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.     

The heterogeneity of the Na+, K(+)-ATPase ouabain sensitivity in microsomal membranes of rat colon smooth muscles

The dose dependence of the Na+, K(+)-ATPase ouabain inhibition in the rat colon smooth muscle permeabilized microsomes has been analyzed according to the model of two independent binding sites of inhibitor to determine the activity of separate molecular forms of the enzyme that differ by affinity for cardiac glycosides. The two-phase inhibition curve with moderate content of the high-affinity activity component was revealed. The apparent inhibition constant of the low-affinity component corresponds to the value for the rat kidney microsomal Na+, K(+)-ATPase (alpha1-isoform). The specific role of the alpha2- and alpha1- Na+, K(+)-ATPase catalytic subunit isoforms in colonic smooth muscle electromechanical coupling is considered.     

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.     

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.     

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 5β,14β-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.     

Regulation of Na+-K+-ATPase: effect of Mg and Ca ions

The participation of Mg2+ and Ca2+ in complicated mechanisms of Na+, K(+)-ATPase regulation is discussed in the survey. The regulatory actions of Mg2+ on Na+, K(+)-ATPase such as its participation in phosphorylation and dephosphorylation of the enzyme, ADP/ATP-exchange inhibition, cardiac glycosides and vanadate binding with the enzyme, conformational changes induction during ATPase cycle are reviewed in detail. Some current views of mechanisms of above mentioned Mg2+ regulatory effects are discussed. The experimental evidence of Ca2+ immediate influence on the functional activity of Na+, K(+)-ATPase (catalytic, transport and glycoside-binding) are given. It's noted that these effects are based on the conformational changes in the enzyme and also on the phase transition in membrane induced by Ca2+. Unimmediate action of Ca2+ on Na+, K(+)-ATPase is also discussed, especially due to its effect on other membrane systems functionally linked with Na(+)-pump (for instance, due to Na+/Ca(+)-exchanger activation). It's concluded that Mg2+ and Ca2+ as "universal regulators" of the cell effectively influence the functional activity and conformational states of Na+, K(+)-ATPase.     

Ouabain Binding, ATP Hydrolysis, and Na+,K+-Pump Activity During Chemical Modification of Brain and Muscle Na+,K+-ATPase

The effects of 16 group-specific, amino acid-modifying agents were tested on ouabain binding, catalytical activity of membrane-bound (rat brain microsomal), sodium dodecyl sulfate-treated Na+,K(+)-ATPase, and Na+,K(+)-pump activity in intact muscle cells. With few exceptions, the potency of various tryptophan, tyrosine, histidine, amino, and carboxy group-oriented drugs to suppress ouabain binding and Na+,K(+)-ATPase activity correlated with inhibition of the Na+,K(+)-pump electrogenic effect. ATP hydrolysis was more sensitive to inhibition elicited by chemical modification than ouabain binding (membrane-bound or isolated enzyme) and than Na+,K(+)-pump activity. The efficiency of various drugs belonging to the same "specificity" group differed markedly. Tyrosine-oriented tetranitromethane was the only reagent that interfered directly with the cardiac receptor binding site as its inhibition of ouabain binding was completely protected by ouabagenin preincubation. The inhibition elicited by all other reagents was not, or only partially, protected by ouabagenin. It is surprising that agents like diethyl pyrocarbonate (histidine groups) or butanedione (arginine groups), whose action should be oriented to amino acids not involved in the putative ouabain binding site (represented by the -Glu-Tyr-Thr-Trp-Leu-Glu- sequence), are equally effective as agents acting on amino acids present directly in the ouabain binding site. These results support the proposal of long-distance regulation of Na+,K(+)-ATPase active sites.     

