Digitalis: new actions for an old drug

The mechanisms by which digitalis causes its therapeutic and toxic actions have been studied for nearly a half century, revealing a great deal about cardiac cell regulation of intracellular ions via the Na-K-ATPase (NKA) and how it is altered by cardiac glycosides. However, recent observations suggest that digitalis may have additional effects on cardiac cell function in both the short and long term that include intracellular effects, interactions with specific NKA isoforms in different cellular locations, effects on intracellular (including nuclear) signaling, and long-term regulation of intracellular ionic balances through circulating ouabain-like compounds. The purpose of this review is to examine the current status of a number of the newest and most interesting developments in the study of digitalis with a particular focus on cardiac function, although we will also discuss some of the new advances in other relevant cardiovascular effects. This new information has important implications for both our understanding of ionic regulation in normal and diseased hearts as well as for potential avenues for the development of future therapeutic interventions for the treatment of heart failure.     

Liquid membrane phenomenon in the actions of digitalis

The liquid membrane phenomenon in the actions of digitalis glycosides (digitoxin, digoxin and ouabain) has been studied. Formation of liquid membranes, in series with a supporting membrane, by digitalis alone and by digitalis in association with lecithin and cholesterol has been demonstrated. The results obtained on the transport of relevant permeants, viz. sodium, potassium and calcium ions and dopamine, adrenaline, noradrenaline and serotonin, in the presence of the liquid membrane generated by digitalis in association with lecithin and cholesterol indicate that the liquid membrane barrier to transport may have a relevance with the biological actions of digitalis.     

Clinical Experience with a New Diuretic,Triamterene, in Congestive Heart Failure

Triamterene therapy was evaluated in 35 patients with congestive heart failure over a period of two and one-half years. The parameters used were: clinical assessment; daily 24-hour urine sodium, potassium, chloride, and total volume; bi-weekly serum sodium, potassium, chloride, uric acid, and SGOT; hemogram, and BUN.Triamterene is a moderately potent diuretic and natriuretic, with the added desirable property of potassium conservation. It acts synergistically with spironolactone and not only potentiates the effects of hydrochlorothiazide but greatly minimizes its kaluretic effect.It is particularly useful in patients in whom cardiac arrhythmias are associated with digitalis intoxication or with inadvertently induced hypokalemia. Its main therapeutic value, used either alone or in combination with other diuretics, is in the longterm management of chronic edema, especially in certain patients refractory to the currently used diuretics.No significant undesirable side effects were noted.     

Potassium chloride: absorption and excretion

The absorption of potassium chloride in liquid form has been studied, using urinary excretion as an index of absorption. The excretion of potassium chloride was observed after inducing a water diuresis and administering a single dose in liquid form. There is evidence that potassium chloride in liquid form is absorbed rapidly, probably from the stomach, and hence there is a good rationale for its use where rapid absorption is needed, as in digitalis intoxication.     

Effects of digitalis intoxication on the phosphoglucomutase and creatine phosphokinase of guinea pig livers

A study was made of the behavior of the phosphoglucomutase (PGluM) and creatine phosphokinase (CPK) in the liver tissue of guinea pigs after chronic digitalis intoxication. It was found that the latter causes a statistically significant decrease of both enzymatic activities. The results obtained by the authors with this investigation, together with those of previous researchers, show that the toxic effect of digitalis on the enzymatic activities is not limited to the reported effect on the ATPase but also involves, at least at the doses and under the conditions used, other enzymatic activities.     

Differential inactivation of inotropic and toxic digitalis receptors in ischemic dog heart. Molecular basis of the deleterious effects of digitalis

When applied to ischemic hearts digitalis exhibits depressed inotropic effect and increased toxicity. The molecular basis of these effects was investigated at the level of the digitalis receptors characterized by Na,K-ATPase assays and [3H]ouabain-binding measurements. In sarcolemma obtained from dog hearts rendered ischemic for 15, 30, and 60 min (left anterior descending), two populations (high and low affinity) of digitalis receptors were detected. The apparent affinity (KD, 300 nM) and the binding capacity of the low-affinity sites (responsible for toxicity) remained constant and similar to those found in normal hearts. The KD value of the high-affinity sites, "responsible for inotropy," remained unchanged (2 nM), but the site number sharply decreased (up to 90%). These inotropic sites that account for 66% of the total binding in normals are gradually inactivated, as the duration of ischemia increases. This inactivation would occur in situ since it was detectable in homogenates and was not depressed by the isolation procedure per se. The loss of function of the inotropic sites and the increased contribution of the low-affinity toxic sites represent the setting of a new distribution of the digitalis receptors in the ischemic heart before reperfusion is instituted. This constitutes the molecular basis of the deleterious pharmacological effects observed with digitalis.     

