3H-ouabain Binding Research Articles

Effects of human urine-derived digitalis-like factor on cultured renal tubular epithelial cells.

We purified a polar digitalis-like factor to apparent homogeneity from human urine using inhibition of 3H-ouabain binding to intact human erythrocytes to monitor digitalis-like activity. Since endogenous digitalis-like factor may act as a natriuretic hormone, we tested the binding characteristics of this urine-derived ouabain-displacing compound to the sodium pump in intact renal epithelial cells, in order to assess its potential physiological significance. Cultured canine and porcine epithelial cell lines, MDCK and LLC-PK1, had a high sodium pump density as estimated from 3H-ouabain binding sites. Human urine-derived ouabain-displacing compound showed a dose-related inhibition of 3H-ouabain binding to both of these cells, similar to the inhibition of unlabelled ouabain. These findings indicate that the ouabain-displacing compound is capable of acting on the sodium pump in intact renal epithelial cells and may be the circulating regulator of Na+,K+-ATPase.

Sodium pump quantity and turnover in rabbit descending colon at different rates of sodium absorption

3H-Ouabain binding to isolated epithelia and basolateral membrane vesicles of Na+-transporting epithelial cells of rabbit descending colon was determined to quantify the number of operative Na+-pump sites at different rates of transcellular Na+ transport which was varied over a wide range by chronic dietary Na+ restriction or Na+ loading. Both in intact epithelia and in basolateral membrane vesicles the maximal number of specific ouabain binding sites was higher in preparations from animals transporting Na+ at high rates than in preparations from animals transporting Na+ at low rates. The affinity of ouabain to its binding site and the association and dissociation rate constants were not dependent on the rate of Na+ transport. In intact epithelia the Na+ turnover rate per pump unit was twice as high in tissues with high Na+ transport than in tissues with low Na+ transport. In basolateral membrane vesicles the Na+ turnover rate was considerably higher than in intact epithelia and there was no difference in turnover rate between vesicle preparations obtained from tissues transporting Na+ at high or low rates. Hence, factors within the intact cell appear to control the turnover rate of the Na+-pump.

Erythrocyte sodium transport in Bartter's syndrome.

Erythrocyte sodium transport was evaluated by measurement of intracellular Na concentration (ICNa), 22Na efflux rate constant (NaERC) and 3H-ouabain binding (BMax) (reflecting the number of Na/K ATPase pump sites) in 9 children with Bartter's syndrome compared to controls (children and adults) and children with various forms of salt wasting disease. There were no differences between control children and adults. In untreated Bartter's syndrome ICNa was significantly increased with NaERC and BMax significantly decreased compared to findings in controls and patients with other salt wasting disease. On prostaglandin synthetase inhibitor (Indomethacin) therapy, ICNa decreased but remained higher than in controls, NaERC increased to normal values but BMax remained low. These data support the view that there is a widespread defect in membrane electrolyte transport in Bartter's syndrome but suggest that the benefit of indomethacin therapy is not manifest via an effect on Na/K ATPase.

Voltage dependence of the Na-K ATPase: measurements of ouabain-dependent membrane current and ouabain binding in oocytes of Xenopus laevis.

Ouabain- or dihydroouabain(DHO)-sensitive membrane currents and binding of 3H-ouabain were investigated under voltage-clamp conditions in full-grown prophase-arrested oocytes of Xenopus laevis. (1) The ouabain-sensitive current is outwardly directed and usually exhibits a maximum at about +20 mV. The occurrence of the maximum is not affected by application of blockers for passive K+ currents. In the presence of Ba2+ as a K+ channel blocker, the KI value for inhibition of the Na-K ATPase by ouabain is reduced by an order of magnitude, the number of binding sites is not affected. In K+-free solution (which inhibits the normal reaction cycle of the Na-K ATPase), addition of DHO has no significant effect on the remaining currents. (2) The voltage-dependence of the ouabain-sensitive current can be modulated. Reduction of extracellular Na+ increases the pump current at the resting potential and reduces the positive slope of the I-V curve. Simultaneously, the number of binding sites for ouabain is reduced by about 25%. Seasonal variations of an unknown factor affect the negative slope. (3) Modulation of the voltage-clamp conditions has no effect on the number of binding sites for ouabain, on current inhibition by ouabain, or on ouabain binding at different concentrations of the inhibitor. It is concluded that the ouabain-sensitive current is not significantly affected by passive permeabilities and that its current-voltage dependence reflects the voltage dependence of current generated by the Na-K pump. Since ouabain binding, also at non-maximal binding concentrations, is not affected by membrane potential, steps that affect the probability of the E2P state should be voltage-insensitive.

