Erland Erdmann

Ouabain-receptor interactions in (Na+ + K+)-ATPase preparations from different tissues and species Determination of kinetic constants and dissociation constants

Abstract 1. 1. [3H]Ouabain-receptor association in (Na+ + K+)-ATPase preparations from beef (kidney, brain, and heart), from dog (heart), and guinea pig (kidney) in the presence of Mg2+ + Pi is a bimolecular reaction, while the dissociation is strictly a first-order process. For the ouabain receptor subunit of the enzyme from beef kidney, rate constants at 37 °C for the association are 1.85 · 104 M−1 · s−1 and for the dissociation 0.94 · 10−4 · s−1. A dissociation constant of 0.47 · 10−8 M was calculated from these rate constants and is determined from a Scatchard plot under equilibrium conditions. The dissociation constant determined in the presence of Na+, Mg2+, and ATP was 1.1 · 10−8 M. Scatchard plots indicate one single type of receptor. The ouabain-receptor dissociation constants varied with the tissue source of (Na+ + K+)-ATPase. The considerable variation of the dissociation constant in guinea pig kidney (1.6 · 10−7 M) was preferentially caused by changes of the dissociation rate constants. 2. 2. From the dissociation constant at 37°C a ΔG° of −11 kcal·mole−1 is calculated for the ouabain-receptor subunit of the enzymes from beef kidney brain, and heart and from dog heart. For the beef kidney enzyme ΔH° is −5 kcal·mole−1 and ΔS° 21 cal·mole−1·deg−1. For the guinea pig subunit thermodynamic calculations reveal a ΔG° of −9 kcal·mole−1, a ΔH° of −11 kcal·mole−1, and a ΔS° of −4 cal·mole−1·deg−1. 3. 3. Dissociation constants of the ouabain-receptor complex are 10- to 100-fold lower than the ouabain concentrations necessary for half-maximal inhibition of the (Na+ + K+)-ATPase. The stoichiometry of [3H]ouabain-binding sites: phosphorylated intermediate varied between 4 (guinea pig kidney) and 1 (beef enzymes). It is assumed that the ouabain receptor and the ATP hydrolysing subunit are not tightly linked.

Effects of ribose on exercise-induced ischaemia in stable coronary artery disease.

There is no established treatment specifically aimed at protecting or restoring cardiac energy metabolism, which is greatly impaired by ischaemia. Even after reperfusion, myocardial content of ATP remains low for more than 72 h. Long-term post-ischaemic dysfunction and irreversibility of ischaemic damage have been associated with low ATP content. Evidence that the pentose sugar ribose stimulates ATP synthesis and improves cardiac function led us to test the possibility that ribose increases tolerance to myocardial ischaemia in patients with coronary artery disease (CAD). 20 men with documented severe CAD underwent two symptom-limited treadmill exercise tests on 2 consecutive days; we postulated that the ischaemia induced might bring about changes in ATP metabolism lasting for several days. Patients whose baseline tests showed reproducibility were randomly allocated 3 days of treatment with placebo or ribose 60 g daily in four doses by mouth. Exercise testing was repeated after treatment on day 5. At that time mean (95% confidence interval) treadmill walking time until 1 mm ST-segment depression was significantly greater in the ribose than in the placebo group (276 [220-331] vs 223 [188-259] s; p = 0.002). The groups did not differ significantly in time to moderate angina. In the ribose-treated group the changes from baseline to day 5 in both time to ST depression and time to moderate angina were significant (p less than 0.005), but these changes were not significant in the placebo group. In patients with CAD, administration of ribose by mouth for 3 days improved the hearts tolerance to ischaemia. The presumed effects on cardiac energy metabolism offer new possibilities for adjunctive medical treatment of myocardial ischaemia.

