Effect of ouabain on macromolecular synthesis during the cell cycle in mitogen-stimulated human lymphocytes

Ouabain inhibited in a concentration-dependent and completely reversible way, the synthesis of DNA, RNA and protein in phytohemagglutinin and concanavalin A-stimulated human lymphocytes without affecting the uptake of nucleosides and amino acids into the cells. On the other hand, ouabain even at very high concentrations was unable to interfere with the binding of [³H]concanavalin A. No correlation was found between the inhibition by ouabain of macromolecular synthesis and that of K⁺ transport. The inhibitor effect of ouabain on the stimulation of macromolecular synthesis could be partially reversed by higher concentrations of K⁺, due to the direct inhibition of ouabain binding. Ouabain added to the cultures at different stages of cell growth suppressed the incorporation of thymidine to various extents. Both ouabain sensitive stages fell in a period preceding the onset of mitosis and were characterized by very active thymidine incorporation. Lymphocytes were most sensitive to ouabain within the S phase. The results suggest that ouabain interferes with mitogen-triggered membrane-associated events, other than K⁺ transport, controlling mitosis at distinct phases of the cell cycle.     

Effects of ouabain and isoproterenol on potassium influx in the turkey erythrocyte. Quantitative relation to ligand binding and cyclic AMP generation

Studies have been carried out in the turkey erythrocyte to examine: (1) the influence of external K+ concentration on both [3H]ouabain binding and the sensitivity of potassium influx to inhibition by ouabain and (2) the quantitative relation between beta-adrenergic receptor site occupancy, agonist-directed cyclic AMP generation and potassium influx rate. Both [3H]ouabain binding and the ability of ouabain to inhibit potassium influx are markedly reduced at increasing external K+ concentrations, and at each K+ concentration the concentrations of ouabain required for half-maximal binding to the erythrocyte membrane and for half-maximal inhibition of potassium influx are identical. Both basal and isoproterenol-stimulated potassium influx rise with increasing external K+ concentrations. In contrast to basal potassium influx, which is 50-70% inhibitable by ouabain, the isoproterenol-stimulated component of potassium influx is entirely insensitive to ouabain. At all concentrations of K+, inhibition of basal potassium influx by ouabain is linear with ouabain binding, indicating that the rate of transport per unoccupied ouabain binding site is unaffected by simultaneous occupancy of other sites by ouabain. Similarly, the rate of isoproterenol-stimulated cyclic AMP synthesis is directly proportional to beta-adrenergic receptor occupany over the entire concentration-response relationship for isoproterenol, showing that at all levels of occupancy beta-adrenergic receptor sites function independently of each other. Analysis of the relation of catecholamine-dependent potassium transport to the number of beta-adrenergic receptor sites occupied indicates an extremely sensitive physiological system, in which 50%-maximal stimulation of potassium transport is achieved at less than 3% receptor occupancy, corresponding to fewer than ten occupied receptors per cell.     

ON THE MECHANISM OF OUABAIN INHIBITION OF SYNAPTOSOME PROTEIN SYNTHESIS

Abstract— Ouabain (200μ m ) inhibited incorporation of radiolabelled leucine or glycine into the protein of neonatal synaptosome fractions but had minimal effect on preparations from adult rats. Leucine uptake into synaptosomes was rapid but not influenced by 200μ m -ouabain in contrast to ouabain inhibition of [¹⁴C]glycine and [¹⁴C]γ-aminobutyric acid uptake. Ouabain blocked the Na⁺ -dependent (stimulated) component of synaptosome fraction protein synthesis in the presence of 25m m -K⁺. Ouabain inhibition was not alleviated by addition of ADP or ATP. 100μ m -atractylate failed to influence [³H]leucine uptake or incorporation. Synergistic inhibition by ouabain was observed with the cycloheximide-sensitive component of protein synthesis and the chloramphenicol sensitive phase. Increasing the medium Ca²⁺ concentration stimulated protein synthesis and this stimulated component was inhibited by ouabain. Ouabain inhibition was associated with decreasing intraterminal K⁺ concentration and [K]_i was linearly related to the protein synthesis rate in control and ouabain treated preparations.     

