Steroid antagonism of the hypertensinogenic activity of adrenocortical steroids

Previous studies in sheep have provided evidence for a separate "hypertensinogenic" class of adrenocortical steroid activity which is not simply related to their classical mineralocorticoid (MC) and/or glucocorticoid (GC) actions. This study investigated the structure-activity relationships of the effects of structural analogues of prednisolone on mean arterial pressure (MAP), and MC and GC actions in sheep. Infusions of these synthetic GC at 0.6 and 24 mg/day produced variable pressor effects which were dissociated from their MC and GC actions. In other experiments, the minimum adrenocortical steroid requirement to reproduce the onset of ACTH-dependent hypertension was determined. Infusion of cortisol, aldosterone, 17 alpha-hydroxy progesterone and 17 alpha,20 alpha-dihydroxy-4-pregnene-3-one was found to be sufficient to reproduce the hypertensive response to ACTH administration in sheep. A subsequent experiment showed that substitution of cortisol by the more potent synthetic GC, prednisolone had no effect on MAP. Therefore, cortisol appears to exert an essential action in ACTH hypertension which is not dependent on its GC activity. Other studies have found that prednisolone (100 mg/day) antagonized 9 alpha-fluoro-prednisolone (0.6 mg/day) induced hypertension but not its MC effects. The effect of progesterone (500 mg/day) and the progesterone analogues, norethisterone, medroxy-progesterone and 16 alpha-methyl progesterone on ACTH (5 micrograms/kg per day) hypertension was investigated. Progesterone completely blocked the hypertension and MC effects of ACTH infusion, while medroxy-progesterone partially blocked the increase in MAP. These data support our concept of a "hypertensinogenic" class of steroid activity.     

Essential roles of endogenous glucocorticoids and TNF/TNFR1 in promoting bone-marrow eosinopoiesis in ovalbumin-sensitized,airway-challenged mice

Aims

Stress mechanisms paradoxically contribute to allergic episodes in humans and mice. Glucocorticoids (GC) and interleukin (IL)-5 synergically upregulate murine bone-marrow eosinophil production. Here we explored the role of endogenous GC in allergen-stimulated bone-marrow eosinophil production in ovalbumin-sensitized/challenged mice.

Main methods

In BALB/c or C57BL/6 mice, sensitized and intranasally challenged with ovalbumin, we monitored eosinophil numbers in freshly harvested or cultured bone-marrow, and plasma corticosterone levels. Metyrapone (MET) was used to inhibit GC synthesis, and RU486 to block GC actions. In sensitized mice challenged intraperitoneally, we examined the relationship between eosinophilia of bone-marrow and peritoneal cavity, in the absence or presence of RU486. In experiments involving in vivo neutralization of tumor necrosis factor-α (TNF) by specific antibodies, or using mice which lack functional type I TNF receptors (TNFRI), we evaluated the relationship between TNF blockade, corticosterone levels, RU486 or MET treatment and challenge-induced bone-marrow eosinophilia.

Key findings

RU486 or MET pretreatments abolished challenge-induced increases in eosinophil numbers in bone-marrow (in vivo and ex vivo), and in the peritoneal cavity. MET, but not RU486, prevented the challenge-induced increase in corticosterone levels. Challenge-induced bone-marrow eosinophilia and corticosterone surge were abolished in TNFRI-deficient mice. Anti-TNF-treatment very effectively prevented challenge-induced bone-marrow eosinophilia, in the absence of RU486 or MET, but had no independent effect in the presence of either drug.

Significance

Endogenous GC was essential for allergen challenge-induced increases in eosinophil numbers inside bone-marrow. This effect required TNF and TNFRI, which suggests an immunoendocrine mechanism.     

Dominance of the strongest: Inflammatory cytokines versus glucocorticoids

Pro-inflammatory cytokines are involved in the pathogenesis of many inflammatory diseases, and the excessive expression of many of them is normally counteracted by glucocorticoids (GCs), which are steroids that bind to the glucocorticoid receptor (GR). Hence, GCs are potent inhibitors of inflammation, and they are widely used to treat inflammatory diseases, such as asthma, rheumatoid arthritis and inflammatory bowel disease. However, despite the success of GC therapy, many patients show some degree of GC unresponsiveness, called GC resistance (GCR). This is a serious problem because it limits the full therapeutic exploitation of the anti-inflammatory power of GCs. Patients with reduced GC responses often have higher cytokine levels, and there is a complex interplay between GCs and cytokines: GCs downregulate pro-inflammatory cytokines while cytokines limit GC action. Treatment of inflammatory diseases with GCs is successful when GCs dominate. But when cytokines overrule the anti-inflammatory actions of GCs, patients become GC insensitive. New insights into the molecular mechanisms of GR-mediated actions and GCR are needed for the design of more effective GC-based therapies.     