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.     

Use of digitalis in general practice. Liverpool Therapeutics Group.

A study, which arose out of a general-practitioner teaching programme in clinical pharmacology, was designed to assess the use of digitalis in 391 patients in general practice. Clinical, biochemical, and pharmacological data were used to assess whether digitalis treatment should be discontinued (89 patients; 22.8%); the dose kept unchanged (47; 12%); or the dose increased (47; 12%) or decreased (24; 6.1%). Serum concentrations of cardiac glycoside were below a defined therapeutic range in 159 patients (40.7%), above this range in 37 (9.7%), and within the range in 195 (49.3%). Studies of this kind may help to promote a more critical attitude to prescribing widely used drugs such as digitalis.     

THE CONSERVATIVE TREATMENT OF MYASTHENIA GRAVIS

Neostigmine is at present the mainstay in treatment of myasthenia gravis. Adjuvant therapy with ephedrine, potassium chloride, or guanidine is recommended. Urecholine,® also used as an adjuvant, is still in the experimental stage and should only be given by one acquainted with its properties and toxic effects. Di-isopropylfluorophosphate and tetraethyl pyrophosphate likewise are still in the experimental stage. Since they are extremely toxic and unstable, general use of them is discouraged until further experimental and therapeutic studies are completed. The results of thymectomy and radiation of the thymus are equivocal and these procedures should be reserved for severe cases which should be selected only after a thorough study.     

Comparative proteomics analysis reveals role of heat shock protein 60 in digoxin-induced toxicity in human endothelial cells

Although digitalis has been used in clinical treatment extensively, the precise mechanism of its toxic actions on cardiovascular system remained unclear, it would be of interest to study the differential proteomic analysis of vascular endothelial cells in response to toxic concentrations of digitalis thus to provide new agents for treatment of digitalis-induced cytotoxicity. We employed human umbilical vein endothelial cells (HUVEC) as our model system. HUVEC were exposed to increasing concentrations (0.1 nM-10 microM) of digoxin at 12-96 h intervals. Cell viability tests revealed that digoxin played dual effects on cell growth. Apoptosis detection confirmed that apoptosis was primarily responsible for digoxin-induced cell death. Proteomics analysis further revealed that the digoxin-induced apoptosis was accompanied by regulated expression of ATP synthase beta chain, cystatin A, electron transfer flavoprotein, heterogeneous nuclear ribonucleoproteins H3, lamin A, profilin-1, proteasome subunit 5, succinyl-CoA ligase beta chain and heat shock protein 60 (HSP60). Deep study on the overexpression of HSP60 confirmed that HSP60 exerted a protective role in digoxin-induced apoptosis through inhibition of caspase-3 activity in HUVEC. These results provided an impetus for further delineation of mechanism of digoxin-induced cytotoxicity and offered new agents that help attenuate its toxicity.     

Radioimmunoassay of an Antibiotic: Gentamicin

GENTAMICIN is an important antibiotic with potentially serious toxicity. The range between therapeutic effectiveness and toxicity is narrow and the dosage must be carefully monitored. Ototoxicity and nephrotoxicity are more common if the peak serum levels are greater than 12 µg/ml.¹, while peak serum levels below 4 µg/ml. are, in some instances², inadequate for bacterial control. Existing methods for the measurement of this and other antibiotics, rely on microbiological assays which determine antibacterial activity in dilutions of the test fluid. These assays lack sensitivity, specificity and precision and have not been extensively used in clinical medicine. Radioimmunoassays have provided practical and widely applied methods for the measurement of hormones³ and digitalis glycosides⁴. These assays possess the technical simplicity, sensitivity and specificity lacking in microbiological assays. This report describes the development of a radioimmunoassay for gentamicin, a potentially toxic antibiotic.     

