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Steroid-binding globulins (SBGs) such as sex hormone binding globulin, corticosteroid binding globulin, and vitamin-D binding protein are receiving increasing notice as being actively involved in steroid actions. This paper reviews data of all three of these SBGs, focusing on their presence and possible activity in the brain and nose. We have found all three proteins in the brain in limbic areas such as the paraventricular (PVN) and supraoptic nuclei (SON) as well as other areas of the hypothalamus, hippocampus, and medial preoptic area. There is also evidence that all three are made in the PVN and SON, in conjunction with the neuropeptides oxytocin and vasopressin. The localization of these three SBGs is more variable within areas of the main olfactory area and the vomeronasal organ. However, all three are found in the mucus of these areas, suggesting that one of their functions is to sequester aerosol steroids, such as pheromones, and deliver them to sensory cells and then to deeper sensory areas. In this manuscript, we present multiple models of SBG action including: A) SBG binding to a membrane receptor, B) this SBG receptor being associated with a larger protein complex including cytoplasmic steroid receptors, C) when the SBGs binds to their SBG receptors, second messengers within the cells respond, D) after SBG binding to its receptor, it releases its associated steroid into the membrane's lipid bilayer, from which it gains access into the cell only when bound by an internal protein, E) the SBG, possibly with its bound SBG receptor, is internalized into the cell from which it can gain access to numerous organelles and possibly the cell's nucleus or F) associate with intracellular steroid receptors, G) SBGs produced in target cells are released from those cells upon specific stimulation, and H) according to the Free Steroid Hypothesis steroids released from the extracellular SBG passively diffuse across the plasma membrane of the cell. These models move the area of steroid endocrinology forward by providing important paths of steroid activity within many steroid target cells.

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We studied the expression of vitamin D receptor and of vitamin D binding protein in the rat vomeronasal organ. With immunofluorescence, in situ hybridization and with reverse transcriptase PCR we found both proteins in sensory as well as in non-sensory cells. Sensory neurons contained immunoreactivity for vitamin D3 receptor in nuclei, in portions of the cytoplasm, and in apical dendrites and their microvilli. Vitamin D binding protein was observed in sensory neuron axons and cytoplasm, mostly confined to dendrites. Colocalization appeared in the contact zone of supporting cells and sensory dendrites. Both proteins were also found in single ciliated cells within the non-sensory epithelium. Vitamin D binding protein was also localized in secretory vesicles in a portion of the vomeronasal glands. Our findings suggest that the rat vomeronasal organ is a vitamin D target.

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Olfactory marker protein (OMP) may act as a modulator within the olfactory signal-transduction cascade. It has also been shown to have some importance in development of olfactory sensory organs. Here we used high resolution immunocytochemistry to localize OMP in the rat vomeronasal organ (VNO). Immunofluorescence for OMP was abundant in cilia and in apical dendrites of sensory cells, mostly associated with intraepithelial capillaries. Perikarya were stained to a lesser extent while intense OMP immunoreactivity was seen in axons of sensory neurons. Single cells within the non-sensory portion of the VNO exhibited intense OMP immunofluorescence in apical cilia and weak cytoplasmic staining. Some of the exocrine cells in the vomeronasal glands contained OMP positive secretory granules. Electron microscopy revealed that non-sensory ciliated cells had short rod like kinocilia as well as microvilli. These cells contained secretory vesicles. Their basal portion was in close apposition to nerve endings. Our findings suggest that the sensory part of the VNO contains OMP positive sensory neurons and that the non-sensory epithelium may contain secondary sensory cells. In addition OMP may be liberated from secretory glands into vomeronasal secretions.

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Clearly, we have presented here evidence of a very complex set of mechanisms and proteins involved with various and intricate actions of steroids at the plasma membrane. Steroids do MUCH more at the plasma membrane than simply passing passively through it. They may sit in the membrane; they are bound by numerous proteins in the membrane, including ERs, SHBG, steroid-binding globulin receptors, and perhaps elements of cellular architecture such as tubulin. It also seems likely that the membrane itself responds graphically to the presence of steroids by actually changing its shape as well, perhaps, as accumulating steroids. Clara Szego suggested in the 1980s that actions of E2 at one level would act synergistically with its actions at another level (e.g. membrane actions would complement nuclear actions). Given the sheer number of proteins involved in steroid actions, just at the membrane level, it seems unlikely that every action of a steroid on every potential protein effector will act to the same end. It seems more likely that these multiple effects and sites of effect of steroids contribute to the confusion that exists as to what actions steroids always have. For example, there is confusion with regard to synthetic agents (SERMs etc.) that have different and often opposite actions depending on which organ they act upon. A better understanding of the basic actions of steroids should aid in understanding the variability of their clinical effects.