Ouabain is a potent promoter of growth and activator of ERK1/2 in ouabain-resistant rat renal epithelial cells

Endogenous cardiotonic steroids (ECS) are putative ligands of the inhibitory binding site of the membrane sodium pump (Na+, K+-ATPase). There is growing evidence that cardiotonic steroids may promote the growth of cardiac and vascular myocytes, including evidence indicating growth stimulation at concentrations in the same range as circulating ECS concentrations. We investigated four parameters to determine whether ouabain, a proposed ECS, promotes growth of immortalized rat proximal tubule epithelial cells: cell count by hemocytometer; metabolic activity as reflected in the mitochondrial conversion of the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, to its formazan product (MA); DNA synthesis reflected as bromodeoxyuridine incorporation (DNA); and mitosis reflected as histone phosphorylation state detected using anti-phosphohistone 3 antibody (HP). Maximum stimulatory responses were observed at 1 nm ouabain (MA, 20.3% increase, p < 0.01; DNA, 28.4% increase, p < 0.001; HP, maximum response at 0.5 h, 50% increase, p < 0.001). We observed that growth stimulation was associated with stimulation of ERK1/2 phosphorylation (ERK-P), and both growth and ERK-P could be blocked by the MEK inhibitor (U0126, 100 nm). Western blot analysis revealed that the only alpha isoform of Na+, K+-ATPase that could be detected in these cultures was the highly ouabain-resistant alpha1 isoform. Measurement of ouabain inhibition of ion transport in these cultures using 86Rb+ uptake revealed the predominance of the expected ouabain-resistant isoform (IC50 = 24 microm) and an additional minor ( approximately 15%) ouabain-sensitive inhibition with IC50 approximately 30 pm. Similar bimodal transport inhibition curves were obtained in freshly dissected rat proximal tubules. These results indicate that renal epithelial cells may be a sensitive target of the ERK1/2-activating and growth-promoting effects of ouabain even in the presence of ouabain-resistant Na+, K+-ATPase.     

Autoradiographic localisation of [3H]ouabain bound to cultured epithelial cell monolayers of MDCK cells

[3H]Ouabain binding to intact MDCK (cultured monolayers of dog kidney) cells of 60 serial passages is dependent upon ouabain concentration, time and medium K+. By utilising high K+ incubations to estimate non-specific [3H]ouabain-binding, the concentration of ouabain giving half maximal specific binding was estimated to be 1.0 . 10(-7) M and the total maximum binding to be 2.33 . 10(5) sites/cell. Ouabain inhibition of (Na+, K+)-pump function was monitored by the cellular uptake of 86Rb over 5 min. The larger fraction of 86Rb uptake was ouabain sensitive and the ouabain concentration giving half-maximal inhibition was 2 . 10(-7) M. The cellular distribution of the (Na+ + K+)-ATPase was investigated using [3H]ouabain autoradiography of intact freeze-dried epithelial monolayers of MDCK cells grown upon millipore filter supports. Binding of [3H]ouabain is localised over the lateral cellular membranes. Autoradiographic silver grain density is close to background levels over both the apical and basal (attachment) membranes.     

The static magnetic field effects on ouabain H3 binding by cancer tissue

Radioactive labeled ouabain was used for estimating the static magnetic field (SMF) induced cell volume changes. Ouabain is a specific inhibitor of Na+/K+ ATPase, and can be used for estimating its quantity--thus giving information about the cell volume changes. Ouabain binding by cancer and normal glandular tissues of breast cancer patients and normal glandular tissues of healthy women was measured after exposure of tissues to SMF 0.2T. SMF exposure led to a decrease of ouabain binding in both normal and cancer tissues when ouabain concentration in the external medium was 10(-9) M, while in the case of higher concentrations of ouabain (10(-7) M, 10(-6) M) an increase of ouabain binding was seen. The normal glandular tissues of healthy women were sensitive to SMF only at the highest concentrations of ouabain used in our experiments. The SMF-induced decrease of binding at low ouabain concentrations was considered as an evidence for the dehydration effect of SMF. It is suggested that the SMF could influence the cancer cell metabolism through cell hydration changes.     

