David Lichtstein

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.

Endogenous ‘ouabain like’ activity in rat brain

The presence of an endogenous ‘ouabain like’ compound in rat brain is demonstrated based on the ability of acid acetone extracts of brain to inhibit [3H] ouabain binding and Na+,K+-ATPase activity. Partial purification of the inhibitory activity was achieved by methanol and trichloroacetic acid fractionations followed by Sephadex G-25 chromatography. The results are discussed with relation to the possible role of the endogenous ‘ouabain like’ compound in the regulation of the Na+,K+ pump activity.

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.

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.

Association Between Sodium- and Potassium-Activated Adenosine Triphosphatase α Isoforms and Bipolar Disorders

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.

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.

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.

Identification of a ouabain-like compound in toad skin and plasma as a bufodienolide derivative

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.

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.

Effect of salt acclimation on digitalis-like compounds in the toad

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.