Ouabain-induced Inhibition Research Articles

Unveiling the Role of Cholesterol in Subnanomolar Ouabain Rescue of Cortical Neurons from Calcium Overload Caused by Excitotoxic Insults.

Na/K-ATPase maintains transmembrane ionic gradients and acts as a signal transducer when bound to endogenous cardiotonic steroids. At subnanomolar concentrations, ouabain induces neuroprotection against calcium overload and apoptosis of neurons during excitotoxic stress. Here, the role of lipid rafts in interactions between Na/K-ATPase, sodium-calcium exchanger (NCX), and N-methy-D-aspartate receptors (NMDARs) was investigated. We analyzed 0.5-1-nanometer ouabain's effects on calcium responses and miniature post-synaptic current (mEPSCs) frequencies of cortical neurons during the action of NMDA in rat primary culture and brain slices. In both objects, ouabain attenuated NMDA-evoked calcium responses and prevented an increase in mEPSC frequency, while the cholesterol extraction by methyl-β-cyclodextrin prevented the effects. The data support the conclusions that (i) ouabain-induced inhibition of NMDA-elicited calcium response involves both pre- and post-synapse, (ii) the presence of astrocytes in the tripartite synapse is not critical for the ouabain effects, which are found to be similar in cell cultures and brain slices, and (iii) ouabain action requires the integrity of cholesterol-rich membrane microdomains in which the colocalization and functional interaction of NMDAR-transferred calcium influx, calcium extrusion by NCX, and Na/K-ATPase modulation of the exchanger occur. This regulation of the molecules by cardiotonic steroids may influence synaptic transmission, prevent excitotoxic neuronal death, and interfere with the pharmacological actions of neurological medicines.

Inhibition of Na+, K+ -ATPase with ouabain is detrimental to equine blastocysts.

Although equine blastocysts ≤ 300 µm in diameter can be successfully vitrified, larger equine blastocysts are not good candidates for cryopreservation. As Na+, K+-ATPase is involved in maintaining blastocyst expansion, perhaps inhibition of this enzyme would be a viable method of reducing blastocyst diameter prior to cryopreservation. Objectives were to evaluate effects of ouabain-induced inhibition of Na+, K+-ATPase in equine blastocysts. Sixteen mares were ultrasonographically monitored, given deslorelin acetate to induce ovulation, and inseminated. Embryos (D7 and D9) were harvested and Na+, K+-ATPase inhibited for 1 or 6 h by exposure to 10-6 M ouabain, either natural ouabain or conjugated to fluorescein (OuabainFL), during incubation at 37° C. Evaluations included morphometric characteristics (bright field microscopy) and viability (Hoescht 33342 + propidium iodide). Blastocysts incubated for 6 h in Holding medium + ouabain (n=3) had, on average, a 45.7% reduction in diameter, with adverse morphologic features and no re-expansion after subsequent incubation in Holding medium for 12 h. In subsequent studies, even a 1-h exposure to Ouabain or OuabainFL, caused similar reductions, namely 38.7 ± 6.7% (n=5) and 33.6 ± 3.3% (n=7) for D7 and D9 blastocysts, respectively. Ouabain binding was confirmed after OuabainFL exposition and all embryos (n=12) lost viability. We concluded that Na+, K+-ATPase inhibition with ouabain caused death of equine blastocysts and therefore was not a viable method of reducing blastocyst size prior to cryopreservation.

Modification of Ca2+-handling in cardiomyocytes by redox sensitive mechanisms in response to ouabain

