Simplified digitalis-like compounds acting on Na(+), K(+)-ATPase

Digitalis compounds are used in the treatment of congestive heart failure as positive inotropic agents; their action is mainly due to the inhibition of Na(+),K(+)-ATPase. A well-known drawback is their arrhythmogenic potential. Attempts to find safer digitalis-like compounds by means of molecular simplifications of the typical 5beta,14beta-steroidal skeleton, which appeared in the medicinal chemistry literature from 1990 until 2002, are briefly reviewed. Several novel achievements were obtained in order to better understand the requisites of the digitalis binding site on Na(+), K(+)-ATPase. Only minor simplification, such as cleavage of the D ring of the digitalis skeleton, could preserve the desired inotropic activity, while highly simplified digitalis-like compounds failed to give sufficiently high inotropic potency, even in the presence of a powerful pharmacophore, such as the O-aminoalkyloxime group.     

A quantitative structure-activity relationship study on a series of Na+, K+-ATPase inhibitors

A quantitative structure-activity relationship (QSAR) study has been made on a new series of digitalis-like Na+,K+-ATPase inhibitors in which the guanylhydrazone group has been replaced by an aminoalkyloxime group. The correlations obtained have shown that the oxime moiety, primary amine group, overall size, and polarizability of the new type of substituents are higly beneficial to the Na+,K+-ATPase inhibition potency of the compounds and that their effect can be quantitatively assessed. The study also showed that the inotropic activity of the compounds is very well correlated with their Na+,K+-ATPase inhibition potency.     

Endogenous digitalis-like substance in pig left ventricle

A crude fraction was isolated from pig heart left ventricle (150 g) homogenates after extraction of lipids, chromatographic separation and desalting. The extract contained an ionic content of 0.21, 0.27, 0.33 and 1.7 mM respectively for Mg2+, Ca2+, K+, and Na+. The albumin extract, used as a reference control, contained an ionic content of 0.88 and 2.1 mM respectively for K+ and Na+ and negligible amounts of Mg2+ and Ca2+. The isolated fraction exhibited digitalis-like properties in the inhibition of sarcolemmal Na+, K+-ATPase in a dose dependent manner, the displacement of [3H]-ouabain binding from membrane receptor sites and produced +ve inotropic response in isolated perfused heart in a dose dependent manner. The albumin extract tested in the same manner showed no digitalis-like properties. The ventricular fraction was unable to displace (-) 3H-DHA binding from membrane sites and its inotropic action was not blocked by propranolol. The data suggests that the fraction isolated from pig heart left ventricle contains a substance which has some properties like digitalis.     

Further biochemical characterization of an Na+ pump inhibitor purified from human urine

An increase in endogenous Na+,K+-ATPase inhibitor(s) with digitalis-like properties has been reported in chronic renal insufficiency, in Na+-dependent experimental hypertension and in some essential hypertensive patients. The present study specifies some properties and some biochemical characteristics of a semipurified compound from human urine having digitalis-like properties. The urine-derived inhibitor (endalin) inhibits Na+,K+-ATPase activity and [3H]-ouabain binding, and cross-reacts with anti-digoxin antibodies. The inhibitory effect on ATPases of endalin is higher on Na+,K+-ATPase than on Mg2+-ATPase and Ca2+-ATPase. The mechanism of endalin action on highly purified Na+,K+-ATPase was compared to that of ouabain and was similar in that it reversibly inhibited Na+,K+-ATPase activity; it inhibited Na+,K+-ATPase non-competitively with ATP; its inhibitory effect was facilitated by Na+; K+ decreased its inhibitory effect on Na+,K+-ATPase; it competitively inhibited ouabain binding to the enzyme; its binding was maximal in the presence of Mg2+ and Pi; it decreased the Na+ pump activity in human erythrocytes; it reduced serotonin uptake by human platelets; and it was diuretic and natriuretic in rat bioassay. The endalin differed from ouabain in only three aspects: its inhibitory effect was not really specific for Na+,K+-ATPase; its binding to the enzyme was undetectable in the presence of Mg2+ and ATP; it was not kaliuretic in rat bioassay. Endalin is a reversible and partial specific inhibitor of Na+,K+-ATPase, its Na+,K+-ATPase inhibition closely resembles that of ouabain and it could be considered as one of the natriuretic hormones.     