The specific 3H-ouabain binding to Na +/K +-ATPase of the human placental tissue is inhibited by newborn plasma extracts

The specific 3H-ouabain binding to Na +/K +-ATPase of the human placental tissue is inhibited by newborn plasma extracts

Relation of left ventricular function and Na,K-pump concentration in suspected idiopathic dilated cardiomyopathy

The possible relation between Na,K-pump concentration and left ventricular (LV) function was studied in 24 patients with suspected idiopathic dilated cardiomyopathy. This was done by measurement of3H-ouabain binding to biopsies obtained during left-sided heart catheterization. In all patients light microscopy of biopsies was compatible with dilated cardiomyopathy. Nineteen patients had impaired LV function as defined by NYHA/WHO and a Na,K-pump concentration of 331 ± 19 pmol/g wet weight, wheras 5 patients had normal LV function and a Na,K-pump concentration of 559 ± 62 pmol/ g wet weight (p < 0.001). The correlation between Na,K-pump concentration and ejection fraction was highly significant (n = 24, r = 0.81, p < 0.001). There was no correlation between volume fraction of collagen tissue and Na,K-pump concentration in the biopsies (n = 24, r = −0.08, p < 0.80), indicating that the decrease in Na,K-pump concentration with dilated cardiomyopathy is not the simple outcome of increased fibrosis in the myocardium. The results indicate that the decrease in Na,K-pump concentration may be of importance for myocardial dysfunction and suggest a simple biochemical assessment of dilated cardiomyopathy by measurement of 3H-ouabain binding.

Inhibition of the electrogenic Na,K pump and Na,K-ATPase activity by tetraethylammonium, tetrabutylammonium, and apamin.

The K+-induced hyperpolarization of Na-loaded mouse diaphragm muscle, enzymatic activity of Na,K-ATPase and 3H-ouabain binding to rat brain microsomes was measured in the presence of K+ channel blockers tetraethylammonium (TEA), tetrabutylammonium (TBA) and apamin. TBA, and to a lesser extent TEA in millimolar concentrations, inhibited the electrogenic effect of the Na,K pump, Na,K-ATPase activity, and 3H-ouabain binding. The inhibition of 3H-ouabain binding by TEA or TBA was more evident in the presence of ATP and Na+ ions. Apamin in nanomolar concentrations inhibited the electrogenic effect of Na,K pump and Na,K-ATPase but not the 3H-ouabain binding. The hyperpolarizing effects of insulin and NADH, but not that of noradrenaline, were also prevented by apamin. The inhibition of Na,K pump by TEA and TBA is apparently due to both competition with K+ for a binding site on the Na,K-ATPase and a reduction in the number of transporting sites. The site of action of apamin on Na,K-ATPase is different from that of tetra-alkylammonium compounds; it apparently decreases the turnover rate of the enzyme.

