3H-ouabain Binding Research Articles

No upregulation of digitalis glycoside receptor (Na,K-ATPase) concentration in human heart left ventricle samples obtained at necropsy after long term digitalisation

The aim was to evaluate the hypothesis that digitalis glycosides increase the concentration of their specific receptor (Na,K-ATPase) in human myocardial tissue, thereby possibly reducing the inotropic effect of long term digitalis treatment. Intact samples of left ventricle were obtained at necropsy from patients who had been on long term treatment with digoxin and from patients not previously given digoxin. Digitalis glycoside receptors were quantified using vanadate facilitated 3H-ouabain binding before and after washing samples in buffer containing excess digoxin antibody fragments for 16 h at 30 degrees C. This washing procedure has previously been shown to reduce prior specific digoxin binding in human left ventricle by 95% and to allow subsequent vanadate facilitated complete quantification of 3H-ouabain binding sites. In this context it was performed to reduce occupancy of digitalis glycoside receptors by digoxin, caused by digitalisation before 3H-ouabain binding. 11 patients who had been on long term treatment with digoxin and eight who had not previously been given digoxin were studied. Left ventricle samples were obtained at necropsy at around 15 h after death. Standard 3H-ouabain binding was 39% less in samples from digitalised than from undigitalised subjects (p less than 0.001). Washing samples in buffer containing excess digoxin antibody fragments induced an increase in 3H-ouabain binding from 174(SEM 10) to 265(20) pmol.g-1 wet weight (n = 11, p less than 0.001) in samples from digitalised patients. After washing, the digitalis glycoside receptor concentration in left ventricle samples showed a tendency to a lower value (14%, p greater than 0.10) in patients exposed to digoxin compared to left ventricle samples from individuals unexposed to digitalis glycoside treatment. Calculating 3H-ouabain binding relative to dry ventricular muscle weight confirmed the results obtained using wet weight as reference. The results suggest that digoxin treatment in life is associated with a 34% occupancy of digitalis glycoside receptors with digoxin. In the human heart there was no evidence for upregulation of digitalis glycoside receptor concentration due to long term digitalisation. Thus at receptor level there was no evidence for development of tolerance to digoxin therapy. The lower digitalis glycoside receptor concentration in the left ventricle observed in the heart failure patients may support the report of a relationship between Na,K-ATPase concentration as evaluated by 3H-ouabain binding and left ventricular function.

Doppler blood flow measurements of umbilical artery of the in vitro perfused human placenta

Lymphoblastoid cell lines established from patients suffering from bipolar manic depression have been used to study the possible involvement of cation transport in the aetiology of this illness. No significant difference was found in the K+ fluxes mediated by the ouabain-sensitive sodium pump, the diuretic-sensitive cotransport system and the passive leak pathway of cell lines established from either control or bipolar subjects. The mean value for the specific binding of 3H-ouabain (sodium pump site number) was significantly higher in the bipolar group (approximately 30%) than in the control group.

Modulation of Ouabain Binding in Beating Ventricular Myocardium from Guinea-Pigs: Effects of Lidocaine and Monensin

To further elucidate the interdependence between digitalis sensitivity and the cellular Na+ load, the influence of two Na+ load modifying drugs, monensin and lidocaine, on the concentration-dependence of ouabain binding and ouabain effects was studied in beating ventricular strips from guinea-pig heart. Monensin (3 x 10(-6)M), a Na+ ionophore known to elevate Na+ influx, enhanced 3H-ouabain binding (by approximately 40%) as well as the ouabain effect at non-toxic ouabain concentrations, and it shifted the threshold for toxicity towards threefold lower ouabain concentrations. Lidocaine (2 x 10(-4)M), a Na+ channel blocker, lowered ouabain binding by about one third, and it extended the ouabain concentration range tolerated without toxicity by a factor of three. In the concentrations used, neither compound exerted any direct effect on ouabain binding studied in isolated cardiac membranes. The binding data obtained in the muscle strips were well fitted by a mathematical model which quantifies the dependence of ouabain binding on the underlying (Na+ +K+)-ATPase activity. These findings provide evidence for an indirect drug-induced modulation of ouabain binding via the interference with the cellular Na+ load.

