Involvement of the Na+, K+-ATPase α1 Isoform and Endogenous Cardiac Steroids in Depression- and Manic-like Behaviors.

Bipolar disorder (BD) is a severe and common chronic mental illness characterized by recurrent mood swings between depression and mania. The biological basis of the disease is poorly understood, and its treatment is unsatisfactory. Na+, K+-ATPase is a major plasma membrane transporter and signal transducer. The catalytic α subunit of this enzyme is the binding site for cardiac steroids. Three α isoforms of the Na+, K+-ATPase are present in the brain. Previous studies have supported the involvement of the Na+, K+-ATPase and endogenous cardiac steroids (ECS) in the etiology of BD. Decreased brain ECS has been found to elicit anti-manic and anti-depressive-like behaviors in mice and rats. However, the identity of the specific α isoform involved in these behavioral effects is unknown. Here, we demonstrated that decreasing ECS through intracerebroventricular (i.c.v.) administration of anti-ouabain antibodies (anti-Ou-Ab) decreased the activity of α1+/- mice in forced swimming tests but did not change the activity in wild type (wt) mice. This treatment also affected exploratory and anxiety behaviors in α1+/- but not wt mice, as measured in open field tests. The i.c.v. administration of anti-Ou-Ab decreased brain ECS and increased brain Na+, K+-ATPase activity in wt and α1+/- mice. The serum ECS was lower in α1+/- than wt mice. In addition, a study in human participants demonstrated that serum ECS significantly decreased after treatment. These results suggest that the Na+, K+-ATPase α1 isoform is involved in depressive- and manic-like behaviors and support that the Na+, K+-ATPase/ECS system participates in the etiology of BD.

Na+, K+-ATPase α Isoforms and Endogenous Cardiac Steroids in Prefrontal Cortex of Bipolar Patients and Controls.

Bipolar disorder is a chronic multifactorial psychiatric illness that affects the mood, cognition, and functioning of about 1-2% of the world's population. Its biological basis is unknown, and its treatment is unsatisfactory. The α1, α2, and α3 isoforms of the Na+, K+-ATPase, an essential membrane transporter, are vital for neuronal and glial function. The enzyme and its regulators, endogenous cardiac steroids like ouabain and marinobufagenin, are implicated in neuropsychiatric disorders, bipolar disorder in particular. Here, we address the hypothesis that the α isoforms of the Na+, K+-ATPase and its regulators are altered in the prefrontal cortex of bipolar disease patients. The α isoforms were determined by Western blot and ouabain and marinobufagenin by specific and sensitive immunoassays. We found that the α2 and α3 isoforms were significantly higher and marinobufagenin levels were significantly lower in the prefrontal cortex of the bipolar disease patients compared with those in the control. A positive correlation was found between the levels of the three α isoforms in all samples and between the α1 isoform and ouabain levels in the controls. These results are in accordance with the notion that the Na+, K+-ATPase-endogenous cardiac steroids system is involved in bipolar disease and suggest that it may be used as a target for drug development.

Na⁺, K⁺-ATPase Signaling and Bipolar Disorder.

Bipolar disorder (BD) is a severe and common chronic mental illness characterized by recurrent mood swings between depression and mania. The biological basis of the disease is poorly understood and its treatment is unsatisfactory. Although in past decades the "monoamine hypothesis" has dominated our understanding of both the pathophysiology of depressive disorders and the action of pharmacological treatments, recent studies focus on the involvement of additional neurotransmitters/neuromodulators systems and cellular processes in BD. Here, evidence for the participation of Na⁺, K⁺-ATPase and its endogenous regulators, the endogenous cardiac steroids (ECS), in the etiology of BD is reviewed. Proof for the involvement of brain Na⁺, K⁺-ATPase and ECS in behavior is summarized and it is hypothesized that ECS-Na⁺, K⁺-ATPase-induced activation of intracellular signaling participates in the mechanisms underlying BD. We propose that the activation of ERK, AKT, and NFκB, resulting from ECS-Na⁺, K⁺-ATPase interaction, modifies neuronal activity and neurotransmission which, in turn, participate in the regulation of behavior and BD. These observations suggest Na⁺, K⁺-ATPase-mediated signaling is a potential target for drug development for the treatment of BD.

Essential Opposite Roles of ERK and Akt Signaling in Cardiac Steroid-Induced Increase in Heart Contractility.

