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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.

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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'β-dihydroxy-5'β-estran-17'β-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., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: The developmental origin of the c-kit expressing progenitor cell pool in the adult heart has remained elusive. Recently, it has been discovered that the injured heart is enriched with c-kit(+) cells, which also express the hematopoietic marker CD45., Methods and Results: In this study, we characterize the phenotype and transcriptome of the c-kit+/CD45+/CD11b+/Flk-1+/Sca-1±(B-type) cell population, originating from the left atrial appendage. These cells are defined as cardiac macrophage progenitors. We also demonstrate that the CD45+ progenitor cell population activates heart development, neural crest and pluripotency-associated pathways in vitro, in conjunction with CD45 down-regulation, and acquire a c-kit+/CD45-/CD11b-/Flk-1-/Sca-1+ (A-type) phenotype through cell fusion and asymmetric division. This putative spontaneous reprogramming evolves into a highly proliferative, partially myogenic phenotype (C-type)., Conclusions: Our data suggests that A-type cells and cardiac macrophage precursor cells (B-type) have a common lineage origin, possibly resolving some current conundrums in the field of cardiac regeneration., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

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., (Copyright © 2015 by the American Society of Nephrology.)

The cardiac steroid ouabain binds to Na(+), K(+)-ATPase and inhibits its activity. Administration of the compound to animals and humans causes an increase in the force of contraction of heart muscle and stabilizes heart rate. In addition, this steroid promotes the growth of cardiac, vascular, and neuronal cells both in vitro and in vivo. We studied the effects of ouabain on mouse recovery following closed head injury (CHI), a model for traumatic brain injury. We show that chronic (three times a week), but not acute, intraperitoneal administration of a low dose (1 μg/kg) of ouabain significantly improves mouse recovery and functional outcome. The improvement in mouse performance was accompanied by a decrease in lesion size, estimated 43 d following the trauma. In addition, mice that underwent CHI and were treated with ouabain showed an increase in the number of proliferating cells in the subventricular zone and in the area surrounding the site of injury. Determination of the identity of the proliferating cells in the area surrounding the trauma showed that whereas there was no change in the proliferation of endothelial cells or astrocytes, neuronal cell proliferation almost doubled in the ouabain-treated mice in comparison with that of the vehicle animals. These results point to a neuroprotective effects of low doses of ouabain and imply its involvement in brain recovery and neuronal regeneration. This suggests that ouabain and maybe other cardiac steroids may be used for the treatment of traumatic brain injury.

Natriuretic hormones (NH) include three groups of compounds: the natriuretic peptides (ANP, BNP and CNP), the gastrointestinal peptides (guanylin and uroguanylin), and endogenous cardiac steroids. These substances induce the kidney to excrete sodium and therefore participate in the regulation of sodium and water homeostasis, blood volume, and blood pressure (BP). In addition to their peripheral functions, these hormones act as neurotransmitters or neuromodulators in the brain. In this review, the established information on the biosynthesis, release and function of NH is discussed, with particular focus on their role in brain function. The available literature on the expression patterns of each of the NH and their receptors in the brain is summarized, followed by the evidence for their roles in modulating brain function. Although numerous open questions exist regarding this issue, the available data support the notion that NH participate in the central regulation of BP, neuroprotection, satiety, and various psychiatric conditions, including anxiety, addiction, and depressive disorders. In addition, the interactions between the different NH in the periphery and the brain are discussed.