We examined the role of redox-sensitive signal transduction mechanisms in modifying the changes in [Ca(2+)](i) produced by ouabain upon incubating adult rat cardiomyocytes with antioxidants or inhibitors of different protein kinases and monitoring alterations in fura-2 fluorescence. Ouabain increased basal [Ca(2+)](i), augmented the KCl-induced increase in [Ca(2+)](i), and promoted oxyradical production in cardiomyocytes. These actions of ouabain were attenuated by an oxyradical scavenging mixture (superoxide dismutase plus catalase), and the antioxidants (N-acetyl-L-cysteine and N-(2-mercaptoproprionyl)glycine). An inhibitor of MAP kinase (PD98059) depressed the ouabain-induced increase in [Ca(2+)], whereas inhibitors of tyrosine kinase (tyrphostin and genistein) and PI3 kinase (Wortmannin and LV294002) enhanced the ouabain-induced increase in [Ca(2+)](i). Inhibitors of protein kinase C (calphostin and bisindolylmalaimide) augmented the ouabain-induced increase in [Ca(2+)](i), whereas stimulation of protein kinase C by a phorbol ester (phorbol 12-myristate 13-acetate) depressed the action of ouabain. These results suggest that ouabain-induced inhibition of Na (+)-K(+) ATPase may alter the redox status of cardiomyocytes through the production of oxyradicals, and increase the activities of various protein kinases. Thus, these redox-sensitive signal transduction mechanisms involving different protein kinases may modify Ca(2+)-handling sites in cardiomyocytes and determine the magnitude of net increase in [Ca(2+)](i) in response to ouabain.

Ouabain-stimulated trafficking regulation of the Na/K-ATPase and NHE3 in renal proximal tubule cells

We have demonstrated that ouabain regulates protein trafficking of the Na/K-ATPase α1 subunit and NHE3 (Na/H exchanger, isoform 3) via ouabain-activated Na/K-ATPase signaling in porcine LLC-PK1 cells. To investigate whether this mechanism is species-specific, ouabain-induced regulation of the α1 subunit and NHE3 as well as transcellular (22)Na(+) transport were compared in three renal proximal tubular cell lines (human HK-2, porcine LLC-PK1, and AAC-19 originated from LLC-PK1 in which the pig α1 was replaced by ouabain-resistant rat α1). Ouabain-induced inhibition of transcellular (22)Na(+) transport is due to an ouabain-induced redistribution of the α1 subunit and NHE3. In LLC-PK1 cells, ouabain also inhibited the endocytic recycling of internalized NHE3, but has no significant effect on recycling of endocytosed α1 subunit. These data indicated that the ouabain-induced redistribution of the α1 subunit and NHE3 is not a species-specific phenomenon, and ouabain-activated Na/K-ATPase signaling influences NHE3 regulation.

Dual activity of the H1-H2 domain of the (Na++K+)-ATPase

(Na++K+)-ATPase is a target receptor of digitalis (cardiac glycoside) drugs. It has been demonstrated that the H1-H2 domain of the α-subunit of the (Na++K+)-ATPase is one of the digitalis drug interaction sites of the enzyme. Despite the extensive studies of the inhibitory effect of digitalis on the (Na++K+)-ATPase, the functional property of the H1-H2 domain of the enzyme and its role in regulating enzyme activity is not completely understood. Here we report a surprise finding: instead of inhibiting the enzyme, binding of a specific monoclonal antibody SSA78 to the H1-H2 domain of the (Na++K+)-ATPase elevates the catalytic activity of the enzyme. In the presence of low concentration of ouabain, monoclonal antibody SSA78 significantly protects enzyme function against ouabain-induced inhibition. However, higher concentration of ouabain completely inactivates the (Na++K+)-ATPase even in the presence of SSA78. These results suggest that the H1-H2 domain of the (Na++K+)-ATPase is capable of regulating enzyme function in two distinct ways for both ouabain-sensitive and -resistant forms of the enzyme: it increases the activity of the (Na++K+)-ATPase during its interaction with an activator; it also participates in the mechanism of digitalis or ouabain-induced inhibition of the enzyme. Understanding the dual activity of the H1-H2 domain will help better understand the structure–function relationships of the (Na++K+)-ATPase and the biological processes mediated by the enzyme.