Reduced concentrations of potassium, magnesium, and sodium-potassium pumps in human skeletal muscle during treatment with diuretics

Animal studies have shown that potassium depletion induced by diuretics or potassium deficient fodder leads to a selective decrease in the concentrations of potassium and in the concentration of sodium-potassium pumps in skeletal muscle. In 25 patients who had received diuretics for 2-14 years the mean concentrations of potassium, magnesium, and sodium-potassium pumps were measured in skeletal muscle biopsy specimens and were significantly lower than in those from a group of age matched controls. The reductions in all three variables were significant in those patients receiving diuretics for arterial hypertension as well as in those being treated for congestive heart failure. In 14 patients the mean muscle potassium concentration was below the control range, but only one of those was hypokalaemic (3.4 mmol/l), and 13 were receiving potassium supplements. In 15 patients the mean muscle magnesium concentration was below normal, and the mean muscle potassium and magnesium concentrations showed a linear correlation. In 12 patients in whom the mean muscle potassium concentration was below 80 mumol/g wet weight there was a linear correlation between the cellular potassium:sodium ratio and the concentration of 3H-ouabain binding sites indicating that potassium deficiency also leads to a down regulation of sodium-potassium pumps in human skeletal muscle. In spite of potassium supplements long term treatment with diuretics may lead to potassium and magnesium deficiencies, which are not detectable using the standard methods of serum analysis. The changes in concentrations of electrolytes and sodium-potassium pumps associated with treatment with diuretics may impair muscle function and potassium homoeostasis and interfere with the distribution of digitalis glycosides.

Na+,K+-ATPase concentration in rodent and human heart and skeletal muscle: apparent relation to muscle performance.

To investigate whether the sodium-potassium pump or Na+,K+-ATPase concentration in muscles is related to the capacity for muscle performance, the 3H-ouabain binding site concentration or 3-O-methylflourescein phosphatase activity was determined in samples of heart ventricular muscles from trained rats, cardiomyopathic hamsters, and human subjects. These methods have earlier been shown to quantify the Na+,K+-ATPase concentration in muscle tissue with high accuracy. When rats were swim trained for six weeks the heart ventricular muscle Na+,K+-ATPase concentration was increased by 20% (p less than 0.02) and the heart to body weight ratio by 14% (p less than 0.005). The increase in Na+,K+-ATPase concentration was only slowly reversible. After three weeks of deconditioning an increase of 12% (p less than 0.05) was still observed. In comparison skeletal muscle Na+,K+-ATPase concentration was increased by up to 46% (p less than 0.001) and decreased by up to 30% (p less than 0.005) after training and immobilisation respectively. Cardiomyopathic hamsters showed a reduction of 33% (p less than 0.005) in the heart ventricular Na+,K+-ATPase concentration compared with normal hamsters. This decrease was associated with a heart to body weight increase of 19% (p less than 0.01) and congestive heart failure. In six patients with a mean ventricular ejection fraction of 68% the mean Na+,K+-ATPase concentration in endomyocardial biopsy specimens obtained by left heart catheterisation was 505(41) compared with 322(32) pmol.g-1 wet wt (p less than 0.001) in nine patients with a mean ejection fraction of 29%. Taken together the present data indicate a relation between the Na+,K+-ATPase concentration and the capacity for muscle performance.

Clinical studies on assay for Na-K ATPase in human blood cells. I. Erythrocyte Na-K ATPase assay in patients with thyroid dysfunction and in those with chronic renal failure