Context: Many encourage service learning and health advocacy training in medical student education, but related evaluation is limited., Objectives: To assess (1) impact of a required community health advocacy training for medical students on student attitudes, knowledge, and skills; (2) student characteristics associated with higher advocacy knowledge and skills; and (3) perspectives of community-based organizations (CBOs)., Design: Cross-sectional surveys., Setting: University of Miami Miller School of Medicine (UMMSM) Regional Medical Campus and main campus., Participants: Medical students at both UMMSM campuses., Intervention: Required community health advocacy training for first- and second-year students including classroom experiences and hands-on project in partnership with a CBO., Main Outcome Measures: Student characteristics, health advocacy-related attitudes, self-reported and objective knowledge, and skills. Scores were compared between campuses, with multivariable modeling adjusting for individual student characteristics. Community-based organization perspectives were assessed via separate surveys., Results: Ninety-eight (77%) regional campus students (intervention group) and 139 (30%) main campus students (comparison group) completed surveys. Versus the comparison group, the intervention group reported greater: mean knowledge of community health needs: 34.6 versus 31.1 (range: 11-44, P < .01), knowledge about CBOs: 3.0 versus 2.7 (range 1-4, P < .01) and knowledge of community resources: 5.4 versus 2.3 (range, 0-11, P < .01), and mean skill scores: 12.7 versus 10.5 (score range: 4-16, P < .01), following the intervention. Using adjusted analysis across both groups, female gender was associated with higher attitudes score. High level of previous community involvement was associated with higher attitude and skill scores. Higher self-reported educational debt was associated with higher skill scores. Community-based organization perspectives included high satisfaction and a desire to influence the training of future physicians., Conclusions: Medical student advocacy training in partnership with community-based organizations could be beneficial in improving student advocacy knowledge and skills in addressing community health issues and in developing sustainable community partnerships.

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.

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.

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.

Depressive disorders are among the world's greatest public health problems. Na(+), K(+)-ATPase is the established receptor for the steroidal digitalis-like compounds (DLC). Alteration in brain Na(+), K(+)-ATPase and DLC have been detected in depressive disorders raising the hypothesis of their involvement in these pathology. The present study was designed to further elaborate this hypothesis by investigating the behavioral and biochemical consequences of neutralization in brain DLC activity attained by anti-ouabain antibodies administrations, in normal Sprague-Dawley (SD) and in the Flinders Sensitive Line (FSL) of genetically depressed rats. Chronic i.c.v. administration of anti-ouabain antibodies to FSL rats elicited anti-depressive behavior. Administration of anti-ouabain antibodies intracerebroventriculary (i.c.v.) to SD rats significantly changed the levels of catecholamines and their metabolites in the hippocampus, ventral tegmentum and nucleus accumbence. These results are in accordance with the notion that endogenous DLC may be involved in the manifestation of depressive disorders and suggests that alteration in their levels may be of significant therapeutic value., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Plasma membrane (PM) Na(+), K(+)-ATPase, plays crucial roles in numerous physiological processes. Cardiac steroids (CS), such as ouabain and bufalin, specifically bind to the Na(+), K(+)-ATPase and affect ionic homeostasis, signal transduction, and endocytosed membrane traffic. CS-like compounds, synthesized in and released from the adrenal gland, are considered a new family of steroid hormones. Previous studies showed that ouabain induces slow Ca(2+) oscillations in COS-7 cells by enhancing the interactions between Na(+), K(+)-ATPase, inositol 1,4,5-trisphosphate receptor (IP(3)R) and Ankyrin B (Ank-B) to form a Ca(2+) signaling micro-domain. The activation of this micro-domain, however, is independent of InsP3 generation. Thus, the mechanism underlying the induction of these slow Ca(2+) oscillations remained largely unclear. We now show that other CS, such as bufalin, can also induce Ca(2+) oscillations. These oscillations depend on extracellular Ca(2+) concentrations [Ca(2+)](out) and are inhibited by Ni(2+). Furthermore, we found that these slow oscillations are Na(+)(out) dependent, abolished by Na(+)/Ca(2+) exchanger1 (NCX1)-specific inhibitors and markedly attenuated by NCX1 siRNA knockdown. Based on these results, a model is presented for the CS-induced slow Ca(2+) oscillations in COS-7 cells., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Although the detailed mechanism of spontaneous generation and regulation of rapid eye movement sleep (REMS) is yet unknown, it has been reported that noradrenergic REM-OFF neurons in the locus coeruleus (LC) cease firing during REMS and, if they are kept active, REMS is significantly reduced. On the other hand, the activity as well as expression of Na-K ATPase has been shown to increase in the LC following REMS deprivation. Ouabain is a specific inhibitor of Na-K ATPase, and endogenous ouabain-like compounds are present in the brain. These findings led us to propose that a decrease in the level of ouabain-like compounds spontaneously available in and around the LC would stimulate and increase the REM-OFF neuronal activities in this region and thus would reduce REMS. To test this hypothesis, we generated anti-ouabain antibodies and then microinjected it bilaterally into the LC in freely moving chronically prepared rats and recorded electrophysiological signals for evaluation of sleep-wakefulness states; suitable control experiments were also conducted. Injection of anti-ouabain antibodies into the LC, but not into adjacent brain areas, significantly reduced percent REMS (mean +/- SEM) from 7.12 (+/-0.74) to 3.63 (+/-0.65). The decrease in REMS was due to reduction in the mean frequency of REMS episode, which is likely due to increased excitation of the LC REM-OFF neurons. Control microinjections of normal IgG did not elicit this effect. These results support our hypothesis that interactions of naturally available endogenous ouabain-like compounds with the Na-K ATPase in the LC modulate spontaneous REMS.