Ouabain receptor interactions in (Na+ + K+)-ATPase preparations. II. Effect of cations and nucleotides on rate constants and dissociation constants

Abstract The action of ATP and its analogs as well as the effects of alkali ions were studied in their action on the ouabain receptor. One single ouabain receptor with a dissociation constant ( K D ) of 13 nM was found in the presence of ( Mg 2+ + P i ) and ( Na + + Mg 2+ + ATP ). pH changes below pH 7.4 did not affect the ouabain receptor. Ouabain binding required Mg2+, where a curved line in the Scatchard plot appeared. The affinity of the receptor for ouabain was decreased by K+ and its congeners, by Na+ in the presence of ( Mg 2+ + P i ), and by ATP analogs (ADP-C-P, ATP-OCH3). Ca2+ antagonized the action of K+ on ouabain binding. It was concluded that the ouabain receptor exists in a low affinity (Rα) and a high affinity conformational state (Rβ). The equilibrium between both states is influenced by ligands of (Na + + K + )-ATPase . With 3 mM Mg2+ a mixture between both conformational states is assumed to exist (curved line in the Scatchard plot).

Ouabain-receptor interactions in (Na++K+)-ATPase preparations

Summary1.Dissociation constants (KD) of the drug receptor complexes of 28 different cardiac glycosides have been determined either with [3H] labelled cardio-active steroids or from displacement of (3H] ouabain with unlabelled cardiac clycosides from the receptor. There is only one single type of cardiac glycoside receptor.2.Structure-affinity relationship of the different cardiac glycosides indicate that cardiac glycosides are recognized from interactions witha)the unsaturated lactone groupb)the steroid nucleus with a cis-configuration of the A∶B ring junctionc)the sugar component.3.Diphenylhydantoin displaces cardiac glycosides from the receptor, and so does prednison-3,20-bisguanylhydrazone.4.The cardiac glycoside receptor concentration increases proportionally with (Na++K+)-activated ATPase activity. Occupation of the cardiac glycoside receptor results in a proportional inhibition of (Na++K+)-ATPase activity.1. n nDissociation constants (K D ) of the drug receptor complexes of 28 different cardiac glycosides have been determined either with [3H] labelled cardio-active steroids or from displacement of (3H] ouabain with unlabelled cardiac clycosides from the receptor. There is only one single type of cardiac glycoside receptor. n n n n n2. n nStructure-affinity relationship of the different cardiac glycosides indicate that cardiac glycosides are recognized from interactions with

Ouabain receptor interactions in (Na+ + K+)-ATPase preparations. III. On the stability of the ouabain receptor against physical treatment, hydrolases and SH reagents

Abstract The stability of partial reactions of (Na + + K + )-ATPase (nucleotide binding, phosphorylation, ouabain binding and potassium phosphatase) against the inactivation of (Na + + K + )-ATPse by 5,5′-dithio-bis-(2-nitrobenzoic acid), ethacrynic acid, phospholipase A and trypsin were investigated. The properties of the ouabain receptor (affinity for the glycoside and binding capacity) were found to be relatively stable after pretreatment with the inactivating substances. The sensitivity of potassium phosphatase against pretreatment by SH reagents and hydrolytic enzymes was comparable tot hat of the (Na + + K + )-ATPase activity. These findings confirm the concept that (Na + + K + )-ATPase may be composed of subunits.

ADP binding to (Na+ + K+)-activated ATPase

Abstract [14C]ADP binding to EDTA-washed ox brain cell membranes was increased by Na+, but decreased by K+, Mg2+ and Ca2+. Na+ abolished the effect of K+ on ADP binding by a competitive mechanism, but could not reverse the inhibitory action of Mg2+ and Ca2+. It is concluded that the cation-induced changes in ADP binding reflect properties of (Na+ + K+)-activated ATPase.

