Digitalis Receptor Research Articles

Endogenous Digitalis-Like Factors: An Overview of the History

The sodium pump is a ubiquitous cell surface enzyme, a Na, K ATPase, which maintains ion gradients between cells and the extracellular fluid (ECF). The extracellular domain of this enzyme contains a highly conserved binding site, a receptor for a plant derived family of compounds, the digitalis glycosides. These compounds inhibit the enzyme and are used in the treatment of congestive heart failure and certain cardiac arrhythmias. The highly conserved nature of this enzyme and its digitalis receptor led to early suggestions that endogenous regulators might exist. Recent examination of this hypothesis emerged from research in two separate areas: the regulation of ECF volume by a natriuretic hormone (NH), and the regulation of peripheral vascular resistance by a circulating inhibitor of vascular Na, K ATPase. These two areas merged with the hypothesis that NH and the vascular Na, K ATPase inhibitor were in fact the same entity, and that it played a causative role in the pathophysiology of certain types of hypertension. The possibility that multiple endogenous digitalis-like factors (EDLFs) exist emerged from efforts to characterize the circulating enzyme inhibitory activity. In this review, the development of this field from its beginnings is traced, the current status of the structure of EDLFs is briefly discussed, and areas for future development are suggested.

Antidigoxin antiserum prevents endogenous digitalis-like compound-mediated reperfusion injury via modulating sodium pump isoform gene expression

Endogenous digitalis-like compound (EDLC) is an endogenous ligand of the digitalis receptor and can remarkably inhibit Na+/K+-ATPase activity. Antidigoxin antiserum (ADA), a selective EDLC antagonist, may lessen myocardial reperfusion injury; however, the molecular mechanisms underlying the effect remain unclear. Therefore, this study investigated whether ADA may prevent myocardial reperfusion injury and modulate gene expression of sodium pump alpha isoforms. Cardiac function was examined in isolated rat hearts subjected to ischemia and reperfusion (I/R). The infarct size, EDLC level, Na+/K+-ATPase activity, and the levels of mRNA for sodium pump alpha isoforms were measured in vivo I/R rat hearts in the presence or absence of ADA. It was found that ADA significantly improved the recovery of cardiac function, decreased infarct size, decreased EDLC level, and recovered Na+/K+-ATPase activity in I/R hearts. Further studies showed that sodium pump alpha1, alpha2, and alpha3 isoform mRNA levels were significantly reduced in I/R hearts, and pretreatment with ADA induced a large increase in the mRNA levels. These results indicate that EDLC may participate in depressing Na+/K+-ATPase activity and sodium pump alpha isoform gene expression in I/R heart. It is suggested that treatment with ADA may prevent EDLC-mediated reperfusion injury via modulating sodium pump isoform gene expression.

Binding of Progesterone to Specific Sites in Isolated Hepatic Cells and Purified Plasma Membrane Fraction

Specific binding for progesterone has been determined in rat hepatocytes and mouse liver purified plasma membranes. The binding is saturable, reversible and temperature dependent. Two types of binding sites have been characterized in hepatocytes. The first is of high affinity and low binding capacity and the other one is of low affinity and high capacity of binding. In plasma membranes one type of specific binding site only exists. These high affinity sites are different from nuclear progesterone receptor, nuclear glucocorticoid receptor, digitalis receptor of Na+, K(+)-ATPase, transcortine and from corticoid binding sites determined previously in plasma membrane. We also have observed that specific progesterone binding to hepatocytes and plasma membrane is independent from the alpha and beta adrenergic receptors and from P-site adenosine receptor.

Direct studies of ligand-receptor interactions and ion channel blocking (Review).

