Ouabain attenuates cardiotoxicity induced by other cardiac steroids.

BACKGROUND AND PURPOSE: All cardiac steroids have a similar structure, bind to and inhibit the ubiquitous transmembrane protein Na(+), K(+)-ATPase and increase the force of contraction of heart muscle. However, there are diverse biological responses to different cardiac steroids both at the cellular and at the molecular level. Moreover, we have recently shown that ouabain inhibits digoxin- and bufalin-induced changes in membrane traffic. The present study was designed to test the hypothesis that ouabain also has an inhibitory effect on cardiotoxicity induced by other cardiac steroids. EXPERIMENTAL APPROACH: The hypothesis was tested in isolated heart muscle preparations and in an in vivo model of cardiotoxicity in guinea pigs. KEY RESULTS: Ouabain at a low dose attenuated the toxicity induced by bufalin and digoxin in heart muscle preparations. In addition, ouabain at the low dose (91 ng.kg(-1).h(-1)), but not at a higher dose (182 ng.kg(-1).h(-1)), delayed the development of digoxin-induced (500 microg.kg(-1).h(-1)) cardiotoxicity in anaesthetized guinea pigs, as manifested by delayed arrhythmia and terminal ventricular fibrillation, as well as a reduced heart rate. In addition, as observed with ouabain, the phosphoinositide 3-kinase inhibitor wortmannin (100 microg.kg(-1).h(-1)) delayed the digoxin-induced arrhythmia in anaesthetized guinea pigs. CONCLUSIONS AND IMPLICATIONS: The present study demonstrates the inhibitory effect, probably through signal transduction pathways, of ouabain on digoxin- and bufalin-induced cardiotoxicity in guinea pigs. Further understanding of this phenomenon could be beneficial for increasing the therapeutic window for cardiac steroids in the treatment of chronic heart failure.

Evidence of a parathyroid hormone-independent chronic effect of estrogen on renal phosphate handling and sodium-dependent phosphate cotransporter type IIa expression.

The effects of estrogen on phosphate metabolism are not well understood. To better define the chronic effects of estrogen on phosphate balance and on renal phosphate handling, the following groups were examined: A. young male and female rats, age- and weight-matched (age 8-10 weeks, 1 (st) study), and B. ovariectomized female rats (OVX), 22 weeks old, ovariectomized aged-matched rats receiving estrogen replacement (15 micromol x 3/week) for 14 weeks (OVX+E), control female rats (intact ovaries), and male rats, both age matched to OVX and OVX+E (2 (nd) Study). In younger females (1 (st) study), plasma phosphate was lower, whereas the urinary excretion of phosphate was higher than in males. In adult intact females and in OVX+E urinary excretion of phosphate was higher than in males and OVX (2 (nd) Study). In these rats, a significant correlation between plasma phosphate and estrogen level was found. Sodium-dependent phosphate cotransporter (NaPiIIa) mRNA expression and protein abundance were higher in the renal cortex of younger male rats than in age- and weight-matched females. In adult rats, NaPiIIa mRNA and protein abundance were higher in OVX than in OVX+E, and in mature males as compared with age-matched females. These differences were not related to the parathyroid hormone (PTH) levels. Chronic estrogen administration was also associated with increased plasma calcium level and urinary calcium excretion. These results suggest that chronic estrogen treatment is associated with an inhibitory, PTH-independent effect on the expression of NaPiIIa in the kidney, leading to sex-related differences in phosphate balance.

Atrial natriuretic Peptide in young and elderly children with mild gastroenteritis.

OBJECTIVE: Atrial Natriuretic Peptide (ANP) has natriuretic and diuretic effects, synthesized and stored in the atrial cells, released in response to stretch of the atrial muscle during increase venous return. Acute gastroenteritis (AGE) causes dehydration. We intend to determine whether the decrease in venous return due to dehydration would lead to a decrease in ANP levels. PATIENTS AND METHODS: This is a prospective observational controlled study. Blood collected from 30 children with AGE and ANP's levels were compared with 25 controls. ANP levels were determined by radioimmunoassay. RESULTS: The study group was in mild dehydration. As a significant difference was found in ANP levels between children in the 3mo-3y group and older children 3y-14y. We analyzed the results according to age. No difference was found between children with AGE and control, in the 3mo-3y, ANP was 12.1 +/- 11 pg/ml versus 13.4 +/- 12 pg/ml respectively, and 3 +/- 2 versus 3.8 +/- 3 pg/ml in the 3y-14y groups, respectively. CONCLUSION: Dehydration due to AGE does not change the ANP's plasma levels. A weak positive correlation between sodium levels and ANP was found r = 0.29. The significant finding of our study is the difference in ANP levels related to age, in the control as well as the GE group.