Evidence that mammalian lignans show endogenous digitalis-like activities

Enterolactone, a lignan that has been identified in biological samples from man and several mammals, shares with ascorbic acid and cardiac glycosides a gamma-butyrolactone. It displaces 3H-ouabain from its binding sites on cardiac digitalis receptor and inhibits, dose dependently, the Na+, K+-ATPase activity of human and guinea-pig heart. The time dependence of this inhibition resembles that of dihydroouabain, a cardiac glycoside in which the lactone ring does not contain conjugated double bonds. The active concentrations of enterolactone as inhibitor of Na+,K+-ATPase are in the 10(-4) M range and, at those concentrations, the cross-reactivity with antidigoxin antibodies is low. Lignans may contribute to the putative digitalis-like activity found in tissues, blood and urine of several mammals including man.     

Digitalis sensitivity of Na+,K(+)-ATPase, myocytes and the heart.

Cardiac Na+,K(+)-ATPase, the receptor molecule for digitalis glycosides, have isoforms with different intrinsic affinities for the glycosides. Expression of these isoforms are under developmental and hormonal regulation. Switching in isoforms to those with lower intrinsic affinity may decrease digitalis sensitivity of the heart. In addition to the intrinsic affinity of the cardiac Na+,K(+)-ATPase for the glycoside, increases in the rate of Na+ influx and decreases in extracellular K+ concentrations increase glycoside sensitivity of the heart and also reduces the margin of safety by reducing reserve capacity of the sodium pump. Reserve capacity of the sodium pump is also reduced by pathological conditions or aging, resulting in reduced margin of safety for the glycoside. Events that follow sodium pump inhibition also affect sensitivity of the heart to digitalis toxicity. These are hypercalcemia and magnesium depletion. It is now feasible to predict digitalis sensitivity of the heart, not empirically but based on the understanding of the mechanisms responsible for the positive inotropic and toxic actions of the glycoside.     

Endogenous Digitalis-Like Na+, K+-ATPase Inhibitors,and Brain Function

Digitalis-like compounds are recently identified steroids synthesized by the adrenal gland, which resemble the structure of plant cardiac glycosides. These compounds, like the plant steroids, bind to and inhibit the activity of the Na⁺, K⁺-ATPase. The possible function of the endogenous digitalis-like compounds has to be evaluated in view of the presence of different isoforms of the Na⁺, K⁺-ATPase, which differ in their sensitivity to digitalis. This review focuses on recent published data on the Na⁺, K⁺-ATPase inhibitors, the digitalis-like compounds, regarding their structure, biosynthesis and secretion from the adrenal gland, physiological role and pathological implications in diseases such as hypertension and depression. Emphasis is given to studies describing the involvement of these compounds in brain function.     

A QSAR study on a series of simplified digitalis-like compounds acting on Na+,K(+)-ATPase

Among the cardiotonics (agents against congestive heart failure), the most important group is of the digitalis cardiac glycosides, but since these compounds suffer from a low therapeutic index, attention has been paid to investigating safer cardiotonic agents through the inhibition of Na+,K(+)-ATPase, the mechanism by which the digitalis cardiac glycosides elicit their action. Recently, a series of perhydroindenes were studied for their Na+,K(+)-ATPase inhibition activity. We report here a QSAR study on them to investigate the physicochemical and structural properties of the molecules that govern their activity in order to rationalize the structural modification to have more potent drugs. A multiple regression analysis reveals a significant correlation between the Na+,K(+)-ATPase inhibition activity of the compounds and Kier's first order valence molecular connectivity index of their R5-substituents and some indicator parameters, suggesting that the R5-substituents of the compounds containing atoms with low valence and high saturation and the R1-substituents having =N-O- moiety will be conducive to the activity.     