Ottawa's Fluoridation Battle

The possibility that cardiac failure may be an important contributory or additive factor has led to the sporadic use of digitalis in the treatment of the respiratory distress syndrome in newborn infants. To assess the value of such medication a double-blind controlled study was conducted on 196 newborn infants, using digoxin and a placebo. As a result of the findings in this study the routine use of digoxin for the prevention of the respiratory distress syndrome is not recommended. The toxic effects of digitalis are outlined.     

The cellular pathways that maintain the quality control and transport of diverse potassium channels

Potassium channels are multi-subunit transmembrane proteins that permit the selective passage of potassium and play fundamental roles in physiological processes, such as action potentials in the nervous system and organismal salt and water homeostasis, which is mediated by the kidney. Like all ion channels, newly translated potassium channels enter the endoplasmic reticulum (ER) and undergo the error-prone process of acquiring post-translational modifications, folding into their native conformations, assembling with other subunits, and trafficking through the secretory pathway to reach their final destinations, most commonly the plasma membrane. Disruptions in these processes can result in detrimental consequences, including various human diseases. Thus, multiple quality control checkpoints evolved to guide potassium channels through the secretory pathway and clear potentially toxic, aggregation-prone misfolded species. We will summarize current knowledge on the mechanisms underlying potassium channel quality control in the secretory pathway, highlight diseases associated with channel misfolding, and suggest potential therapeutic routes.     

Reduced concentrations of potassium,magnesium, and sodium-potassium pumps in human skeletal muscle during treatment with diuretics

Animal studies have shown that potassium depletion induced by diuretics or potassium deficient fodder leads to a selective decrease in the concentrations of potassium and in the concentration of sodium-potassium pumps in skeletal muscle. In 25 patients who had received diuretics for 2-14 years the mean concentrations of potassium, magnesium, and sodium-potassium pumps were measured in skeletal muscle biopsy specimens and were significantly lower than in those from a group of age matched controls. The reductions in all three variables were significant in those patients receiving diuretics for arterial hypertension as well as in those being treated for congestive heart failure. In 14 patients the mean muscle potassium concentration was below the control range, but only one of those was hypokalaemic (3·4 mmol/l), and 13 were receiving potassium supplements. In 15 patients the mean muscle magnesium concentration was below normal, and the mean muscle potassium and magnesium concentrations showed a linear correlation. In 12 patients in whom the mean muscle potassium concentration was below 80 μmol/g wet weight there was a linear correlation between the cellular potassium:sodium ratio and the concentration of ³H-ouabain binding sites indicating that potassium deficiency also leads to a down regulation of sodium-potassium pumps in human skeletal muscle.In spite of potassium supplements long term treatment with diuretics may lead to potassium and magnesium deficiencies, which are not detectable using the standard methods of serum analysis. The changes in concentrations of electrolytes and sodium-potassium pumps associated with treatment with diuretics may impair muscle function and potassium homoeostasis and interfere with the distribution of digitalis glycosides.     

Pathology of structured water and associated cations in cells (ther tissue damage syndrome) and its medical treatment

Modern experimental evidence indicates that the cell should be regarded as analogous to an ion exchanger resin granule with structured water in the interstices and with potassium and sodium ions associated with fixed negative charges on the protein matrix. In tissues damaged by disease or trauma, a similar set of changes in properties of cell cations and water is to be expected, for which a similar set of therapies is appropriate. Tissue damage causes a configurational change of the protein matrix from the normal to the damaged state. This leads to loss of association preference for potassium vs. sodium ions and to loss of water structuring, resulting in replacement of cell potassium by sodium and abnormal uptake of water by the cell. Appropriate therapies for reestablishment of the normal configurational state of the proteins of the cell are reestablishment of normal cell ATP production, for which prostaglandin PGBx is the rational approach, plus diets or drugs that decrease sodium and/or increase potassium concentrations in the body. Partial normalization of cell protein configuration by digitalis compounds may also be possible.     