Clearance of Extracellular K+ during Muscle Contraction—Roles of Membrane Transport and Diffusion

Excitation of muscle often leads to a net loss of cellular K+ and a rise in extracellular K+ ([ K+ ]o), which in turn inhibits excitability and contractility. It is important, therefore, to determine how this K+ is cleared by diffusion into the surroundings or by reaccumulation into the muscle cells. The inhibitory effects of the rise in [K+ ]o may be assessed from the time course of changes in tetanic force in isolated muscles where diffusional clearance of K+ is eliminated by removing the incubation medium and allowing the muscles to contract in air. Measurements of tetanic force, endurance, and force recovery showed that in rat soleus and extensor digitorum longus (EDL) muscles there was no significant difference between the performance of muscles contracting in buffer or in air for up to 8 min. Ouabain-induced inhibition of K+ clearance via the Na+,K+ pumps markedly reduced contractile endurance and force recovery in air. Incubation in buffer containing 10 mM K+ clearly inhibited force development and endurance, and these effects were considerably reduced by stimulating Na+,K+ pumps with the β2-agonist salbutamol. Following 30–60 s of continuous stimulation at 60 Hz, the amount of K+ released into the extracellular space was assessed from washout experiments. The release of intracellular K+ per pulse was fourfold larger in EDL than in soleus, and in the two muscles, the average [K+ ]o reached 52.4 and 26.0 mM, respectively, appreciably higher than previously detected. In conclusion, prevention of diffusion of K+ from the extracellular space of isolated working muscles causes only modest interference with contractile performance. The Na+,K+ pumps play a major role in the clearance of K+ and the maintenance of force. This new information is important for the evaluation of K+-induced inhibition in muscles, where diffusional clearance of K+ is reduced by tension development sufficient to suppress circulation.

Sodium–calcium exchange does not require allosteric calcium activation at high cytosolic sodium concentrations

The activity of the cardiac Na(+)-Ca(2+) exchanger (NCX1.1) is allosterically regulated by Ca(2+), which binds to two acidic regions in the cytosolically disposed central hydrophilic domain of the NCX protein. A mutation in one of the regulatory Ca(2+) binding regions (D447V) increases the half-activation constant (K(h)) for allosteric Ca(2+) activation from approximately 0.3 to > 1.8 microm. Chinese hamster ovary cells expressing the D447V exchanger showed little or no activity under physiological ionic conditions unless cytosolic [Ca(2+)] was elevated to > 1 microm. However, when cytosolic [Na(+)] was increased to 20 mm or more (using ouabain-induced inhibition of the Na(+),K(+)-ATPase or the ionophore gramicidin), cells expressing the D447V mutant rapidly accumulated Ca(2+) or Ba(2+) when the reverse (Ca(2+) influx) mode of NCX activity was initiated, although initial cytosolic [Ca(2+)] was < 100 nm. Importantly, the time course of Ca(2+) uptake did not display the lag phase that reflects allosteric Ca(2+) activation of NCX activity in the wild-type NCX1.1; indeed, at elevated [Na(+)], the D447V mutant behaved similarly to the constitutively active deletion mutant Delta(241-680), which lacks the regulatory Ca(2+) binding sites. In cells expressing wild-type NCX1.1, increasing concentrations of cytosolic Na(+) led to a progressive shortening of the lag phase for Ca(2+) uptake. The effects of elevated [Na(+)] developed rapidly and were fully reversible. The activity of the D447V mutant was markedly inhibited when phosphatidylinositol 4,5-bisphosphate (PIP2) levels were reduced. We conclude that when PIP2 levels are high, elevated cytosolic [Na(+)] induces a mode of exchange activity that does not require allosteric Ca(2+) activation.

Sodium‐calcium exchanger (NCX‐1) and calcium modulation: NCX protein expression patterns and regulation of early heart development