Transport functions of the liver. Lack of correlation between hepatocellular ouabain uptake and binding to (Na+ + K+)-ATPase

Ouabain uptake was studied on isolated rat hepatocytes. Hepatocellular uptake of the glycoside is saturable (Km = 348 mumol/l, Vmax = 1.4 nmol/mg cell protein per min), energy dependent and accumulative. Concentrative ouabain uptake is not present on permeable hepatocytes, Ehrlich ascites tumor cells and AS-30D ascites hepatoma cells. There is no correlation between ouabain binding to rat liver (Na+ + K+)ATPase and ouabain uptake into isolated rat hepatocytes. While ouabain uptake is competitively inhibited by cevadine, binding to (Na+ + K+)-ATPase is not affected by the alkaloid. Although the affinities of digitoxin and ouabain to (Na+ + K+)-ATPase are similar, digitoxin is 10000-times more potent in inhibiting [3H]ouabain uptake as compared to ouabain. That binding to (Na+ + K+)-ATPase appears to be no precondition for ouabain uptake was also found in experiments with plasmamembranes derived from Ehrlich ascites tumor cells and AS-30D hepatoma cells. While tumor cell (Na+ + K+)-ATPase is ouabain sensitive, the intact cells are transport deficient. Hepatic ouabain uptake might be related to bile acid transport. Several inhibitors of the bile acid uptake system also inhibit ouabain uptake.     

Regulation of cellular growth by sodium pump activity.

Cellular growth has been found to be directly related to the amount of sodium pumping activity in mouse lymphoblasts (L5178-Y) cultured in varying concentrations of the cardiac glycoside, ouabain. No short-term adaptation (within one generation) occured; i.e., neither growth rate nor (Na+ + K+)-ATPase activity increased in cells cultured for 1-2 days in ouabain. Growth inhibition commenced after two hours, occurring concomitantly with decreased 3H-leucine incorporation into protein. The time course of this inhibition of protein synthesis, measured by leucine incorporation was similar to, but slightly slower than the time course or the dissipation of the sodium gradient. On the other hand, 3H-thymidine incorporation is unaffected by ouabain treatment over the same period. The uptake of 3H-alanine, a neutral amino acid thought to be transported via a Na+-dependent carri-r, was depressed concurrently with the sodium gradient dissipation. It is suggested, therefore, that ouabain inhibition of cellular growth results primarily from the dissipation of the sodium gradient leading to decreased Na+-dependent transport of amino acids (e.g., alanine) and, therefore, decreased protein synthesis, as observed by leucine incorporation. A sensitive and rapid method for determining ouabain inhibition of cell volume regulation is also described, which may prove potentially useful for assaying Na pump activity.     

Enhancement (by ATP, insulin, and lack of divalent cations) of ouabain inhibition of cation transport and ouabain binding in frog skeletal muscle; effect of insulin and ouabain on sarcolemmal (Na + K)MgATPase.