Interaction of (Na+,K+)-ATPases and digitalis genins. A general model for inhibitory activity

Previous models of digitalis genin interaction with the (Na+,K+)-ATPase system (the putative receptor for such drugs) were deficient in explaining the (Na+,K+)-ATPase inhibitory activity of a number of digitalis genin analogues. With rat brain (Na+,K+)-ATPase we observed that the C-17 side chain carbonyl (C = O) oxygen distance of a given genin in relation to its position in the reference compound digitoxigenin was the primary determinant of its biological activity. With a number of genin analogues, we observed a strict correlation of this structural parameter with its binding site compatibility as well as inhibitory potency with respect to the (Na+,K+)-ATPase. In every case the correlation to inhibition data was obtained using a minimum energy conformation for the genin structure. The general applicability of that model is now proposed based on the following observations. The carbonyl oxygen position versus the biological activity relationship fully holds with (Na+,K+)-ATPase preparations from other tissues and species and also when different binding conditions are used for the enzyme genin interaction. The relationship is equally valid for the K+-p-nitrophenyl phosphatase activity. Correlations of the data obtained under these various conditions provide further support for this relationship and for the concept that altered affinities of the enzyme for a given genin under different binding conditions reflect conformational variations of a single binding site.     

The thermodynamic essence of the reversible inactivation of Na+/K+-transporting ATPase by various digitalis derivatives is relaxation of enzyme conformational energy

This paper reports on the kinetic and thermodynamic parameters describing the interaction of selected digitalis derivatives with hog and guinea-pig cardiac (Na+ + K+)-ATPase (Na+/K+-transporting ATPase EC 3.6.1.37). 32 digitalis derivatives were characterized as to the values of the delta G0', delta G----not equal to, and delta G----not equal to quantities in their interaction with (Na+ + K+)-ATPase from hog cardiac muscle in the presence of ATP, Mg2+, Na+ and K+. Nine derivatives were additionally characterized as to the values of the delta H0', delta S0', delta H----not equal to, delta S----not equal to, delta H not equal to, and delta S not equal to quantities in their interaction with the hog enzyme promoted by ATP, Mg2+ and Na+ in the presence or absence of K+. The formation of the inhibitory complexes is in any case an endothermic, entropically driven process. The Gibbs energy barriers in the formation and dissociation of the complexes, delta G----not equal to and delta G----not equal to, are imposed by large, unfavourable delta H not equal to values. K+ decreases the delta G0' value by increasing the delta G----not equal to value more than the delta G----not equal to value. In comparison with hog (Na+ + K+)-ATPase, the interaction of three derivatives with guinea-pig cardiac enzyme in the presence of ATP, Mg2+, Na+ and K+ is characterized by lower delta G0' values caused by lower favourable delta S0' values, and is accompanied by lower delta G----not equal to values. The magnitude of the kinetic parameters and the characteristic of the thermodynamic quantities describing the interaction between various digitalis derivatives and (Na+ + K+)-ATPase, indicate the induction of substantial conformational changes in the enzyme protein. A large entropy gain in the enzyme protein, observed irrespective of enzyme origin and ligation, appears to be the common denominator of the inhibitory action of all digitalis derivatives studied, suggesting that the digitalis-elicited relaxation of high conformational energy (negentropy strain) of the enzyme protein is the thermodynamic essence of the reversible inactivation of (Na+ + K+)-ATPase.     

The endogenous digitalis-like factor

Intensive search into the presence of endogenous digitalis-like factor (EDLF) started shortly after identification of the alpha subunit of the Na,K,-ATPase as being receptor for digitalis glycosides. After years of skepticism, present data testify EDLF really exists. Most probably, the EDLF has chemical structure of either ouabain or of one of its isomers. It is secreted by the adrenal cortex, and, under conditions of stress, it's secretion is regulated differently from the secretion of both gluco- and mineralocorticoids. The physiological role of the EDLF has not been fully understood yet. In the newborn's kidneys, the inhibition of the Na,K-ATPase may assist to increase elimination of surplus sodum from the organism. In individuals of any age, the inhibitory influence of EDLF upon Na,K-ATPase in the arterial wall smooth muscle cells increases peripheral vascular resistance and thus, blood pressure. In the tissue culture, direct positive inotropic influences of EDLF upon rat cardiomyocytes was observed. However, the importance of positive inotropic effect of the EDLF upon the heart in clinical medicine remains to be elucidated. (Mol Cell Biochem 160/161: 111–115, 1996)     