Short-term effects of cardiac steroids on intracellular membrane traffic in neuronal NT2 cells.

Cardiac steroids (CS) are specific inhibitors of Na+, K+-ATPase activity. Although the presence of CS-like compounds in animal tissues has been established, their physiological role is not clear. In a previous study we showed that in pulse-chase membrane-labeling experiments, long term (hours) interaction of CS at physiological concentrations (nM) with Na+, K+-ATPase, caused changes in endocytosed membrane traffic in human NT2 cells. This was associated with the accumulation of large vesicles adjacent to the nucleus. For this sequence of events to function in the physiological setting, however, CS would be expected to modify membrane traffic upon short term (min) exposure and membrane labeling. We now demonstrate that CS affects membrane traffic also following a short exposure. This was reflected by the CS-induced accumulation of FM1-43 and transferrin in the cells, as well as by changes in their colocalization with Na+, K+-ATPase. We also show that the CS-induced changes in membrane traffic following up to 2 hrs exposure are reversible, whereas longer treatment induces irreversible effects. Based on these observations, we propose that endogenous CS-like compounds are physiological regulators of the recycling of endocytosed membrane proteins and cargo in neuronal cells, and may affect basic mechanisms such as neurotransmitter release and reuptake.

Diverse effects of stress and additional adrenocorticotropic hormone on digitalis-like compounds in normal and nude mice.

Digitalis-like compounds (DLC) are steroidal hormones that are synthesized in, and released from, the adrenal gland, whose regulation may be directed by the hypothalamic-pituitary-adrenal (HPA) axis. Increasing evidence points to antitumour properties of these compounds and we hypothesized that the establishment of tumours in athymic nude mice may be facilitated by an abnormal synthesis or secretion of DLC. To explore this hypothesis, DLC concentrations were determined in the plasma, and in adrenal and hypothalamic tissues of nude compared to normal mice under basal conditions, and 30 min after a stress stimulus (i.p. injection of 100 micro l saline) with or without additional adrenocorticotropic hormone (ACTH) 1 micro g/per animal. Simultaneously, plasma corticosterone and serum adrenocorticotropic hormone (ACTH) concentrations were analysed. The basal DLC concentrations were similar in the plasma and the hypothalamus of both strains, whereas the basal adrenal DLC concentration was significantly lower in the nude mice compared to normal mice. The stress stimulus induced in normal mice a significant increase in DLC concentrations in the adrenal gland, the plasma and the hypothalamus. However, in nude mice, it caused an increase only in the adrenal gland and the hypothalamus, whereas the plasma DLC concentration was not affected. In both strains, the administration of ACTH in addition to injection stress did not provoke a further increase in DLC concentrations while inducing a significant increase in plasma corticosterone concentration. Regardless of the applied stimulus, the nude mice expressed significant lower DLC concentrations in the adrenal gland and the plasma compared to normal mice. The low basal adrenal DLC concentration in nude mice and their impaired DLC response towards stress- and ACTH stimulation both support an involvement of DLC in tumorigenesis.

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.

Dietary phosphate-dependent growth is not mediated by changes in plasma phosphate concentration.