Using small, intact frog muscles, the basic properties of Na+ and K+ transport were shown to resemble those of the (Na+ + K+)Mg2+ATPase (EC 3.6.1.3) isolated from skeletal muscle. (a) External K+ is essential for Na+ exit and K+ entry after the muscles are Na+-loaded and K+-depleted; (b) the ouabain concentration causing maximum inhibition of recovery is the same for transport as for the inhibition of the isolated enzyme. Ouabain causes a decrease in the sorbitol space and causes muscle fibre swelling. Absence of Ca2+ and Mg2+ inhibits recovery of normal Na+ and K+ concentrations and increases the sorbitol space. Insulin stimulates K+ uptake and Na+ loss in intact muscles but has no effect on the isolated sarcolemmal (Na+ + K+)Mg2+ATPase. Absence of divalent cations, addition of external ATP and of insulin enhance the ouabain inhibition of recovery. Bound ouabain was measured using [3H]ouabain and [14C]sorbitol (to measure the extracellular space). The process of binding was slowly reversible and was saturable within a range of ouabain concentrations from 1.48 X 10(-7) to 5.96 X 10(-7) M. From the nonexchangeable ouabain bound, the density of glycoside receptors was estimated to be 650 molecules per square micrometre of membrane surface. The absence of divalent cations, addition of external ATP and of insulin significantly enhanced the amount of ouabain bound. Substitution of Na+ and K+ by choline greatly reduced the bound ouabain.     

Inhibition by colchicine of the mitogenic stimulation of lymphocytes prior to the S phase

Colchicine, vinblastine, and vincristine inhibit the mitogenic stimulation of lymphocytes by concanavalin A as measured by the incorporation of [3H]thymidine and the appearance of blast cells. The inhibitory effect of colchicine could not be accounted for by diminution in cell viability or by metaphase arrest of mitosis in the stimulated cells. Moreover, the inhibition of [3H]thymidine incorporation was not due to blockage of thymidine transport or inhibition of DNA synthesis inasmuch as addition of colchicine had no effect on cells in the S phase of the cell cycle. The time of inhibition was correlated with the kinetics of cellular commitment to lectin activation and the kinetic data indicated that colchicine blocks stimulation early in the sequence of events following addition of the mitogen. These findings support the hypothesis that cytoplasmic microtubular function plays a role in the commitment of resting cells to undergo mitotic division.     

The apparent discrepancy of ouabain inhibition of cation transport and of lymphocyte proliferation is explained by time-dependency of ouabain binding

Mitogenesis of human blood lymphocytes in culture is inhibited by concentrations of ouabain that are approximately one order of magnitude lower than those that block Na and K transport. For example, the 50% inhibition (ID₅₀) of Na-K transport, 280 nM, is seven-fold greater than the ID₅₀ for RNA synthesis, DNA synthesis, or blastogenesis, ?40 nM. Yet, inhibition of transport and consequent reduction in cell K is considered responsible for the effects of ouabain on mitogenesis. Since synthetic processes are assessed at least 24 hours after lymphocyte stimulation, this discrepancy could be explained by either 1) a progressive increase in K leak, or 2) a progressive inhibition of Na-K transport by ouabain during 24 hours of PHA treatment. We found that the lymphocyte membrane leak rate of K increased immediately after PHA treatment but did not increase further from 4 to 24 hours. In contrast, the ouabain sensitivity of ⁴²K uptake was markedly increased with time: ID₅₀ for ⁴²K uptake of 35 nM at 24 hours as compared to 280 nM at 30 minutes. Measurement of ouabain binding revealed a seven-fold increase in the lymphocyte-associated ouabain after 24 hours compared to binding at 1 hour. These data indicate that the dose response of ouabain inhibition of active K transport and lymphocyte proliferation are closely correlated if one considers the slow membrane binding of ouabain at low concentrations.     