Ouabain-induced inhibition of early heart development indicated that Na/K-ATPase plays an important role in maintaining normal ionic balances during differentiation of cardiomyocytes (Linask and Gui [1995] Dev Dyn 203:93-105). Inhibition of the sodium pump is generally accepted to affect the activity of the Na(+)-Ca(++) exchanger (NCX) to increase intracellular [Ca(++)]. These previous findings suggested that Ca(++) signaling may be an important modulator during differentiation of cardiomyocytes. In order to identify a connection between heart development and NCX-mediated Ca(++) regulation, we determined the embryonic spatiotemporal protein expression pattern of NCX-1 during early developmental stages. In both chick and mouse embryos, NCX-1 (the cardiac NCX isoform) is asymmetrically expressed during gastrulation; in the right side of the Hensen's node in the chick, in the right lateral mesoderm in the mouse. At slightly later stages, NCX-1 is expressed in the heart fields at comparable stages of heart development, in the chick at stage 7 and in the mouse at embryonic day (ED) 7.5. By ED 8 in the mouse, the exchanger protein displays a rostrocaudal difference in cardiac expression and an outer curvature-inner curvature ventricular difference. By ED 9.5, cardiac expression has increased from that seen at ED8 and NCX-1 is distributed throughout the myocardium consistent with the possibility that it is important in regulating initial cardiac contractile function. Only a low level of expression is detected in inflow and outflow regions. To substantiate a role for the involvement of calcium-mediated signaling, using pharmacologic approaches, ionomycin (a Ca(++) ionophore) was shown to perturb cardiac cell differentiation in a manner similar to ouabain as assayed by cNkx2.5 and sarcomeric myosin heavy chain expression. In addition, we show that an inhibitor of NCX, KB-R7943, can similarly and adversely affect early cardiac development at stage 4/5 and arrests cardiac cell contractility in 12-somite embryos. Thus, based upon NCX-1 protein expression patterns in the embryo, experimental Ca(++) modulation, and inhibition of NCX activity by KB-R7943, these results suggest an early and central role for calcium-mediated signaling in cardiac cell differentiation and NCX's regulation of the initial heartbeats in the embryo.

Intracellular Reactive Oxygen Species Mediate the Linkage of Na+/K+-ATPase to Hypertrophy and Its Marker Genes in Cardiac Myocytes

We showed before that in cardiac myocytes partial inhibition of Na+/K+-ATPase by nontoxic concentrations of ouabain causes hypertrophy and transcriptional regulations of growth-related marker genes through multiple Ca2+-dependent signal pathways many of which involve Ras and p42/44 mitogen-activated protein kinases. The aim of this work was to explore the roles of intracellular reactive oxygen species (ROS) in these ouabain-initiated pathways. Ouabain caused a rapid generation of ROS within the myocytes that was prevented by preexposure of cells to N-acetylcysteine (NAC) or vitamin E. These antioxidants also blocked or attenuated the following actions of ouabain: inductions of the genes of skeletal alpha-actin and atrial natriuretic factor, repression of the gene of the alpha3-subunit of Na+/K+-ATPase, activation of mitogen-activated protein kinases, activation of Ras-dependent protein synthesis, and activation of transcription factor NF-kappaB. Induction of c-fos and activation of AP-1 by ouabain were not sensitive to NAC. Ouabain-induced inhibition of active Rb+ uptake through Na+/K+-ATPase and the resulting rise in intracellular Ca2+ were also not prevented by NAC. A phorbol ester that also causes myocyte hypertrophy did not increase ROS generation, and its effects on marker genes and protein synthesis were not affected by NAC. We conclude the following: (a) ROS are essential second messengers within some but not all signal pathways that are activated by the effect of ouabain on Na+/K+-ATPase; (b) the ROS-dependent pathways are involved in ouabain-induced hypertrophy; (c) increased ROS generation is not a common response of the myocyte to all hypertrophic stimuli; and (d) it may be possible to dissociate the positive inotropic effect of ouabain from its growth-related effects by alteration of the redox state of the cardiac myocyte.

Extracellular Na+ electrode for monitoring net Na+ flux in cell suspensions.

A computer-linked extracellular sodium-sensitive electrode system is described that is suitable for the routine measurement of net Na+ transport in cell suspensions. The commercially available Na+ electrode exhibited high selectivity for Na+ over other cations and a rapid response time (less than 3 s). This system resolved changes of 0.4 mM in the presence of 147 mM extracellular Na+. Measurements of Na+ transport in suspensions of rabbit proximal tubules showed that ouabain caused a dose-dependent net Na+ influx with an inhibitor constant (Ki) of 2.5 +/- 0.2 microM and a maximal velocity (Vmax) of 229 +/- 7 nmol Na+.min-1.mg protein-1. This compared favorably with the ouabain-induced K+ efflux (Ki = 2.4 microM; Vmax = 160 +/- 3.3 nmol K+.min-1.mg protein-1) and the ouabain-induced inhibition of respiration (Ki = 3.3 microM; Vmax = 11.8 nmol O2.min-1.mg protein-1). In addition, Ba2+, a K+ channel blocker known to depolarize the cell, caused a net Na+ efflux, whereas glucose, a Na+-cotransported solute, promoted a net Na+ influx. This system should be a powerful tool for continuous monitoring of net Na+ fluxes in cell suspensions.