Interaction of cardiac steroids (CS) with the Na(+), K(+)-ATPase elicits, in addition to inhibition of the enzyme's activity, the activation of intracellular signaling such as extracellular signal-regulated (ERK) and protein kinase B (Akt). We hypothesized that the activities of these pathways are involved in CS-induced increase in heart contractility. This hypothesis was tested using in vivo and ex vivo wild type (WT) and sarcoplasmic reticulum Ca(2+) atpase1a-deficient zebrafish (accordion, acc mutant) experimental model. Heart contractility was measured in vivo and in primary cardiomyocytes in WT zebrafish larvae and acc mutant. Ca(2+) transients were determined ex vivo in adult zebrafish hearts. CS dose dependently augmented the force of contraction of larvae heart muscle and cardiomyocytes and increased Ca(2+) transients in WT but not in acc mutant. CS in vivo increased the phosphorylation rate of ERK and Akt in the adult zebrafish heart of the two strains. Pretreatment of WT zebrafish larvae or cardiomyocytes with specific MAPK inhibitors completely abolished the CS-induced increase in contractility. On the contrary, pretreatment with Akt inhibitor significantly enhanced the CS-induced increase in heart contractility both in vivo and ex vivo without affecting CS-induced Ca(2+) transients. Furthermore, pretreatment of the acc mutant larvae or cardiomyocytes with Akt inhibitor restored the CS-induced increase in heart contractility also without affecting Ca(2+) transients. These results support the notion that the activity of MAPK pathway is obligatory for CS-induced increases in heart muscle contractility. Akt activity, on the other hand, plays a negative role, via Ca(2+) independent mechanisms, in CS action. These findings point to novel potential pharmacological intervention to increase CS efficacy.

Endogenous cardiac steroids in animal models of mania.

OBJECTIVES: Bipolar disorder (BD) is a complex psychiatric disorder characterized by mania and depression. Alterations in brain Na(+) , K(+) -ATPase and cardiac steroids (CSs) have been detected in BD, raising the hypothesis of their involvement in this pathology. The present study investigated the behavioral and biochemical consequences of a reduction in endogenous brain CS activity in animal models of mania. METHODS: Amphetamine (AMPH)-induced hyperactivity in BALB/c and black Swiss mice served as a model of mania. Behavior was evaluated in the open-field test in naïve mice or in mice treated with anti-ouabain antibodies. CS levels were determined by enzyme-linked immunosorbent assay (ELISA), using sensitive and specific anti-ouabain antibodies. Extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) phosphorylation levels in the frontal cortex were determined by western blot analysis. RESULTS: Administration of AMPH to BALB/c and black Swiss mice resulted in a marked increase in locomotor activity, accompanied by a threefold increase in brain CSs. The lowering of brain CSs by the administration of anti-ouabain antibodies prevented the hyperactivity and the increase in brain CS levels. AMPH caused an increase in phosphorylated ERK (p-ERK) and phosphorylated Akt (p-Akt) levels in the frontal cortex, which was significantly reduced by administration of the antibodies. A synthetic 'functional antagonist' of CSs, 4-(3'α-15'ß-dihydroxy-5'ß-estran-17'ß-yl) furan-2-methyl alcohol, also resulted in attenuation of AMPH-induced hyperactivity. CONCLUSIONS: These results are in accordance with the notion that malfunctioning of the Na(+) , K(+) -ATPase/CS system may be involved in the manifestation of mania and identify this system as a potential new target for drug development.

Reduction in maternal circulating ouabain impairs offspring growth and kidney development.

Ouabain, a steroid present in the circulation and in various tissues, was shown to affect the growth and viability of various cells in culture. To test for the possible influence of this steroid on growth and viability in vivo, we investigated the involvement of maternal circulating ouabain in the regulation of fetal growth and organ development. We show that intraperitoneal administration of anti-ouabain antibodies to pregnant mice resulted in a >80% decline in the circulating ouabain level. This reduction caused a significant decrease in offspring body weight, accompanied by enlargement of the offspring heart and inhibition of kidney and liver growth. Kidney growth inhibition was manifested by a decrease in the size and number of nephrons. After the reduction in maternal circulating ouabain, kidney expression of cyclin D1 was reduced and the expression of the α1 isoform of the Na(+), K(+)-ATPase was increased. In addition, the elevation of proliferation signals including ERK1/2, p-90RSK, Akt, PCNA, and Ki-67, and a reduction in apoptotic factors such as Bax, caspase-3, and TUNEL were detected. During human pregnancy, the circulating maternal ouabain level increased and the highest concentration of the steroid was found in the placenta. Furthermore, circulating ouabain levels in women with small-for-gestational age neonates were significantly lower than the levels in women with normal-for-gestational age newborns. These results support the notion that ouabain is a growth factor and suggest that a reduction in the concentration of this hormone during pregnancy may increase the risk of impaired growth and kidney development.

