Endogenous cardiac glycosides, a new class of steroid hormones.

The search for endogenous digitalis has led to the isolation of ouabain as well as several additional cardiotonic steroids of the cardenolide and bufadienolide type from blood, adrenals, and hypothalamus. The concentration of endogenous ouabain is elevated in blood upon increased Na(+) uptake, hypoxia, and physical exercise. Changes in blood levels of ouabain upon physical exercise occur rapidly. Adrenal cortical cells in tissue culture release ouabain upon addition of angiotensin II and epinephrine, and it is thought that ouabain is released from adrenal cortex in vivo. Ouabain levels in blood are elevated in 50% of Caucasians with low-renin hypertension. Infusion over several weeks of low concentrations of ouabain, but not of digoxin, induces hypertension in rats. A digoxin-like compound, which has been isolated from human urine and adrenals, as well various other endogenous cardiac glycosides may counterbalance their actions within a regulatory framework of water and salt metabolism. Marinobufagenin, for instance, whose concentration is increased after cardiac infarction, may show natriuretic properties because it inhibits the alpha1 isoform of Na(+)/K(+)-ATPase, the main sodium pump isoform of the kidney, much better than other sodium pump isoforms. In analogy to other steroid hormones, cardiotonic steroid hormones in blood are bound to a specific cardiac glycoside binding globulin. The discovery of ouabain as a new adrenal hormone affecting Na(+) metabolism and the development of the new ouabain antagonist PST 2238 allows for new possibilities for the therapy of hypertension and congestive heart failure. This will lead in turn to a better understanding of the disease on a physiological and endocrinological level and of the action of ouabain on the cellular level as a signal that is transduced to the plasma membrane as well as to the cell nucleus.     

Ouabain stimulates endothelin release and expression in human endothelial cells without inhibiting the sodium pump.

Ouabain, a sodium pump (Na+/ K+-ATPase) inhibitor, has been shown to act as a hormone and is possibly involved in the pathogenesis of hypertension. The mechanism by which ouabain may act was investigated using primary cultures of human umbilical artery endothelial cells (HUAECs), which are known to express and release the vasoconstrictive hormone endothelin (ET-1). Five minutes after application, low concentrations of ouabain induced Ca2+ oscillations and stimulated ET-1 release from endothelial cells into the medium. To investigate whether the observed effects were due to inhibition of the sodium pump, the effects of ouabain on the uptake of 86Rb+ by HUAECs were examined. Unexpectedly, ouabain concentrations below 10 nm stimulated 86Rb+ uptake by 15-20%, and in some experiments by 50%, results that are consistent with a stimulation of the pump. Within the concentration range 1-10 nm, ouabain induced a 2.5-fold stimulation (phosphorylation) of mitogen-activated protein kinase (MAP kinase). After incubation of HUAECs with ouabain for 12 h, the glycoside stimulated cell growth by 49 +/- 4%, as measured by cell number, with a maximum response at 5 nm. At similar concentrations, ouabain also increased ET-1 mRNA abundance by 19.5 +/- 3.1%. The results indicate that, by influencing ET-1 expression and release, ouabain may contribute to the regulation of vascular tone. The data also confirm that it is not a global inhibition of the sodium pump that is involved in the mechanism of action of this cardiac glycoside.     

Effects of Ca2+ on the sodium pump observed in cardiac myocytes isolated from guinea pigs