The protective action of R56865 against ouabain-induced intoxication in rat heart isolated atria and ventricles

The action of R56865 has been examined on the contractile effects produced by ouabain concentrations interacting with high (3 μM) and low (300 μM) affinity digitalis receptors on electrically stimulated ventricular strips isolated from rat. R56865 1 μM reduced the increase in resting tension produced by ouabain 300 μM and left unalterated the inotropic effect evoked by ouabain 3 and 300 μM was reduced by higher concentrations (3 and 6 μM) of R56865. The action of R56865 was also studied on ouabain-induced intoxication in electrically stimulated and spontaneously beating atria of rat. On electrically stimulated (3 Hz) whole atria, R56865 0.3 μM reduced the maximal increase in resting tension produced by ouabain 300 μM and delayed the time to onset of the ouabain-induced arrhythmias but did not affect ouabain's inotropic effect. Higher concentrations of R56865 were required to reduce the inotropic effect of ouabain. The protective action of R56865 against ouabain-induced intoxication was most pronounced on spontaneously beating atria where it reduced spontaneous rate of beats. Experiments in electrically driven left atria indicated that only a part of the protective effect of R56865 could be related to its bradycardic action. The effect of R56865 was also examined on ouabain-induced inhibition of sodium pump in human red blood cells. R56865 μM did not modify the inhibition produced by ouabain (from 0.3 to 10 nM), this indicates that the protective action of R56865 against ouabain-induced intoxication is not due to an interaction with the inhibitory effect of ouabain on sodium pump.

培養血管内皮細胞のＰＧＩ２産生遊離機構におけるＮａ｀＋´動態の関与

The regulatory mechanism of prostacyclin (PGI2) generation and its enhancement by ouabain and monensin in cultured human vascular endothelial cells was investigated with references to the kinetics of Ca++ and Na+. Na+-K+ ATPase activity was assayed as ouabain-sensitive 86Rb uptake. Cytosolic free calcium ion concentration ([Ca++]i) was measured using fura-2. The kinetics of 45Ca of 22Na, and [Ca++]i preceded PGI2 generation. Ouabain inhibited Na+-K+ ATPase activity (inhibition of 22Na release and 86Rb uptake) and increased 22Na uptake, while monensin enhanced 22Na uptake and Na+-K+ ATPase activity at 2.5min after starting incubation. Both of these reagents decreased 45Ca release and increased 45Ca uptake (inhibition of Na+-Ca++ exchange systems) at 2.5min. And all of these effects were attenuated at 10min. Ouabain and monensin increased [Ca++]i. Through these results it was concluded that the enhanced PGI2 generation by ouabain or monensin was speculated to be brought about by the increased [Ca++]i and Ca++ uptake which was based on the decreased Ca++ release via the inhibition of Na+-Ca++ exchange systems, and that enhanced PGI2 generation might be autoregulated by the attenuation of increased [Na+]i, which was derived from the attenuation of ouabain-induced inhibition of Na+-K+ ATPase activity, or from the enhancement of monensin-induced increase of its activity.

Adaptational Changes of Sarcomere and Sarcolemma During Chronic Cardiac Overloading in Rats and in Humans

In response to increasing demand, the cardiac muscle has developed several adaptational mechanisms. Gene expression is modified in a quantitative and a qualitative way since the heart hypertrophies and since its structure changes to improve the efficiency of the contraction. The sarcomere modifications are both species- and tissue-specific. An isoenzymic shift of myosin from high adenosine triphosphatase (ATPase) activity form V-1 to low activity form V-3 occurs in all conditions in which V-1 is initially predominant, i.e., in rat (and also rabbit) ventricles and the atria of other species, including humans. It was not observed in conditions in which V-3 was predominant, as in human ventricles (and also in those of cats and pigs). Another shift from creatine kinase (CK) monomer M to CK B, the form that predominates in the fetal heart, is also observed. The sarcolemma is also modified, at least in rats. The digitalis receptor was characterized by studying the inotropic effect of the drug on an isolated heart preparation and on a purified preparation of sarcolemma with a high Na+,K(+)-ATPase activity by binding [3H]ouabain and ouabain-induced inhibition of the enzymatic activity. In hypertrophied heart, both the recovery of normal contractility after ouabain infusion and the release of previously bound ouabain infusion and the release of previously bound ouabain were slowed, as for fetal hearts. Changes in other inotropic receptors have also been reported. From a practical point of view, this means that screening of new inotropic agents has to be done on hypertrophied hearts and not, as usual, on normal tissue.