Hypo-osmotic stimulation of active Na+ transport in frog muscle: Apparent upregulation of Na+ pumps

The purpose of this work was to determine if hypotonicity, in addition to the stimulation of active Na+ transport (Venosa, R.A., 1978, Biochim. Biophys. Acta 510:378-383), promoted changes in (i) active K+ influx, (ii) passive Na+ and K+ fluxes, and (iii) the number of 3H-ouabain binding sites. The results indicate that a reduction of external osmotic pressure (pi) to one-half of its normal value (pi = 0.5) produced the following effects: (i) an increase in active K+ influx on the order of 160%, (ii) a 20% reduction in Na+ influx and K+ permeability (PK), and (iii) a 40% increase in the apparent density of ouabain binding sites. These data suggest that the hypotonic stimulation of the Na+ pump is not caused by an increased leak of either Na+ (inward) or K+ (outward). It is unlikely that the stimulation of active Na+ extrusion and the rise in the apparent number of pump sites produced by hypotonicity were due to a reduction of the intracellular ionic strength. It appears that, at least in part, the stimulation of active Na+ transport takes place whenever muscles are transferred from one medium to another of lower tonicity even if neither one was hypotonic (for instance pi = 2 to pi = 1 transfer). Comparison of the present results with those previously reported indicate that in addition to the number of pump sites, the cycling rate of the pump is increased by hypotonicity. Active Na+ and K+ fluxes were not significantly altered by hypertonicity (pi = 2).

The developing corneal endothelium: correlation of morphology, hydration and Na/K ATPase pump site density.

The physiology and anatomy of the cornea of the New Zealand white rabbit were studied from birth to young adulthood (3 months). The main objective of the study was to follow the ontogeny of the corneal endothelium and correlate its maturation with the establishment of stromal transparency. With maturity, central corneal thickness increases as do corneal diameter and surface area. Endothelial morphology undergoes marked changes including an increase in cell hexagonality and cell surface area, along with a decrease in cell density and coefficient of variation of cell area. Corneal hydration decreases from a high value at birth to the adult level by 20 days after birth, the time of the onset of stromal transparency. By transmission electron microscopy, corneas of newborn rabbits exhibit an endothelium of irregular cell height with some overlap at the bases of adjacent cells. Apical junctions are incomplete in the neonates. With time the endothelium thins and cells becomes more regular in height, overlap of adjacent cells diminishes, and apical junctions develop. Descemet's membrane is thin in newborns and thickens and becomes more homogenous in appearance with maturation. The abundance of Na/K ATPase pump sites per endothelial cell, as determined by 3H-ouabain binding, increases progressively with age even after the establishment of corneal transparency at 20 days. Scatchard and LIGAND analyses of 3H-ouabain binding data indicate that there is a progressive increase in Bmax with no change in the KD from 7 days to 3 months.

Exercise‐induced hyperkalaemia can be reduced in human subjects by moderate training without change in skeletal muscle Na, K‐ATPase concentration

In 15 conscripts, venous plasma potassium was followed during exercise on a training bicycle before and after 10 weeks of moderate physical training and a putative relationship with skeletal muscle Na,K-ATPase was evaluated. Peak plasma potassium concentration obtained at exhaustion was 6.1 +/- 0.2 and 5.6 +/- 0.2 mmol l-1 (mean +/- SEM, n = 14, P less than 0.05) before and after training, respectively. Throughout the exercise period and within the first minutes of rest plasma potassium concentration was 0.2-0.5 mmol l-1 higher before than after training. Neither peak values nor peak rises in plasma potassium concentration before nor after training were correlated to the 3H-ouabain binding site (Na,K-ATPase) concentration in vastus lateralis muscle. The results indicate that net loss of potassium from the skeletal muscle pool during exercise is reduced after training, that the heart during exercise may be exposed to a smaller rise in plasma potassium concentration after training than before, and that moderate improvement of capacity to clear extracellular potassium during exercise may be due to increased activity of existing Na,K-pumps in resting skeletal muscle fibres. This may reduce muscle fatigue, increase physical performance and explain the paradoxical observation that, despite an increased catecholamine response, there is a reduced risk of cardiac events after training.