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Glucocorticoids are known to be involved in myocardial regeneration and destruction. Cardiomyocytes are mostly devoid of nuclear glucocorticoid receptors (GRs) and it is generally assumed that effects of adrenal steroids in heart are mediated through the mineralocorticoid receptor (MR). Here we used immunocytochemistry to study localization of corticosteroid binding globulin (CBG) in semithin sections of human cardiac tissue samples. With staining of consecutive sections we examined colocalization with GR and MR immunoreactivities. While GR staining was almost undetectable, a portion of myocytes with MR immunostained nuclei was found. Almost all cardiomyocytes exhibited CBG immunostaining in cytoplasm and on the cell membrane. Most pronounced CBG immunoreactivities were found in Purkinje fibers and in smooth muscle cells of arterial walls. With RT-PCR, we found in homogenates of cardiac tissue detectable levels of CBG encoding mRNA. Our findings indicate that CBG is expressed in human heart. Known cardiac effects of adrenal steroids may in part be mediated through the binding globulin and its putative membrane receptor in addition to nuclear steroid receptors and direct genomic action. Highlights of our study: Human cardiomyocytes express mineralocorticoid receptors, but are mostly free of nuclear glucocorticoid receptors. CBG is expressed in myocardium and in Purkinje fibers. CBG in heart is colocalized with mineralocorticoid receptor. Endothelia and smooth muscle cells of arterial walls show colocalization of CBG and MR.

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Volatile and non-volatile derivates of gonadal steroids are known to act as pheromones in many mammalian species. Pheromones have multiple effects on the brain via the olfactory system. Their primary port of entry seems to be the vomeronasal organ (VNO) but the underlying cellular and molecular mechanisms are unclear so far. Recently we localized sex hormone binding globulin (SHBG) in both the main and the accessory olfactory system of rat with immunocytochemistry and RT-PCR. The accessory olfactory system consisting of VNO and accessory olfactory bulb showed high expression of SHBG. In the present paper we studied SHBG expression in the VNO in greater detail. In semithin sections we found SHBG immunostaining in the perinuclear cytoplasm of some of the sensory neurons, in sensory cilia and in their axons. A portion of the basal cells and some of the goblet cells in the non-sensory epithelium showed intense SHBG staining. SHBG was abundant in exocrine cells of the vomeronasal glands, perhaps compartimentalized in secretory vesicles. In situ hybridization revealed specific signals in sensory and non-sensory cells of the VNO. Our findings indicate that SHBG expressed in the VNO may be liberated into nasal secretions to bind aerosolic steroids. SHBG in sensory cells may be involved in signaling actions of pheromones.

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Ovarian steroids are known to act on the olfactory system. Their mode of action, however, is mostly unclear to date since nuclear receptors are lacking in sensory neurons. Here we used immunocytochemistry and RT-PCR to study expression and distribution of sex hormone binding globulin (SHBG) in the rat olfactory system. Single sensory cells in the olfactory mucosa and their projections in the olfactory bulb showed specific SHBG immunostaining as determined by double immunofluorescence with olfactory marker protein OMP. Larger groups of SHBG stained sensory cells occurred in the vomeronasal organ (VNO). A portion of the olfactory glomeruli in the accessory olfactory bulb showed large networks of SHBG positive nerve fibres. Some of the mitral cells showed SHBG immune fluorescence. RT-PCR revealed SHBG encoding mRNA in the olfactory mucosa, in the VNO and in the olfactory bulbs indicating intrinsic expression of the binding globulin. The VNO and its related projections within the limbic system are known to be sensitive to gonadal steroid hormones. We conclude that SHBG may be of functional importance for rapid effects of olfactory steroids on limbic functions including the control of reproductive behaviours through pheromones.