Formation of new high density glycogen-microtubule structures is induced by cardiac steroids.

Cardiac steroids (CS), an important class of naturally occurring compounds, are synthesized in plants and animals. The only established receptor for CS is the ubiquitous Na(+),K(+)-ATPase, a major plasma membrane transporter. The binding of CS to Na(+),K(+)-ATPase causes the inhibition of Na(+) and K(+) transport and elicits cell-specific activation of several intracellular signaling mechanisms. It is well documented that the interaction of CS with Na(+),K(+)-ATPase is responsible for numerous changes in basic cellular physiological properties, such as electrical plasma membrane potential, cell volume, intracellular [Ca(2+)] and pH, endocytosed membrane traffic, and the transport of other solutes. In the present study we show that CS induces the formation of dark structures adjacent to the nucleus in human NT2 and ACHN cells. These structures, which are not surrounded by membranes, are clusters of glycogen and a distorted microtubule network. Formation of these clusters results from a relocation of glycogen and microtubules in the cells, two processes that are independent of one another. The molecular mechanisms underlying the formation of the clusters are mediated by the Na(+),K(+)-ATPase, ERK1/2 signaling pathway, and an additional unknown factor. Similar glycogen clusters are induced by hypoxia, suggesting that the CS-induced structural change, described in this study, may be part of a new type of cellular stress response.

Endogenous ouabain regulates cell viability.

The endogenous cardiac steroid-like compounds, endogenous ouabain (EO) in particular, are present in the human circulation and are considered putative ligands of the inhibitory binding site of the plasma membrane Na(+)-K(+)-ATPase. A vast amount of data shows that, when added to cell cultures, these steroids promote the growth of cardiac, vascular, and epithelial cells. However, the involvement of the endogenous compounds in the regulation of cell viability and proliferation has never been addressed experimentally. In this study, we show that EO is present in mammalian sera and cerebral spinal fluid, as well as in commercial bovine and horse sera. The lowering of serum EO concentration by the addition of specific anti-ouabain antibodies caused a decrease in the viability of several cultured cell lines. Among these, neuronal NT2 cells were mostly affected, whereas no reduction in viability was seen in rat neuroendocrine PC12 and monkey kidney COS-7 cells. The anti-ouabain antibody-induced reduction in NT2 cell viability was significantly attenuated by the addition of ouabain and was not observed in cells growing in serum-free media. Furthermore, the addition to the medium of low concentrations (nM) of the cardenolide ouabain, but not of the bufadienolide bufalin, increased NT2 and PC12 cell viability and proliferation. In addition, at these concentrations both ouabain and bufalin caused the activation of ERK1/2 in the NT2 cells. The specific ERK1/2 inhibitor U0126 inhibited both the ouabain-induced activation of the enzyme and the increase in cell viability. Furthermore, anti-ouabain antibodies attenuated serum-stimulated ERK1/2 activity in NT2 but not in PC12 cells. Cumulatively, our results suggest that EO plays a significant role in the regulation of cell viability. In addition, our findings support the notion that activation of the ERK1/2 signaling pathway is obligatory but not sufficient for the induction of cell viability by EO.

Interaction of atrial natriuretic peptide and ouabain in the myocardium.

Natriuretic peptides and digitalis-like compounds serve as regulators of homeostasis, including control of volume expansion and blood pressure. The aim of the present study was to explore possible interactions between atrial natriuretic peptide (ANP) and ouabain in the heart. ANP (1 nmol/L) had no effect in papillary muscle preparations from guinea pigs. Ouabain (1 µmol/L) induced positive inotropic effect. The addition of ANP prior to ouabain resulted in a significant decrease in the ouabain-induced positive inotropic effect, manifested as an attenuated increase in twitch maximal upward force slope and resting muscular tension. In addition, ANP caused an increase in Na⁺-K⁺-ATPase activity in heart microsomal preparations. The effect of ouabain on Na⁺-K⁺-ATPase activity was shown in a biphasic manner. Ouabain (0.01-1 nmol/L) had a small but significant increase on pump activity, but higher doses of ouabain inhibited activity. ANP attenuated ouabain-induced Na⁺-K⁺-ATPase activity. Furthermore, ouabain (50 nmol/L) or ANP (10 nmol/L) alone induced Akt activation in cardiomyocytes. However, ANP blocked ouabain-induced Akt activation. These results point to the existence of interactions between ANP and ouabain on Na⁺-K⁺-ATPase signaling and function in the heart, which may be mediated by regulation of Na⁺-K⁺-ATPase activity and (or) signal transduction mechanisms.