Changes in intracellular cations during the cell cycle in HeLa cells

Intracellular Na+, K+, and Mg2+ concentrations have been measured during the HeLa cell cycle and compared with changes in oxygen utilization and macromolecular synthesis. Cell water content remains relatively constant at 79 +/- 1% during the cell cycle. A biphasic change in intracellular Na+ occurs with low values as cells reach peak S phase and again in early G1. The decrease in S coincides with an increase in cell volume during increased macromolecular synthesis. The fall in intracellular Na+ during mitosis/early G1 coincides with decreased energy utilization as macromolecular synthesis decreases with a continued decrease in [Na+]i in G1 corresponding to a period of increasing cell volume and an increase in protein synthesis. Intracellular Na+ is relatively high during late S/G2 when phosphate incorporation into protein and phospholipid is maximal. Intracellular K+ concentrations largely parallel intracellular Na+ levels although the intracellular K+:Na+ ratio is significantly lower as the cell volume increases during late G2/mitosis. Additions of a Na+-pump inhibitor (strophanthidin) not only caused a rise in [Na+]i and fall in [K+]i but also inhibited protein synthesis. Conversely, addition of a protein synthesis inhibitor (cycloheximide) blocked amino acid incorporation and produces a fall in intracellular Na+ levels. These findings indicate that intracellular Na+ and K+ play an important role in regulating cell hydration during the cell cycle and that changes in Na+, K+-ATPase activity, synthesis and/or utilization of high energy phosphate compounds, fluid phase turnover (endocytosis), Na+:H+ exchange (pHi), Donnan forces, and ionic adsorption may all be involved.     

Inhibition of lymphocyte activation by ouabain Interference with the early activation of membrane phospholipid metabolism

Activation of lymphocytes by antigens and mitogens can effectively be prevented by ouabain, a known inhibitor of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-ATPase}$. Recently it was shown that lowering of intracellular levels of monovalent cations is not involved in the inhibitory effect of ouabain. $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-ATPase}$ was found to be closely associated with acylCoA: lysophosphatidylcholine acyltransferase in the plasma membrane of lymphocytes. Both enzymes are activated as an immediate consequence of mitogen binding. Human peripheral lymphocytes were stimulated with concanavalin A. Ouabain suppressed the induction of RNA and DNA synthesis in a concentration-dependent way. Increase of RNA synthesis was suppressed only if the glycoside were added within the first hours of activation. If ouabain was added later, incorporation of uridine remained at the rate that was reached at the time of glycoside administration, pointing to an early event where ouabain may be operative. Ouabain, in a dose-dependent manner similar to that affecting RNA and DNA synthesis, inhibited the increase in the incorporation of oleate into phospholipids in stimulated lymphocytes, whereas the turnover of phospholipid fatty acids in resting lymphocytes was unaffected. Increasing extracellular K⁺ concentrations reversed the binding of ouabain to lymphocytes. Simultaneously, the inhibition of stimulated RNA synthesis was decreased and the inhibition of oleate incorporation was reversed. These results suggest that the suppression of lymphocyte activation by ouabain is due to the inhibition of membrane phospholipid metabolism mediated by the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-ATPase}$.     

Persistent effects of ouabain treatment on human lymphocytes: synthesis of DNA, RNA and protein in stimulated and unstimulated cells.

Pretreatment of human lymphocytes for 2 days in 2 X 10(-6)M ouabain caused irreversible loss of their subsequent capacity to stimulate in the mixed lymphocyte reaction (MLR). Pretreatment for the same period with 10(-7)M ouabain resulted in an enhanced incorporation of thymidine into DNA of the responding cells in the MLR; this effect was also on the stimulating cells, as previously reported by Christen et al. (Cell, Immunol. 19, 137-142 (1975)). Pretreatment of stimulating lymphocytes with 10(-7)M ouabain caused a persistent but reversible inhibition of the synthesis of RNA and protein in the MLR; peak incorporation of labelled uridine or alanine reached the same level as that of the control cultures, but 24 h later. Exactly the same persistent but reversible inhibition was found in the case of DNA syntheis of cells pretreated with 10(-7)M ouabain and then stimulated by antigens (streptolysin-O and varidase) or by mitogens (phytohemagglutinin and concanavalin A); the same level of incorporation of labelled thymidine occurred but 24-48 h later than in the case of the controls. Pretreatment with the cardiotonic steroid under these conditions also resulted in a pronounced inhibition of the basal, unstimulated levels of RNA and protein synthesis in the case of both control lymphocytes and those which had been treated with mitomycin C. The effects of ouabain pretreatment on basal RNA and protein synthesis were identical for both 2 X 10(-6)M and for 10(-7)M; the effect of pretreatment of stimulating cells with these two concentrations was completely opposite: irreversible inhibition of the proliferative response of allogeneic responding cells at the former concentration and delayed activation at the latter.     

