Presence of digitalis-like factor in mammalian plasma

We attempted to purify a digitalis-like factor from volume expanded dog plasma using an inhibitory effect on the binding of [3H]ouabain to intact human erythrocytes to monitor digitalis-like activity. A highly polar [3H] ouabain displacing compound was purified to a high degree using a combination of chromatographic procedures including reverse phase and gel filtration high performance liquid chromatography. This compound, a reversible inhibitor of [3H]ouabain binding, closely resembles ouabain in its polarity and significantly increases during saline infusion. Its molecular weight was estimated to be 343Da. Moreover, similar compound was consistently detected in other mammalian plasma.     

Increased digitalis-like activity in human cerebrospinal fluid after expansion of the extracellular fluid volume

The present study was designed to determine whether acute expansion of the extracellular fluid volume influenced the digitalis-like activity of human cerebrospinal fluid (CSF), previously described by our laboratory. Human CSF samples, drawn before and 30 minutes after the intravenous infusion of 1 liter of either saline or glucose solutions, were assayed for digitalis-like activity by inhibition of either the 86Rb+ uptake into human erythrocytes or by the activity of a purified Na+ - K+ ATPase. The CSF inhibitory activity on both systems significantly increased after the infusion of sodium solutions but did not change after the infusion of glucose. These results indicate that the digitalis-like factor of human CSF might be involved in the regulation of the extracellular fluid volume and electrolyte content and thereby in some of the physiological responses to sodium loading.     

Tissue distribution of an endogenous ligand to the Na, K ATPase molecule

A variety of evidence indicates the presence of a circulating ligand to the Na, K ATPase molecule that is involved in the regulation of extracellular sodium metabolism. To examine the potential role of endogenous ligands to the Na, K ATPase molecule in the regulation of intracellular sodium metabolism, the tissue distribution of digitalis-like activity was quantitated in several brain regions and peripheral organs. The digitalis-like activity of desalted and delipidated extracts of tissue was widely distributed and produced a displacement of tritiated ouabain that was parallel to the displacement produced by cold ouabain. These results suggest that tissue contains an endogenous ligand to the Na, K ATPase molecule and that this ligand may regulate intracellular sodium metabolism in an autocoid-like manner.     

Existence of a polar digitalis-like factor in mammalian hypothalamus

We were able to partially purify a polar digitalis-like factor from rat and bovine hypothalami based on the capacity to inhibit [3H]ouabain binding to intact human erythrocytes. This factor was characterized in reference to the digitalis-like factor that we have isolated and reported on. Hypothalamic factor shared digitalis-like activities and physicochemical properties with the one derived from human urine and mammalian plasma. These findings strongly suggest that a polar digitalis-like factor identical to the circulatory factor does exist in mammalian hypothalamus.     

Identification and preliminary characterization of two human digitalis-like substances that are structurally related to digoxin and ouabain.

In order to characterize the structure of endogenous digitalis-like immunoreactive factor (DLIF), we utilized peritoneal dialysis fluid from patients with chronic renal failure as a source of endogenous digitalis-like immunoreactive factor (DLIF), and subjected it to one-step ion exchange chromatography, followed by one step reverse HPLC. Crude dialysis fluid contained 0.09 ng/ml of DLIF, and using Amberlite XAD-2 chromatography we extracted 110 ng of DLIF from 800 ml of dialysis fluid. By applying this partially purified DLIF to our HPLC system, we discerned three peaks of DLIF activity, with retention times of 34, 58 and 63 minutes. The first peak overlapped the elution profile of ouabain, and the third peak co-eluted precisely with digoxin. The second DLIF peak was not in proximity to any of the digitalis-like markers employed. Thus, our results indicate that DLIF isolated from peritoneal dialysis fluid exists in three distinct forms, one of which resembles ouabain, and one which is identical to digoxin.     

Digitalis-like activity in human plasma. Purification, affinity, and mechanism

A factor having digitalis-like characteristics has been isolated from human plasma and its mechanism of action compared with the commonly used cardenolide, ouabain. The purification scheme involved dialysis of human plasma, lyophilization of dialysate, extraction of methanol-soluble components, and flash evaporation, followed by preparative, semipreparative, and analytical scale reverse-phase chromatography. One peak of biologically active material was obtained and shown to possess digitalis-like activity in assays of sodium pump activity, receptor binding, and Na,K-ATPase activity. Results from (i) the determination of the ligand conditions supporting binding, (ii) kinetics of association and dissociation from the Na,K-ATPase, (iii) affinity titration, (iv) selectivity, and (v) competition studies, when taken together, show that the endogenous digitalis-like factor is a specific inhibitor of the sodium pump that stabilizes the E2P form of the enzyme in a manner analogous to ouabain. The endogenous digitalis-like factor binds competitively in or near the receptor site for cardiac glycosides with an apparent affinity 8-20-fold greater than any known cardioactive steroid. The presence of digitalis-like activity in the circulation of individuals with no known intake of these compounds suggests that the material characterized here is an endogenous counterpart to the cardenolides. This factor may regulate sodium pump activity and provide a rationale for the existence of gene and tissue-specific forms of the Na,K-ATPase having distinct sensitivity to the cardenolides.     