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Sepsis is known to affect neuroendocrine circuits: injections of lipopolysaccaride are potent stimulators of oxytocin secretion from the posterior lobe, acute sepsis leads to uterus contractions and spontaneous abort. Here, we report changes in expression and distribution of hypothalamic oxytocin in rats that had been subjected to caecal ligation and puncture which led to acute sepsis. Septic animals showed loss of oxytocin immunostaining in perikarya of the supraoptic and paraventricular nuclei and an increase of oxytocin positive fibres, suggesting a shift of oxytocin pools into the axonal compartment. Immunostaining of the posterior lobe revealed reduction of oxytocin in septic rats. Magnocellular neurons in supraoptic- and to a lesser extent in paraventricular nuclei showed nuclear immunoreactivity for the protooncogene c-Fos, indicating stimulation of transcriptional activity upon sepsis. Contrary to magnocellular oxytocin immunoreactivity, we observed increased oxytocin immunoreactivity in cell bodies and processes of periventricular nucleus and in perivascular neurons. Oxytocin neurons in other regions of the hypothalamus and the preoptic region did not appear to be affected by acute sepsis. Our findings suggest a differential activation of neurohypophyseal and cerebrospinal fluid contacting oxytocin systems while centrally projecting oxytocin neurons may not be affected. Systemic oxytocin levels may serve as additional diagnostic marker for sepsis.

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It has been 7 years (can it really be that long?) since we co-edited a volume (#38) of Hormone and Metabolic Research that focused on evidence that steroid-binding globulins play an active role in the actions of steroids. There has been considerable progress in identifying the location, the physiological actions, and of determining the role of binding globulins in the actions of steroids and identifying a membrane-associated receptor for a binding protein since then and this review will discuss this progress.

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The hypothalamic-pituitary-adrenal axis is supposed to be involved in the pathogenesis of the metabolic disorders. Differences in adipose tissues and parameters of insulin resistance are linked to steroid homeostasis. We assessed the correlation of fat tissue distribution, gender, and glucose control with levels of systemic corticosteroid-binding globulin (CBG), free cortisol (FuF), and total cortisol (FuM). Data of 1 114 patients with overweight, lipid disorders, and impaired glucose tolerance were collected. Blood samples were sorted according to gender and anthropometric measures. Variable-association was calculated using the Spearman Rank Correlation coefficient and tested for significance (p<0.05 and p<0.01). CBG and FuF were consistently negatively correlated to each weight parameter. Especially in women, fat mass index (FMI) was significantly negatively correlated with CBG-levels. While CBG levels dropped with increasing age, FuF showed an inverse behavior. Glycohemoglobin levels showed negative correlations with CBG while fasting glucose did not. Both changes were associated with significant increases in FuF. All negative correlations to cortisol and its binding globulin with regards to weigh- and glucose-control parameters were absent in smokers compared to nonsmokers. Our observations suggest that different weight parameters correspond to adrenal steroids and their buffer systems. Especially in women, CBG levels might serve as prognostic marker for the fat mass. In addition, CBG levels may predict long term blood glucose control more reliably than FG. However, the value of CBG as an indirect surrogate-marker for obesity and glucose is limited in smokers.

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With combined immunoperoxidase and immunofluorescence, we observed colocalization of cytochrome P450 aromatase with the posterior lobe peptide oxytocin and its associated neurophysin 1 in adult male rats. P450 was most abundant in the anterior hypothalamus. Colocalization of OT with P450 was observed in the preoptic region, the periventricular nucleus of the hypothalamus, the lateral subcommissural nucleus, and in the zona incerta. Magnocellular perikarya in the supraoptic and in the paraventricular nuclei contained only occasionally both antigens. P450 immunostaining overlapped to a great extent with known estrogen target regions. Oxytocinergic functions are controlled by estradiol while androgen receptors are mostly absent in neuroendocrine hypothalamic nuclei. Our findings suggest that systemic androgens may be aromatized to estrogens in male oxytocinergic neurons linked to the limbic system.

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The hypothalamic nonapeptide vasopressin is a known player in the pathogenesis of chronic heart failure. According to the large body of clinical evidence, vasopressin has an impact on salt and water imbalance, hyponatremia, and subsequent renal insufficiency - the most common and destructive co-morbidity of patients afflicted with chronic heart failure. Despite the well-documented elevated levels of vasopressin in the blood of such patients, its expression in the magnocellular hypothalamic nuclei and transport to the posterior pituitary has not yet been investigated. In addition, the literature almost lacks the information on the contribution of another member of nonapeptide family, oxytocin, in the pathogenesis of this disease. Here we present a postmortem analysis of vasopressin and oxytocin-immunoreactive neurons and their terminals in the posterior pituitary of 8 male patients (53.8+/-9.3 years) who had died from CHF and 9 male controls (54.6+/-11.8 years). In line with previous clinical reports, our study on hypothalami of chronic heart failure patients revealed a significant increase in the relative profile density (+29%) of vasopressin-positive neurons in the hypothalamic supraoptic nucleus. Consistently we found a significant increase in the relative optic density of vasopressin-immunoreactivity in the posterior pituitary (+33%) of these patients. In contrast, the similar analysis applied for oxytocin neurons revealed no statistically significant differences to controls. In conclusion, our study provides the morphological evidence for activation of vasopressin (but not oxytocin) expression and vasopressin transport to the posterior pituitary in patients with chronic heart failure.