A number of erythrocyte Na-K ATPase units were measured in 22 patients with hyperthyroid Graves' disease, 3 with primary hypothyroidism, 3 with simple obesity, 13 with chronic renal failure on hemodialysis, and 20 normal controls, using ouabain binding assay as described by DeLuise et al. The number of Na-K ATPase units, derived by maximal binding of 3H-ouabain, was decreased in patients with simple obesity (Mean +/- SD, 0.26 +/- 0.07 pmol/10(9) RBC), as compared with that in normal controls (0.39 +/- 0.10), and a significant negative correlation between the number of the binding sites and the ratio of the measured body weight to the optimal body weight calculated by the modified Broca's method was observed in normal controls and patients with obesity (r = -0.51, p less than 0.05). The results agreed closely with that reported by DeLuise et al and provided validation of our estimates of the erythrocyte Na-K pump units. The maximal 3H-ouabain binding was significantly diminished in patients with hyperthyroid Graves' disease (0.28 +/- 0.07) when compared with that in normal controls, while the bindings were significantly elevated in patients with hypothyroidism (0.91 +/- 0.26). These results were in disagreement with those previously reported by animal studies where Na-K ATPase was found to be stimulated by thyroid hormones. It might be possible to partly explain this discrepancy by the degradation of Na-K ATPase in erythrocytes in addition to the apparent differences between erythrocytes and the other tissues and by the length of time that the tissue was exposed to the action of the hormones. Therefore, erythrocyte from normal controls and patients with hyperthyroid Graves' disease were divided into low and high density portions by a discontinuous 'percoll' density gradient centrifugation, and the bindings of the erythrocytes in two portions were separately measured. The bindings of erythrocyte in the higher density portion, representing relatively old-aged erythrocyte, were diminished to 92 +/- 19% of the bindings of the original whole erythrocytes in normal controls. An even more marked reduction of the maximal bindings of 3H-ouabain in old-aged erythrocytes was observed in patients with hyperthyroid Graves' disease (72 +/- 26%). Moreover, this % reduction based on aging related significantly to serum T4 concentrations in those patients (r = 0.85, p less than 0.05). These findings suggest that the number of erythrocyte Na-K ATPase units may reflect the overall peripheral metabolic state, regulated by thyroid hormone-dependent thermogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanism of inhibition of rat brain (Na+-K+)-stimulated adenosine triphosphatase reaction by cadmium and methyl mercury.

The mechanisms of inhibition of rat brain Na+-K+-ATPase by cadmium chloride (CdCl2) and methylmercuric chloride (CH3HgCl) were studied in vitro by assessing the effects of these heavy metals on this enzyme and associated component parameters. Both the heavy metals significantly inhibited the overall Na+-K+-ATPase in a concentration-dependent manner with an estimated median inhibitory concentration (IC-50) of 3.2 X 10(-5) M for CdCl2 and 6 X 10(-6) M for CH3HgCl. Protection of enzyme against heavy metal inhibition by 5 X 10(-5) M to 1 X 10(-4) M dithiothreitol (DTT) and glutathione (GSH) or cysteine (CST) indicates that both monothiols and dithiols have the same ability in regenerating sulfhydryl (-SH) groups or chelating the metals. Inhibition of K+-p-nitrophenyl phosphatase (K+-PNPPase), the component enzyme catalyzing the K+-dependent dephosphorylation in the overall Na+-K+-ATPase reaction by these heavy metals, indicates that the mechanism of inhibition involves binding to this phosphatase. Reversal of K+-PNPPase inhibition by DTT, GSH, and CST suggests sulfhydryl groups as binding sites. Binding of 3H-ouabain, a cardiac glycocide and inhibitor of both phosphorylation and dephosphorylation, to brain fraction was significantly decreased by CH3HgCl, and this inhibition was reversed by the three thiol compounds, suggesting presence of -SH group(s) in the ouabain receptor site. Cadmium chloride failed to inhibit the binding of this receptor, indicating that the mechanics of inhibition of ATPase by CH3HgCl and CdCl2 are different from each other. The results suggest that the critical conformational property of enzyme common to both kinase (E1) and phosphatase (E2) is susceptible to CH3HgCl whereas only phosphatase is sensitive to CdCl2.

Erythrocyte 3H-ouabain binding and digitalis treatment in ethanol addicted patients

The binding of 3H-oubain to human erythrocytes was analyzed in a population of hospitalized male ethanol addicted patients under long term digitalis treatment. In the non-alcoholic patient group the long term digitalis treatment induced an increase in Bmax and Kd values; such modification was not observed in the alcoholic patients. Chronic alcohol intake itself induced an increase in 3H-ouabain kinetic parameters. These observations confirm that ouabain binding to human erythrocytes is subject to pharmacological and toxicological regulation and that adaptive changes in peripheral tissues can be useful in predicting possible parallel modifications in other less accessible tissues.