Small molecules can activate membrane bound receptors to elicit cellular responses and approximately 80% of all such responses are mediated through membranes. Solid state nuclear magnetic resonance (NMR) methods have been developed for the resolution of specifically labelled ligands, designed to report back structural, dynamic and electronic details of the ligand whilst at its site of action. In particular, ion channel blockers, drugs, inhibitors, prosthetic groups and solutes have been studied when at their target site in functionally competent membranes. A blocker of the channel in the anion transporter of erythrocytes (AE1) has been studied to show that considerable motional freedom within the channel site occurs. The activating agonist of the ligand gated ion channel, the nicotinic acetylcholine receptor, is constrained at its site of action and binds through a cation-pi interaction as revealed in the NMR spectra. The footprint for the inhibitor binding site of the digitalis receptor, the Na(+)/K(+)-ATPase has been resolved as well as the molecular conformation of digitalis showing a bent structure whilst at its site of action. For the gastric proton pump, however, a planar conformation is observed for a reversible substituted imidazopyridine. In all cases not only the structure, but also some indications about the electronic interactions of ligand and target have been resolved using these approaches.

17 alpha-O-(aminoalkyl)oxime derivatives of 3 beta,14 beta-dihydroxy-5 beta-androstane and 3 beta-hydroxy-14-oxoseco-D-5 beta-androstane as inhibitors of Na(+),K(+)-ATPase at the digitalis receptor.

The synthesis and binding affinities to the digitalis Na(+),K(+)-ATPase receptor of a series of 3 beta,14 beta-dihydroxy-5 beta-androstane and 3 beta-hydroxy-14-oxoseco-D-5 beta-androstane derivatives bearing a 17 alpha-(aminoalkoxy)imino chain are reported; some derivatives were also studied for their inotropic activity. Our recently proposed model of interaction of molecules with the digitalis receptor was used to design these compounds. On that basis, the possibility to design novel potent inhibitors of Na(+),K(+)-ATPase without being constrained by the stereochemistry of the classical digitalis skeleton in the D-ring region was predicted. The binding affinities of the most potent compounds in the two series, (EZ)-17 alpha-[2-[(2-aminoethoxy)imino]ethyl]-5 beta-androstane-3 beta,14 beta-diol (6f) and (EZ)-3 beta-hydroxy-17 alpha-[2-[(2-aminoethoxy)imino]ethyl]-14,15-seco-5 beta-androstan-14-one (24c) are higher than that of the potent natural compound digitoxigenin, despite the unusual alpha-exit of the substituent in position 17 of 6f or the disruption of the D-ring in 24c. These results further support the validity of our recently proposed model of binding at the digitalis receptor. Results of the inotropic tests on guinea pig atrium deserve further investigation on the pharmacological profile of these derivatives.

Structural insights into the binding of cardiac glycosides to the digitalis receptor revealed by solid-state NMR.

Several biologically active derivatives of the cardiotonic steroid ouabain have been made containing NMR isotopes ((13)C, (2)H, and (19)F) in the rhamnose sugar and steroid moieties, and examined at the digitalis receptor site of renal Na(+)/K(+)-ATPase by a combination of solid-state NMR methods. Deuterium NMR spectra of (2)H-labeled inhibitors revealed that the sugar group was only loosely associated with the binding site, whereas the steroid group was more constrained, probably because of hydrogen bonding to residues around the K(+)-channel region. Crosspolarization magic-angle spinning NMR showed that chemical shifts of inhibitors (13)C-labeled in the sugar group moved downfield by 0.5 ppm after binding to the digitalis site, suggesting that the sugar was close to aromatic side groups. A (19)F, (13)C- rotational-echo double-resonance NMR strategy was used to determine the structure of an inhibitor in the digitalis receptor site, and it showed that the ouabain derivatives adopt a conformation in which the sugar extends out of the plane of the steroid ring system. The combined structural and dynamic information favors a model for inhibition in which the ouabain analogues lie across the surface of the Na(+)/K(+)-ATPase alpha-subunit with the sugar group facing away from the surface of the membrane but free to move into contact with one or more aromatic residues.

Receptor occupancy with digoxin vs receptor occupancy with a putative endogenous digitalislike factor.