Digitalis-like activity in human plasma: Relation to blood pressure and sodium balance

purpose: On the assumption that renal tubular cells are more important as the target cells for a natriuretic factor than blood cells, we used a well-characterized cultured renal tubular cell line, Madin-Darby canine kidney (MDCK) cells, to monitor the circulating digitalis-like factor in human plasma and examine its role in the regulation of blood pressure and sodium balance. subjects and methods: We investigated the effects of plasma on binding of radioactive ouabain to monolayered MDCK cells in order to determine the level of a circulating digitalis-like factor. First, we measured specific 3H-ouabain binding to MDCK cells in the presence of plasma from 71 outpatients (34 normotensive subjects and 37 hypertensive patients) after incubation for 4 hours. Second, we measured specific 3H-ouabain binding after incubation of cells with plasma from 16 hospitalized subjects (eight normotensive subjects and eight hypertensive patients) receiving low and high sodium diets. results: In Study 1, ouabain binding was lower by 30% with plasma from hypertensive patients than with plasma from normotensive subjects (p <0.01). There was a significant negative correlation between individual subject's systolic or mean blood pressure and ouabain binding (r = −0.34, p <0.01 or r = −0.29, p <0.01). In Study 2, ouabain binding was also significantly reduced by 25% in the presence of plasma from hypertensive subjects as compared with plasma from normotensive subjects irrespective of sodium intake (p <0.01). A significant negative correlation was also found for all subjects between either systolic, diastolic, or mean blood pressure and ouabain binding (r = −0.58, p <0.01, r = −0.51, p <0.01, or r = −0.55, p <0.01, respectively). With the changes from low to high sodium intake, there was a corresponding decrease in ouabain binding (p <0.01) and an increase in sodium excretion (p <0.01). A significant negative correlation was observed between these two parameters (r = −0.47, p <0.05). conclusions: These findings suggest that a circulating digitalis-like factor, which may act on renal tubular cells as the ouabain-displacing compound, is increased in patients with essential hypertension and also demonstrate that plasma levels may be influenced by changes in dietary sodium intake.

Sodium pump numbers and cation transport of lymphocytes in pregnancy-induced hypertension

Pregnancy-induced hypertension may be linked with sodium pump inhibition and an increase in vascular myocytic tone and, hence, flow impedance. All of the findings of studies on circulating plasma and blood cells are not, however, consistent with this hypothesis. We therefore assessed sodium pump numbers and cation transport in lymphocytes from 23 women with untreated pregnancy-induced hypertension, 28 normotensive pregnant women and 28 healthy non-pregnant women. We measured the maximum 3H-ouabain binding capacity to determine the sodium pump activity and the apparent dissociation constant (the reciprocal of which estimates binding affinity) by Scatchard analysis, ouabain-sensitive (pump-mediated) 86rubidium influx and ouabain-resistant (pump-independent) influx in lymphocytes in vitro. Pregnant women, whether normotensive or hypertensive, had significantly more sodium pump activity and a higher pump-mediated and pump-independent 86rubidium influx than non-pregnant women. Sodium pump activity and the pump-mediated and pump-independent 86rubidium influx all reached normal, non-pregnant levels in normotensive pregnant women 6 weeks after delivery, but remained high in women with pregnancy-induced hypertension. The normotensive and hypertensive pregnant women and non-pregnant women all had similar ouabain binding affinity. The results of our study do not support the circulating sodium pump inhibitor hypothesis in pregnancy-induced hypertension.