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.

Background: The sodium- and potassium-activated adenosine triphosphatase (Na+, K+-ATPase) is a major plasma membrane transporter for sodium and potassium. We recently suggested that bipolar disorders (BD) may be associated with alterations in brain Na+, K+-ATPase. We further conjectured that the differences in Na+, K+-ATPase in BD patients could result partially from genetic variations in Na+, K+-ATPase alpha isoforms., Methods: To test our hypothesis, we undertook a comprehensive study of 13 tagged single nucleotide polymorphisms (SNPs) across the three genes of the brain alpha isoforms of Na+, K+- ATPase (ATP1A1, ATP1A2, and ATP1A3, which encode the three alpha isoforms, alpha1, alpha2, and alpha3, respectively) identified using HapMap data and the Haploview algorithm. Altogether, 126 subjects diagnosed with BD from 118 families were genotyped (parents and affected siblings). Both individual SNPs and haplotypes were tested for association using family-based association tests as provided in the UNPHASED and PBAT set of programs., Results: Significant nominal association with BD was observed for six single SNPs (alpha1: rs11805078; alpha2: rs2070704, rs1016732, rs2854248, and rs2295623; alpha3: rs919390) in the three genes of Na+, K+-ATPase alpha isoforms. Haplotype analysis of the alpha2 isoform (ATP1A2 gene) showed a significant association with two loci haplotypes with BD (rs2295623: rs2070704; global p value = .0198, following a permutation test)., Conclusions: This study demonstrates for the first time that genetic variations in Na+, K+-ATPase are associated with BD, suggesting a role of this enzyme in the etiology of this disease.

Natriuretic peptides (NP), including atrial natriuretic peptide (ANP), induce potent natriuresis and vasodilation and thereby generate hypotension in vivo. Despite intensive efforts, clinical application of NP as an antihypertensive agent is limited because of their short biological half-life and poor bioavailability. Recently, we have developed a strategy that facilitates slow release of peptides from PEG-peptide inactive conjugates, based on reversible pegylation. Peptides prepared by this approach undergo slow, spontaneous chemical hydrolysis at physiological conditions, releasing the native active peptide/protein drug from the inactive conjugates over prolonged periods. A PEG chain of 30 kDa was linked covalently to the alpha-amino side chain of the hormone via a MAL-Fmoc-NHS spacer, yielding PEG 30-Fmoc-ANP, a prodrug that releases the native hormone upon incubation at physiological conditions. Bolus administration of native ANP to Wistar rats receiving adrenaline yields a short, transitory effect in lowering blood pressure (BP), reaching a maximum at 2 min, and then returning to control values after 12 to 25 min. In contrast, administration of PEG 30-Fmoc-ANP lowered BP following a lag period of 50 min, and maintained low BP for a period exceeding 60 min. Saline or PEG 30-Fmoc-Alanine were not effective in lowering BP in Wistar rats. These results show that the novel compound, PEG 30-Fmoc-ANP, is a reversible pegylated prodrug derivative that facilitates a prolonged BP lowering effect in rats and may be considered as a candidate for development into an antihypertensive drug.