The effect of ethanol and acetaldehyde on Na pump function in cultured rat heart cells

To further define the sarcolemmal effects of ethanol and acetaldehyde, their effects on Na pump function were studied in synchronously contracting monolayers of neonatal rat myocardial cells. The effects of ethanol (10 mg/dl to 1000 mg/dl: 2 X 10(-3) M-0.2 M) and acetaldehyde (10(-6) M to 10(-4) M) on total 42K influx, ouabain-sensitive 42K influx, Na pump density (from specific 3H-ouabain binding) and pump turnover rates were measured. Applied acutely ethanol had no effect on 42K influx but after 30 min of treatment 42K influx was decreased by 13%, 23% and 48% in 100 mg/dl, 300 mg/dl and 1000 mg/dl ethanol respectively. This primarily reflected a decrease in mean ouabain-sensitive K+ influx from a control of 12.54 to 9.90, 8.95 and 6.68 (p-mol/cm2/s) in 100, 300 and 1000 mg/dl (2 X 10(-2) M, 6 X 10(-2) M) ethanol. Acetaldehyde in the concentrations tested had no effect on K+ influx. Ethanol treatment produced a decrease in Na pump density, maximum within 30 min and dose-dependent, at concentrations of 100 mg/dl (22%), 300 mg/dl (37%) and 1000 mg/dl (55%). Acetaldehyde had no effect on Na pump density. In the presence of ethanol (300 mg/dl and 1000 mg/dl) intracellular Na+ increased significantly and the Na+ efflux declined in parallel with the K+ influx. From the ouabain-sensitive K+ and Na+ fluxes and the Na pump density individual pump turnover rates were calculated at 62.5/s in control cells and 66/s and 84/s in cells treated with 300 mg/dl (6 X 10(-2) M) and 1000 mg/dl (0.2 M) respectively. We conclude that ethanol, but not acetaldehyde has a depressant effect on sarcolemmal Na pump function. The results suggest this is due primarily to a decrease in the number of sarcolemmal Na pump sites.

The role of pump number and intracellular sodium and potassium in determining Na,K pump activity in human erythrocytes

The factors that determine the activity of the Na,K pump in vivo were investigated by measuring Na,K pump activity under in vivo conditions in human red cells and relating it to the intracellular content of sodium ([Na] i) and potassium ([K] i) and the number of pump units per cell (pump number). Na,K pump activity was measured as ouabain-sensitive K + influx, pump number was determined from the maximal binding of 3H-ouabain to intact cells, and [Na] i and [K] i were measured by atomic absorption spectrophotometry in washed, packed cells. In the 81 samples studied, pump activity per cell was significantly correlated with pump number ( r = .64, P < 0.001), but was negatively correlated with [Na] i ( r = −.28, P < 0.02) and was not correlated with [K] i. An inverse relationship was found between pump number and [Na] i. When pump activity was expressed as activity per pump unit, rather than per cell, a significant relationship was seen between pump activity and [Na] i ( r = .50, P < 0.001), and a negative correlation existed between the activity per pump unit and [K] i ( r = −.29, P < 0.01). The effect of intracellular Na + at physiologic levels on pump activity was not strong, with the activity per pump unit increasing only 25% with a doubling of [Na] i. These results indicate that pump number is the major determinant of pump activity in human red cells in vivo, while [Na] i and [K] i are of secondary importance. Since the Na,K pump may account for 5% to 40% of cellular energy expenditure, pump number may be an important determinant of energy expenditure in cells and of metabolic rate in organisms.

Receptor radioligand system for measuring digoxin activity.

