energy levels of ESWL delivered, included interstitial fibrosis, tubular atrophy, glomeruli destruction, capsular thickening, perivascular fibrosis and mild arteriole wall thickening. Changes were proportional to the number of shocks received. We conclude that ESWL delivered to immature animals does not significantly affect renal growth and function but it can cause significant permanent histological renal changes even at low doses and may result in an increase in adult mean arterial blood pressure.

Role of protein phosphatase in activation of KCl cotransport in human erythrocytes.

We investigated the effects of okadaic acid, a novel highly specific inhibitor of protein phosphatases on swelling-activated transport in human erythrocytes. Nanomolar concentrations of this compound inhibited swelling-activated K transport. Complete inhibition of this transport was observed at 1 microM. Analysis of the time course of activation of K transport upon swelling revealed that okadaic acid not only decreased the final steady-state flux but also markedly increased the time lag for activation. These results suggest that okadaic acid decreases the rate constant for the conversion of KCl transporters from the resting to the active form. Okadaic acid also inhibited N-ethylmaleimide (NEM)-stimulated K transport, and this inhibition was also observed when cells were first treated with NEM before exposure to okadaic acid. The latter finding suggests that NEM activation of KCl transport is reversible and that a later step for this activation may involve the net dephosphorylation of the KCl transport protein. These results provide the first evidence that activation of KCl cotransport in human erythrocytes is regulated by phosphoprotein phosphatase.

Nifedipine inhibits calcium-activated K transport in human erythrocytes.

The effect of nifedipine on K transport across human erythrocytes was investigated. Nifedipine had no effect on K influx mediated by the Na-K pump, Na-K-2Cl cotransport, or the passive residual K flux. However, it inhibited the K and water loss from ATP-depleted cells in the presence of external Ca (Cao). Similar inhibition of Ca-activated K [K(Ca)] efflux was observed in fresh cells exposed to Cao and A23187 or ionomycin. The inhibition was observed even when nifedipine was added after initiation of the K(Ca) efflux and was not readily reversed by washing cells with drug-free media. When K(Ca) efflux was plotted as a function of external free Ca, nifedipine reduced the maximum K(Ca) efflux but had no effect on the Ca concentration required for half-maximum K(Ca) efflux. The inhibition of K(Ca) efflux by nifedipine was not consequent to its effect on conductive Cl permeability, because valinomycin-induced K efflux in Cl media was enhanced rather than reduced by nifedipine and because the inhibition was also seen with SCN, a nonlimiting anion. Nifedipine inhibited the K(Ca) efflux with a dissociation constant (Kd) of 4 microM. The inhibitory capacity of nifedipine was reduced by increasing external K. Nifedipine reduced not only the basic conductance but also the zero-current K conductance with a Kd of 23 microM. Other Ca-channel blockers, such as verapamil and diltiazem, did not inhibit K(Ca) efflux, but other dihydropyridines, including BAY K 8644, a Ca-channel agonist, were effective in inhibiting K(Ca) efflux.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics of volume-sensitive K transport in human erythrocytes: evidence for asymmetry.

The kinetic properties of volume-sensitive K fluxes in swollen human erythrocytes were investigated. Swelling-activated Cl-dependent K influx was a saturable function of external K concentration with a low affinity (apparent Km of 115-130 mM) and high capacity [maximal velocity (Vmax) = 20-30 mmol.l original cells-1.h-1 (mmol.loc-1.h-1)]. The Vmax and apparent Km for Cl-dependent K efflux were lower (Km = 47 mM; Vmax = 2.2 mmol.loc-1.h-1). The Hill coefficients for both K efflux and influx were close to unity, suggesting a single binding site for K. The increase of external K trans-stimulated K efflux, but the increase of intracellular K had no effect on Cl-dependent K influx in swollen cells. Under zero trans conditions, the Vmax (18 vs. 3 mmol.loc-1.h-1) and Km (138 vs. 32) were markedly different for influx and efflux, respectively. These results provide evidence for intrinsic functional asymmetry, such that the transporter is more prevalent and stable in the outward-facing conformation. The mean ratio of Km to Vmax for efflux (12.1) was 1.56 times larger than the same ratio for influx (7.8), but the difference between the means did not reach statistical significance. These kinetic observations are analyzed in terms of the simple carrier and the cotransport models.

Mechanism of alteration of sodium potassium pump of erythrocytes from patients with chronic renal failure.