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.

Plasma membrane Na(+)-K(+)-ATPase, which drives potassium into and sodium out of the cell, has important roles in numerous physiological processes. Cardiac steroids (CS), such as ouabain and bufalin, specifically interact with the pump and affect ionic homeostasis, signal transduction, and endocytosed membrane traffic. CS-like compounds are present in mammalian tissues, synthesized in the adrenal gland, and considered to be new family of steroid hormones. In this study, the mechanism of Na(+)-K(+)-ATPase involvement in the regulation of endocytosis is explored. We show that the effects of various CS on changes in endosomal pH are mediated by the pump and correspond to their effects on endosomal membrane traffic. In addition, it was found that CS-induced changes in endocytosed membrane traffic were dependent on alterations in [Na(+)] and [H(+)] in the endosome. Furthermore, we show that various CS differentially regulate endosomal pH and membrane traffic. The results suggest that these differences are due to specific binding characteristics. Based on our observations, we propose that Na(+)-K(+)-ATPase is a key player in the regulation of endosomal pH and endocytosed membrane traffic. Furthermore, our results raise the possibility that CS-like hormones regulate differentially intracellular membrane traffic.

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.

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.

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.

Cholestasis is a common feature of several malignant diseases, including pancreatic, hepatic, gallbladder, and ampullary carcinomas. It is usually secondary to main bile duct obstruction or widespread hepatic metastasis, but it can also be a paraneoplastic syndrome of other underlying malignancies. Stauffer's syndrome is a rare paraneoplastic manifestation of renal cell carcinoma (RCC) that is characterized by elevated alkaline phosphatase, erythrocyte sedimentation rate, alpha-2-globulin, and gamma-glutamyl transferase, thrombocytosis, prolongation of prothrombin time, and hepatosplenomegaly, in the absence of hepatic metastasis and jaundice. A rare variant of this syndrome with jaundice has recently been described in 3 cases in the literature. We report a patient who presented with abdominal pain and cholestatic jaundice in whom RCC was incidentally found during initial workup. Jaundice and liver dysfunction resolved completely after surgical resection of the tumor. This case illustrates the protean manifestations of RCC, and the importance of considering Stauffer's syndrome and its variant in the differential diagnosis of anicteric and icteric cholestasis, which may allow early recognition and treatment of an underlying malignancy.

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.

Atrial natriuretic peptide (ANP) has known natriuretic, diuretic, and vasodilatatory effects. It is synthesized and stored in the atrial cells. Stretching of the atrial muscle fibers during an increase in venous return sets a response of ANP release into the blood stream. High levels of ANP were measured in a number of lung diseases. Pneumonia in children is frequently accompanied by the hyponatremia of the syndrome of inappropriate antidiuretic hormone (ADH) secretion (SIADH). High levels of ANP were found among patients with SIADH. Our objective was to determine if ANP plasma levels are altered in children with pneumonia, and to evaluate a possible correlation between severity of pneumonia and ANP levels. Blood samples from 28 children diagnosed with pneumonia were collected. Plasma ANP levels were determined by radioimmunoassay and compared to levels in 25 children without pneumonia. ANP levels in the pneumonia group (mean +/- SD, 16.02 +/- 11.69 pg/ml) increased significantly (P < 0.01) compared to levels in the control group (mean +/- SD, 7.44 +/- 9.29 pg/ml). Children in the pneumonia group also exhibited low levels of plasma sodium (mean +/- SD, 134.88 +/- 2.5 mmol/l) compared to levels in children without pneumonia (mean +/- SD, 139.77 +/- 4.15 mmol/l) (P < 0.01). There was no correlation between ANP plasma levels and severity of pneumonia. In conclusion, ANP levels in children with pneumonia, as in other lung diseases, are increased. High ANP levels may play a role in maintaining water and electrolyte equilibrium during a state of inappropriate ADH secretion accompanying pneumonia., (2005 Wiley-Liss, Inc.)