The purpose of this study was to develop and evaluate a receptor radioligand assay (RRA) for the measurement of digoxin, its metabolites, and endoxin and to compare the results of this assay with those of a fluorescence polarization immunoassay (FPIA) for digoxin. The within-run coefficients of variations for the RRA at digoxin concentrations of 2.0 and 5.0 nmol/L were 21 and 10%, respectively. Serum samples collected from 33 volunteers (not on digoxin medication) measured in the RRA resulted in levels less than 1.23 nmol/L digoxin equivalents (1.23 nmol/L is the lower level of sensitivity for this system). Serum samples collected from 29 patients receiving digoxin for therapeutic purposes and measured in the RRA gave a mean of 3.39 nmol/L digoxin equivalents. The samples collected from the patients receiving digoxin were also measured using FPIA with a mean of 1.75 nmol/L digoxin. The interference of progesterone, 11-alpha-hydroxyprogesterone, cortisone, and six digoxin metabolites on the binding of 3H-ouabain in the RRA was also evaluated.

Retinoic-acid-induced differentiation of HL-60 cells is associated with biphasic activation of the Na + — K + pump

The human promyelocytic leukemia cell line, HL-60, can be induced to differentiate into granulocyte-like cells when cultured in the presence of 10 -6 M retinoic acid (RA) for several days. Following the addition of RA two kinds of changes occur. First, there are early changes that comprise an increase in the intracellular concentration of sodium ions [Na] i, which reaches its maximum after 6 h, and an increase in the activity of the Na +-pump, which is reflected by an ouabain-sensitive K + influx that peaks at 8 h (170% of the control value) and that occurs without any change in the number of pump molecules, as measured by the binding of 3H-ouabain. Second, beginning after 12 h of culture with RA, a decrease in the number of ouabain-binding sites occurs, this being accompanied by an increase in the number of K + ions actively transported by each site. The effect of modulation of Na + -pump activity on the RA-induced differentiation of HL-60 cells was studied using low, noncytotoxic concentrations of ouabain which, although alone having no differentiating effect, accelerated and potentiated the effect of RA on differentiation. When added in combination. these drugs induced rapid stimulation of the Na + -pump, which reached its peak after 2 h. These results indicate that a concomitant increase in the level of [Na +] i and in the activity of the Na + -pump constitute primary events in the interaction between RA and HL-60 cells, and that cation fluxes may play a role in the initiation of the process of differentiation.

(3H)ouabain binding to leukaemic cells and intralymphocytic sodium content in chronic lymphocytic leukaemia; no evidence for alterations of the Na+/K+-pump.

The number of specific (3H)ouabain binding sites and dissociation constants (Kd) were determined by Scatchard analysis of values for leucocytes from patients with B-cell chronic lymphocytic leukaemia (CLL), chronic myeloid leukaemia (CML), acute blastic leukaemia (AL) and healthy subjects. CLL lymphocytes and normal B-cells bound significantly less (3H)ouabain than did normal T-lymphocytes. CML granulocytes showed the same binding characteristics as normal granulocytes, while blast cells from AL patients bound significantly more (3H)ouabain than did normal granulocytes or B-cells. The increased binding capacity in blast cells might, at least partly, reflect their larger cell size. A decrease in Kd values was only found in CLL lymphocytes, as compared with normal B-cells. Intralymphocytic sodium content in CLL lymphocytes was significantly increased, as compared with that in T-cell-enriched normal lymphocytes. (3H)ouabain binding did not show any relationship to different prognostic variables in CLL. The present data mainly argue against altered Na+/K+-ATPase enzyme activity as an indicator of malignancy.

Retinoic-acid-induced differentiation of HL-60 cells is associated with biphasic activation of the Na + − K + pump