Eigenschaften des Receptors für Herzglykoside

SummarySpecific binding of cardioactive glycosides to their receptor in cell membranes was measured. Structural requirements for optimal binding and effects of cations on this binding were investigated. Furthermore, a simple assay is described to evaluate the saturation of ouabain receptor sites of erythrocytes.n 1.Binding of cardiac glycosides to their receptor is a reversible process:cardiac steroid + receptor cardiac steroid-receptor-complex. For ouabain the affinity of the receptor in beef heart was determined from the ratio of the rate constants and from equilibrium binding at different temperatures. The dissociation constant of the ouabain-receptor-complex at 37° C was 0.3×10−8 M.2The receptor for cardiac glycosides is part of the (Na++K+)-activated ATPase. The number of receptor sites is directly related to (Na++K+)-ATPase activity of the cell membranes.3Cardiac glycoside receptors from different species have different affinities for ouabain. Receptors from different organs but of the same species, however, show the same affinity for the drug. The respective dissociation constants were determined in beef (heart, kidney, brain, erythrocyte), dog (heart), guinea pig (kidney), man (erythrocyte).4Thermodynamic studies of ouabain binding to its receptor from beef heart revealedaΔG°=−11 Kcal·Mol−1,ΔH°=−5 Kcal·Mol−1,ΔS°=21 cal·deg−1·Mol−1.5Dissociation constants of 25 structurally different cardiac glycoside-receptor-complexes were measured on the basis of displacement of [3H] ouabain from the receptor. Equilibrium binding of [3H]digoxin and [3H]β-acetyldigoxin to the receptor was used as control.6Certain structures of the glycoside molecule greatly influenced its affinity to the receptor—from these studies it is concluded that the cardiac glycoside must be attached to at least three counterparts at the receptor:a)the lactone groupb)the steroid nucleusc)the sugar component.7The affinity of the receptor for the cardiac steroid is directly correlated to the (Na++K+)-ATPase activity. Binding of ouabain to the receptor causes a proportional inhibition of the enzyme.8Potassium decreases the affinity of the receptor for the drug. In hypokalaemia, more cardioactive steroid is bound due to the higher affinity under these conditions. There is a Ca++-K+-antagonism in respect to the effect on ouabain binding. In the presence of K+ the affinity of the receptor is increased back to normal by Ca++.9The individual affinity of human receptors can be evaluated by measuring glycoside binding to human erythrocyte membranes. Changes in the affinity and differences in the amount of receptor sites per erythrocyte can be determined.10The percentage of erythrocyte receptor sites occupied by the cardiac steroid can be measured in treated patients, since the drug receptor-complex does not dissociate rapidly at 4° C. 10–30% of the receptor sites are occupied at therapeutic serum digoxin concentrations.11It is possible to test new drugs for their cardiac glycoside-like effects and their affinity to the receptor by in vitro measuring the displacement of [3H]ouabain. Drugs displacing ouabain from the receptor without inhibiting (Na++K+)-ATPase might be used in the treatment of glycoside intoxication.ZusammenfassungEs wurden die Bindung von Herzglykosiden an ihren Receptor, ihre Steroidspezifität, die Beeinflussung durch Kationen untersucht und ein Test zur Bestimmung der Sättigung des Receptors am Erythrocyten entwickelt.n 1.Die Bindung von Herzglykosiden an ihren Receptor ist ein reversibler Prozeß:Herzglykosid + Receptor Herzglykosid-Receptor-Komplex.Es wurde die Affinität des Receptors aus Rinderhirn zum Herzglykosid aus den Geschwindigkeitskonstanten und unter Gleichgewichtsbedingungen