Membrane domains of intestinal epithelial cells: distribution of Na+,K+- ATPase and the membrane skeleton in adult rat intestine during fetal development and after epithelial isolation

The organization of the basolateral membrane domain of highly polarized intestinal absorptive cells was studied in adult rat intestinal mucosa, during development of polarity in fetal intestine, and in isolated epithelial sheets. Semi-thin frozen sections of these tissues were stained with a monoclonal antibody (mAb 4C4) directed against Na+,K+-ATPase, and with other reagents to visualize distributions of the membrane skeleton (fodrin), an epithelial cell adhesion molecule (uvomorulin), an apical membrane enzyme (aminopeptidase), and filamentous actin. In intact adult epithelium, Na+,K+-ATPase, membrane-associated fodrin, and uvomorulin were concentrated in the lateral, but not basal, subdomain. In the stratified epithelium of fetal intestine, both fodrin and uvomorulin were localized in areas of cell-cell contact at 16 and 17 d gestation, a stage when Na+,K+-ATPase was not yet expressed. These molecules were excluded from apical domains and from cell surfaces in contact with basal lamina. When Na+,K+-ATPase appeared at 18-19 d, it was codistributed with fodrin. Detachment of epithelial sheets from adult intestinal mucosa did not disrupt intercellular junctions or lateral cell contacts, but cytoplasmic blebs appeared at basal cell surfaces, and a diffuse pool of fodrin and actin accumulated in them. At the same time, Na+,K+-ATPase moved into the basal membrane subdomain, and extensive endocytosis of basolateral membrane, including Na+,K+-ATPase, occurred. Endocytosis of uvomorulin was not detected and no fodrin was associated with endocytic vesicles. Uvomorulin, along with some membrane-associated fodrin and some Na+,K+-ATPase, remained in the lateral membrane as long as intercellular contacts were maintained. Thus, in this polarized epithelium, interaction of lateral cell-cell adhesion molecules as well as basal cell-substrate interactions are required for maintaining the stability of the lateral membrane skeleton and the position of resident membrane proteins concentrated in the lateral membrane domain.     

Inhibition of hamster sperm Na+, K+-ATPase activity by taurine and hypotaurine

Taurine, hypotaurine and the structural analogue, beta-alanine, were tested for their effects on Na+, K+-ATPase activity of crude homogenates prepared from washed cauda epididymal hamster sperm. Preincubation with 0.1-10 mM taurine or hypotaurine inhibited Na+, K+-ATPase in a dose-dependent manner, while beta-alanine had an inhibitory effect only at 10 mM. The results of this study are the first evidence to demonstrate inhibition of Na+, K+-ATPase activity by taurine and hypotaurine and are discussed in relation to the ability of these compounds to sustain hamster sperm motility and fertility.     

Endogenous inotropic substance from heart tissue has digitalis-like properties.

In the past few years, we developed an extraction procedure which we successfully used to isolate a crude fraction containing digitalis-like substance (DLS) from porcine left ventricular tissue. In this study, the crude fraction was found to cross-react with digoxin antibodies and showed immunoreactivity of 4.25 +/- 0.6 ng digoxin equivalent/ml. On further purification of the crude fraction using silica gel G column chromatography, a fraction C was obtained, which was highly positive inotropic on canine trabeculae and it dose-dependently inhibited ouabain sensitive 86Rb+ uptake in rat heart slices. A 50% inhibition of uptake was obtained by 25 microliters of fraction C. Fraction C also inhibited canine kidney Na+, K(+)-ATPase (Sigma, U.S.A.) dose-dependently and a 50% inhibition of this enzyme required 17 microliters of fraction C. Ashing of the fraction C at 500 degrees C resulted in loss of inotropic and enzyme inhibitory activities, indicating an organic nature of the unknown digitalis-like substance.     