Human Skeletal Muscle Na, K-ATPase Concentration Quantified by3H-Ouabain Binding to Intact Biopsies Before and After Moderate Physical Conditioning

The putative effect of moderate training on human skeletal muscle Na,K-ATPase concentration and thus on the capacity for active uptake of potassium was evaluated. In 15 conscripts the Na,K-pump concentration was determined in vastus lateralis muscle by measurement of 3H-ouabain binding to intact muscle biopsies before and after 10 weeks of physical training. All subjects had improved physical fitness, body weight was reduced by 3% (P less than 0.001), Cooper's test showed an improvement by 7% (P less than 0.05) and leg circumference 10 cm above the knee joint had increased by 3% (P less than 0.001). Mean Na,K-pump concentration +/- S.E.M. in vastus lateralis muscles was 308 +/- 13 (N = 15) and 300 +/- 7 (N = 15) pmol x g wet wt.-1 (P less than 0.60) before and after training, respectively. Thus, in human subjects moderate improvement of physical performance may occur without any change in skeletal muscle Na,K-pump concentration. It may be, however, that change in the acute regulation of skeletal muscle Na,K-ATPase--i.e. augmented activity of existing Na,K-pumps--may reduce exercise-induced rise in plasma potassium concentration after moderate physical conditioning. Since the circumference of the legs had increased, the total amount of Na,K-ATPase in the legs had probably increased. Hence, moderate training may induce muscle hypertrophy with a balanced synthesis of muscle mass and Na,K-pumps.

Quantification of the total Na,K-ATPase concentration in atria and ventricles from mammalian species by measuring3H-ouabain binding to intact myocardial samples. Stability to short term ischemia reperfusion

Na,K-ATPase concentration was measured by vanadate facilitated 3H-ouabain binding to intact samples taken from various parts of porcine and canine myocardium. In porcine and canine heart 3H-ouabain binding site concentration in ventricles was 1.4-2.5 times larger than in atria. Evaluation of 3H-ouabain binding kinetics revealed no major difference between atria and ventricles: Equilibrium was obtained after the same incubation time in right atrium (RA) as in left ventricle (LV), both in porcine and canine heart. Unspecific uptake and retention of 3H-ouabain was for porcine heart RA and LV 1.5 and 1.4, respectively, and for canine heart RA and LV, both 1.2% filling (i.e., volume (ml) of incubation medium 3H-radioactivity taken up per mass unit (g wet wt.) of tissue multiplied by 100). The apparent dissociation constant (KD) was 1.4 x 10(-8) and 1.9 x 10(-8) in porcine RA and LV and 2.6 x 10(-8) and 6.1 x 10(-8) mol/l in canine RA and LV. Loss of specifically bound 3H-ouabain during the washout procedure occurred with a half-life time (T1/2) of 16.7 and 28.6 in RA and LV of porcine heart and 91.2 and 151.6 h in RA and LV of canine heart. Duly corrected for these errors of the method--factor 1.16 and 1.13, respectively, for porcine RA and LV, and factor 1.11 and 1.13 for canine RA and LV, total 3H-ouabain binding site concentration was found to be 553 +/- 74 and 1037 +/- 45 pmol/g wet wt. (means +/- SEM, n = 5) in porcine RA and LV, and 569 +/- 37 and 1410 +/- 40 pmol/g wet wt. (means +/- SEM, n = 5) in the canine RA and LV. These values were confirmed by measurements of 3H-digoxin binding to the porcine heart. The present quantification of myocardial Na,K-ATPase gives values up to 154 times higher than measurements based upon Na,K-ATPase activities in membrane fractions where the recovery of Na,K-ATPase may be less than 1% due to loss during purification. A higher Na,K-ATPase concentration is found in small animals than in large animals. A relationship between higher concentration of Na,K-ATPase and larger pressure work in ventricles compared to atria is suggested. Myocardial 3H-ouabain binding sites were found to be stable for 20 min of ischemia, followed by 1 h of reperfusion, supporting the concept that myocyte injury induced by short term ischemia may be reversible and that reperfusion may result in normalization.