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Postmortem paraffin hypothalamic blocks from male subjects with chronic heart failure (CHF) were used for morphometric and immunohistochemical studies of the supraoptical nucleus (SON). Morphometric analysis revealed a significant enlargement of pericarya and nuclei in SON of CHF subjects compared with controls. In addition, eccentrically positioned nucleoli were more frequently found in SON of the subjects with CHF. Moreover, a significant rise in relative entropy of all studied parameters of SON was documented in CHF subjects. The immunohistochemical study revealed a substantial increase of vasopressin immunoreactivity in SON neurons of the CHF subjects in comparison with control ones. Taken together, these results suggest considerable enhancement of synthetic activity of SON neurons in patients with CHF consistent with clinical observations that demonstrate significant elevation of blood vasopressin levels in patients with chronic cardiac insufficiency.

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Neuroendocrine factors play an important role in the pathogenesis of chronic heart failure. Despite numerous clinical and experimental studies, the role of the hypothalamic-pituitary-adrenal axis and glucocorticoid hormones is not fully characterised. Here we present a study of plasma cortisol concentration in 74 chronic heart failure patients, divided into four groups based on NYHA functional classes I-IV, and in 17 control subjects. In parallel, we performed morphological analysis of the hypothalamic-pituitary-adrenal axis components from 8 male patients who had died from chronic heart failure, and 9 male controls. In our study we applied immunohistochemical method and quantitative analysis to investigate an expression of hypothalamic neurohormones (corticotropin-releasing hormone, vasopressin) and adrenocorticotropin hormone in the pituitary, as well as performed general histological examination of the adrenal cortex. Measurement of morning cortisol concentration in plasma of chronic heart failure patients revealed neither difference compared to controls nor with the severity of the disease. Despite this, a two-fold increase in the density of corticotropin-releasing hormone-immunoreactive neurons as well as a two-fold increase in the number of corticotropin-releasing hormone neurons co-expressing vasopressin in the hypothalamic paraventricular nucleus were found. In the anterior pituitary the density of adrenocorticotropin hormone-immunoreactive cells was significantly increased. General histological analysis of the adrenal cortex revealed a drastic thinning of the zona fasciculata and dystrophic changes in corticocytes. Structural changes, observed in the adrenal cortex, suggest a relative glucocorticoid deficiency, which may contribute to corticotropin-releasing hormone and adrenocorticotropin hormone upregulation in hypothalamus and pituitary of chronic heart failure patients.

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New and more active concepts of steroid binding globulin action are emerging from recent research. As a result, examination of steroid levels in aging humans and the role of steroid binding globulins need to be re-visited. This review will discuss the possibility that sex hormone binding globulin (SHBG) plays an active role in the aging process. It will discuss the changes in blood levels of SHBG in aging humans in association with sexual activity, prostate hypertrophy and cancer, uterine leiomyoma, breast cancer, obesity and particularly the relationship between SHBG and HDL-cholesterol, Alzheimer's disease, osteoporosis, and cardiovascular disease. Starting with the idea that SHBG is an active participant in steroid action demands a re-evaluation of data demonstrating a primary change in blood SHBG levels in association with various pathologies. Here we discuss the postulate that SHBG may act at its own receptor at the plasma membrane level to influence other receptors such as scavenger receptors and HDL-cholesterol receptors. We will also suggest that SHBG is a critical marker for mating and thus may be an important physiological molecule in control of aging.

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The long-term effect of betamethasone on the myelination of commissural and associational fibres was investigated in fetal sheep. We studied the corpus callosum and subcortical white matter by electron microscopy. Axons were subdivided into classes according to their axonal diameter: class I: < or =0.65 microm; class II: 0.66-0.84 microm; class III: > or =0.85 microm. Under control conditions, the different functions of the white matter tracts examined were reflected by three morphological criteria: (1) there was a diverse percentage of axonal classes in the investigated areas. In corpus callosum the axons of class II predominate (47.1%). In the subcortical white matter, class I axons with small diameter are in majority (40.8%). (2) In the subcortical white matter more axons are present, with especially large diameter and hence of axonal class III. (3) The axons of subcortical white matter have thicker myelin sheaths than those of the corpus callosum. Betamethasone administration caused a significant decrease of class II axons in the corpus callosum (36.9%). In corpus callosum, axons of all classes present thicker myelin sheaths. Betamethasone administration resulted in a change in the formation of the myelin sheath in the commissural fibres of the corpus callosum but not in the associational fibres of the subcortical white matter. This could be the morphological correlate to behavioral and cognitive changes known to occur in humans after prenatal glucocorticoid treatment.