Ouabain binding sites and (Na+,K+)-ATPase activity in rat cardiac hypertrophy. Expression of the neonatal forms.

The adaptation of the myocardium to mechanical overload which results in cardiac hypertrophy involves several membrane functions. The digitalis receptor in sarcolemma vesicles from hypertrophied rat hearts is characterized by binding of [3H]ouabain and ouabain-induced inhibition of (Na+,K+)-ATPase. The results show the existence of two families of ouabain binding sites with apparent dissociation constants (Kd) of 1.8-3.2 X 10(-8) M and 1-8 X 10(-6) M, respectively, which are similar to those found in normal hearts. The presence of the high affinity receptor in hypertrophied rat heart is correlated to a detectable inhibition of the (Na+,K+)-ATPase (IC50 = 1-3 X 10(-8) M). However, the high and low affinity sites in hypertrophied hearts bind and release ouabain at 4-5-fold slower rates than the corresponding sites in normal hearts. These properties are similar to that we observed in newborn rat cardiac preparations. Taken together with the expression of myosin isoforms (Schwartz, K., Lompre, A.M., Bouveret, P., Wisnewsky, C., and Whalen, R.G. (1982) J. Biol. Chem. 23, 14412-14418), our data show that the physiological adaptation of the heart also involves the resurgence of the neonatal forms of the digitalis receptor.

Modulation of glycoside sensitivity of the cardiac sodium pump

Glycoside sensitivity of isolated cardiac Na+,K+-ATPase, i.e., the enzymatic representation of the sodium pump, is dependent on the chemical structure of the glycoside and the animal species from which the enzyme is obtained (1). The sensitivity, or affinity, of the isolated enzyme for the cardiac glycoside can be estimated either from the KD value (dissociation constant) for the [3H]ouabain binding reaction or the IC50 value (concentration required to cause a 50% inhibition) for ouabain-induced inhibition of Na+,K+-ATPase activity. For example, the KD value for Na+,K+-ATPase obtained from ventricular muscle of guinea-pig heart for [3H]ouabain is 0.1 to 0.15 μM. The IC50 value is several-fold higher; 0.5 to 0.7 μM for the same combination of enzyme and glycoside. This difference results from the fact that [3H]ouabain binding is usually estimated in the presence of Na+, Mg2+ and ATP (or Mg2+ and inorganic phosphate) whereas Na+,K+-ATPase activity is assayed in the presence of these ligands plus K+. All of these ligands influence the glycoside sensitivity of Na+,K+-ATPase.

Effect of sodium-potassium pump inhibitors and membrane-depolarizing agents on sodium nitroprusside-induced relaxation and cyclic guanosine monophosphate accumulation in rat aorta.