Purification of a [formula omitted] ATPase inhibitor from borderline hypertensives' plasma

Increasing experimental evidences suggest an involvement of an endogenous Na + K + ATPase inhibitor in regulating water and electrolytes balance as well as in the pathogenesis of hypertension. However, conflicting results on the nature and the chemical structure of this substance still make it difficult to understand exactly its physiological mechanism of action. In the present study an attempt was made to purify a Na + K + ATPase inhibitor from hypertensives' plasma by solid phase extraction followed by 2 HPLC steps using reverse and normal phase columns. The fractions, from both columns, were able to inhibit Na + K + ATPase, 3H-ouabain binding to enzyme, ouabain sensitive 86Rb uptake and pNPPase activity in a manner not affected by boiling. Ultrafiltration experiments demonstrate that inhibitory activity is largely due to a low-molecular weight substance. These findings seem to confirm the presence in hypertensives plasma of a Na + K + ATPase inhibitor with some similarities with ouabain.

Evidence against spare or uncoupled β-adrenoceptors in the human heart

It is well established that increasing degrees of heart failure are accompanied by a reduced density of myocardial beta-adrenoceptors. It is unclear, however, whether all beta-adrenoceptors in the cardiac cell membrane are coupled to the effector system or whether "spare receptors" or "uncoupled" beta-adrenoceptors also exist. To investigate this, we measured the density of beta-adrenoceptors and the positive inotropic response to isoprenaline in preparations from the same human hearts. The myocardium from nonfailing hearts had significantly (p less than 0.01) higher numbers of beta-adrenoceptors (104 +/- 7 fmol/mg protein) compared with tissue from moderately (mitral valve disease, New York Heart Association [NYHA] class II to III, 60 +/- 2.8 fmol/mg protein) and terminally (dilated cardiomyopathy, NYHA class IV, 35 +/- 2.7 fmol/mg protein) failing human hearts. The KD values of the drug-receptor complexes did not differ within the different patient groups. There was a linear relationship (r = 0.97) between the beta-adrenoceptor density measured and the maximally obtainable positive inotropic effect elicited by isoprenaline in the three groups tested. Thus there seem to be no spare beta-adrenoceptors, that is, receptors not required for the production of the maximal inotropic response in the left ventricular human myocardium, and there are no uncoupled beta-adrenoceptors. The beta-adrenoceptors associated with the plasma membrane (marker: 3H-ouabain binding sites) remained functionally active. In addition, these results indicate that either there is no amplifier system behind the receptor level or it remains unchanged in the failing left ventricular human myocardium under the conditions tested.

Effect of amiodarone on 3H-ouabain binding sites in human skeletal muscle

Na,K-ATPase, or the Na,K-pump, is essential for the excitability and contractility of muscle tissue. Hypothyroidism in associated with a marked decrease in the Na,K-pump concentration in skeletal muscle and myocardium. In 7 patients on long-term amiodarone treatment there was a 36% reduction in the concentration of 3H-ouabain binding sites in skeletal muscle biopsies compared to 7 healthy subjects. This decrease during long-term amiodarone treatment may represent an equivalent reduction in the concentration of the functional Na,K-pump and it may be important in the adverse effect of amiodarone on muscle.

The concentration of the Na,K-pump in skeletal and heart muscle in congestive heart failure

Na,K-ATPase (or the Na,K-pump) is essential for excitability and contractility of muscle tissue. Previous studies have shown a decrease in the concentration of this pump in endomyocardial biopsies from patients with dilated cardiomyopathy. The effect of congestive heart failure on the concentration of Na,K-ATPase in skeletal muscle was assessed in 16 patients by measurement of binding of 3H-ouabain to biopsies of the vastus lateralis muscle. Ten patients had impaired left ventricular function with an ejection fraction of 0.32 ± 0.03 and a concentration of the Na,K-pump of 229 ± 15 pmol/g wet weight in the skeletal muscle, whereas 6 patients had an ejection fraction of 0.66 ± 0.05 ( P < 0.001) and a concentration of 307 ± 17 pmol/g wet weight ( P < 0.01). In endomyocardial biopsies, the concentration of Na,K-ATPase was 340 ± 37 and 500 ± 39 pmol/g wet weight ( P < 0.025) in patients with impaired and normal ventricular function, respectively. There was a significant correlation between the concentration of the Na,K-pump in the biopsies of the skeletal muscle and ejection fraction, as well as between its concentration in the endomyocardial and skeletal muscular biopsies ( r = 0.56, P < 0.025 and r = 0.72, P < 0.005, respectively). The decrease in concentration of the pump in skeletal muscle may contribute to the limitation of exercise capacity in congestive heart failure.

