Haim Rosen

Prenatal stress selectively alters the reactivity of the hypothalamic-pituitary adrenal system in the female rat

A study was made of the effects of prenatal stress on the reactivity of the hypothalamic-pituitary adrenal (HPA) axis in male and female offspring. Rat dams were subjected to noise and light stress on an unpredictable basis throughout pregnancy. At 28 days of age mRNA for POMC, proenkephalin and prodynorphin were measured in the hypothalamus of the offspring. A marked reduction was found in POMC mRNA in PS females (PSF) but not in males (PSM), but the other mRNAs did not differ from controls (C). At 60 days of age, PSF has 3 times higher resting levels of serum corticosterone (COR) and significantly lower dexamethasone (DEX)3H hippocampal binding sites than CF. Overnight adrenalectomy abolished the difference in DEX binding. After 10 min exposure to open field PS males and females voided more fecal pellets and made fewer center entries than C offspring, testifying to increased emotionality. Open field stress caused a 3-5-fold rise in circulating COR in all groups within 15 min, which returned to baseline by 90 min in all rats except PSF. These data show that prenatal stress can cause permanent alterations in the behavior of both sexes in stressful situations but appears to cause a selective effect on the HPA axis in the female rat.

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.

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.

Behavioral effects of lipopolysaccharide in rats: involvement of endogenous opioids

Activation of the immune system in response to either infection or lipopolysaccharide (LPS) produces neurophysiological, neuroendocrine and behavioral changes. Some of the physiological consequences of LPS are mediated by endogenous opioid peptides. The following studies were designed to characterize the effects of LPS in several behavioral paradigms, and to determine the role of opioids in mediating these effects. The effects of LPS on locomotor and self-care activity were assessed in the open field test. Rats were injected with either saline or a dose of LPS (25, 50, 100, or 1000 micrograms/kg). 4 h later, the animals were placed in an open field and the numbers of line crossings, rearings and grooming episodes were counted. LPS significantly suppressed the three open field behaviors in a dose-related manner. The effect of LPS on sensitivity to pain was determined using the hot-plate and tail-flick tests. Administration of LPS (200 micrograms/kg) increased pain sensitivity in the hot plate test 30 min after drug administration, but produced a significant analgesic response 4 h after drug administration in both tests. Further characterization of LPS-induced analgesia demonstrated that it began about 2 h after and disappeared 30 h after the administration of LPS. Administration of naltrexone completely blocked the analgesic effects of LPS 4 h after its administration, but had no effect on LPS-induced suppression of activity in the open field. The effect of LPS on body temperature was biphasic, producing hypothermia at 2 h and hyperthermia at 8-30 h after its administration. Naltrexone had no effect on the body temperature changes induced by LPS.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Transcriptional regulation of the human sterol 27-hydroxylase gene (CYP27) and promoter mapping.

Recent evidence suggests that sterol 27-hydroxylase may play a role in cholesterol homeostasis and affect atherogenesis. The major objective of the study was to map and characterize the sterol 27-hydroxylase (CYP27) promoter region. Here we show that CYP27 gene has a TATA-less promoter and transcription initiates at a cluster of sites. The basic promoter is located between -166 and -187 bp from the translation initiation site. Possible positive transcription regulation sites are located at position -187 to -320 and -857 to -1087 bp. A negative transcription regulator site is located in position -320 to -413 bp. An enhancer sequence is located upstream to position -1087. CYP27 is upregulated by dexamethasone and downregulated by cyclosporin A and cholic acid. The dexamethasone responsive element is located between 1087 and 678 bp upstream to the putative ATG. Cyclosporin A affects bile acid metabolism by repressing CYP27 at the transcriptional level. The cyclosporin A- responsive element is mapped to between 1087 and 4000 bp upstream of the ATG. Cholic acid represses sterol 27-hydroxylase mRNA level by affecting the stability of its mRNA. The results obtained here imply that CYP27 has a potentially important role in cholesterol homeostasis in human cells, and is regulated by several substances that were previously shown to affect bile acid metabolism.

Translational Control of Specific Genes during Differentiation of HL-60 Cells

Eukaryotic gene expression can be regulated through selective translation of specific mRNA species. Nevertheless, the limited number of known examples hampers the identification of common mechanisms that regulate translation of specific groups of genes in mammalian cells. We developed a method to identify translationally regulated genes. This method was used to examine the regulation of protein synthesis in HL-60 cells undergoing monocytic differentiation. A partial screening of cellular mRNAs identified five mRNAs whose translation was specifically inhibited and five others that were activated as was indicated by their mobilization onto polysomes. The specifically inhibited mRNAs encoded ribosomal proteins, identified as members of the 5′-terminal oligopyrimidine tract mRNA family. Most of the activated transcripts represented uncharacterized genes. The most actively mobilized transcript (termed TA-40) was an untranslated 1.3-kilobase polyadenylated RNA with unusual structural features, including two Alu-like elements. Following differentiation, a significant change in the cytoplasmic distribution of Alu-containing mRNAs was observed, namely, the enhancement of Alu-containing mRNAs in the polysomes. Our findings support the notion that protein synthesis is regulated during differentiation of HL-60 cells by both global and gene-specific mechanisms and that Alu-like sequences within cytoplasmic mRNAs are involved in such specific regulation.