The present study was undertaken in order to assess the role of dietary phosphate in growth. A diet deficient in phosphate led to a suppression of growth in juvenile rats. The phenomenon is similar to that described for diets deficient in other essential single components such as Mg, Zn or K. However, unlike the other constituents, dietary phosphate restriction affected the growth rate much more than it altered the serum phosphate concentration; addition of phosphate to the drinking water of rats fed a low-phosphate diet restored the growth rate without a concomitant change in serum phosphate concentration. The suppression of growth rate by the deletion of phosphate was associated with a delayed decrease in food intake. This finding implies that the variation in appetite was secondary to the change in growth. The increase in body weight following phosphate supplementation was associated with a concomitant increment in food intake. The phosphate-dependent growth was, however, evident also in rats that were pair-fed with those that were not supplied with phosphate. It is concluded that dietary phosphate-dependent growth is not mediated by changes in phosphate concentrations in the extracellular fluid. It is plausible that signals arising from receptors for phosphate in the digestive system constitute part of the growth control apparatus in rats.

Digitalis and digitalislike compounds down-regulate gene expression of the intracellular signaling protein 14-3-3 in rat lens.

Na+,K+-ATPase activity in the epithelial layer is fundamental to the maintenance of ionic concentration gradients and transparency of the lens. Recently we have identified endogenous digitalislike compounds (DLC), 19-norbufalin and its peptide derivatives, in human cataractous lenses (Lichtstein et al. Eur J Biochem 216: 261-268, 1993). Lenses were treated with 10 nM ouabain, bufalin or 19-norbufalin derivative for 24 h and were compared to control lenses. Differential display analysis revealed that one of the down-regulated genes was 14-3-3 theta. Down-regulation was confirmed by Northern blot and by RT-PCR analysis. RT-PCR of additional 14-3-3 isoforms revealed that the eta and gamma isoforms of 14-3-3 are also down-regulated by ouabain, bufalin and 19-norbufalin derivative, whereas the zeta isoform is down-regulated only by bufalin. These results demonstrate that one of the consequences of Na+,K+-ATPase inhibition by exogenous or endogenous inhibitors is the down-regulation of mRNA transcripts encoding several isoforms of 14-3-3. Since the 14-3-3 proteins are multifunctional regulatory proteins, the reduction in the abundance of various isoforms will have profound effects on cell function. Furthermore, These results, together with the demonstration of digitalislike compounds in the normal lens, and their increased level in human cataractous lenses, strongly suggests their involvement in the molecular mechanisms responsible for cataract formation.

Use of a lipophilic cation to monitor electrical membrane potential in the intact rat lens.

PURPOSE: Tetraphenylphosphonium (TPP+) is a permeant lipophilic cation that accumulates in cultured cells and tissues as a function of the electrical membrane potential across the plasma membrane. This study was undertaken to determine whether TPP+ can be used for assessing membrane potential in intact lenses in organ culture. METHODS: Rat lenses were cultured in media containing 10 microM TPP+ and a tracer level of 3H-TPP+ for various times. 3H-TPP+ levels in whole lenses or dissected portions of lenses were determined by liquid scintillation counting. Ionophores, transport inhibitors, and neurotransmitters were also added to investigate their effects on TPP+ uptake. RESULTS. Incubation of lenses in low-K+ balanced salt solution and TC-199 medium, containing physiological concentrations of Na+ and K+, led to a biphasic accumulation of TPP+ in the lens that approached equilibrium by 12 to 16 hours of culture. The TPP+ equilibrated within 1 hour in the epithelium but penetrated more slowly into the fiber mass. The steady state level of TPP+ accumulation in the lens was depressed by 90% when the lenses were cultured in a medium containing high K+. The calculated membrane potential for the normal rat lens in TC-199 was -75 +/- 3 mV. Monensin (1 microM) and nigericin (1 microM), Na+H+ and K+H+ exchangers respectively, as well as the protonophore carbonylcyanide-m-chlorophenylhydrazone (CCCP, 10 microM) and the calcium ionophore A23187 (10 microM), abolished TPP+ accumulation and caused cloudiness of the lenses. The neurotransmitter acetylcholine at 50 microM decreased TPP+ accumulation in the lens, but this effect could be prevented by simultaneous application of 1 mM atropine. CONCLUSIONS: TPP+ accumulation can be used as an indicator of changes in membrane potential in intact lenses, but because of the long time required to reach steady state, its utility is limited. The slow accumulation of TPP+ and its slow efflux from the lens under conditions known to depolarize membranes are consistent with a diffusion barrier in the deep cortex and nucleus of the lens.