Specific photoaffinity labeling of the digitalis binding site of the sodium and potassium ion activated adenosinetriphosphatase induced by energy transfer

A ouabain p-aminobenzenediazonium derivative with a high specific radioactivity has been synthesized from ouabain and used as a photolabel for the (sodium plus potassium)-activated adenosinetriphosphatase from Electrophorus electricus electric organ and from dog kidney. In the dark it binds reversibly to the digitalis receptor site, with binding characteristics comparable to those of ouabain. The photoactivation of the ouabain derivative to produced covalent labeling of the receptor was obtained by energy transfer from a tryptophan residue in the (Na+,K+)ATPase to the ouabain p-aminobenzenediazonium molecule bound at the active site. The great advantage of this procedure compared to previous methods is that free molecules of the photoactivatable derivative are not photodecomposed. Analysis of the photolabeled polypeptides on sodium dodecyl sulfate gel electrophoresis showed that over 90% of the total radioactivity incorporated was found in the large molecular weight alpha-chain of the kidney enzyme (Mr 93 000). The same specific labeling of the alpha-subunit was obtained with a crude microsomal fraction from Electrophorus electricus. A mild tryptic fragmentation of the subunit into two peptide fragments of Mr 58 000 and 41 000, respectively, shows that the digitalis receptor is located in the N-terminal 41 000 fragment.     

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.     

Association of the positive inotropic action of ouabain with a second species of digitalis receptors

Studies were conducted to determine whether Na-K ATPase or a second species of digitalis receptors in canine cardiac sarcolemma membrane preparations is associated with the positive inotropic action of nontoxic concentrations of ouabain. [3H]ouabain association and dissociation experiments using highly enriched sarcolemma preparations from canine ventricle indicate the presence of two species of ouabain binding receptors. Ouabain binding to Na-K ATPase of the sarcolemma preparation requires supporting ligands and is characterized by fast association and very slow dissociation in vitro. The second species of digitalis receptor does not require supporting ligands for ouabain binding and is characterized by slow association and fast dissociation. To determine which species of digitalis receptor is associated with the positive inotropic action of digitalis, ouabain washout experiments were conducted using various isolated canine myocardial preparations. Washout of the positive inotropic effects of 1.2-2.4 X 10(-7) M ouabain gave half-life values of 1.5-2.0 h for the various myocardial preparations. [3H]ouabain dissociation from the second species of digitalis receptors gave half-life values of 1.7-1.8 h, whereas dissociation from the sarcolemma Na-K ATPase gave half-life values of 8.9-9.3 h for the various sarcolemma preparations utilized. Therefore, based on similarities in half-life values between ouabain inotropy and [3H]ouabain dissociation from the second class of digitalis receptors, it is postulated that the positive inotropic action of digitalis glycosides is associated with the second species of digitalis receptors in the sarcolemma and not with the digitalis inhibitory receptor of Na-K ATPase for nontoxic concentrations of digitalis.     

The Combined Effect of Shungite and Heavy Metals on the Growth of Microalgae Роpulation

The combined effect of 3 mg/L potassium dichromate, 1.5 mg/L cadmium sulfate, and 100 g/L shungite on the growth of chlorococcales green microalgae culture Scenedesmus quadricauda (Turp.) Bréb. is studied. The toxic effect of potassium dichromate and cadmium sulfate on S. quadricauda is estimated by calculating the share of living and dead cells and physiological parameters. The toxic effect of heavy metals does not manifest itself under the combined action of potassium dichromate or cadmium sulfate and shungite on S. quadricauda. The best growth of the algae culture occurred only when only shungite was added to the culture medium. Shungite can be used to neutralize the toxic effect of heavy metals.     

Localization of neurally mediated arrhythmogenic and coronary vasoconstrictor properties of digitalis

Studies of the cardiotoxicity of digitalis have revealed an appreciable role of the autonomic nervous system in modulating these toxicities. Neuraxis transection experiments and studies with digitalis compounds that fail to enter the central nervous system suggest that centers localized to the floor of the fourth cerebral ventricle play a substantial part in neural arrhythmogenic effects of the digitalis glycosides. Stimulation of the area postrema (AP) in animals partially digitalized, but not otherwise manifesting ventricular arrhythmias, elicits ventricular tachycardia. Neuraxis transection experiments and studies with a charged digitalis derivative also suggest localization of the coronary vasoconstrictive properties of digitalis to the AP. The results suggest that this region of the brain stem is sensitive to neurally active substances that cause peripheral sympathetic effects of considerable consequence.