It is presently unknown whether Ca2+ plays a role in the physiological control of Na+/K+-ATPase or sodium pump activity. Because the enzyme is exposed to markedly different intra- and extracellular Ca2+ concentrations, tissue homogenates or purified enzyme preparations may not provide pertinent information regarding this question. Therefore, the effects of Ca2+ on the sodium pump were examined with studies of [3H]ouabain binding and 86Rb+ uptake using viable myocytes isolated from guinea-pig heart and apparently maintaining ion gradients. In the presence of K+, a reduction of the extracellular Ca2+ increased specific [3H]ouabain binding observed at apparent binding equilibria: a half-maximal stimulation was observed when extracellular Ca2+ was lowered to about 50 microM. The change in [3H]ouabain binding was caused by a change in the number of binding sites accessible by ouabain instead of a change in their affinity for the glycoside. Ouabain-sensitive 86Rb+ uptake was increased by a reduction of extracellular Ca2+ concentration. Benzocaine in concentrations reported to reduce the rate of Na+ influx failed to influence the inhibitory effect of Ca2+ on glycoside binding. When [3H]ouabain binding was at equilibrium, the addition of Ca2+ decreased and that of EGTA increased the glycoside binding. Mn2+, which does not penetrate the cell membrane, had effects similar to Ca2+. In the absence of K+, cells lose their tolerance to Ca2+. Reducing Ca2+ concentration prevented the loss of rod-shaped cells but failed to affect specific [3H]ouabain binding observed in the absence of K+. These results indicate that a large change in extracellular Ca2+ directly affects the sodium pump in cardiac myocytes isolated from guinea pigs.     

How does salt retention raise blood pressure?

A critical question in hypertension research is: How is long-term blood pressure controlled? Excessive NaCl ingestion or NaCl retention by the kidneys and the consequent tendency toward plasma volume expansion lead to hypertension. Nevertheless, the precise mechanisms linking salt to high blood pressure are unresolved. The discovery of endogenous ouabain, an adrenocortical hormone, provided an important clue. Ouabain, a selective Na+ pump inhibitor, has cardiotonic and vasotonic effects. Plasma endogenous ouabain levels are significantly elevated in approximately 40% of patients with essential hypertension and in animals with several forms of salt-dependent hypertension. Also, prolonged ouabain administration induces hypertension in rodents. Mice with mutant Na+ pumps or Na/Ca exchangers (NCX) and studies with a ouabain antagonist and an NCX blocker are revealing the missing molecular mechanisms. These data demonstrate that alpha2 Na+ pumps and NCX1 participate in long-term regulation of vascular tone and blood pressure. Pharmacological agents or mutations in the alpha2 Na+ pump that interfere with the action of ouabain on the pump, and reduced NCX1 expression or agents that block NCX all impede the development of salt-dependent or ouabain-induced hypertension. Conversely, nanomolar ouabain, reduced alpha2 Na+ pump expression, and smooth muscle-specific overexpression of NCX1 all induce hypertension. Furthermore, ouabain and reduced alpha2 Na+ pump expression increase myogenic tone in isolated mesenteric small arteries in vitro, thereby tying these effects directly to the elevation of blood pressure. Thus, endogenous ouabain, and vascular alpha2 Na+ pumps and NCX1, are critical links between salt and hypertension. New pharmacological agents that act on these molecular links have potential in the clinical management of hypertension.     

Endogenous and exogenous cardiac glycosides: their roles in hypertension, salt metabolism, and cell growth

Cardiotonic steroids (CTS), long used to treat heart failure, are endogenously produced in mammals. Among them are the hydrophilic cardenolide ouabain and the more hydrophobic cardenolide digoxin, as well as the bufadienolides marinobufagenin and telecinobufagin. The physiological effects of endogenous ouabain on blood pressure and cardiac activity are consistent with the "Na⁺-lag" hypothesis. This hypothesis assumes that, in cardiac and arterial myocytes, a CTS-induced local increase of Na⁺ concentration due to inhibition of Na⁺/K⁺-ATPase leads to an increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i) via a backward-running Na⁺/Ca²⁺ exchanger. The increase in [Ca²⁺]_i then activates muscle contraction. The Na⁺-lag hypothesis may best explain short-term and inotropic actions of CTS. Yet all data on the CTS-induced alteration of gene expression are consistent with another hypothesis, based on the Na⁺/K⁺-ATPase "signalosome," that describes the interaction of cardiac glycosides with the Na⁺ pump as machinery activating various signaling pathways via intramembrane and cytosolic protein-protein interactions. These pathways, which may be activated simultaneously or selectively, elevate [Ca²⁺]_i, activate Src and the ERK1/2 kinase pathways, and activate phosphoinositide 3-kinase and protein kinase B (Akt), NF-B, and reactive oxygen species. A recent development indicates that new pharmaceuticals with antihypertensive and anticancer activities may be found among CTS and their derivatives: the antihypertensive rostafuroxin suppresses Na⁺ resorption and the Src-epidermal growth factor receptor-ERK pathway in kidney tubule cells. It may be the parent compound of a new principle of antihypertensive therapy. Bufalin and oleandrin or the cardenolide analog UNBS-1450 block tumor cell proliferation and induce apoptosis at low concentrations in tumors with constitutive activation of NF-B. endogenous cardiotonic steroids; ouabain; marinobufagenin; rostafuroxin; bufalin; oleandrin; sodium pump; sodium/potassium-adenosinetriphosphatase; arterial hypertension; sodium metabolism; cell proliferation; cancer therapy     