Pathogenic Stress Induces Human Monocyte to Express an Extracellular Web of Tunneling Nanotubes.

Actin-based tunneling nanotubes are a means of intercellular communication between remote cells. In the last decade, this type of nanotube was described in a wide variety of cell types and it became widely accepted that communication through these nanotubes is related to response to environmental changes. Few reports, however, are available regarding the expression of similar nanotubes in vivo or in primary cells. Moreover, the functional significance of this intercellular communication for health and disease is largely unknown. In this context, and as a first step in unraveling these questions, we examined the formation of similar nanotubes in primary peripheral human monocytes. To that end, we combined the use of a live cell imaging system along with advanced methods of fluorescent and scanning electron microscopy. This experimental approach reveals for the first time that the bacterial lipopolysaccharide endotoxin induces a transient expression of an unexpected abundance of actin-based tunneling nanotubes associated with vesicles. In addition, it was found that a similar response can be achieved by treating human monocytes with various bacterial and yeast membrane components, as well as with a viral component analog. In all these cases, this response is mediated by distinct complexes of toll-like receptors. Therefore, we suggest that the observed phenomena are related to a broad type of monocyte pathogen response, and raise the possibility that the phenomena described above may be involved in many clinical situations related to inflammation as a new topic of study.

Physiological roles of endogenous ouabain in normal rats.

Endogenous ouabain (EO)-like compounds are synthesized in and released from the adrenal gland. Although EO has been implicated in several pathological states such as hypertension and heart and kidney failure, its physiological roles in normal animal have not been elucidated. To address this issue, we studied the effects of reduction in plasma EO resulting from antiouabain antibody administration. Normal rats were treated for 28 days with antiouabain antibodies or rabbit IgG as control. Infusions were delivered through a jugular vein cannula by osmotic pumps, and blood pressure was monitored by tail-cuff plethysmography. The animals were housed in metabolic cages to measure water and food consumption and urine excretion. After 28 days, the thoracic aorta was isolated and used to study phenylephrine-induced contraction and atrial natriuretic peptide (ANP)-induced vasorelaxation. The adrenal gland cortex was enlarged in the antiouabain antibody-treated rats. Moreover, on the second day of treatment, there was a significant transient reduction in natriuresis in the antiouabain antibody-treated rats, suggesting that EO is a natriuretic hormone. Reduction in natriuresis was also observed when EO levels were reduced by active immunization resulting from sequential injection of ouabain-albumin. Furthermore, following 28 days of treatment, the response to phenylephrine was significantly lowered and that to ANP was significantly increased in aortic rings from antiouabain antibody-treated rats. These findings show for the first time that circulatory ouabain plausibly originating in the adrenal has physiological roles controlling vasculature tone and sodium homeostasis in normal rats.

Na+, K+-ATPase α3 isoform in frontal cortex GABAergic neurons in psychiatric diseases.

Na+, K+-ATPase is an essential membrane transporter. In the brain, the α3 isoform of Na+, K+-ATPase is vital for neuronal function. The enzyme and its regulators, endogenous cardiac steroids (ECS), were implicated in neuropsychiatric disorders. GABAergic neurotransmission was also studied extensively in diseases such as schizophrenia and bipolar disorder (BD). Post mortem brain samples from subjects with depression, schizophrenia or BD and non-psychiatric controls were provided by the Stanley Medical Research Institute. ECS levels were determined by ELISA. Expression levels of the three Na+, K+-ATPase-α isoforms, α1, α2 and α3, were determined by Western blot analysis. The α3 levels in GABAergic neurons in different regions of the brain were quantified by fluorescence immunohistochemistry. The results show that Na+, K+ -ATPase α3 isoform levels were lower in GABAergic neurons in the frontal cortex in BD and schizophrenia as compared with the controls (n = 15 subjects per group). A study on a 'mini-cohort' (n = 3 subjects per group) showed that the α3 isoform levels were also lower in GABAergic neurons in the hippocampus, but not amygdala, of bipolar and schizophrenic subjects. In the temporal cortex, higher Na+, K+ -ATPase α3 protein levels were found in the three psychiatric groups. No significant differences in ECS levels were found in this brain area. This is the first report on the distribution of α3 in specific neurons in the human brain in association with mental illness. These results strengthen the hypothesis for the involvement of Na+, K+ -ATPase in neuropsychiatric diseases.