The human promyelocytic leukemia cell line, HL-60, can be induced to differentiate into granulocyte-like cells when cultured in the presence of 10 –6 M retinoic acid (RA) for several days. Following the addition of RA two kinds of changes occur. First, there are early changes that comprise an increase in the intracellular concentration of sodium ions [Na]j, which reaches its maximum after 6 h, and an increase in the activity of the Na +-pump, which is reflected by an ouabain-sensitive K + influx that peaks at 8 h (170% of the control value) and that occurs without any change in the number of pump molecules, as measured by the binding of 3H-ouabain. Second, beginning after 12 h of culture with RA, a decrease in the number of ouabainbinding sites occurs, this being accompanied by an increase in the number of K + ions actively transported by each site. The effect of modulation of Na +-pump activity on the RA-induced differentiation of HL-60 cells was studied using low, noncytotoxic concentrations of ouabain which, although alone having no differentiating effect, accelerated and potentiated the effect of RA on differentiation. When added in combination, these drugs induced rapid stimulation of the Na + -pump, which reached its peak after 2 h. These results indicate that a concomitant increase in the level of [Na +]i and in the activity of the Na +-pump constitute primary events in the interaction between RA and HL-60 cells, and that cation fluxes may play a role in the initiation of the process of differentiation.

Frequent stimulation of the guinea-pig myocardium raises the inotropic efficacy of tissue-bound ouabain.

3H-Ouabain binding to frequently (1 Hz) stimulated papillary muscles from reserpine-pretreated guinea pigs was evaluated at ouabain concentrations of 18.5 and 200 nmol/l. Myocardial activity increased the amount of 3H-ouabain bound to the tissue in comparison with quiescent preparations. Since the shape of the time course of ouabain binding changed with frequent stimulation, a greater number of ouabain-accessible binding sites of the Na pump as induced by the rise in intracellular Na with frequent stimulation cannot be the sole mechanism of the frequency dependence. In view of their stimulatory properties on the Na pump the effects of intracellular Na and extracellular K could be equivalent. By contrast, both interventions were differently effective. The K antagonism on 3H-ouabain binding was independent from stimulation frequency. Furthermore, the shape of the time course of binding was not altered by [K]o. As evidenced by the dependence of half-times to steady-state effect on muscle diameter, the apparent rate of diffusion of ouabain was accelerated with the frequency of contractions. This acceleration could have interfered with the time course of binding at frequent stimulation. After correlating the time courses of positive inotropic effect and ouabain binding (concentration of ouabain in the medium 200 nmol/l), frequent stimulation was found to raise the inotropic efficacy of tissue-bound ouabain. The relation of excitation-dependent Na influx to the saturable, ouabain-inhibited, Na pump explained the frequency dependence of the intropic efficacy of ouabain; that is, the observed change of efficacy was consistent with Na-pump saturation in dependence on intracellular Na.

Cyclic AMP-dependent stimulation of Na,K-ATPase in shark rectal gland.

Scatchard analysis of 3H ouabain bound to isolated rectal gland cells as a function of increasing ouabain concentrations produced a concave curvilinear plot that was resolved into two specific sites with either a high (I) or low (II) affinity for ouabain. Cyclic cAMP/theophylline (+/- furosemide, 10(-4) M) increased the amount of 3H ouabain bound to the high-affinity site I. Vanadate, a phosphate congener which promotes formation of the ouabain-binding state of the enzyme, mimicked the effects of cAMP/theophylline at low concentrations of ouabain, suggesting that cAMP/theophylline increases binding to site I by enhancing the rate of turnover of resident enzyme. Enhanced 86Rb uptake seen following cAMP/theophylline administration was primarily associated with increased flux through the high-affinity ouabain site, and this stimulation was not obliterated by the co-administration of furosemide. A model was presented which suggested the presence of two noninteracting pools of enzyme or isozymes which exhibit either a high or low affinity for ouabain. Cyclic AMP both stimulated turnover via site I, and modified the kinetics of binding of 3H ouabain to site II. The (ave) Kd of 3H ouabain for site II was increased from 3.6 microM (controls) to 0.5 microM (cAMP/theophylline) and the Hill coefficient was modified from 0.45 (controls) to 1.12 (cAMP/theophylline), suggesting a transition from a negative- to a noncooperative binding state. While furosemide reversed the effects of cAMP/theophylline on site II kinetics, it did not obliterate cAMP/theophylline effects on site I. This suggests that cAMP may alter the intrinsic turnover rate of this particular pool of Na,K-ATPase in shark rectal gland.