The purpose of this study was to investigate the relationship between sodium nitroprusside-induced relaxation, inhibition of the sodium-potassium pump, and cyclic guanosine monophosphate. Exposure of rat thoracic aorta to ouabain, or potassium- or magnesium-free Krebs-Ringer bicarbonate solution, procedures which presumably inhibit the sodium-potassium pump, or to potassium chloride or tetraethylammonium, membrane-depolarizing agents, inhibited relaxation to nitroprusside. These conditions had little or no effect on the elevated cyclic guanosine monophosphate levels at a concentration of nitroprusside (0.1 microM) that relaxed norepinephrine contracted tissues by 80%. However, at a maximum relaxant concentration of nitroprusside (1.0 microM), these conditions decreased the elevation of cyclic guanosine monophosphate. The inhibition of elevated cyclic guanosine monophosphate levels was independent of the endothelium, extracellular calcium, and the cyclic guanosine monophosphate phosphodiesterase inhibitor, M&B 22,948. The inhibitory effects of ouabain and of potassium- and magnesium-free solution on the increased levels of cyclic guanosine monophosphate caused by 1.0 microM nitroprusside were abolished when tissues were incubated without norepinephrine, or with norepinephrine in the presence of the alpha-adrenergic blocker, phentolamine. In contrast, a beta-adrenergic blocker, propranolol, had no effect on the ouabain-induced inhibition of elevated cyclic guanosine monophosphate levels, with norepinephrine present. These results are consistent with the hypothesis that membrane events regulate cyclic guanosine monophosphate synthesis. At nitroprusside concentrations greater than 0.1 microM, the formation of cyclic guanosine monophosphate appears to be coupled to the status of the smooth muscle cell membrane and integrity of the sodium-potassium pump.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac glycoside receptors in cultured heart cells—II: Characterization of a high affinity and a low affinity binding site in heart muscle cells from neonatal rats

The binding of [ 3H]ouabain has been studied in (Na + + K +)-ATPase enriched cardiac cell membranes, as well as in cardiac muscle and non-muscle cells in culture—all obtained from hearts of neonatal rats. The binding has been correlated with ouabain-induced inhibition of (Na + + K +)-ATPase (cardiac cell membranes) and the inhibition of active ( 86Rb + + K +)-influx (cardiac muscle and non-muscle cells in culture). Furthermore, the effect of ouabain on the amplitude of cell-wall motion and contraction velocity has been studied in electrically driven cardiac muscle cells. In muscle and non-muscle cells, two classes of ouabain binding sites have been identified. In rat heart muscle cells, the high affinity binding site has a dissociation constant ( K D ) of 3.2 × 10 −8 M and a binding capacity ( B) of 0.2 pmole/mg protein (80,000 sites/cell); the values for the low affinity binding site are: K D = 7.1 × 10 −6 M; B = 2.6 pmole/mg protein (10 6 sites/cell). The binding to both types of binding sites is depressed by K + and abolished after heat denaturation of the cells. The kinetics of [ 3H]ouabain binding to rat heart muscle cells (association and dissociation rate constants, K +- and temperature-dependence of association and dissociation processes) have been characterized. In rat heart muscle and non-muscle cells, the binding of [ 3H]ouabain to the low affinity site results in inhibition of the ( 86Rb + + K +)-influx ( EC 50 = 1.3 and 1.5 × 10 −5 M ouabain), a decrease in cell-K + ( EC 50 = 1.9 and 1.4 × 10 −5 M ) and an increase in cell-Na + (10 −5-10 −4 M). The ouabain-induced positive inotropic effect (increase in amplitude of cell-wall motion, increase in contraction velocity) in cardiac muscle cells is observed only at ouabain concentrations ⩾5 × 10 −6 M, and it is therefore probably attributed to occupation of the low affinity binding site. Coupling of occupation of the low affinity site by ouabain with drug-induced inhibition of the sodium pump and with drug-induced positive inotropic action is further substantiated by kinetic measurements. In contrast, occupation of the high affinity binding site does not produce any measurable inhibition of the sodium pump activity or positive inotropy. In agreement with the findings in intact cells, two classes of ouabain binding sites ( K D = 2.2 × 10 −8 and 1.3 × 10 −4 M have been characterized in (Na + + K +)-ATPase enriched cardiac cell membranes from neonatal rats; only the binding of ouabain to the low affinity binding site produces a measurable inhibition of (Na + + K +)-ATPase activity ( EC 50 = 4.8 × 10 −5 M ouabain). Our results support the finding of different types of ouabain binding sites in rat heart. The nature of the high affinity/low capacity ouabain binding site remains to be elucidated.