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Androgen-binding protein (ABP) and oxytocin (OT) are among the factors that control smooth muscle proliferation and tumour growth through oxytocin receptor (OTR). A close functional interaction of OTR and caveolin 1 has been shown to modulate cell growth and proliferation. We investigated samples from 10 patients (mean age 68.3) who underwent transurethral prostate resection because of benign prostate hyperplasia (BPH) by immunohistochemistry. Post-mortem prostate samples of three young men (age 18, 28, 33) were used as controls. Tissue samples were embedded in epoxy resin and cut into serial 1 microm sections for colocalization of ABP, OTR, proliferation marker p21 and caveolin 1. ABP was found in stroma of the smooth muscle cells in all studied samples. OTR staining occurred in most of these cells in BPH while controls contained only scattered cells positive for OTR. There were no apparent differences in immunostaining for p21 while immunoreactivity for caveolin 1 was observed in most cells in BPH and only in few cells in controls. Caveolin 1 was mostly colocalized with ABP and OTR in BPH samples while controls did only occasionally show this colocalization. Our observations indicate an interaction of ABP and OTR, associated with caveolin 1, which may account in part for known non-genomic actions of gonadal steroids. Androgen dependent prostate growth in BPH may be linked to these mechanisms.

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Several observations suggest that caveolin-1 has an important role in control of cell proliferation and cancerogenesis. For instance, oxytocin provokes a proliferative response in the prostate tissue when the oxytocin receptor is localized mainly in caveolin-1-enriched domains and an anti-proliferative effect when the same receptor is not localized in caveolae. Moreover, oxytocin concentrations are elevated in prostate tissue of patients with benign prostatic hyperplasia (BPH). In this study the expression pattern of the molecules caveolin-1, oxytocin receptor, androgen receptor and p21 (cell cycle arrest indicator) was investigated in the prostate tissue of BPH patients and of young controls. We found that both caveolin-1 and oxytocin receptor expression is drastically increased with age in both smooth muscle and epithelium of the prostate. We also found a significantly increased co-localization of the oxytocin receptor with caveolin-1 in both the muscle and the epithelium, especially in BPH patients. Androgen receptor and p21 staining was found throughout the prostate but did not change significantly with age or in BPH patients. We conclude that oxytocin may have a proliferative effect on the prostate tissue through the caveolae-associated receptors and thus contribute to BPH. This process seems to be androgen receptor independent.

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BACKGROUND: Sex hormone-binding globulin (SHBG) is a 94-kDa homodimer that binds steroids and is made in the hypothalamus. We have demonstrated that infusions of SHBG into the hypothalami of rats increase their female sexual receptivity except when SHBG is coupled to dihydrotestosterone (DHT) suggesting that SHBG has an active function in behavioral neuroendocrinology. METHODS: This study examines the possibility that SHBG is internalized by neuronal and/or non-neuronal brain cells as one possible mode of action using in vitro and in vivo techniques. RESULTS: First, analysis of the uptake of radiolabeled SHBG ((125)I-SHBG) found (125)I-SHBG uptake in HT22 hippocampal cells stably transfected with cDNA for ER beta (HT22-ER beta). The addition of DHT to (125)I-SHBG significantly inhibited (125)I-SHBG uptake in HT22-ER beta cells but not in HT22-ER alpha or HT22 wild-type cells. SHBG internalization was specific as it did not occur in either the human neuroblastoma cell line SK-N-SH or the glioma cell line C6. Second, SHBG was labeled with a fluor (Alexa-555), and infused into the lateral cerebroventricles of ovariectomized rats. Optimal SHBG uptake was seen 10 min after these infusions. SHBG uptake was seen in specific parts of the choroid plexus and periventricular cells as well as into cells in the paraventricular nucleus, the medial forebrain bundle, and the habenula. CONCLUSIONS: These studies suggest that SHBG is internalized by brain cells, which may be affected by the presence of ER beta. The gonadal steroids have numerous effects in brain and the discovery that the steroid-binding protein SHBG is taken up into neurons and brain cells may demand a change in thinking about how steroids are delivered to brain cells to affect neurophysiology.

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