Na+, K+-ATPase of the canine mesenteric artery

Na+,K+-ATPase, the enzymatic moiety that operates as the electrogenic sodium-potassium pump of the cell plasma membrane, is inhibited by cardiac glycosides, and this specific interaction of a drug with an enzyme has been considered to be responsible for digitalis-induced vascular smooth muscle contraction. Although studies aimed at localization, isolation, and measurement of the Na+,K+-ATPase activity (or Na+, K- pump activity) indicate its presence in vascular smooth muscle sarcolemma, its characterization as the putative vasopressor receptor site for cardiac glycosides has depended on pharmacological studies of vascular response in vivo and on isolated artery contractile responses in vitro. More recently, radioligand-binding studies using [3H]ouabain have aided in the characterization of drug-enzyme interaction. Such studies indicate that in canine superior mesenteric artery (SMA), Na+,K+-ATPase is the only specific site of interaction of ouabain with resultant inhibition of the enzyme. The characteristics of [3H]ouabain binding to this site are similar to those of purified or partially purified Na+,K+-ATPase of other tissues, which suggests that if Na+,K+-ATPase inhibition is causally related to digitalis-mediated effects on vascular smooth muscle contraction, then therapeutic concentrations of cardiac glycosides could act to cause SMA vasoconstriction. The additional finding from radioligand-binding studies that Na+,K+-ATPase exists in much smaller quantities (density of sites per cell) in SMA than in either heart or kidney may have implications concerning its physiological, biochemical or pharmacological role in modulating vascular muscle tone.     

Production and characterization of antibodies to ouabain.

Two monoclonal antibodies (249F8 and 278A9) to ouabain were produced by somatic cell fusion. They reacted in dose-dependent manners with ouabain and digoxin. These antibodies were supposed to recognize the partial structure important for cardiac compounds to show their pharmacological activity, Na+, K(+)-ATPase inhibition in a competitive mode against K+. These monoclonal antibodies may be useful for the immunochemical isolation, the structural elucidation, and the quantitative measurement of putative endogenous digoxin-like factors.     

Interaction of palytoxin and cardiac glycosides on erythrocyte membrane and (Na+ + K+) ATPase

Palytoxin (PTX), at extremely low concentrations (0.01-1 nM), caused K+ release from rabbit erythrocytes. Among the various chemical compounds tested, cardiac glycosides potently inhibited the PTX-induced K+ release. The order of inhibitory potency (IC50) was cymarin (0.42 microM) greater than convallatoxin (0.9 microM) greater than ouabain (2.3 microM) greater than digitoxin (88 microM) greater than digoxin (90 microM). Their corresponding aglycones, even at 10 microM, did not inhibit the K+ release, but competitively antagonized the inhibitory effect of the glycosides. All these cardiotonic steroids inhibited the activity of (Na+ + K+)-ATPase prepared from hog cerebral cortex in narrow concentration ranges (IC50 = 0.15-2.4 microM), suggesting that the inhibition of K+ release is not related to their inhibitory potency on the (Na+ + K+)-ATPase activity, and the sugar moiety of cardiac glycosides is involved in the inhibition. On the other hand PTX, at higher concentrations (greater than 0.1 microM), inhibited the (Na+ + K+)-ATPase activity. However, this inhibitory effect of PTX was not antagonized by ouabain. It is suggested that, compared with ouabain, PTX has additional binding site(s) on the (Na+ + K+)-ATPase.     

Na+,K(+)-ATPase inhibition by an endogenous peptide, SPAI-1, isolated from porcine duodenum.

SPAI-1, a peptide isolated from porcine duodenum, has been shown to inhibit Na+,K(+)-ATPase in vitro (Araki et al. (1989) Biochem. Biophys. Res. Commun. 164, 496-502). The characteristics of ATPase inhibition by this novel peptide were examined. SPAI-1 inhibited Na+,K(+)-ATPase preparations isolated from various organs of dog or rat or from sheep kidney with similar potency. Three isoforms of rat Na+,K(+)-ATPase had similar sensitivity to inhibition by SPAI-1 although these isoforms had remarkable differences in their sensitivity to the inhibitory effect of ouabain. Ca(2+)-ATPase isolated from the sarcoplasmic reticulum of rabbit skeletal muscle was insensitive to inhibition by SPAI-1. Ouabain-insensitive Mg(2+)-ATPase activity was unaffected by low concentrations of SPAI-1, but was stimulated at high concentrations. SPAI-1 inhibited H+,K(+)-ATPase from hog stomach in concentrations similar to that required for Na+,K(+)-ATPase inhibition. These results indicate that SPAI-1 is a specific inhibitor for monovalent cation transporting ATPases.     