Evidence for the existence of the same endogenous digitalis-like factor in several mammalian species

1. Endogenous digitalis-like activity was studied comparatively in four mammalian species: guinea pig, dog, cow and rat. 2. Water extracts were prepared from guinea pig, dog, cow and rat hearts and assayed by ouabain radioreceptorassay, digoxin radioimmunoassay and digitoxin radioimmunoassay. Extracts were further analysed by fractionation by gel permeation chromatography with Sephadex G-25. 3. A similar behaviour was observed with the four species in the three assays. Extracts displaced tritiated ouabain binding to its receptor and labeled digoxin analogue binding to antidigoxin antibodies in a competitive manner. Displacement of labeled digitoxin analogue to antidigitoxin antibodies did not follow Michaelis-Menten kinetics. IC50 ratios between assays were similar for the four species studied. 4. Extracts from the four species exhibited a similar pattern when fractionated with Sephadex G-25. Endogenous digoxin-like immunoreactivity eluted after the salts, suggesting that the active material is of a molecular weight of less than 1000. 5. Results suggest that a similar endogenous factor endowed with digitalis-like characteristics is present in all mammalian species.     

Characterization of digitalis-like factors in human plasma. Interactions with NaK-ATPase and cross-reactivity with cardiac glycoside-specific antibodies

Much of the evidence for a physiologically important endogenous inhibitor of the sodium pump has been either contradictory or indirect. We have identified three discrete fractions in desalted deproteinized plasma from normal humans that resemble the digitalis glycosides in that they: are of low molecular weight; are resistant to acid and enzymatic proteolysis; inhibit NaK-ATPase activity; inhibit Na+ pump activity in human erythrocytes; displace [3H]ouabain bound to the enzyme; and cross-react with high-affinity polyclonal and monoclonal digoxin-specific antibodies but not with anti-ouabain or anti-digitoxin antibodies. An additional fraction cross-reacted with digoxin-specific antibodies but had no detectable activity against NaK-ATPase. The three inhibitory fractions differed from cardiac glycosides in that their concentration-effect curves in a NaK-ATPase inhibition and [3H]ouabain radioreceptor assays were steeper than unlabeled ouabain. This suggests that these inhibitors are not simple competitive ligands for binding to NaK-ATPase. In the presence of sodium, no fraction required ATP for binding to NaK-ATPase, and in the presence of potassium, only one fraction had the reduced affinity for the enzyme that is characteristic of cardiac glycosides. Unlike digitalis, all three NaK-ATPase inhibitory fractions stimulated the activity of skeletal muscle sarcoplasmic reticulum Ca-ATPase. The presence of at least three fractions in human plasma that inhibit NaK-ATPase and cross-react to a variable degree with different digoxin-specific antibody populations could explain much of the conflicting evidence for the existence of endogenous digitalis-like compounds in plasma.     

Relationships among endogenous digitalis-like factors in essential hypertension

Elements of a hypothesis that relate endogenous digitalis-like factors to both natriuretic hormone and hypertension are briefly reviewed. The stimulus for secretion of these factors appears to involve a tendency toward a state of extracellular fluid volume expansion as a consequence of an inherited or an acquired defect in renal function. Several studies implicate the brain and, in particular, the hypothalamus in the control of the secretion. The digitalis-like factors are thought to act by partial inhibition of active sodium transport, thereby promoting increased intracellular levels of Na+ and Ca2+ in a variety of cell types. In the kidney, inhibition of sodium transport leads to a compensatory natriuresis to correct the tendency for volume overload. In smooth muscle, the inhibition of sodium transport will indirectly increase intracellular calcium levels. The increased availability of Ca2+ will elevate muscle tone and increase peripheral vascular resistance. Also presented are criteria that may be used to characterize digitalis-like activity in samples and extracts obtained from purification procedures. Finally, we review our measurements of the 6-h integrated plasma levels of digitalis-like factors and other hormones for normotensive subjects and patients with essential hypertension. The data indicate the presence of two classes of digitalis-like factors with potentially different roles in electrolyte metabolism and hypertension.     

Antidigoxin antibodies neutralize the effect of newborn endogenous digitalis-like factor on erythrocyte 86Rb uptake.