The possibility that an endogenous ligand for the digitalis receptor might exist has been a source of speculation resulting in efforts over the past decades to identify such a hormone. In the current context it is of interest that prolonged wash of myocardial and skeletal muscular samples from subjects who were not in digoxin treatment generally resulted in small tendencies to increase 3H-ouabain binding ranging from 2 to 9% and from -2 to 7%, respectively. It may be appreciated that neither wet weight nor water content of left ventricular or skeletal muscular samples have been found to change as a result of the prolonged wash. Although these tendencies most likely are a mere play of chance, it may be argued that the studies did not entirely rule out the possibility of the existence of a quantitatively small amount of endogenous digitalislike factor. However, based on the evaluations of receptor occupancy with digoxin during treatment of 24-34% in the left ventricle and 9-13% in skeletal muscle, it would seem reasonable to expect that a comparatively larger fraction of digitalis receptors should be occupied by a putative endogenous digitalislike factor, if such a factor were to be of any physiological significance.

Synthesis and biological evaluation of 2-Hydroxy derivatives of digitoxigenin and 3-Epidigitoxigenin

The four stereoisomers of the 2-hydroxy derivatives of digitoxigenin and 3-epidigitoxigenin have been synthesized, their structures established by NMR, and their binding affinity for the digitalis receptor on Na+, K+-ATPase evaluated. These derivatives showed lower affinities than the parent compounds. The hydrophilic hydroxy groups in the alpha position are more detrimental to the affinity than hydroxy groups in the beta position.

A new approach to the design of novel inhibitors of Na+,K+-ATPase: 17alpha-substituted seco-D 5beta-androstane as cassaine analogues.

A new three-dimensional model for the relative binding mode of cassaine 1 and digitoxigenin 2 at the digitalis receptor site is proposed on the basis of the structural and conformational similarities among 1, 2 and its 14,15-seco analogues 3 and 4. Accordingly, the speculation that also 17alpha-substituted derivatives of the digitalis 5beta,14beta-androstane skeleton could efficiently bind to the Na+,K+-ATPase receptor is put forward and verified through the synthesis of some related compounds. The binding affinity shown by 2-(N,N-dimethylamino)ethyl 3beta, 14-dihydroxy-5beta,14beta-androstane-17alpha-acrylate 6 (IC50 = 5.89 microM) and, much more significantly, by the corresponding 14, 15-seco-14-oxo derivative 9 (IC50 = 0.12 microM) substantiates the new hypothesis and opens new prospects to the design of novel inhibitors of Na+,K+-ATPase as potential positive inotropic compounds.

Synthesis and Biological Evaluation of 14 -Methoxy Digitalis Derivatives

The synthesis and biological evaluation of 14β-methoxy derivatives of digitoxigenin and of other digitalis-like compounds are reported. These compounds have a 14β-oxygen, which can be a hydrogen bonding acceptor, as is the case of 14β,15β-epoxide derivatives, but not a hydrogen bonding donor as is the case of 14β-hydroxy derivatives. All the new 14β-methoxy derivatives show a considerable reduced binding affinity on Na+,K+-ATPase when compared with the 14β-hydroxy analogues and also with the 14β,15β-epoxy derivatives. These results could mean that the digitalis receptor does not permit the presence of a bulky substituent in the 14β region, even of relatively small volume like the methyl group

Synthetic candidates for EDLF. Activity on human placenta digitalis receptors.

Synthetic candidates for EDLF. Activity on human placenta digitalis receptors.

Characterization of specific corticosterone binding sites in adrenal cortex plasma membrane and their localization by autoradiographic studies.

Specific corticosterone binding to calf adrenal cortex plasma membrane was measured using the biologically active radioligand [3H]corticosterone. Corticosterone binding was found to be time-dependent, saturable and reversible, and was reduced by more than 70% when membranes were pretreated with proteases. The population of corticosterone binding sites in calf adrenal cortex plasma membrane was homogeneous and displayed the following characteristics: equilibrium dissociation constant Kd = 77 +/- 8 nM and maximum specific binding capacity Bmax = 70,378 +/- 6,385 fmol/mg protein. The relative affinities of several structural analogues of steroids were deduced from competition assays. From these experiments we can conclude that the plasma membrane binding site characterized is selective for corticosterone and progesterone derivatives, and different from nuclear glucocorticoid, mineralocorticoid, estrogen and progestin receptors. Likewise, this corticosterone binding site is independent of mineralocorticoid and Na+, K(+)-ATPase digitalis receptors. From autoradiographic studies we suggest these corticosterone binding sites are located in the whole adrenal cortex.