The mood cycle hypothesis: possible involvement of steroid hormones in mood regulation by means of Na+, K+-ATPase inhibition.

The mood cycle hypothesis attempts to propose a model for mood regulation based on current data. The hypothesis contends that steroid hormones inhibit sodium-potassium adenosine triphosphatase (Na+, K+-ATPase; Na+ pump) in the hypothalamus, either directly or by converting into digitalis-like compounds. This inhibition stimulates beta-endorphin (beta-E) secretion, which is normally construed as elevated mood. In turn, beta-E inhibits steroid secretion, thus completing negative feedback loops. These loops are collectively termed the mood cycle.

Reducing the risk of malpractice claims.

Many physicians will be sued for malpractice at some time during their careers. Risk of litigation can be reduced by adopting practices that include keeping thorough medical records, educating office personnel, and fostering good patient-physician relationships. The last is important because patients who view their physicians as caring tend not to sue even if an adverse outcome occurs.

Na+, K+-ATPase inhibitors down-regulate gene expression of the intracellular signaling protein 14-3-3 in rat lens.

To identify genes that are differentially expressed by Na+, K+-ATPase inhibitors, we used the differential display technique to compare mRNA expression patterns in rat lens. Lenses were treated with 10 microM ouabain, bufalin, or 19-norbufalin derivative for 24 h and were compared with control lenses. Differential display analysis revealed that one of the down-regulated genes was 14-3-3 theta. Down-regulation was confirmed by Northern blot and by reverse transcription-polymerase chain reaction analysis. Reverse transcription-polymerase chain reaction of additional 14-3-3 isoforms revealed that the eta and gamma isoforms of 14-3-3 are also down-regulated by ouabain, bufalin, and 19-norbufalin derivative, whereas the zeta isoform is down-regulated only by bufalin. Down-regulation of the 14-3-3 isoforms occurred without a significant change in gamma-crystallin gene expression. These results demonstrate that one of the consequences of Na+, K+-ATPase inhibition by exogenous or endogenous inhibitors is the down-regulation of mRNA transcripts encoding several isoforms of 14-3-3. Because the 14-3-3 proteins are multifunctional regulatory proteins, the reduction in the abundance of various isoforms will have profound effects on cell function.

The effects of digitalis-like compounds on rat lenses.

PURPOSE: Fundamental to the maintenance of ionic concentration gradients and transparency of the lens is the activity of Na+,K+-adenosine triphosphatase (ATPase) in the epithelial layer. Recent studies have identified endogenous digitalis-like compounds (DLCs) and 19-norbufalin and its peptide derivatives in human cataractous lenses. These compounds inhibit the activity of Na+,K+-ATPase and have been suggested to be involved in cataract formation. The present experiments were designed to test this hypothesis by determining the ability of digitalis and DLCs to induce changes in protein composition and leakage from rat lenses in organ culture. METHODS: DLCs were determined in rat lenses using three independent assays: interaction with ouabain antibodies, interaction with bufalin antibodies, and inhibition of [3H]-ouabain binding to red blood cells. Rat lenses were incubated in modified TC-199 medium in 5% CO2 atmosphere at 37 degrees C for the time of the experiment. The onset of cataractogenesis was assessed by measuring protein leakage from lenses and by crystallin composition in the lens and media. RESULTS: DLCs were present in rat lens with concentrations 7 to 30 times higher in the capsular-epithelial layer than in the lens fibers regions. Ouabain, bufalin, digoxin, and DLC induced dose- and time-dependent leakage of protein from rat lenses. Lenses incubated with these compounds showed alterations in crystallin content consistent with changes that initiate opacity. All the compounds caused a multilayering of epithelial cells in the region surrounding the mitotic area and, at the same time, cell death in the central anterior region. CONCLUSIONS: Digitalis and endogenous DLCs are cataractogenic factors. These results, together with the demonstration of DLCs in the normal lens and their increased levels in human cataractous lenses, strongly suggest their involvement in the molecular mechanisms responsible for cataract formation.

Massive gastrointestinal bleeding from Meckel's diverticulum in a 91-year-old man.