Cat Heart Muscle In Vitro : IV. Inhibition of transport in quiescent muscles

Cellular concentrations, [K]_i, [Na]_i, and [Cl]_i, and cell water contents were measured in vitro at 27°C in cat papillary muscles. Measurements were made with and without ouabain at varying concentrations of K and ouabain, at pH 5.2 and 9.0, in absence of O₂, and in NaCl-free solution. Large losses of cell K and increases of cell Na occurred in presence of ouabain, at 2–3°C, and in K-free medium. The dependence of inhibition of cation transport by ouabain on external K concentration, studied at constant initial [K]_i, was consistent with a competition between K and ouabain localized to the external face of the membrane. In NaCl-free sucrose solution [K]_i remained at its physiological value and was not affected by exposure to ouabain or low temperature, except when Ca was also omitted. Ouabain inhibition persisted at pH 9.0 and in Ca-poor media. Cells swelled and lost K at pH 5.2, and residual ouabain effect was small. At pH 9.0, or in absence of O₂, or in Ca-poor solutions cells became permeable to mannitol. The ion movements observed after inhibition of active transport are compatible either with a passive K distribution and a primary inhibition of Na extrusion or with inhibition of a coupled active transport of both K and Na.     

Effect of ouabain on the lactogenic action of prolactin and on the level of mammary prolactin receptors

Ouabain added to the culture medium of rabbit mammary gland inhibits prolactin action on the initiation of lactose and casein synthesis. The degree of inhibition is a function of the ouabain concentration in the medium. Likewise, ouabain blocks the accumulation of casein mRNA supported by prolactin. In addition, ouabain provokes a rapid disappearance of prolactin receptors. Conversely prolactin keeps its capacity to enhance the concentration of casein mRNA and the parallel casein synthesis when K+ ions are totally absent from the culture medium. These results suggest that although prolactin induces a modification of the K+/Na+ ratio in the mammary cell and ouabain prevents this effect of prolactin, the inhibitory action of ouabain on lactogenesis can be explained essentially by its effect on the hormone receptors.     

[3H]Ouabain binding and Na+, K+-ATPase in resealed human red cell ghosts

The interaction of the cardiac glycoside [3H]ouabain with the Na+, K+ pump of resealed human erythrocyte ghosts was investigated. Binding of [3H]ouabain to high intracellular Na+ ghosts was studied in high extracellular Na+ media, a condition determined to produce maximal ouabain binding rates. Simultaneous examination of both the number of ouabain molecules bound per ghost and the corresponding inhibition of the Na+, K+-ATPase revealed that one molecule of [3H]ouabain inhibited one Na+, K+-ATPase complex. Intracellular magnesium or magnesium plus inorganic phosphate produced the lowest ouabain binding rate. Support of ouabain binding by adenosine diphosphate (ADP) was negligible, provided synthesis of adenosine triphosphate (ATP) through the residual adenylate kinase activity was prevented by the adenylate kinase inhibitor Ap5A. Uridine 5'-triphosphate (UTP) alone did not support ouabain binding after inhibition of the endogenous nucleoside diphosphokinase by trypan blue and depletion of residual ATP by the incorporation of hexokinase and glucose. ATP acting solely at the high- affinity binding site of the Na+, K+ pump (Km approximately 1 microM) promoted maximal [3H]ouabain binding rates. Failure of 5'-adenylyl-beta- gamma-imidophosphate (AMP-PNP) to stimulate significantly the rate of ouabain binding suggests that phosphorylation of the pump was required to expose the ouabain receptor.     