Effect of anthelmintics and phenothiazines on adenosine 5'-triphosphatases of filarial parasite Setaria cervi

S. cervi showed particulate bound Ca2+ ATPase and Na+,K(+)-ATPase activities while Mg2+ ATPase was detected in traces. ATPase of S. cervi was also differentiated from the nonspecific p-nitrophenyl phosphatase activity. Female parasite and microfilariae exhibited higher Ca2+ ATPase and Na+,K(+)-ATPase activities than the male adults and the enzyme Na+,K(+)-ATPase was mainly concentrated in the gastrointestinal tract of the filarial parasite. Na+,K(+)-ATPase of the filariid was ouabain-sensitive while Ca2(+)-ATPase activity was regulated by concentration of Ca2+ ions and inhibited by EGTA. Phenothiazines, viz. trifluoperazine, promethazine and chlorpromazine caused significant inhibition of Ca2+ ATPase and Na+,K(+)-ATPase. Diethylcarbamazine was a potent inhibitor of these ATPases. Mebendazole, levamisole and centperazine also caused significant inhibition of the ATPases indicating this enzyme system as a common target for the action of anthelmintic drugs.     

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.     

Monovalent cation dependence of tissue plasminogen activator synthesis by HeLa cells

HeLa cells synthesize and secrete increased levels of tissue plasminogen activator (tPA) when incubated for 18 h with 10-20 nM phorbol myristate acetate. This response was inhibited by a number of conditions which affect intracellular Na+ and K+ concentrations. Removing extracellular Na+, while maintaining isotonicity with choline+, reduced the secretion of both functional and antigenic tPA in a linear fashion. A series of cardiac glycosides and related compounds strongly inhibited tPA secretion with the following rank order of potency: digitoxin = ouabain greater than digoxin greater than digitoxigenin greater than digoxigenin greater than digitoxose greater than digitonin. These compounds also inhibited cellular Na+/K+-ATPase activity over an identical concentration range. Two compounds which selectively increase cellular permeability to K+, valinomycin, and nigericin, strongly inhibited tPA secretion, with IC50 values of approximately 50 nM. In contrast, monensin, which selectively increases cellular permeability to Na+, was much less active. Valinomycin, but not nigericin, also inhibited cellular Na+/K+-ATPase activity. Phorbol myristate acetate, 5-20 nM, increased Na+/K+-ATPase activity up to 2-fold and tPA secretion up to 15-fold. We conclude that the secretion of tPA by HeLa cells treated with phorbol myristate acetate proceeds via a mechanism which requires extracellular Na+ and a functional Na+/K+-ATPase ("sodium pump") enzyme.     

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.     

Synthesis and evaluation of cardiac glycoside mimics as potential anticancer drugs

The cardiac glycoside digitoxin, consisting of a steroid core linked to a labile trisaccharide, has been used for centuries for the treatment of congestive heart failure. The well known pharmacological effect is a result of the ability of cardiac glycosides to inhibit the Na(+), K(+)-ATPase. Within recent years cardiac glycosides have furthermore been suggested to possess valuable anticancer activity. To mimic the labile trisaccharide of digitoxin with a stabile carbohydrate surrogate, we have used sulfur linked ethylene glycol moieties of varying length (mono-, di-, tri- or tetra-ethylene glycol), and furthermore used these linkers as handles for the synthesis of bivalent steroids. The prepared compounds were evaluated for their potencies to inhibit the Na(+), K(+)-ATPase and for their cytotoxic effect on cancerous MCF-7 cells. A clear trend is observed in both inhibition and cytotoxic effect, where the bioactivity decreases as the size increases. The most potent Na(+), K(+)-ATPase inhibitors are the compounds with the shortest ethylene glycol chain (K(app) 0.48 μM) and thiodigitoxigenin (K(app) 0.42 μM), which both are comparable with digitoxigenin (K(app) 0.52 μM). For the cancer cell viability assay the shortest mimics were found to have highest efficacy, with the best ligand having a monoethylene glycol unit (IC(50) 0.24 μM), which was slightly better than digitoxigenin (IC(50) 0.64 μM), while none of the novel cardiac glycoside mimics display an in vitro effect as high as digitoxin (IC(50) 0.02 μM).     