bei verschiedenen Temperaturen bestimmt. Die Dissoziationskonstante des Strophanthin-Receptor-Komplexes aus Rinderherz beträgt 0,3·10−8 M bei 37°C.2Der Herzglykosid-Receptor ist ein Teil der (Na++K+)-aktivierten ATPase. Die Menge an Herzglykosid-Receptor ist direkt proportional der spezifischen (Na++K+)-ATPase-Aktivität (U/mg).3Die Herzglykosid-Receptoren verschiedener Tierspecies haben unterschiedliche Affinität zu g-Strophanthin (Ouabain). Dagegen zeigen dieReceptoren aller Organe einer Tierspecies die gleiche Affinität für ein Herzglykosid. Es wurden die Dissoziationskonstanten des Herzglykosid-Receptors vom Rind (Herz, Niere, Hirn, Erythrocyt), Hund (Herz), Meerschweinchen (Niere), Mensch (Erythrocyt) bestimmt.4Es wurden die thermodynamischen Parameter für die Strophanthinbindung an den Receptor aus Rinderherz ermittelt alsΔG°=−11 Kcal·Mol−1,ΔH°=−5 Kcal·Mol−1,ΔS°=21 cal·grad−1·Mol−1.5An (Na++K+)-ATPase aus Rinderhirn wurde aus der Verdrängung von [3H]-g-Strophanthin die Dissoziationskonstanten des Herzglykosid-Receptor-Komplexes für 25 Herzglykoside bestimmt. Die Bestimmungsmethode wurde durch die Verwendung von [3H]markiertem Digoxin undβ-Acetyldigoxin überprüft.6Aus den Spezifitätsstudien wird geschlossen, daß der Herzglykosid-Receptor 3 Bindungsstellen hat. Das Herzglykosid wird erkannt aufgrunda)des Steroid-Anteils,b)des Lacton-Ringes,c)des Zuckeranteils.7Die Affinität des Receptors zu den Herzglykosiden kann direkt mit der halbmaximalen Hemmung der (Na++K+)-ATPase korreliert werden: Je höher die Dissoziationskonstante (KD) des Herzglykosid-Receptor-Komplexes desto höher ist die Herzglykosidkonzentration, die für eine halbmaximale Hemmung der (Na++K+)-ATPase benötigt wird.8Durch Erniedrigung der Affinität bindet der Herzglykosid-Receptor in Gegenwart von K+ weniger Herzglykosid. Die Herzglykosid-Sensitivität unter Hypokaliämie ist somit auf eine Zunahme der Affinität des Receptors zum Herzglykosid zurückzuführen. Ca2+-Ionen wirken antagonistisch zu K+; sie erhöhen in Abwesenheit und Gegenwart von K+ die Affinität des Receptors zu Herzglykosiden.9Die Eigenschaften des menschlichen Herzglykosid-Receptors lassen sich an mit Aqua dest. gewaschenen Erythrocyten messen. Damit ergibt sich die Möglichkeit, präventiv vor Herzglykosidgaben zu messen, ob der Herzglykosid-Receptor des Patienten vom Normalkollektiv abweicht. Es lassen sich sowohl Abnahmen der Zahl der Herzglykosid-Receptoren wie Änderungen der Affinität erfassen.10Da Herzglykoside bei +4° C praktisch nicht vom Glykosid-Receptor abdissoziieren, kann das Ausmaß der Beladung des Erythrocyten-Receptors mit radioaktiv markiertem Strophanthin gemessen werden. Ein Test für die Messung der Beladung wird beschrieben. Therapeutische Herzglykosid-Konzentrationen liegen bei 10–30% Beladung des Receptors.11Unbekannte Pharmaka können aufgrund der Verdrängung von [3H]-Strophanthin vom Herzglykosid-Receptor der (Na++K+)-ATPase auf ihre Herzglykosid-Wirksamkeitin vitro getestet werden. Die Affinität zum Receptor kann aus der Verdrängung berechnet werden. Dieser „Verdrängungstest“ kann für die Beurteilung neuer Herzglykosidevor der klinischen Erprobung wie für das Auffinden von Pharmaka für die Bekämpfung der Herzglykosid-Intoxikation hilfreich sein.

Interaktionen zwischen Diuretika und herzwirksamen Glykosiden an Myokard und Reizleitungssystem

Die Wirkungen von Diuretika interferieren, beim Patienten angewendet, in vielfaltiger Weise mit denen von herzwirksamen Glykosiden [1]. Solche Diuretika, die etwa, wie Spironolactone, Triamteren oder Amilorid, am Tubulussystem der Niere eine Kaliumrctcntion hervorrufen und die Natriurcsc steigern [2], lassen daruber hinaus Interaktionen mit Glykosiden am Reizleitungssystem und Arbeitsmyokard unmittelbar vermuten.