Action of ouabain on sodium transport in toad urinary bladder, Evidence for two pathways for sodium entry

The cardiac glycoside ouabain inhibits transepithelial sodium transport in the toad urinary bladder. It is shown that this drug reduces the rate coefficient for sodium exit at the serosal pump site. In addition, ouabain inhibits entry across the mucosal border whenever the electrochemical potential gradient for sodium is made less favorable. The data are interpreted as indicating the existence of two separate pathways for sodium entry, one of which is ouabain inhibitable.     

Noradrenaline: a circulating inhibitor of sodium transport.

Leucocytes were isolated from venous blood of 11 normotensive volunteers with no family history of hypertension and the sodium efflux rate constants determined both alone and in the presence of increasing physiological concentrations of noradrenaline. There was a significant dose dependent reduction of total sodium efflux rate constant due to a reduction in ouabain sensitive sodium pump activity, glycoside insensitive efflux rate constants being unaffected. The magnitude of this effect was similar to the reduction in leucocyte sodium efflux rate constants observed in hypertensive patients (and their normotensive relatives). The noradrenaline induced depression of sodium pump activity was prevented by propranolol in a further seven experiments, suggesting that the effect was mediated by beta adrenoceptors. Catecholamines possibly functioning as circulating inhibitors of sodium transport may contribute to some of the disturbances in membrane electrolyte handling both in essential hypertension in man and in some experimental models of hypertension.     

Na+ channel and Na+-K+ ATPase involvement in norepinephrine- and veratridine-stimulated metabolism in perfused rat hind limb.

In the constant flow perfused rat hind limb, norepinephrine (NE) evoked increases in oxygen uptake (VO2) and lactate efflux (LE) were inhibited by the cardiac glycoside ouabain (1 mM), without interrupting the NE-mediated vasoconstriction. The membrane labilizer veratridine, previously shown to increase VO2 and LE, without increasing perfusion pressure, was also shown to be inhibited by the cardiac glycoside ouabain, as well as by the ouabain analogues digitoxin and digoxin. The stimulatory actions of veratridine on VO2 were inhibitable by low doses of the specific sodium channel blocker tetrodotoxin (TTX), while NE effects were unaffected, suggesting that NE may be acting via a TTX-insensitive sodium channel. It is concluded that agents such as NE (a vasoconstrictor) or veratridine (a membrane labilizer), which stimulate VO2 in the perfused rat hind limb, do so by increasing Na+ influx. The observed increases in oxygen consumption and LE are due to Na+-K+ ATPase activity to pump Na+ out of the cell at the expense of ATP turnover. Energy dissipation due to Na+ cycling may be a form of facultative thermogenesis attributable to NE that can be stimulated by membrane labilizers such as veratridine in the constant flow perfused rat hind limb.     

Progesterone derivatives that bind to the digitalis receptor: effects on Na+,K+-ATPase and isolated tissues