Plasma membrane studies on drug-sensitive and -resistant cell lines. II. Ouabain sensitivity of (Na+ +K+)-stimulated Mg2+-ATPase.

Mutant cell lines have been selected from the murine plasmocytoma MOPC 173 for their resistance to ouabain, dibutyryl cyclic AMP, theophyllin and concanavalin A. We have compared three wild-type cell lines with their seven resistant counterparts. All resistant mutants exhibited a (Na+ + K+)-stimulated Mg2+-ATPase resistance to ouabain inhibition when measured in microsomes. The homogeneity of ouabain binding sites has been found in most of the cell lines; however, two different populations of sites have been detected in one wild-type and in one resistant cell lines. These results led us to hypothetise the (Na+ + K+)-ATPase-ouabain interaction being modulated by a non-specific membrane structure.     

Regulatory changes of membrane transport and ouabain binding during progesterone-induced maturation ofXenopus oocytes

Summary During progesterone-stimulated maturation of defolliculated full-grownXenopus oocytes, the activities of the transport systems forl-alanine, thymidine, chloride, phosphate, and alkali ions decrease. Differences of the extent and time course of these changes suggest that they are controlled by at least partially independent mechanisms.A closer investigation of the Na-K ATPase has shown that in unstimulated oocytes, ouabain produces maximal inhibition when 8–12×10⁹ molecules are bound per cell. This number is bound during the first phase of a diphasic uptake process. Since this phase can be suppressed by increasing the concentration of external K⁺ to 45 mmol/liter or more, it is concluded that it refers to binding to the Na–K pump in the plasma membrane. Ouabain bound prior to progesterone-induced germinal vesicle breakdown (GVBD) remains bound after the breakdown, although the Na–K pump loses the capacity to bind ouabain after GVBD in oocytes that had not been exposed to ouabain preceding GVBD. In the presence of Mg⁺⁺ membranes isolated before regulatory inhibition of pumping and ouabain binding show a Na⁺-dependent incorporation of³²P from -[³²P]-ATP that can be reversed by the addition of K⁺. The phosphorylation site migrates on LiDS-polyacrylamide gel electropherograms at about 98,000 daltons and can be identified as a Commassie blue-stainable band.     

Effects of ouabain and isoproterenol on potassium influx in the turkey erythrocyte: Quantitative relation to ligand binding and cyclic AMP generation

Studies have been carried out in the turkey erythrocyte to examine: (1) the influence of external K⁺ concentration on both [³H]ouabain binding and the sensitivity of potassium influx to inhibition by ouabain and (2) the quantitative relation between β-adrenergic receptor site occupancy, agonist-directed cyclic AMP generation and potassium influx rate. Both [³H]ouabain binding and the ability of ouabain to inhibit potassium influx are markedly reduced at increasing external K⁺ concentrations, and at each K⁺ concentration the concentrations of ouabain required for half-maximal binding to the erythrocyte membrane and for half-maximal inhibition of potassium influx are identical. Both basal and isoproterenol-stimulated potassium influx rise with increasing external K⁺ concentrations. In contrast to basal potassium influx, which is 50–70% inhibitable by ouabain, the isoproterenol-stimulated component of potassium influx is entirely insensitive to ouabain. At all concentrations of K⁺, inhibition of basal potassium influx by ouabain is linear with ouabain binding, indicating that the rate of transport per unoccupied ouabain binding site is unaffected by simultaneous occupancy of other sites by ouabain. Similarly, the rate of isoproterenol-stimulated cyclic AMP synthesis is directly proportional to β-adrenergic receptor occupancy over the entire concentration-response relationship for isoproterenol, showing that at all levels of occupancy β-adrenergic receptor sites function independently of each other.Analysis of the relation of catecholamine-dependent potassium transport to the number of β-adrenergic receptor sites occupied indicates an extremely sensitive physiological system, in which 50%-maximal stimulation of potassium transport is achieved at less than 3% receptor occupancy, corresponding to fewer than ten occupied receptors per cell.