Augmentation of Ouabain-Induced Increase in Heart Muscle Contractility by Akt Inhibitor MK-2206.

Cardiac steroids (CSs), such as ouabain and digoxin, increase the force of contraction of heart muscle and are used for the treatment of congestive heart failure (CHF). However, their small therapeutic window limits their use. It is well established that Na+, K+-ATPase inhibition mediates CS-induced increase in heart contractility. Recently, the involvement of intracellular signal transduction was implicated in this effect. The aim of the present study was to test the hypothesis that combined treatment with ouabain and Akt inhibitor (MK-2206) augments ouabain-induced inotropy in mammalian models. We demonstrate that the combined treatment led to an ouabain-induced increase in contractility at concentrations at which ouabain alone was ineffective. This was shown in 3 experimental systems: neonatal primary rat cardiomyocytes, a Langendorff preparation, and an in vivo myocardial infarction induced by left anterior descending coronary artery (LAD) ligation. Furthermore, cell viability experiments revealed that this treatment protected primary cardiomyocytes from MK-2206 toxicity and in vivo reduced the size of scar tissue 10 days post-LAD ligation. We propose that Akt activity imposes a constant inhibitory force on muscle contraction, which is attenuated by low concentrations of MK-2206, resulting in potentiation of the ouabain effect. This demonstration of the increase in the CS effect advocates the development of the combined treatment in CHF.

Reduction in endogenous cardiac steroids protects the brain from oxidative stress in a mouse model of mania induced by amphetamine.

OBJECTIVES: Bipolar disorder (BD) is a severe mental illness characterized by episodes of mania and depression. Numerous studies have implicated the involvement of endogenous cardiac steroids (CS), and their receptor, Na+, K+ -ATPase, in BD. The aim of the present study was to examine the role of brain oxidative stress in the CS-induced behavioral effects in mice. METHODS: Amphetamine (AMPH)-induced hyperactivity, assessed in the open-field test, served as a model for manic-like behavior in mice. A reduction in brain CS was obtained by specific and sensitive anti-ouabain antibodies. The level of oxidative stress was tested in the hippocampus and frontal cortex by measuring the activity of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), as well as the levels of antioxidant non-protein thiols (NPSH) and oxidative damage biomarkers thiobarbituric acid reactive substances (TBARS) and protein carbonyl (PC). RESULTS: AMPH administration resulted in a marked hyperactivity and increased oxidative stress, as manifested by increased SOD activity, decreased activities of CAT and GPx, reduced levels of NPSH and increased levels of TBARS and PC. The administration of anti-ouabain antibodies, which reduced the AMPH-induced hyperactivity, protected against the concomitant oxidative stress in the brain. CONCLUSIONS: Our results demonstrate that oxidative stress participates in the effects of endogenous CS on manic-like behavior induced by AMPH. These finding support the notion that CS and oxidative stress may be associated with the pathophysiology of mania and BD.

The digitalis-like steroid hormones: new mechanisms of action and biological significance.

Digitalis-like compounds (DLC) are a family of steroid hormones synthesized in and released from the adrenal gland. DLC, the structure of which resembles that of plant cardiac glycosides, bind to and inhibit the activity of the ubiquitous cell surface enzyme Na(+), K(+)-ATPase. However, there is a large body of evidence suggesting that the regulation of ion transport by Na(+), K(+)-ATPase is not the only physiological role of DLC. The binding of DLC to Na(+), K(+)-ATPase induces the activation of various signal transduction cascades that activate changes in intracellular Ca(++) homeostasis, and in specific gene expression. These, in turn, stimulate endocytosis and affect cell growth and proliferation. At the systemic level, DLC were shown to be involved in the regulation of major physiological parameters including water and salt homeostasis, cardiac contractility and rhythm, systemic blood pressure and behavior. Furthermore, the DLC system has been implicated in several pathological conditions, including cardiac arrhythmias, hypertension, cancer and depressive disorders. This review evaluates the evidence for the different aspects of DLC action and delineates open questions in the field.

Diverse biological responses to different cardiotonic steroids.