The steroid nature of the cardiac glycosides (CG) suggests that an endogenous steroid (or steroids) may be the natural ligand for the specific receptor site, i.e., Na+,K+-ATPase. Derivatives of progesterone (PROG) that compete in a [3H]ouabain radioreceptor binding assay (RRA) are characterized by 17 alpha-acetylation and modifications in the B ring. Chlormadinone acetate (CMA) is the most potent analog identified thus far, having about one-twentieth the RRA potency of ouabain. CMA interacts at the ouabain site on Na+,K+-ATPase, inhibits the enzyme in the same rank order of species sensitivity as do the CG, and inhibits the sodium pump in vitro in guinea pig atrium and perfused heart, cardiac myocytes, rat diaphragm, and red blood cells. CMA does not cross-react with digoxin antiserum, which indicates that CG antibodies are not necessarily directed at molecular determinants of biological activity. By crystallographic analysis, the 20-carbonyl moiety in CMA is seen spatially oriented so as to be equivalent to the lactone 23-oxygen atom in the CG. CMA exerts primarily cardiodepressant effects, in accordance with the often-reported similar action of PROG. Negative inotropy may be mediated other than by Na+,K+-ATPase because PROG itself has no significant CG-like actions. Positive inotropy by CG, cardiodepression by CMA and PROG or low concentrations of CG, occasional transient enhancement of contractility by CMA, and pump stimulation by low concentrations of CMA or CG may reflect different affinities of the compounds for sites that mediate Na+,K+-ATPase/pump inhibition, positive inotropy, and negative inotropy. Thus, PROG derivatives related to CMA appear to be likely candidates for endogenous digitalis-like hormones. Body tissues possess the enzymes for conversion of PROG to derivatives related even more closely than the semisynthetic CMA to the CG configuration.     

The sodium pump and cardiotonic steroids-induced signal transduction protein kinases and calcium-signaling microdomain in regulation of transporter trafficking

The Na/K-ATPase was discovered as an energy transducing ion pump. A major difference between the Na/K-ATPase and other P-type ATPases is its ability to bind a group of chemicals called cardiotonic steroids (CTS). The plant-derived CTS such as digoxin are valuable drugs for the management of cardiac diseases, whereas ouabain and marinobufagenin (MBG) have been identified as a new class of endogenous hormones. Recent studies have demonstrated that the endogenous CTS are important regulators of renal Na⁺ excretion and blood pressure. The Na/K-ATPase is not only an ion pump, but also an important receptor that can transduce the ligand-like effect of CTS on intracellular protein kinases and Ca²⁺ signaling. Significantly, these CTS-provoked signaling events are capable of reducing the surface expression of apical NHE3 (Na/H exchanger isoform 3) and basolateral Na/K-ATPase in renal proximal tubular cells. These findings suggest that endogenous CTS may play an important role in regulation of tubular Na⁺ excretion under physiological conditions; conversely, a defect at either the receptor level (Na/K-ATPase) or receptor–effector coupling would reduce the ability of renal proximal tubular cells to excrete Na⁺, thus culminating/resulting in salt-sensitive hypertension.     

Photoaffinity labeling of erythrocyte membrane (Na+ + K+)-ATPase with high specific activity [125I]iodoazidogalactosyl digitoxigenin

Photoaffinity labeling of (Na+ + K+)-ATPase in erythrocyte membranes with cardiotonic steroid derivatives, followed by gel electrophoresis, requires a radiolabel of very high specific activity, since the enzyme represents less than 0.05% of the total membrane protein. We report the synthesis of a radioiodinated, photosensitive derivative of the cardiac glycoside, 3-beta-O-(4-amino-4,6-dideoxy-beta-D-galactosyl)digitoxigenin, with very high specific activity. The product, [125I]iodoazidogalactosyl digitoxigenin ([125I]IAGD), is carrier-free with a specific activity of 2200 Ci/mmol. Incubation of [125I]IAGD (1.8 nM) with human erythrocyte membranes (300 micrograms protein), followed by photolysis and analysis by SDS-PAGE, showed specific radiolabeling of a polypeptide that had the same molecular weight as catalytic alpha subunit (100,000 Mr) of (Na+ + K+)-ATPase in eel electroplax microsomes. Photoaffinity labeling of erythrocyte and electroplax membranes by [125I]IAGD was specific for the cardiac glycoside binding site of (Na+ + K+)-ATPase since radiolabeling of the alpha subunit was inhibited when ouabain was included in the pre-photolysis incubation. [125I]IAGD can, therefore, be used as a probe in structural studies of human erythrocyte membrane (Na+ + K+)-ATPase.     