Increasing evidence indicates the existence of endogenous digitalis like factor(s) (EDLF). We recently reported on the partial purification of an EDLF from newborn (cord) blood which possesses both digoxin-like immunoreactivity and the ability to inhibit the cell membrane sodium pump measured as the inhibitory activity on erythrocyte 86Rb uptake. We here report that high affinity digoxin-binding antibodies (Fab fragments; Digibind, Burroughs Wellcome Co.) are capable of neutralizing the inhibitory activity on erythrocyte Rb uptake not only of digoxin but also of ouabain and of partially purified newborn EDLF. These results provide, to our knowledge for the first time, direct evidence that antidigitalis antibodies may cross-react with one or more circulating substances which share antigenic determinants with digoxin and ouabain and possess endogenous digitalis-like properties, strongly suggesting that these antibodies may be useful tools both for the assay of EDLF and for the study of its biological effects.     

Digitalis-like biological activity in rat cerebellum

The cytosolic fraction of rat cerebellum possesses a factor(s) which is capable of inhibiting synaptosomal Na,K-ATPase activity, competing with [3H]ouabain binding to rat brain synaptosomes, and inducing positive inotropy in guinea pig atrial strips. These results demonstrate the existence of a ouabain-like principle in rat cerebella. The inhibitory activity of the factor was found to be partially thermolabile and diminished by a proteolytic agent, and the activity could be augmented by increasing concentrations of Mg2+, suggesting a regulatory mechanism for the endogenous digitalis-like principle.     

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.     

Endogenous digitalis-like factor as a stimulator of endothelin secretion from endothelial cells

Effects of human urine-derived endogenous digitalis-like factor (EDLF) and ouabain on endothelin (ET) secretion were examined in cultured endothelial cells. ET was secreted in a linear fashion over 5 hours from bovine pulmonary artery endothelium into serum-free medium. EDLF stimulated ET secretion in a dose-dependent manner. In contrast, ouabain did not affect ET secretion at the concentration of 10(-9)-10(-5) M. These results indicate that human urine-derived EDLF is distinct from plant-derived ouabain and act as a stimulator of ET secretion by endothelial cells.     

Human hypertensive placenta contains an increased amount of Na,K-ATPase with higher affinity for cardiac glycosides

Placentas of women suffering from pregnancy-induced hypertension (PIH) were found to contain a greater amount of Na,K-ATPase molecules, estimated from anthroyl ouabain binding, than normotensive individuals. Both the microsomal fraction of placental cells and purified Na,K-ATPase showed an increased affinity for the specific inhibitor ouabain which, in the case of the microsomes, bound with a dissociation constant of 0.9 nM as compared with 3.4 nM in the controls. Likewise, the dissociation constant of the ouabain complex with purified Na,K-ATPase was about 3.5 times lower in the hypertensive patients. The differences are apparently caused by a different microenvironment of the ouabain-binding site, as reflected in the quantum yield of bound anthroyl ouabain. If an endogenous digitalis-like factor is present in the body fluids to regulate Na,K-ATPase activity, the present results render its role quite plausible.     

Endogenous digoxin-like factor: serum concentration and interrelations with the electrolyte picture in normal subjects

Endogenous Digitalis-Like Factor (DLF) is a putative hypothalamic Na+,K+-ATPase inhibitor that mediates natriuresis in response to intravascular volume expansion or sodium loading. The precise structure of this substance remains unknown; however, it cross-reacts with antibody to digoxin. Using a radioimmunoassay, we measured DLF concentrations in 26 normal subjects: mean value of this factor was 0.512 ng digoxin-equivalents/ml +/- 0.038 SEM; DLF correlated significantly with serum sodium levels (r = 0.59 - p less than 0.01) and daily urinary sodium excretion (r = 0.48 - p less than 0.05). Our results confirm that endogenous digitalis-like factor has a physiological role as regulator of natriuresis, in response to plasma sodium concentrations.     

Recovery rate of the cardiovascular system in rabbits following short-term halothane anesthesia.

Mean arterial pressure, cardiac output and heart rate were determined in eight male New Zealand white rabbits while conscious and after being anesthetized with halothane plus nitrous oxide for 15 minutes. Delivery of the anesthetic agent was stopped and the measurement repeated at 15, 30, 60 and 210 minutes. In a separate experiment blood samples were obtained for plasma renin activity in six rabbits before anesthesia, after 15 minutes of halothane plus nitrous oxide administration, and again 210 minutes after cessation of the anesthesia. Later, this experiment was repeated with the same rabbits except that they were allowed to breathe room air instead of the anesthesia. The halothane anesthesia resulted in decreased mean arterial pressure and cardiac output, but these returned to the preanesthetic levels by 15 minutes after stopping the anesthesia. Heart rate increased during halothane administration, and although it tended to return toward control levels after cessation of the halothane, heart rate was still elevated 210 minutes later. Halothane plus nitrous oxide produced an increase in plasma renin activity, which then subsided to normal by 210 minutes following anesthesia; breathing room air did not result in increases in plasma renin activity. These studies revealed that although short-term anesthesia with halothane plus nitrous oxide resulted in cardiovascular changes in rabbits, after cessation of the anesthetic agent the cardiovascular system quickly returned to normal.     