Age-Dependent Change in Myocardial Cardiac Glycoside Receptor (Na,K-pump) Concentration in Children

Myocardial Na,K-ATPase concentration was quantified in 18 0-8-year-old human subjects by vanadate-facilitated 3H-ouabain binding to intact samples of the left ventricle of the heart obtained at autopsy. Within the first 6 months of life, the Na,K-ATPase concentration showed a rapid decrease. The mean value in the age range from birth to 6 months was 1.6 times the mean value obtained in the age range 6 months to 8 years. The mean values +/- SEM were 1,076 +/- 57 (n = 10) and 671 +/- 28 (n = 8) (p less than 0.001) pmol/g wet weight, respectively. The highest value [1,433 +/- 56 pmol/g wet weight (n = 5)] was found in a 3-month-old child and the lowest value [545 +/- 22 pmol/g wet weight (n = 5)] in an 8-year-old. Evaluation of 3H-ouabain-binding kinetics showed no age-dependent variations. The total amount of Na,K-ATPase found in the heart was approximately 30 and 80 nmol within the first 3 years of life and at 8 years, respectively. The age-dependent change in myocardial Na,K-ATPase concentration can be ascribed to variation in the ratio between the amount of Na,K-ATPase and muscle mass during development. Since myocardial Na,K-ATPase is the receptor for cardiac glycosides, the present results may in part explain the clinical observation that cardiac glycoside sensitivity and toxicity change in young age.

3H-Ouabain binding sites in porcine skeletal muscle as influenced by environmental temperature and energy intake

The influence of environmental temperature and energy intake on 3H-ouabain binding sites in skeletal muscle has been investigated in young growing pigs at 8 weeks of age. Animals lived for several weeks at 35 or 10 degrees C on a high (H) or low (L) level of energy intake. The four treatment groups were thus: 35H, 35L, 10H and 10L. The total number of 3H-ouabain binding sites (Bmax) in longissimus dorsi muscle (mean values +/- SEM) were 221 +/- 66, 214 +/- 61, 350 +/- 76 and 486 +/- 114 pmol/g wet weight for the 35H, 35L, 10H and 10L groups respectively. Bmax was significantly greater in those living in the cold than the warm (P less than 0.001). Moreover, at 10 degrees C energy intake had a significant effect, with Bmax being greater in the 10L than the 10H group (P less than 0.005). Level of energy intake had no influence on Bmax at 35 degrees C. The apparent dissociation constant was not affected by either temperature or intake. The elevated Bmax and hence the increase in number of Na+,K+-pumping sites in the cold is probably related to increased muscular activity associated with shivering. However, thyroid status also influences the number of Na+,K+-pumping sites and this may have been a contributory factor in the present study. In addition, the elevated Bmax suggests a greater potential for non-shivering thermogenesis associated with increased Na+,K+-ATPase concentration in the cold. Differences in relative stage of development between the four groups may help to explain the results for Bmax in relation to level of energy intake.

Inhibition of Na-K pump current in guinea pig ventricular myocytes by dihydroouabain occurs at high- and low-affinity sites.