Endogenous cardiotonic steroids and salt-sensitive hypertension

Endogenous cardiotonic steroids (CTS), also called digitalis like factors, have been postulated to play important roles in pathogenesis of hypertension for nearly half of a century. For the past 50 years biomedical scientists have been in quest of an unidentified factor or hormone that both increases blood pressure and renal sodium excretion; this “natriuretic hormone” was, in fact, postulated to interact with the Na/K-ATPase. Recent discoveries have led to the identification of steroid molecules which are present in humans, rodents and amphibians, and which, in a complex manner, interact with each other and with the other systems that regulate renal salt handling and contribute to the salt-sensitivity of blood pressure.Recent findings include the specific identification of endogenous cardenolide (endogenous ouabain) and bufadienolide (marinobufagenin) CTS in humans along with the delineation of mechanisms by which CTS can signal through the Na/K-ATPase. Although CTS were first considered important in the regulation of renal sodium transport and arterial pressure, more recent work implicates these hormones in the central regulation of blood pressure and regulation of cell growth, and development of cardiovascular and renal fibrosis in particular.     

Physiological role of the alpha1- and alpha2-isoforms of the Na+-K+-ATPase and biological significance of their cardiac glycoside binding site

An interesting feature of Na+-K+-ATPase is that it contains four isoforms of the catalytic alpha-subunit, each with a tissue-specific distribution. Our laboratory has used gene targeting to define the functional role of the alpha1- and alpha2-isoforms. While knockout mice demonstrated the importance of the alpha1- and alpha2-isoforms for survival, the knockin mice, in which each isoform can be individually inhibited by ouabain and its function determined, demonstrated that both isoforms are regulators of cardiac muscle contractility. Another intriguing aspect of the Na+-K+-ATPase is that it contains a binding site for cardiac glycosides, such as digoxin. Conservation of this site suggests that it may have an in vivo role and that a natural ligand must exist to interact with this site. In fact, cardiac glycoside-like compounds have been observed in mammals. Our recent study demonstrates that the cardiac glycoside binding site of the Na+-K+-ATPase plays a role in the regulation of blood pressure and that it mediates both ouabain-induced and ACTH-induced hypertension in mice. Whereas chronic administration of ouabain or ACTH caused hypertension in wild-type mice, it had no effect on blood pressure in mice with a ouabain-resistant alpha2-isoform of Na+-K+-ATPase. Interestingly, animals with the ouabain-sensitive alpha1-isoform and a ouabain-resistant alpha2-isoform develop ACTH-induced hypertension to a greater extent than wild-type animals. Taken together, these results demonstrate that the cardiac glycoside binding of the Na+-K+-ATPase has a physiological role and suggests a function for a naturally occurring ligand that is stimulated by administration of ACTH.     

Two classes of ouabain receptors in chick ventricular cardiac cells and their relation to (Na+,K+)-ATPase inhibition, intracellular Na+ accumulation, Ca2+ influx, and cardiotonic effect

The biochemical and pharmacological properties of the (Na+,K+)-ATPase have been studied at different stages of chick embryonic heart development in ovo and under cell culture conditions. The results show the existence of two families of ouabain binding sites: a low affinity binding site with a dissociation constant (Kd) of 2-6 microM for the ouabain-receptor complex and a high affinity binding site with a Kd of 26-48 nM. Levels of high affinity sites gradually decrease during cardiac ontogenesis to reach a plateau near 14 days of development. Conversely the number of low affinity binding sites is essentially invariant between 5 days and hatching. Cultured cardiac cells display the same binding characteristics as those found in intact ventricles. Inhibition of 86Rb+ uptake in cultured cardiac cells and an increase in intracellular Na+ concentration, due to (Na+,K+)-ATPase blockade, occur in a ouabain concentration range corresponding to the saturation of the low affinity ouabain site. Ouabain-stimulated 45Ca2+ uptake increases in parallel with the increase in the intracellular Na+ concentration. It is suppressed in Na+-free medium or when Na+ is replaced by Li+ suggesting that the increase is due to the indirect activation of the Na+/Ca2+ exchange system in the plasma membrane. Dose-response curves for the inotropic effects of ouabain on papillary muscle and on ventricular cells in culture indicate that the development of the cardiotonic properties is parallel to the saturation of the low affinity binding site for ouabain. Therefore, inhibition of the cardiac (Na+,K+)-ATPase corresponding to low affinity ouabain binding sites seems to be responsible for both the cardiotonic and cardiotoxic effects of the drug.     