Glucocorticoid regulation of astrocytic fate and function

Glial loss in the hippocampus has been suggested as a factor in the pathogenesis of stress-related brain disorders that are characterized by dysregulated glucocorticoid (GC) secretion. However, little is known about the regulation of astrocytic fate by GC. Here, we show that astrocytes derived from the rat hippocampus undergo growth inhibition and display moderate activation of caspase 3 after exposure to GC. Importantly, the latter event, observed both in situ and in primary astrocytic cultures is not followed by either early- or late-stage apoptosis, as monitored by stage I or stage II DNA fragmentation. Thus, unlike hippocampal granule neurons, astrocytes are resistant to GC-induced apoptosis; this resistance is due to lower production of reactive oxygen species (ROS) and a greater buffering capacity against the cytotoxic actions of ROS. We also show that GC influence hippocampal cell fate by inducing the expression of astrocyte-derived growth factors implicated in the control of neural precursor cell proliferation. Together, our results suggest that GC instigate a hitherto unknown dialog between astrocytes and neural progenitors, adding a new facet to understanding how GC influence the cytoarchitecture of the hippocampus.     

Deconstructing the roles of glucocorticoids in adipose tissue biology and the development of central obesity

Central obesity is associated with insulin resistance and dyslipidemia. Thus, the mechanisms that control fat distribution and its impact on systemic metabolism have importance for understanding the risk for diabetes and cardiovascular disease. Hypercortisolemia at the systemic (Cushing's syndrome) or local levels (due to adipose-specific overproduction via 11β-hydroxysteroid dehydrogenase 1) results in the preferential expansion of central, especially visceral fat depots. At the same time, peripheral subcutaneous depots can become depleted. The biochemical and molecular mechanisms underlying the depot-specific actions of glucocorticoids (GCs) on adipose tissue function remain poorly understood. GCs exert pleiotropic effects on adipocyte metabolic, endocrine and immune functions, and dampen adipose tissue inflammation. GCs also regulate multiple steps in the process of adipogenesis. Acting synergistically with insulin, GCs increase the expression of numerous genes involved in fat deposition. Variable effects of GC on lipolysis are reported, and GC can improve or impair insulin action depending on the experimental conditions. Thus, the net effect of GC on fat storage appears to depend on the physiologic context. The preferential effects of GC on visceral adipose tissue have been linked to higher cortisol production and glucocorticoid receptor expression, but the molecular details of the depot-dependent actions of GCs are only beginning to be understood. In addition, increasing evidence underlines the importance of circadian variations in GCs in relationship to the timing of meals for determining their anabolic actions on the adipocyte. In summary, although the molecular mechanisms remain to be fully elucidated, there is increasing evidence that GCs have multiple, depot-dependent effects on adipocyte gene expression and metabolism that promote central fat deposition. This article is part of a Special Issue entitled: Modulation of Adipose Tissue in Health and Disease.     

Metabolic correlations of glucocorticoids and polyamines in inflammation and apoptosis

Glucocorticoid hormones (GC) are essential in all aspects of human health and disease. Their anti-inflammatory and immunosuppressive properties are reasons for therapeutic application in several diseases. GC suppress immune activation and uncontrolled overproduction and release of cytokines. GC inhibit the release of pro-inflammatory cytokines and stimulate the production of anti-inflammatory cytokines. Investigation of GC’s mechanism of action, suggested that polyamines (PA) may act as mediators or messengers of their effects. Beside glucocorticoids, spermine (Spm) is one of endogenous inhibitors of cytokine production. There are many similarities in the metabolic actions of GC and PA. The major mechanism of GC effects involves the regulation of gene expression. PA are essential for maintaining higher order organization of chromatin in vivo. Spermidine and Spm stabilize chromatin and nuclear enzymes, due to their ability to form complexes with negatively charged groups on DNA, RNA and proteins. Also, there is an increasing body of evidence that GC and PA change the chromatin structure especially through acetylation and deacetylation of histones. GC display potent immunomodulatory activities, including the ability to induce T and B lymphocyte apoptosis, mediated via production of reactive oxygen species (ROS) in the mitochondrial pathway. The by-products of PA catabolic pathways (hydrogen peroxide, amino aldehydes, acrolein) produce ROS, well-known cytotoxic agents involved in programmed cell death (PCD) or apoptosis. This review is an attempt in the better understanding of relation between GC and PA, naturally occurring compounds of all eukaryotic cells, anti-inflammatory and apoptotic agents in physiological and pathological conditions connected to oxidative stress or PCD.     