Further characterization of cardiodigin, Na+, K+-ATPase inhibitor extracted from mammalian tissues

This study was undertaken to characterize endogenous digitalis-like activity in water extract from mammalian tissues. Prepurified samples obtained from guinea-pig heart were analysed by reverse-phase HPLC using an acetonitrile gradient. The eluent was assayed for its activity as inhibitor of human heart Na+, K+-ATPase and digoxin-like immunoreactivity. Both activities were recovered in the same fraction after two successive chromatographic steps. These results provide further evidence for the presence of an endogenous digitalis-like factor, cardiodigin, in mammalian heart.     

Purification of Na+,K+-ATPase expressed in Pichia pastoris reveals an essential role of phospholipid-protein interactions

Na+,K+-ATPase (porcine alpha/his10-beta) has been expressed in Pichia Pastoris, solubilized in n-dodecyl-beta-maltoside and purified to 70-80% purity by nickel-nitrilotriacetic acid chromatography combined with size exclusion chromatography. The recombinant protein is inactive if the purification is done without added phospholipids. The neutral phospholipid, dioleoylphosphatidylcholine, preserves Na+,K+-ATPase activity of protein prepared in a Na+-containing medium, but activity is lost in a K+-containing medium. By contrast, the acid phospholipid, dioleoylphosphatidylserine, preserves activity in either Na+- or K+-containing media. In optimal conditions activity is preserved for about 2 weeks at 0 degrees C. Both recombinant Na+,K+-ATPase and native pig kidney Na+,K+-ATPase, dissolved in n-dodecyl-beta-maltoside, appear to be mainly stable monomers (alpha/beta) as judged by size exclusion chromatography and sedimentation velocity. Na+,K+-ATPase activities at 37 degrees C of the size exclusion chromatography-purified recombinant and renal Na+,K+-ATPase are comparable but are lower than that of membrane-bound renal Na+,K+-ATPase. The beta subunit is expressed in Pichia Pastoris as two lightly glycosylated polypeptides and is quantitatively deglycosylated by endoglycosidase-H at 0 degrees C, to a single polypeptide. Deglycosylation inactivates Na+,K+-ATPase prepared with dioleoylphosphatidylcholine, whereas dioleoylphosphatidylserine protects after deglycosylation, and Na+,K+-ATPase activity is preserved. This work demonstrates an essential role of phospholipid interactions with Na+,K+-ATPase, including a direct interaction of dioleoylphosphatidylserine, and possibly another interaction of either the neutral or acid phospholipid. Additional lipid effects are likely. A role for the beta subunit in stabilizing conformations of Na+,K+-ATPase (or H+,K+-ATPase) with occluded K+ ions can also be inferred. Purified recombinant Na+,K+-ATPase could become an important experimental tool for various purposes, including, hopefully, structural work.     

Endogenous digitalis: reality or myth?

Endogenous digitalis is defined as a natural ligand for the digitalis-binding site of the Na+, K(+)-ATPase and is a specific, high-affinity reversible inhibitor of the enzyme activity. Such endogenous digitalis is thought to be involved in sodium homeostasis and blood pressure regulation as a vasoactive and natriuretic substance. The search for endogenous digitalis goes back to the early 1960s. Since then large efforts have been exerted by numerous laboratories worldwide, but little advance has been made until recently except for the identification of nonspecific Na+, K(+)-ATPase inhibitors. Some researchers even doubt the existence of endogenous digitalis. The recognition that assay methodology is associated with many pitfalls and problems has accelerated the rate of recent progress. Chemical identification of endogenous digitalis will be forthcoming in the very near future. In this article, important issues surrounding endogenous digitalis are critically reviewed.     