Although Meckel's diverticulum is the most common congenital abnormality of the gastrointestinal (GI) tract, complications in adults are rare, especially in the elderly. Intestinal obstruction is the most common complication in the adult, and inflammation mimicking acute appendicitis may also occur. Lower GI bleeding as a result of Meckel's diverticulum with ectopic gastric mucosa is distinctly unusual among the elderly, with most previous case reports involving patients under the age of 40. The case we report involved a 91-year-old man with massive lower GI hemorrhage found to be due to a Meckel's diverticulum with ectopic gastric mucosa.

Bufodienolides as endogenous Na+, K+-ATPase inhibitors: biosynthesis in bovine and rat adrenals.

The biosynthesis of digitalis-like compounds (DLC) was determined in bovine and rat adrenal homogenates by following changes in the concentration of DLC using three independent sensitive bioassays: inhibition of [3H]-ouabain binding to red blood cells and competitive ouabain and bufalin ELISA. The amounts of DLC in bovine and rat adrenal homogenates, as measured by the two first bioassays, increased with time when the mixtures were incubated under tissue culture conditions. These results suggest that Na+, K+-ATPase inhibitors which interact with ouabain antibodies, but not those which interact with bufalin antibodies, are synthesized in bovine and rat adrenals.

Biosynthesis of digitalis-like compounds in rat adrenal cells: hydroxycholesterol as possible precursor.

The biosynthesis of digitalis-like compounds (DLC) was determined in bovine and rat adrenal homogenates, as well as in primary rat adrenal cells, by following changes in the concentration of DLC using three independent sensitive bioassays: inhibition of [3H]-ouabain binding to red blood cells and competitive ouabain and bufalin ELISA. The amounts of DLC in bovine and rat adrenal homogenates, as measured by the two first bioassays, increased with time when the mixtures were incubated under tissue culture conditions. Rat primary adrenal cells were incubated in the presence of [1,2-(3)H]-25-hydroxycholesterol, [26,27-(3)H]-25-hydroxycholesterol or [7-(3)H]-pregnenolone. The radioactive products, as well as the digitalis-like activity, were fractionated by three sequential chromatography systems. When [1,2-(3)H]-25-hydroxycholesterol or [7-(3)H]-pregnenolone was added to the culture medium, the radioactivity was co-eluted with digitalis-like activity, suggesting that at least one of the DLC might originate in hydroxycholesterol. In contrast, when the culture medium was supplemented with [26,27-(3)H]-25-hydroxycholesterol, the radioactivity was not co-eluted with the digitalis-like activity, indicating that side chain cleavage is the first step in the synthesis of digitalis-like compounds by rat adrenal.

Ouabain-sensitive Na+,K(+)-ATPase activity in toad brain.

Toads of the genus Bufo are highly resistant to the toxic effects of digitalis glycosides, and the Na+,K(+)-ATPase of all toad tissues studied to date has been relatively insensitive to inhibition by digitalis and related compounds. In studies of brain microsomal preparations from two toad species, Bufo marinus and Bufo viridis, inhibition of ATPase activity and displacement of [3H]ouabain from Na+,K(+)-ATPase occurred over broad ranges of ouabain or bufalin concentrations, consistent with the possibility that more than one Na+,K(+)-ATPase isoform may be present in toad brain. The data could be fitted to one- or two-site models, both of which were consistent with the presence of Na+,K(+)-ATPase activity with high sensitivity to ouabain and bufalin. Ki (concentration capable of producing 50% inhibition of activity) values for ouabain in the one-site model were in the 0.2 to 3.7 microM range, whereas Ki1 values in the two-site model ranged from 0.085 to 0.85 microM, indicating that brain ATPase was at least three orders of magnitude more sensitive to ouabain than B. marinus bladder ATPase (Ki = 5940 microM). Ouabain was also an effective inhibitor of 86Rb+ uptake in B. marinus brain tissue slices (Ki = 3.1 microM in the one-site model; Ki1 = 0.03 microM in the two-site model). However, the relative contribution of the high ouabain-sensitivity site to the total activity was 17% in the transport assay as compared with 63% in the Na+,K(+)-ATPase enzymatic assay. We conclude that a highly ouabain-sensitive Na+,K(+)-ATPase activity is present and functional in toad brain but that its function may be partially inhibited in vivo.