Resolution of Pump and Leak Components of Sodium and Potassium Ion Transport in Human Erythrocytes

Further support for the pump-leak concept was obtained. Net transport was resolved into pump and leak components with the cardiac glycoside, ouabain. The specificity of ouabain as a pump inhibitor was demonstrated by its ineffectiveness when the pump was already inhibited by lack of one of the three pump substrates, sodium ion, potassium ion, or adenosine triphosphate. In the presence of ouabain the rates of passive transport of sodium and potassium ions changed almost in proportion to changes in their extracellular concentrations when one ion was exchanged for the other. In the presence of ouabain and at the extracellular concentrations which produced zero net transport, the ratio of potassium ions to sodium ions was 1.2-fold higher inside the cells than outside. This finding was attributed to a residual pump activity of less than 2% of capacity. The permeability to potassium ions was 10% greater than the permeability to sodium ions. A test was made of the independence of pump and leak. Conditions were chosen to change the rate through each pathway separately or in combination. When both pathways were active, net transport was the sum of the rates observed when each acted separately. A ratio of three sodium ions pumped outward per two potassium ions pumped inward was confirmed.     

乌本苷免疫活性物和组织中钠泵容量的关系

依赖于Na+、K+的Na+-K+-ATP酶(EC3.6.1.3,钠泵),广泛存在于哺乳类动物细胞质膜上,是催化Na+、K+跨膜主动运输的质膜酶,除维持正常的细胞内外离子浓度梯度外,对细胞能量代谢也有重要影响,许多疾病的发生是由于钠泵活性异常引起.研究证明有2种特异性的内源性钠泵抑制因子在?..     

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.     

Structural insights into the high affinity binding of cardiotonic steroids to the Na+,K+-ATPase

The Na+,K+-ATPase belongs to the P-ATPase family, whose characteristic property is the formation of a phosphorylated intermediate. The enzyme is also a defined target for cardiotonic steroids which inhibit its functional activity and initiate intracellular signaling. Here we describe the 4.6 ? resolution crystal structure of the pig kidney Na+,K+-ATPase in its phosphorylated form stabilized by high affinity binding of the cardiotonic steroid ouabain. The steroid binds to a site formed at transmembrane segments αM1-αM6, plugging the ion pathway from the extracellular side. This structure differs from the previously reported low affinity complex with potassium. Most importantly, the A domain has rotated in response to phosphorylation and αM1-2 move towards the ouabain molecule, providing for high affinity interactions and closing the ion pathway from the extracellular side. The observed re-arrangements of the Na+,K+-ATPase stabilized by cardiotonic steroids may affect protein-protein interactions within the intracellular signal transduction networks.     

Digitaloid pregnanes promote potassium-sparing diuresis in the guinea pig.

The synthesis of 17 alpha-acetoxy-3 beta-[(beta-D-glucopyranosyl)oxy]- 6 alpha-methylpregn-4-en-20-one, the glucoside of medroxyprogesterone acetate (MPA-glu), is described. MPA-glu and 14-amino-20 beta-hydroxy-3 beta-[(alpha-L-rhamnopyranosyl)oxy]-5 beta, 14 beta-pregnane (LND 623), pregnane glycosides that bind to the digitalis receptor, and digoxin, a cardiac glycoside, were infused intravenously into the anesthetized guinea pig. Each of the three steroids significantly enhanced urinary volume and sodium excretion without affecting blood pressure and creatinine clearance. Potassium excretion was markedly enhanced by digoxin but unaffected by MPA-glu or LND 623. These observations conform to previous work that demonstrated, in the rat, potassium-sparing diuresis by the glucoside of 14 beta-hydroxyprogesterone, a cardiotonic pregnane. There is a dissociation between potency to inhibit [3H]ouabain binding and the extra ATPase actions of the digitaloid pregnanes.