Synthesis and structure-activity relationships of 17beta-substituted 14beta-hydroxysteroid 3-(alpha-L-rhamnopyranoside)s: steroids that bind to the digitalis receptor.

The preparation of 17beta-substituted 14beta-hydroxysteroid C-3 alpha-L-rhamnopyranosides is described. These derivatives have a 14beta,20-ether, 14beta,20-lactone, or 17beta-CH2CH2OH, -CH2CH2NH2, -CH=CHNO2(E), -CH=CHCOOH(E), -CH(OH)CH2NO2(R), -CH(OMe)CH2NO2(R), -CH2-CH2COOH, or -CH(OH)CH2NH2(R) group. Derivatives were assayed in a radioligand binding assay for [3H]ouabain in membranes from canine heart muscle. The digitalis "receptor" comprises isoenzymes of the ion-pumping enzyme, Na+,K+-ATPase. The 17beta-CH=CHNO2(E), 17beta-CH=CHCOOH(E), and 17beta-CH(OMe)CH2NO2(R) derivatives were the most potent and equivalent to ouabain with low-nanomolar IC50 values. The very potent binding affinity of the disubstituted compound 17beta-CH(OMe)CH2NO2(R) further demonstrates that 17beta-unsaturated substitution is not required for potent binding affinity. This observation may be of value in the separation of cardiotonic and cardiotoxic effects. Tosylation of the 17beta-CH2OH, prepared from the 17beta-CHO by lithium aluminum hydride reduction, yielded the 14beta,17beta-ether. Synthesis of the 17beta-CH2COOH gave the epimeric 14alpha,17alpha- and 14beta,17beta-lactones. Structures have been established by NMR analysis.

Digitalis-like compounds: Synthesis and biological evaluation of 3β-(aminoalkylthio) derivatives

The synthesis and the binding affinities to the digitalis receptor on the Na +,K +-ATPase of a series of 3β-(aminoalkylthio) digitalis derivatives are described. In most cases these derivatives showed a higher binding affinity than that of the corresponding 3β-hydroxy and 3β-mercapto parent compoubds.

Specific Modulation of two Neuronal Digitalis Receptors by Anaesthesia

Three isoenzymes of digitalis receptors (α1, α2, α3) in the brain and only one in the kidney (α1) can be distinguished by their ouabain affinities and their responsiveness to sodium. Since we have reported modulations for these digitalis receptors by their fatty acid membrane environment, anaesthesics could bind on and modulate either directly these receptors or indirectly by disturbing membrane lipids. The aim of this study was to evaluate this anaesthetic action on apparent ouabain affinities and sodium dependence of cerebral and renal Na+,K+-ATPase isoenzymes activities. Rat brain and kidney membrane fractions with pentobarbital-induced anaesthetized state were compared to an unanaesthetized state for their (1) fatty acid composition of total membrane phospholipids, (2) responsiveness to ouabain and (3) Na+ dependence of digitalis receptors. An anaesthesia period of 10 minutes induced (1) a fatty acid modification of brain membranes and (2) a significant sensibilization to ouabain for the α2 and α3 isoforms of digitalis receptors (α2, IC50; 8.2 ± 0.5 × 107 mol/l vs 4.5±0.2 × 107 mol/l; α3, IC50; 6.0±0.3 × 10-8 mol/l vs 2.5±0.1 × 10-8 mol/l). In contrast, the ouabain affinity of the α1 subunit expressed in kidney and brain membranes was unaltered. No anaesthetic effect was observed on the Na+ dependence of the α1 isoenzyme in the brain (4 mmol/l) and the kidney (8 mmol/l). Pentobarbital induced a desensibilization for α2-receptors (8.3±0.5 vs 16.0±1.4 mmol/l Na+) and a sensibilization for α3-receptors (14.4±0.8 vs 10±1.3 mmol/l Na+). These altered properties could be related to a selective modification of the fatty acid composition and/or to the presence of a specific binding site for pentobarbital on these two neuronal digitalis receptors.