A chimeric gastric H+,K+-ATPase inhibitable with both ouabain and SCH 28080

2-Methyl-8-(phenylmethoxy)imidazo(1,2-a)pyridine-3acetonitrile+ ++ (SCH 28080) is a K+ site inhibitor specific for gastric H+,K+-ATPase and seems to be a counterpart of ouabain for Na+,K+-ATPase from the viewpoint of reaction pattern (i.e. reversible binding, K+ antagonism, and binding on the extracellular side). In this study, we constructed several chimeric molecules between H+,K+-ATPase and Na+,K+-ATPase alpha-subunits by using rabbit H+,K+-ATPase as a parental molecule. We found that the entire extracellular loop 1 segment between the first and second transmembrane segments (M1 and M2) and the luminal half of the M1 transmembrane segment of H+, K+-ATPase alpha-subunit were exchangeable with those of Na+, K+-ATPase, respectively, preserving H+,K+-ATPase activity, and that these segments are not essential for SCH 28080 binding. We found that several amino acid residues, including Glu-822, Thr-825, and Pro-829 in the M6 segment of H+,K+-ATPase alpha-subunit are involved in determining the affinity for this inhibitor. Furthermore, we found that a chimeric H+,K+-ATPase acquired ouabain sensitivity and maintained SCH 28080 sensitivity when the loop 1 segment and Cys-815 in the loop 3 segment of the H+,K+-ATPase alpha-subunit were simultaneously replaced by the corresponding segment and amino acid residue (Thr) of Na+,K+-ATPase, respectively, indicating that the binding sites of ouabain and SCH 28080 are separate. In this H+, K+-ATPase chimera, 12 amino acid residues in M1, M4, and loop 1-4 that have been suggested to be involved in ouabain binding of Na+, K+-ATPase alpha-subunit are present; however, the low ouabain sensitivity indicates the possibility that the sensitivity may be increased by additional amino acid substitutions, which shift the overall structural integrity of this chimeric H+,K+-ATPase toward that of Na+,K+-ATPase.     

Impaired Na+,K+-ATPase activity as a mechanism of reactive nitrogen species-induced cytotoxicity in guinea pig liver exposed to lipopolysaccharides

In animal models of endotoxin, the excess production of NO and the reactive nitrogen species (RNS), are potent oxidant and nitrating agents, lead to lipid peroxidation, apoptosis, tissue dysfunction and injury and inactivate enzymes in many cell types. Although liver functions are well known to deteriorate following bacterial infection, the underlying specific mechanism(s) remain a matter of considerable debate. Therefore, the aim of the present study was to determine the in vivo effect of bacterial lipopolysaccharides (LPS) on Na+,K+-ATPase activity of guinea pig liver, and to investigate the possible contribution of RNS by measuring of iNOS activity and 3-nitrotyrosine (nTyr) levels. Liver Na+,K+-ATPase activity were maximally inhibited 6 h after LPS injection (p < 0.001 ). nTyr was not detectable in liver of normal control animals, but was detected markedly in LPS exposed animals. LPS treatment significantly increased iNOS activity of liver (p < 0.001). The regression analysis revealed a very close correlation between Na+,K+-ATPase activity and nTyr levels of LPS treated animals (r = -0.863, p < 0.001). Na+, K+-ATPase activity were also negatively correlated with iNOS activity (r = -0.823, p < 0.003) in inflamed tissues. Our results have strongly suggested that bacterial LPS disturbs activity of membrane Na+,K+-ATPase that may be an important component leading to the pathological consequences such as hepatocyte cell loss and dysfunction in which the production of RNS are increased as in the case of LPS challenge.     

Effect of prostaglandins on rat heart sarcolemmal ATPases

The ability of prostaglandins (PG) D2, E1, E2, F2 alpha and I2 (2.8 X 10(-11) to (2.8 X 10(-7) M) to modify Ca2+, Mg2+ and (Na+ + K+)-ATPase activities of rat heart sarcolemmal membrane fractions was examined. Administration of PGE2, PGF2 alpha, and PGI2 reduced basal (Na + + K+)-ATPase activity by up to 30, 80, and 80%, respectively. PGE1 and PGD2 were ineffective at any concentration. Neither Mg2+ -ATPase nor Ca2+ -ATPase was affected by PG treatment. Kinetic analysis revealed that the (Na+ + K+)-ATPase activity reducing ability of PGE2, PGF2 alpha and PGI2 was of a complex nature involving a reduction in Vmax and an elevation of the respective K values for either substrate or activator. These results demonstrate that some PG's are potent inhibitors of rat heart (Na+ + K+)-ATPase. These PG's produced varied inotropic influences on isolated heart preparations and it is uncertain whether their myocardial actions are dependent on enzyme inhibition.