Wilhelm Schoner

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.

Endogenous and Exogenous Cardiac Glycosides and their Mechanisms of Action

Cardiac glycosides have been used for decades to treat congestive heart failure. The recent identification of cardiotonic steroids such as ouabain, digoxin, marinobufagenin, and telocinobufagin in blood plasma, adrenal glands, and hypothalamus of mammals led to exciting new perspectives in the pathology of heart failure and arterial hypertension. Biosynthesis of ouabain and digoxin occurs in adrenal glands and is under the control of angiotensin II, endothelin, and epinephrine released from cells of the midbrain upon stimulation of brain areas sensing cerebrospinal Na+ concentration and, apparently, the body’;s K+ content. Rapid changes of endogenous ouabain upon physical exercise may favor the economy of the heart by a rise of intracellular Ca2+ levels in cardiac and atrial muscle cells. According to the sodium pump lag hypothesis, this may be accomplished by partial inhibition of the sodium pump and Ca2+ influx via the Na+/Ca2+ exchanger working in reverse mode or via activation of the Na+/K+-ATPase signalosome complex, generating intracellular calcium oscillations, reactive oxygen species, and gene activation via nuclear factor-κB or extracellular signal-regulated kinases 1 and 2. Elevated concentrations of endogenous ouabain and marinobufagenin in the subnanomolar concentration range were found to stimulate proliferation and differentiation of cardiac and smooth muscle cells. They may have a primary role in the development of cardiac dysfunction and failure because (i) offspring of hypertensive patients evidently inherit elevated plasma concentrations of endogenous ouabain; (ii) such elevated concentrations correlate positively with cardiac dysfunction, hypertrophy, and arterial hypertension; (iii) about 40% of Europeans with uncomplicated essential hypertension show increased concentrations of endogenous ouabain associated with reduced heart rate and cardiac hypertrophy; (iv) in patients with advanced arterial hypertension, circulating levels of endogenous ouabain correlate with BP and total peripheral resistance; (v) among patients with idiopathic dilated cardiomyopathy, high circulating levels of endogenous ouabain and marinobufagenin identify those individuals who are predisposed to progressing more rapidly to heart failure, suggesting that endogenous ouabain (and marinobufagenin) may contribute to toxicity upon digoxin therapy.In contrast to endogenous ouabain, endogenous marinobufagenin may act as a natriuretic substance as well. It shows a higher affinity for the ouabain-insensitive α1 isoform of Na+/K+-ATPase of rat kidney tubular cells and its levels are increased in volume expansion and pre-eclampsia. Digoxin, which is synthesized in adrenal glands, seems to counteract the hypertensinogenic action of ouabain in rats, as do antibodies against ouabain, for example, (Digibind®) and rostafuroxin (PST 2238), a selective ouabain antagonist. It lowers BP in ouabain- and adducin-dependent hypertension in rats and is a promising new class of antihypertensive medication in humans.

Bovine Adrenals Contain, in Addition to Ouabain, a Second Inhibitor of the Sodium Pump

In the search for endogenous cardiac glycosides, two different inhibitors of the sodium pump have been isolated from bovine adrenals. Inhibitor A with a molecular mass of 600 Da and a UV maximum at 250 nm was purified from 16 kg of bovine adrenals. The pure substance (<1 ng) inhibited the sodium pump of human red blood cells with an affinity similar to that of ouabain, yet it cross-reacted with antibodies against the bufadienolide proscillaridin A but not against the cardenolide ouabain. Inhibitor A was slightly more hydrophilic than ouabain on RP-C18 high pressure liquid chromatography. Hence, it showed properties similar to the proscillaridin A immunoreactivity (Sich, B., Kirch, U., Tepel, M., Zideck, W., and Schoner, W. (1996) Hypertension 27, 1073–1078) that increased in humans with systolic blood pressure and pulse pressure. Inhibitor B of the sodium pump with a molecular mass of 584 Da was purified 106-fold from 20 kg of bovine adrenals. It cross-reacted with antibodies against ouabain but not with antibodies against proscillaridin A and inhibited the sodium pump of human and rat red blood cells with the same affinity as ouabain. All other properties, such as the retention time in a C18-reversed phase chromatography, molecular mass determination by electrospray mass spectrometry and fragmentation pattern, and UV and 1H NMR spectroscopic data, were identical to ouabain. Hence, sodium pump inhibitor B from bovine adrenals is the cardenolide ouabain.