We examined intracellular electrolytes, K influx, and [3H]ouabain-binding capacity of erythrocytes from 32 normal subjects and 45 patients with end-stage renal failure on dialysis, including 16 with high intracellular Na (mean 17.3 +/- 3.9 mmol/liter cell water). The [3H]ouabain-binding capacity of erythrocytes with high cell Na was markedly reduced as compared with that of erythrocytes from normal subjects (274 +/- 52 vs. 455 +/- 59 sites/cell, P less than 0.001). The mean serum creatinine was higher in the uremic group with high cell Na. There was a significant linear correlation between intracellular Na and [3H]ouabain-binding in both normal and uremic subjects. Cross-incubation of normal cells with uremic plasma for 24 h failed to reduce [3H]ouabain-binding capacity of normal cells. In spite of a substantial increase in cell Na, K pump influx was not higher in uremic erythrocytes with high cell Na. When intracellular Na was altered with nystatin (cell Na equal to 120 mmol/liter cell water in both groups), K pump influx was proportional to the number of Na-K pump sites so that the ion turnover rate per pump site was similar in the two groups. Uremic plasma failed to depress K pump influx of normal erythrocytes. The passive net influx of Na in uremic cells with high intracellular Na was not different from that observed in erythrocytes from normal subjects. When erythrocytes were separated by age on Percoll density gradients, the number of Na-K pump sites of the youngest uremic cells was significantly lower than that of the youngest normal cells, suggesting that decreased synthesis of Na-K pump sites, rather than accelerated loss of Na-K pump sites during aging, was responsible for the decrease in [3H]ouabain-binding capacity of erythrocytes from uremic subjects. Taken together, these findings suggest that a decrease in the number of Na-K pump sites plays a major role in the abnormality of Na-K pump of erythrocytes from patients with chronic renal failure.

Extrarenal potassium transport and the beta 2-adrenergic system.

The distribution of a short-term potassium load was quantitated in three groups of acutely nephrectomized rats infused with KCl at 0.75, 1.50, and 2.25 mEq/kg/hr for 90 minutes. The rate of net potassium transfer from the extracellular fluid compartment to the intracellular fluid compartment was stable from 30 to 90 minutes and proportional to the rate of infusion, with no evidence of saturation of transport mechanisms. In the period 60 to 90 minutes, the calculated increments in intracellular potassium concentration over 10-minute intervals were similar to the respective increments in extracellular potassium concentration. The apparent volume of distribution of the infused potassium over this period was similar to total body water. At the termination of the infusions, the net potassium transfer rates rapidly fell to very low levels. Our studies support the view that the extrarenal modulation of short-term potassium load is dependent on changes in plasma potassium concentration that conform to a first-order linear kinetic model. The infusion of the selective beta 2-adrenergic blocker butoxamine without potassium administration to acutely nephrectomized rats resulted in a net efflux of potassium from the intracellular fluid compartment. The addition of butoxamine to three groups of rats receiving KCl at the previous rates resulted in a fixed decrement in net potassium transfer to the intracellular fluid compartment that was not significantly different from the net efflux of potassium seen with butoxamine alone. Despite beta blockade, the net transfer of potassium remained proportional to the infusion rate. Thus, in our studies, beta 2-adrenergic blockade had a relatively fixed influence on decreasing net potassium transfer, both under basal conditions and during short-term potassium administration.

Glucocorticoid-induced alterations in the sodium potassium pump of the human erythrocyte.

To evaluate the effects of glucocorticoids on the Na-K pump in human subjects, were evaluated the intracellular sodium and potassium, 42K influx across and the [3H]ouabain binding to cell membranes of intact human erythrocytes from a group of subjects taking glucocorticoids and a group of normal subjects. Intracellular sodium concentration was lower (7.2 +/- 0.4 vs. 10.9 +/- 0.2 mmol/liter cell water) and intracellular potassium concentration higher (149.8 +/- 1.5 vs. 137.2 +/- 1.2 mmol/liter cell water) in erythrocytes from steroid-treated patients. In spite of a significantly decrease intracellular sodium which normally diminishes ouabain-sensitive 42K influx, the ouabain-sensitive K influx was unchanged in erythrocytes from the steroid-treated group. Maximum [3H]ouabain binding was markedly higher in the steroid-treated group (835 +/- 44 vs. 449 +/- 11 sites/cell). There was close linear correlation between [3H]ouabain binding and inhibition of K pump, suggesting the specificity of ouabain binding to Na-K pump sites on the cell membrane. Association kinetics for ouabain were similar in the two groups despite the marked difference in the amount of [3H]ouabain binding. External potassium concentration required for half-maximum ouabain-sensitive K influx was identical in the two groups. Thus, the additional Na-K pump sites in the steroid-treated group were qualitatively similar to those in normals. These results suggest that administration of glucocorticoids leads to an increase in the number of Na-K pump sites. The increase in the number of Na-K pump sites may explain the low levels of intracellular sodium and higher cell potassium observed in steroid-treated subjects.