Dietary phosphate has profound effects on growth and renal handling of the compound. On the basis of changes in growth rate and food intake, after alterations in phosphate load, our laboratory previously suggested that these effects are mediated by intestinal signals (Landsman A, Lichtstein D, Bacaner M, and Ilani A. Br J Nutr 86: 217-223, 2001). The aim of this study was to further evaluate the role of dietary phosphate on food intake and appetite and specific organ growth, and to test for the presence of a serum factor that may affect renal phosphate handling in phosphate-resupplied rats. The experimental design was based on a comparison between groups of rats receiving identical low-phosphate diets but drinking water containing either phosphate or chloride. We show that 1) changes in food intake after alterations in phosphate load occurred in parallel with variations in digestive system distention, suggesting that dietary phosphate has also a direct effect on appetite; 2) dietary phosphate-dependent growth has a specific effect on the growth of liver and epididymal fat; and 3) serum of rats supplied with phosphate contains a factor that inhibits sodium-dependent phosphate transport in a model of renal proximal tubule cells. Collectively, these observations are in accord with the hypothesis that factor(s) emanating from the digestive system in response to dietary phosphate load may be involved in growth, appetite and renal handling of phosphate.

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.

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.

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.

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.

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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.

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.

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.

Sodium-dependent phosphate entry into neuronal cells was demonstrated in synaptic plasma membrane vesicles and synaptosomes prepared from rat brains, in PC12 cells and in primary culture of pituitary cells. The extent of the sodium-dependent phosphate transport in the synaptic plasma membrane preparation, at [Na]out = 110 mM and [P(i)]out = 0.1 mM, varied between 0.28 to 1.02 nmol phosphate/mg membrane protein/min. In pituitary cells the value was only about 0.05 nmol P(i)/mg protein/min. In PC12 cells the activity increased from 0.0085 to 0.26 nmol P(i)/mg protein/min in the transit from undifferentiated to differentiated cells. The dependence of phosphate on sodium concentrations fits a model in which two sodium ions are required to transfer the phosphate into the cells with a K[Na]0.5 of 43 mM. The K(m) for the phosphate transport in the synaptic plasma membrane preparations was between 0.1 and 0.45 mM. It is concluded that sodium-driven active transport of phosphate is a ubiquitous activity in various types of neuronal cells.

Atrial natriuretic peptides (ANP) are a family of humoral compounds released from the heart atria and involved in water and salt homeostasis. ANP immunoreactivity and ANP-binding sites were also found in several areas of the central nervous system including the olfactory bulb. In the present study, the possible involvement of ANP in olfaction was tested by measuring the content and distribution of IR-ANP and ANP-binding sites in rat olfactory bulb in control and rats. The results implicate ANP in the processes leading to olfactory perception.

The binding of atrial natriuretic peptide (ANP) to rat olfactory bulb membranes and synaptosomes was examined. [125I]ANP (rat, 99-126) bound specifically to a single class of binding site on olfactory bulb membrane preparation with dissociation constant (Kd) of 106 pM and maximum binding capacity (Bmax) of 13.6 fmol/mg protein. Comparable results were obtained when the binding was characterized using displacement and kinetic experiments. The ring deleted analog of ANP, C-ANP (rat, 4-23) displaced [125I]ANP only minimally from its binding site in the membrane preparation. Saturation, displacement and blocking experiments on [125I]ANP binding to rat olfactory bulb synaptosomes revealed the presence of two distinct binding sites. Simultaneous analysis of homogeneous and heterogeneous displacement curves and blocking experiments revealed the quantitative characteristics of these receptors to be: Kd1 = 44 pM, Bmax1 = 42 fmol/mg protein and Kd2 = 1050 pM, Bmax2 = 173 fmol/mg protein, for the high and low affinity binding sites, respectively. Kinetic experiments further confirmed the differences between the receptors present in the membranes and the synaptosomes preparations. The demonstration of multiple ANP binding sites in olfactory bulb synaptosomes but not membrane preparations raises the possibility of a particular function of ANP in nerve terminals.