Canine Cardiac Digitalis Receptors are Preserved in Congestive Heart Failure Induced by Rapid Ventricular Pacing

In dogs, it has been reported that acute ischemia or severe and terminal heart failure results in a selective reduction of myocardial α3 isoform of Na, K-ATPase activity. The aim of this study was to evaluate if a similar change in the two canine digitalis receptor isoforms occurs following 4 weeks of rapid ventricular pacing-induced heart failure without profound necrosis. Heart failure was induced in dogs by rapid ventricular pacing (240 beats × min-1). Digitalis receptors were quantitated by [3H]-ouabain binding with isolated microsomal membranes from sham-operated (n = 3) and heart failure dogs (n = 4) and by Western blot analysis using specific α1 and α3 polyclonal antibodies. In kinetic studies, similar dissociation rates of 19 to 22 × 10-4 s-1 and 1.3 to 2.4 10-4 s-1 corresponding to high and low affinity sites respectively, were found in sham-operated and CHF dogs. Immunoblotting showed similar abundance of α1 isoform in the two groups; however, levels of α3 were increased by at least 50% in pacing-induced heart failure animals. In conclusion, heart failure selectively modulates the expression of cardiac α3 isoform in dogs.

Digitalis-like compounds: Synthesis and biological evaluation of seco-D and D-homo derivatives

The synthesis of seco-D and D-homo digitalis derivatives, from the carda-14,20(22)-dienolide 1, is described. Selective ozonolysis gave the seco-D 14-ketoaldehyde 2a. Modification of the two carbonyl groups and of the α, β-unsaturated lactone ring of the seco-D 14-ketoaldehyde 2a allowed preparation of derivatives with a broad range of binding affinity to the Na +, K +-ATPase receptor. Some of the seco-D derivatives ( 10, 11b, and 13b) showed a binding affinity similar to that of digitoxigenin, demonstrating that the D-ring is not essential for recognition by the digitalis receptor. In the class of D-homo derivatives the highest binding value, about 15 times lower than that of digitoxigenin, was that of the C >D cis compound 29b; the C D trans analog 28b showed a 7-fold decrease in binding affinity, indicating that the C D configuration plays an important role in D-homo derivatives as in the classical digitalis compounds.

Synthetic steriod derivatives as candidates for endogenous digitalis-like factors: Interaction with human placenta digitalis receptors

Synthetic steriod derivatives as candidates for endogenous digitalis-like factors: Interaction with human placenta digitalis receptors

ChemInform Abstract: Derivatives of 3‐Digitoxigenone Amidinohydrazone: Synthesis and Effect on the Digitalis Receptor of Several Species. Part 7. Compounds with Positive Inotropic Activity

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Marinoic Acid, a Novel Bufadienolide-Related Substance in the Skin of the Giant Toad, Bufo marinus.

We found a novel substance, 3 beta-hydroxy-11,12-seco-5 beta, 14 beta-bufa-20,22-dienolide-11,14-olide-12-oic acid (1), which we called marinoic acid, in the skin of the toad, Bufo marinus. The structure was established from NMR and MS data. Like bufadienolides, marinoic acid contained an A/B ring structure in the cis configuration and a D/alpha-pyrone ring structure, but the structure of the C ring differed considerably from that of bufadienolides. Marinoic acid exhibited biological activity, as demonstrated by inhibition of Na+, K(+)-ATPase enzymatic activity, and by inhibition of [3H]ouabain binding to the digitalis receptor site on Na+, K(+)-ATPase, although marinoic acid was a less effective inhibitor than typical bufadienolides. Although marinoic acid cannot be classified as a bufadienolide, its chemical structure and its Na+, K(+)-ATPase inhibitory activity suggest that it is bufadienolide-related.