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.     

Na+,K(+)-ATPase phosphorylation in the choroid plexus: synergistic regulation by serotonin/protein kinase C and isoproterenol/cAMP-PK/PP-1 pathways.

BACKGROUND: The ion pump Na+,K(+)-ATPase is responsible for the secretion of cerebrospinal fluid from the choroid plexus. In this tissue, the activity of Na+,K(+)-ATPase is inhibited by serotonin via stimulation of protein kinase C-catalyzed phosphorylation. The choroid plexus is highly enriched in two phosphoproteins which act as regulators of protein phosphatase-1 activity, DARPP-32 and inhibitor-1. Phosphorylation catalyzed by cAMP-dependent protein kinase on a single threonyl residue converts DARPP-32 and inhibitor-1 into potent inhibitors of protein phosphatase-1. Previous work has shown that in the choroid plexus, phosphorylation of DARPP-32 and I-1 is enhanced by isoproterenol and other agents that activate cAMP-PK. We have now examined the possible involvement of the cAMP-PK/protein phosphatase-1 pathway in the regulation of Na+,K(+)-ATPase. MATERIALS AND METHODS: The state of phosphorylation of Na+,K(+)-ATPase was measured by determining the amount of radioactivity incorporated into the ion pump following immunoprecipitation from 32P-prelabeled choroid plexuses incubated with various drugs (see below). Two-dimensional phosphopeptide mapping was employed to identify the protein kinase involved in the phosphorylation of Na+,K(+)-ATPase. RESULTS: The serotonin-mediated increase in Na+,K(+)-ATPase phosphorylation is potentiated by okadaic acid, an inhibitor of protein phosphatases-1 and -2A, as well as by forskolin or the beta-adrenergic agonist, isoproterenol, activators of cAMP-dependent protein kinase. Two-dimensional phosphopeptide maps suggest that this potentiating action occurs at the level of a protein kinase C phosphorylation site. Forskolin and isoproterenol also stimulate the phosphorylation of DARPP-32 and protein phosphatase inhibitor-1, which in their phosphorylated form are potent inhibitors of protein phosphatase-1. CONCLUSIONS: The results presented here support a model in which okadaic acid, forskolin, and isoproterenol achieve their synergistic effects with serotonin through phosphorylation of DARPP-32 and inhibitor-1, inhibition of protein phosphatase-1, and a reduction of dephosphorylation of Na+,K(+)-ATPase at a protein kinase C phosphorylation site.     

Relationships between adenylate cyclase and Na+, K(+)-ATPase in rat pancreatic islets

We tested the hypothesis that the adenylate cyclase system and Na+, K(+)-ATPase are reciprocally related in rat pancreatic islets. We studied the effect of theophylline, caffeine, and dibutyryl cyclic AMP on Na+, K(+)-ATPase activity in a membrane preparation from collagenase-isolated rat islets. Theophylline, caffeine, or dibutyryl cyclic AMP, in concentrations of 1 mM, all inhibited Na+, K(+)-ATPase activity (44,62, and 43%, respectively). Kinetic analysis indicated that theophylline and dibutyryl cAMP inhibit Na+, K(+)-ATPase by different mechanisms; theophylline decreased Vmax and decreased apparent Km (ATP), whereas dibutyryl cAMP decreased Vmax and increased apparent Km (ATP). Similar inhibition of Na+, K(+)-ATPase by theophylline or dibutyryl cAMP was noted in a particulate fraction from rat kidney and in a purified porcine brain Na+, K(+)-ATPase preparation. The adenylate cyclase system and Na+, K(+)-ATPase may act reciprocally in pancreatic islets and in other tissues. In the beta cell this relationship may be essential in coordinating consumption of ATP in the stimulated, as opposed to the rest, state.