Cardiac glycoside receptors in cultured heart cells—I. characterization of one single class of high affinity receptors in heart muscle cells from chick embryos

Binding of ( 3H)-ouabain and ouabain-induced inhibition of the sodium pump and of the (Na + + K +)-ATPase have been characterized in cultured cardiac muscle and non muscle cells, as well as in cardiac cell membranes—all obtained from chick embryos. 1. In both cell types, ouabain binds to a single type of binding sites in a temperature-dependent manner. The association rate but not the dissociation rate, is lowered by K +; specific binding is lost after heat-denaturation of the cells. Binding parameters (association and dissociation rate constants, activation energies for association and dissociation) are similar in muscle and non muscle cells. The dissociation constant of specific ouabain binding is 1.5 × 10 −7 M in cardiac muscle cells, and 1.9 × 10 −7 M in cardiac non muscle cells, the binding capacity being 2.6 and 2.1 pmoles/mg protein respectively. 2. Specific binding of ouabain to the cells is coupled to inhibition of the sodium pump, as can be seen from ouabain-induced inhibition of active ( 86Rb + + K +)-uptake, decrease in cellular K +, and increase in cellular Na + (EC 50 = 10 −7−10 −6 M). 3. The data obtained with cardiac cells are in good agreement with results found for ouabain binding (dissociation constant 4.3 × 10 −7 M) and (Na + + K +)-ATPase inhibition (EC 50 = 1.4 × 10 −6 M) in cardiac cell membranes prepared from the same tissue. 4. Due to the experimental evidence it is concluded that the binding site for ouabain is identical with the cardiac glycoside receptor of these cells. 5. In cardiac non muscle cells, binding of ouabain to its receptor is strictly coupled to inhibition of active K +-transport in a stoichiometric manner. In cardiac muscle cells, however, active K +-transport is inhibited by less than 10% when up to 40% of cardiac glycoside receptors have bound ouabain. It is assumed that this non-stoichiometric coupling of receptor occupancy and sodium pump inhibition in cardiac muscle cells may prevent substantial changes of Na +- and K +-contents in the heart in the presence of therapeutic levels of cardiac glycosides.

Isolation of interferon-resistant variants of friend erythroleukemia cells: Effects of interferon and ouabain

Interferon (IFN)-resistant clones of Friend leukemia cells (FLC) have been isolated by continuous growth in IFN-rich medium followed by plating in semisolid medium in the presence of 1500 U/ml of IFN. FLC variants are fully resistant to the IFN-induced antiviral state against lytic viruses (Mengo and vesicular stomatitis virus) and retrovirus (Friend-MuLV) production at least up to IFN doses 1000-fold higher than those needed to protect parental FLC. With respect to erythroid differentiation in DMSO-induced FLC it is emphasized that: (1) the FLC variant tested was also resistant to the enhancement of erythroid differentiation by low doses (15–480 U/ml) of IFN; (2) IFN doses (5000–20,000 U/ml) practically unable to reduce Friend-MuLV release and VSV yields did inhibit, instead, the erythroid differentiation of the DMSO-induced FLC variant tested almost at the same extent as with susceptible FLC. The striking dichotomy of IFN-induced antiviral versus differentiation effects suggests that different mechanisms underlie different IFN effects, the specificity of which was fully ascertained by employing pure IFN preparations. In the FLC system, treatment with ouabain does not compete IFN-induced antiviral state. Yet, two out of three variants tested were partially resistant to ouabain-induced inhibition of Friend-MuLV release while being fully susceptible to other ouabain-induced effects on FLC. In addition, an ouabain-resistant FLC variant was not resistant to IFN.

Effect of ouabain on osmotic resistance and monovalent cation transport of red cells in hereditary spherocytosis.

The effect of ouabain on the osmotic resistance of red cells from 17 splenectomized patients with congenital or hereditary spherocytosis (HS), from 5 of their relatives suspected of having a subclinical form of the disease and from unsplenectomized and splenectomized normal controls was studied. In red cells from the HS patients and the splenectomized controls a small ouabain-induced decrease in osmotic resistance was seen, whereas in the unsplenectomized controls no such change was observed. The osmotic resistance of red cells from the 5 relatives, on the other hand, was significantly decreased by ouabain. The usefulness of ouabain in unmasking HS in its subclinical form was further tested by studying monovalent cation influx into red cells. The active influx of 86Rb+ into red cells from HS suffers was significantly increased. A similar degree of ouabain-induced inhibition of 86Rb+ transport was seen in all groups studied. The passive influx of 22Na+, which was also increased in HS red cells, was not affected by ouabain in any of the four groups.