Binding of cardiac glycosides to the Na+,K+-dependent ATPase has been shown to occur at both high- and low-affinity sites. However, recent reports suggest that glycoside-induced inhibition of electrogenic Na-K pump current occurs with simple first-order binding kinetics at relatively low-affinity sites. This implies that high-affinity binding sites have little to do with Na-K pump inhibition during exposure to cardiac glycosides. To better understand the role of the high-affinity site, we investigated the concentration dependence of Ipump inhibition by dihydroouabain (DHO) in guinea pig ventricular myocytes through use of wide-pore patch pipettes to "fix" internal Na+ activity at approximately 30 mM and to voltage clamp at -40 mV (T = 34 degrees C). DHO was found to have no effect on membrane conductance at a holding potential of -40 mV. Holding current was monitored and the difference between steady-state holding current before and during external exposure to nine concentrations (range, 0.01-1,000 microM) of DHO was measured and normalized to cellular membrane capacitance. The concentration dependence of the inhibition of Na-K pump current was biphasic and well fitted to a two-binding site model with inhibitory KD values of 0.05 microM and 64.5 microM. This is consistent with previously reported 3H-ouabain binding studies in guinea pig myocardium. These findings indicate that the electrogenic properties of the Na-K pump can be inhibited by glycoside binding to both high- and low-affinity sites.

Stability of mammalian heart Na,K-ATPase concentration to short-term ischemia-reperfusion as quantified by vanadate facilitated 3H-ouabain binding

Stability of mammalian heart Na,K-ATPase concentration to short-term ischemia-reperfusion as quantified by vanadate facilitated 3H-ouabain binding

Acute sodium loading alters sodium pump in Caucasian hypertensive subjects.

The effect of an acute saline load on the sodium pump was determined from measurements of intracellular sodium and potassium, ouabain-inhibitable sodium efflux, and the number of sodium-potassium adenosine triphosphatase (Na,K-ATPase) sites per cell (using 3H-ouabain binding) of erythrocytes from 22 hypertensive and 21 normotensive subjects before and after a 2-1 infusion of 0.9% saline over a 4-hour period. Before the infusion, ouabain-inhibitable sodium efflux was the only measured parameter that was significantly (p less than 0.025) different between hypertensive (1.65 +/- 0.21 mmol/l red blood cell [RBC]/hr) and normotensive (1.46 +/- 0.25 mmol/l RBC/hr) subjects. After the saline infusion, there was a significant (p less than 0.001) decrease in the ouabain-sensitive sodium efflux of the hypertensive (1.55 +/- 0.22 mmol/l RBC/hr) but not of the normotensive (1.48 +/- 0.43 mmol/l RBC/hr) subjects. Although the changes in intracellular sodium of the normotensive and hypertensive subjects caused by the saline infusion were not significant, the fact that the change was in opposite directions in the two groups yielded a significant (p less than 0.02) differential response. After the saline infusion there was a significant increase in intracellular potassium (p less than 0.001, paired t test) and in the 3H-ouabain-binding affinity constant (p less than 0.001, paired t test) for both hypertensive and normotensive subjects. A second-order rate constant, which is an estimate of the apparent affinity constant of the sodium pump, was calculated from the ouabain-inhibitable sodium efflux, the intracellular sodium, and the number of Na,K-ATPase sites per cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Dialysis and Transplantation on Red Cell Na Pump Function in Renal Failure

Ion pumping by the erythrocyte Na, K-ATPase has been measured using ouabain-sensitive 86Rb flux in 11 non-dialysed patients with chronic renal failure (CRF), 13 patients on haemodialysis (HD), 13 patients on peritoneal dialysis (CAPD) and 15 patients with functional transplants (FT). Flux measurements were performed in plasma and simultaneous estimates of specific 3H-ouabain binding were made. The results indicate that, compared to normal controls, Na,K pump flux was reduced by 21% in CRF (p less than 0.01), 30% in HD (p less than 0.01), 15% in CAPD (p less than 0.02), and was normal in FT. Mean specific ouabain binding sites per cell (+/- SEM) were; controls 366 +/- 16; CRF, 290 +/- 16; HD, 344 +/- 17; CAPD, 321 +/- 18; FT, 345 +/- 26. Calculation of mean turnover rate per pump site indicated that patients on HD showed a 30% reduction compared to controls (influx 55 K ions/s versus 79 K ions/s, p less than 0.01). Cross-incubation experiments suggest that the lowered pump flux seen in the CRF and HD groups was due to plasma factors. This work shows that erythrocyte Na,K pump number is reduced in CRF, while patients on maintenance HD have normal pump numbers per erythrocyte but reduced pump turnover.