The Na+, K(+)-activated adenosine triphosphatase (ATPase) is present in the membrane of eukaryotic cells and represent a major pathway for Na+ and K+ transport across the plasma membrane. Cardiac glycosides, such as digoxin or ouabain, inhibit this enzyme activity by binding to a specific receptor on the membrane. Studies conducted in this and other laboratories have proven the existence of digitalis-like compounds in animal and human tissues which may serve as regulators, in vivo, of the Na+, K(+)-pump activity. The levels of digitalis-like compounds in the plasma are increased in hypertension and other illnesses. A possible link at the cellular and molecular level between these compounds and etiology of arrhythmias, an important cause of morbidity and mortality in patients with various diseases of the heart, can be postulated: Na+, K(+)-ATPase activity contributes directly and indirectly to the electrical membrane potential of cardiac cells. The inhibition of this pump by the endogenous digitalis-like compounds, in discrete areas of the heart, can induce changes of the membrane potential of these cells. These changes may cause an increase in excitability of the particular cells and contribute to the generation of arrhythmias.

Human cataractous lens nuclei extract inhibited, in a dose-dependent fashion, [3H]ouabain binding to rat brain synaptosomes and microsomal Na(+)- and K(+)-dependent adenosine triphosphate (Na+, K(+)-ATPase) activity and interacted with anti-digoxin antibodies. The compounds responsible for these activities, termed digitalis-like compounds (DLC), were also detected in bovine, rat, cat and rabbit, normal, transparent lenses, but the levels were only 0.7-5.4% of the average levels in the cataractous human lenses. DLC from the human cataractous lenses were purified by a procedure consisting of organic extractions and batch chromatography followed by filtration through a 3000 Da cut-off filter and subsequent separations using reverse-phase high-performance liquid chromatography. The presence of DLC in the different fractions obtained in the chromatograms was monitored by their ability to inhibit [3H]ouabain binding and Na+, K(+)-ATPase activity. Based on chemical ionization mass spectrometry together with ultraviolet spectrometry and biological characterization, it is suggested that new bufodienolides, 19-norbufalin and 19-norbufalin peptide derivatives are responsible for the endogenous DLC activity. It is proposed that these compounds may regulate Na+, K(+)-ATPase activity in the lens under some physiological and pathological conditions.

Digitalis-like compounds (DLC), normal constituents of animal tissues, are possible regulators of the Na+,K(+)-ATPase implicated in water and salt homeostasis. DLC are present in toad (Bufo viridis) tissues. Although DLC highest levels were found in toad skin, it was also detected in plasma and many internal organs. The abundant distribution and the different levels of DLC in various tissues exclude the possibility that toxicity is the only function of these compounds in the toad. The concentration of DLC in toad plasma is 30 microM, out of which 25-30% is bound to plasma proteins. Fractionation of toad plasma proteins on a G-100 Sephadex column followed by the extraction of DLC from the plasma proteins revealed that DLC are bound primarily to proteins of 48,000-53,000 Da. These results establish the existence of bufodienolide-binding protein(s) in animal plasma.