Ouabain-Like Compound Changes Rapidly on Physical Exercise in Humans and Dogs: Effects of β-Blockade and Angiotensin-Converting Enzyme Inhibition

Ouabain, an inhibitor of the sodium pump, has been identified as a constituent of bovine adrenal glands. We were interested whether the release of this cardiotonic steroid is stimulated by physical exercise. Hence, athletes and healthy dogs were subjected to ergometry. Ouabain-like compound (OLC) was measured in venous blood by enzyme-linked immunosorbent assay as well as by 86Rb+ uptake inhibition (as ouabain equivalents). OLC increased in venous blood of athletes after 15 minutes of ergometry from 2.5±0.5 to 86.0±27.2 nmol/L (n=51; P<0.001), as did the concentration of a circulating inhibitor of the sodium pump from 7.3±1.7 to 129.8±51 nmol/L (ouabain equivalents, P<0.05). Half-maximal increase in heart rate and systolic blood pressure occurred at 5.1±1.2 nmol/L and at 30±1 nmol/L OLC, respectively. On rest, OLC decreased in humans and dogs with a half-life of 3 to 5 minutes. In beagles exposed to moderate exercise on a treadmill for 13 minutes, levels of OLC increased 46-fold (from 3.7±0.8 to 166.9±91.8 nmol/L; n=6; P<0.005). This effect was suppressed when the dogs had been treated for 3 weeks with the &bgr;1-adrenergic receptor blocker atenolol or the angiotensin-converting enzyme inhibitor benazepril. We conclude that OLC changes rapidly during exercise and is under the control of norepinephrine and angiotensin II.

A fresh facet for ouabain action

Ouabain signaling through a plasma membrane can produce oscillations of intracellular calcium levels, resulting in translocation of the NF-κB transcription factor into the nucleus and gene activation. This is a previously unrecognized form of steroid action.

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).

Pulse pressure correlates in humans with a proscillaridin A immunoreactive compound

Endogenous digitalis-like factors in humans are presumably cardenolides and bufadienolides. To test whether bufadienolide-like substances may circulate in human blood, we used antibodies from rabbits against the bufadienolide proscillaridin A to measure the concentration of cross-reacting material in human plasma with an indirect enzyme-linked immunosorbent assay. IgG had an apparent affinity of 2 x 10(-9) mol/L for proscillaridin A. It was specific for bufadienolides and did not cross-react with cardenolides or several steroid hormones. Extraction of human plasma with ethanol and fractionation of this extract over a high-performance liquid chromatographic reverse-phase C18 column with a propanol/isopropanol gradient resulted in the separation of three peaks of increasing hydrophobicity (ED1, ED2, ED3) that inhibited the sodium pump of human red blood cells and cross-reacted with proscillaridin A antibodies. The concentration of the proscillaridin A immunoreactivity ED1 in normotensive subjects had a geometric mean of 0.1 nmol/L, with a dispersion factor of 8.77. ED1 correlated positively in a group of 60 normotensive subjects, 22 patients with hypertension, and 19 patients with chronic renal failure with mean arterial blood pressure (log ED1 [nmol/L] = 0.013 x mm Hg-2.17, r = .25, P < .05), systolic pressure (log ED1 [nmol/L] = 0.010 x mm Hg-2.23, r = .32, P < .01), and pulse pressure (log ED1 [nmol/L] = 0.019 x mm Hg-1.80, r = .38, P < .0001). There was no correlation with other parameters of the donors. We conclude that several substances cross-reacting with proscillaridin A antibodies and inhibiting the sodium pump of human red blood cells circulate in human blood. The level of one of these substances (ED1) correlates with mean arterial and pulse pressures.

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. Dissociation 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. 2. Structure-affinity relationship of the different cardiac glycosides indicate that cardiac glycosides are recognized from interactions with

Ouabain as a Mammalian Hormone

Abstract: Endogenous ouabain changes rapidly in humans and dogs upon physical exercise and is under the control of epinephrine and angiotensin II. Hence, the steroid acts as a rapidly acting hormone. A search for a specific binding globulin for cardiac glycosides in bovine plasma resulted in the identification of the d allotype of the μ chain of IgM whose hydrophobic surfaces interact with cardiotonic steroids and cholesterol. Such IgM complexes might be involved in the hepatic elimination of cardiotonic steroids. Thus, differences in the signaling cascade starting at Na+,K+‐ATPase must explain any differences in the action of ouabain and digoxin in the genesis of arterial hypertension.

Endogenous Digitalis-Like Factors

The postulate of a natriuretic factor inhibiting the sodium pump in the kidney led to the detection of increased concentrations of endogenous digitalis-like factors in blood after salt loading, in essential hypertension, in pregnancy-induced hypertension and in chronic hypervolaemia. The recent isolation of ouabain or a close isomer thereof from human plasma and the demonstration of a compound similar if not identical to digoxin in adrenals and human urine shows that mammals like non-vertebrates and toads may synthesize cardiac glycosides in their adrenals and possibly in hypothalamus. The hypothalamus also forms other compounds of unknown structure which bind to the cardiac glycoside receptor site. The differential functions of endogenously formed ouabain and of a digoxin-like substance are unclear. The detailed knowledge of the physiological role of both endogenously formed cardiac glycosides in the regulation of blood pressure has still to be worked out.