Cardiotonic steroids (CS) such as ouabain, digoxin and bufalin, are steroidal drugs prepared from the seeds and dried leaves of the genus Digitalis, and the skin and parotid gland of amphibians, are used as a cardiac stimulant. Steroids similar or identical to the cardiotonic steroids were identified in human tissues. The available literature unequivocally supports the notion that these endogenous CS function as hormones in mammals. Recent studies show that although similar in structure, the different CS exhibit diverse biological responses. This was shown at the molecular, cellular, tissue and whole animal levels. This review summarizes these diversities, raises a possible explanation for their presence and discusses their implication on the physiological role of the different steroids.

Cardiac steroids induce changes in recycling of the plasma membrane in human NT2 cells.

Cardiac steroids (CSs) 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 evident. In the present study, treatment of human NT2 cells with physiological concentrations (nanomolar) of CSs caused the accumulation of large vesicles adjacent to the nucleus. Experiments using N-(3-triethylammonium propyl)-4-(dibutilamino)styryl-pyrodinum dibromide, transferrin, low-density lipoprotein, and selected anti-transferrin receptor and Rab protein antibodies revealed that CSs induced changes in endocytosis-dependent membrane traffic. Our data indicate that the CS-induced accumulation of cytoplasmic membrane components is a result of inhibited recycling within the late endocytic pathway. Furthermore, our results support the notion that the CS-induced changes in membrane traffic is mediated by the Na+, K(+)-ATPase. These phenomena were apparent in NT2 cells at nanomolar concentrations of CSs and were observed also in other human cell lines, pointing to the generality of this phenomenon. Based on these observations, we propose that the endogenous CS-like compounds are physiological regulators of recycling of endocytosed membrane proteins and cargo.

Involvement of Na(+), K(+)-ATPase and endogenous digitalis-like compounds in depressive disorders.

BACKGROUND: Sodium and potassium-activated adenosine triphosphatase (Na(+), K(+)-ATPase) and endogenous digitalis-like compounds (DLC) in the brain have been implicated in the pathogenesis of mood disorders. This hypothesis was examined by the determination of Na(+), K(+)-ATPase/DLC system in parietal cortex of patients with different mood disorders and two animal models of depression. METHODS: Na(+), K(+)-ATPase concentrations in human brain synaptosomal fractions, from patients with mood disorders, schizophrenia, and normal individuals, were determined by (3)H-ouabain binding assay. Alpha isoforms were quantified by Western blotting. Brain DLC were measured using sensitive enzyme linked immunosorbant assay (ELISA). The effects of ouabain and ouabain-antibodies on behavior were determined in two animal models of depression. RESULTS: (3)H-ouabain binding in bipolar patients was significantly lower than in major depressed and schizophrenic patients. Na(+), K(+)-ATPase alpha isoforms in synaptosomal fractions were not different among the groups. DLC levels in the parietal cortex of bipolar patients were significantly higher than in normal individuals and depressed patients. Injection of lipopolysaccharide (intraperitoneally) to rats elicited depression-like symptoms, which were significantly attenuated by pre-injection of ouabain-antibodies. Injection of ouabain and ouabain-antibodies (intracerebroventricular) reduced depression-like symptoms in the forced swimming test in rats. CONCLUSIONS: The results support the possibility that Na(+), K(+)-ATPase and endogenous DLC participate in the pathogenesis of depressive disorders.

4-(3'alpha15'beta-dihydroxy-5'beta-estran-17'beta-yl)furan-2-methyl alcohol: an anti-digoxin agent with a novel mechanism of action.

The synthesis and some pharmacological properties of 4-(3'alpha-15'beta-dihydroxy-5beta-estran-17'beta-yl)furan-2-methyl alcohol (16) have been described. The compound was synthesized by reacting a synthetic 3alpha- benzyloxy-5beta-estr-15-en-17-one with the ethylene acetal of 4-bromo-2-furancarboxyaldehyde, followed by hydrolysis of the ethylene acetal and reduction of the aldehyde. Despite its resemblance to the structure of cardiac steroids (CS), 16 does not bind to the CS receptor on Na(+),K(+)-ATPase and does not increase the force of contraction of heart muscle. However, 16 inhibited the digoxin-induced increase in the force of contraction and arrhythmias in guinea pig papillary muscle and human atrial appendages. The steroid also inhibited digoxin-induced alteration in endocytosed membrane traffic, indicating a novel mechanism of action.