Identification of NaK-ATPase inhibitors in human plasma as nonesterified fatty acids and lysophospholipids

Elevated plasma levels of factors with cardiac glycoside-like activity have been implicated in the response to volume expansion in animals and in the pathogenesis of certain human diseases. We recently described four fractions (IR1, EI1, EI2, EI3) from normal human plasma that inhibit NaK-ATPase, displace ouabain from the enzyme, and exhibit digoxin-like immunoreactivity (Kelly, R. A., O'Hara, D. S., Canessa, M. L., Mitch, W. E., and Smith, T. W. (1985) J. Biol. Chem. 260, 11396-11405). In this report, we identify the active component of these plasma fractions as long-chain nonesterified fatty acids (NEFA) and lysophospholipids. These lipids were present in fractions EI1, EI2, and EI3 in quantities sufficient to account for all of the NaK-ATPase inhibitory activity. The digoxin-like immunoreactivity in fraction IR1 could be attributed to hydrocortisone and other endogenous steroids. To explore the nature of the lipid-NaK-ATPase interactions, we examined the effects of various ATP or sodium concentrations on the NaK-ATPase activity measured in the presence of NEFA. Varying sodium did not affect the inhibition of NaK-ATPase by linoleic acid. At less than 0.15 mM ATP, linoleic acid stimulated NaK-ATPase, but at higher ATP concentrations, the enzyme was progressively inhibited. In summary, NEFA and lysophospholipids, at levels similar to those occurring in human plasma, may account for all of the NaK-ATPase inhibitory activity observed in human plasma fractions. These lipids probably do not directly regulate NaK-ATPase in vivo under normal physiologic conditions, but may alter the sodium pump in disease states characterized by abnormalities in lipid metabolism or plasma protein binding.     

Purification and characterization of human urine-derived digitalis-like factor

We were able to purify a digitalis-like factor to apparent homogeneity from human urine based on the inhibitory effect on [3H]-ouabain binding to intact human erythrocytes. This ouabain displacing compound closely resembles ouabain in its polarity, molecular weight, non-peptidic nature and mode of action except for its UV absorbance spectrum. This compound sharing many biological activities of ouabain may be the endogenous ligand for the Na+, K+-ATPase and serve as a specific regulator of the sodium pump.     

Increase in plasma digitalis-like activity during percutaneous transluminal coronary angioplasty in patients with coronary stenosis

Despite the fact that numerous studies have been published regarding the possible presence in plasma of an endogenous Na-K pump inhibitor with a digitalis-like structure in essential hypertension, very little is known about this factor in heart disease in general, and in situations characterized by low cardiac output. We measured the ability of plasma obtained from the femoral vein to inhibit a human renal Na(+)-K+ ATPase before and immediately after percutaneous transluminal coronary angioplasty (PTCA) in 6 patients suffering from angina pectoris and severe coronary stenosis. Intraerythrocyte sodium and potassium concentrations were also measured simultaneously. Na(+)-K+ ATPase inhibition proved significantly greater after angioplasty as compared to basal activity (percentage inhibition: 31.5 +/- 7.8 vs 16.1 +/- 12.2). No significant changes in intraerythrocyte sodium and potassium were detected. Though we are not in a position to define the mechanism underlying the increase in the digitalis-like factor, a plausible hypothesis may be that the reduction in cardiac output during PTCA by raising cardiac pressures may stimulate the production of a factor of compensatory inotropic significance.     

The possible role of endogenous digitalis-like substance in the regulation of circadian changes in urinary electrolyte excretion in man

The urinary volume (U.V.), Na excretion (UNaV) and K excretion (UKV) have been reported to show a circadian rhythm in man, but the mechanism of this rhythm has not been made clear. To investigate how atrial natriuretic peptide (ANP) and endogenous digitalis-like substance (DLS) participate in the circadian change in urinary electrolyte, the circadian changes in ANP and DLS (digoxin-like immunoactivity: DLI, Na-K-ATPase inhibitor: ATPI, ouabain binding inhibitor to Na-K-ATPase: OBI) were evaluated in 5 normal man. ANP, DLI and OBI showed no significant correlation with urinary electrolyte excretion, but there was a significant positive correlation between plasma ATPI and urinary Na excretion. From these results it is suggested that circulating Na-K-ATPase inhibitor (plasma ATPI) may be involved in the regulation of the circadian rhythm of urinary Na excretion.