Digitalis-like compounds (DLC), constituents of animal tissues, are possible regulators of the Na+, K(+)-ATPase implicated in water and salt homeostasis. The distribution of DLC in the toad (Bufo viridis) was determined following methanol extraction and partial purification. DLC highest levels were found in the skin but it was also detected in the plasma and many internal organs. Short term (hours) exposure of the toad to hypertonic shock (1.5% NaCl) induced an increase in plasma osmolarity due to an increase in Na+ and Cl- levels. This treatment induced a transient, three fold, increase of DLC levels in the brain and transient reduction of its levels in the ventral skin. Acclimation of the toads to burrowing conditions for six weeks resulted in an increase in plasma osmolarity due to a large increase in plasma urea with a small increase in ion concentrations. Under these conditions DLC levels in the dorsal skin increased by 100% without alteration of its levels in the plasma, brain and ventral skin. DLC levels in the toad brain of control animals, showed a significant dependence on season, being highest in the summer and lowest in the winter. DLC levels in the skin peaked in May while the levels in the plasma were season independent. The changes in DLC levels induced by the short- as well as long-term perturbations in the animal environmental salinity together with the seasonal differences suggest that DLC in the toad is involved in water and salt homeostasis of these animals, but may also participate in other unknown functions.

Digitalis-like compounds (DLC) were shown to be a normal constituent of the skin and plasma of toads. In order to assess the possible physiological role of these compounds in the toad, their levels were determined in the brain, plasma and skin following acclimation in different NaCl solutions. We demonstrate that an increase in salt concentrations in the animal medium from 0 to 1.2% decreased the levels of DLC in the brain by 50% without altering significantly its levels in the plasma and skin. An increase in medium salt concentration to 1.5% resulted in a 50% increase of DLC levels in the skin without changing its levels in the plasma or brain. These results suggest that skin and brain DLC may participate in the long-term salt and water homeostasis in the toad, while the plasma compound either participates in the short-term regulations of salt and water homeostasis or have some other, unknown, function.

Cardiac glycosides such as digoxin or ouabain have long been known to influence the strength of contraction of cardiac muscle. Although the mechanism of action of these compounds remains unknown, all the proposed modes of action are based on initial binding to specific membrane receptors which are part of the (Na+ + K+)ATPase complex. These receptors, well characterized and defined, suggest the existence of an endogenous substance capable of binding to them, in analogy with endogenous opiates, discovered long after morphine and its receptors. As glycosides affect (Na+ + K+)ATPase activity, an endogenous substance may be a regulator of this important enzyme. Indeed, the search for endogenous regulators of the (Na+ + K+)ATPase or ouabain-like compounds (OLC) has recently intensified. These compounds, extracted and partially purified from mammalian brain, heart, blood and urine, and from toad skin and plasma, have been shown to inhibit 3H-ouabain binding and (Na+ + K+)ATPase activity. We report here that in addition to these effects, the OLC, highly purified from toad skin and sheep brain, increases the force of contraction of frog and guinea pig atrium.

An endogenous inhibitor of the sodium pump has been detected and concentrated 1000-fold from bovine plasma. The steps of purification included deproteinization and extraction with methanol, removal of lipids by coextractions with a lipophilic solvent, desalting and further concentration by adsorption on C18-SepPack cartridges and HPLC fractionation on a weak anionic exchange column. The material isolated displaces 3H-ouabain from brain synaptosomes, inhibits red cell membrane Na,K-ATPase without inhibiting Mg-ATPase or Ca,Mg-ATPase. Deproteinization of plasma by boiling may lead to appearance of non-specific inhibitors. The procedures developed should now permit isolation of sufficient amount of material for further purification and structural characterization.

An ouabain-like compound (OLC) was purified from toad skin. The purification procedure consisted of three sequential separations on HPLC using amino and reverse phase chromatography. Using UV, NMR and Mass spectroscopy the structure of the purified material is suggested to be mono-hydroxy-14,15-epoxy-20,22-dienolide glycoside (resibufogenin). Evidence is presented that this compound is also present in the toad plasma. It is suggested that the endogenous bufodienolide derivative participates in the physiological regulation of the Na+,K+-ATPase activity.