Aldosterone and the kidney: rapid regulation of renal microcirculation

Recent studies provide evidence that aldosterone (Aldo) accelerates hypertension, proteinuria and glomerulosclerosis in animal models of chronic renal failure. Although the underlying mechanisms are not well defined, Aldo may exert these deleterious renal effects by elevating renal vascular resistance (RVR) and glomerular capillary pressure (P(GC)). To test this possibility, we studied the action of Aldo on rabbit afferent (Af-) and efferent arterioles (Ef-Arts), crucial vascular segments to the control of glomerular hemodynamics. Aldo caused rapid (within 5 min) constriction in both arterioles. The constriction was not affected by spironolactone but was reproduced by membrane-impermeable albumin-conjugated Aldo, suggesting that vasoconstrictor actions are non-genomic. This notion was further supported by the finding that neither actinomycin D nor cycloheximide had effect. The vasoconstrictor action of Aldo on Af-Arts was inhibited by nifedipine (L-type calcium channel blocker), whereas that on Ef-Arts was inhibited by efonidipine (both L- and T-type calcium channel blocker) but not nifedipine. Disrupting the endothelium or nitric oxide (NO) synthesis inhibition augmented the vasoconstriction in Af-Arts, demonstrating that endothelium-derived NO modulates the vasoconstrictor actions of Aldo. Thus, Aldo causes non-genomic vasoconstriction via calcium mobilization thorough L- or T-type calcium channels in Af- or Ef-Arts, respectively. These vasoconstrictor actions on the glomerular microcirculation may play an important role in the pathophysiology and progression of renal diseases by elevating RVR and P(GC), especially when endothelium functions are impaired. In addition to our study, this review describes recent findings on the rapid cardiovascular actions of Aldo, with a particular attention to the renal hemodynamics.     

Putative target sites for mobile G+C rich clusters in yeast mitochondrial DNA: Single elements and tandem arrays

Summary GC clusters constitute the major repetitive elements in the mitochondrial (mt) genome of the yeast Saccharomyces cerevisiae. Many of these clusters are optional and thus contribute much to the polymorphism of yeast mtDNAs. We have made a systematic search for polymorphic sites by comparing mtDNA sequences of various yeast strains. Most of the 26 di- or polymorphic sites found differ by the presence or absence of a GC cluster of the majority class, here referred to as the M class, which terminate with an AGGAG motif. Comparison of sequences with and without the GC clusters reveal that elements of the subclasses M1 and M2 are inserted 3 to a TAG, flanked by A+T rich sequences. M3 elements, in contrast, only occur in tandem arrays of two to four GC clusters; they are consistently inserted 3 to the AGGAG terminal sequence of a preexisting cluster. The TAG or the terminal AGGAG, therefore, are regarded as being part of the target sites for M1 and M2 or M3 elements, respectively. The dinucleotide AG is in common to both target sites; it also occurs at the 3 terminus (AGGAG). This suggests its duplication during GC cluster insertion. This notion is supported by the observation that GC clusters of the minor classes G and V similarily repeat at their 3 terminus a GT or an AA dinucleotide, respectively, from their putative target sites.     

CD-NP: A Novel Engineered Dual Guanylyl Cyclase Activator with Anti-Fibrotic Actions in the Heart

Natriuretic peptides (NPs) are cardioprotective through the activation of guanylyl cyclase (GC) receptors A and B. CD-NP, also known as cenderitide, is a novel engineered NP that was designed to uniquely serve as a first-in-class dual GC receptor agonist. Recognizing the aldosterone suppressing actions of GC-A activation and the potent inhibitory actions on collagen synthesis and fibroblast proliferation through GC-B activation, the current study was designed to establish the anti-fibrotic actions of CD-NP, administered subcutaneously, in an experimental rat model of early cardiac fibrosis induced by unilateral nephrectomy (UNX). Our results demonstrate that a two week subcutaneous infusion of CD-NP significantly suppresses left ventricular fibrosis and circulating aldosterone, while preserving both systolic and diastolic function, in UNX rats compared to vehicle treated UNX rats. Additionally we also confirmed, in vitro, that CD-NP significantly generates the second messenger, cGMP, through both the GC-A and GC-B receptors. Taken together, this novel dual GC receptor activator may represent an innovative anti-fibrotic therapeutic agent.     

<Superscript>1</Superscript>H NMR metabolic profiling of gastric cancer patients with lymph node metastasis

Introduction

Gastric cancer (GC) is a malignant tumor worldwide. As primary pathway for metastasis, the lymphatic system is an important prognostic factor for GC patients. Although the metabolic changes of gastric cancer have been investigated in extensive studies, little effort focused on the metabolic profiling of lymph node metastasis (LNM)-positive or negative GC patients.

Objectives

We performed ¹H NMR spectrum of GC tissue samples with and without LNM to identify novel potential metabolic biomarkers in the process of LNM of GC.

Methods

¹H NMR-based untargeted metabolomics approach combined with multivariate statistical analyses were used to study the metabolic profiling of tissue samples from LNM-positive GC patients (n?=?40), LNM-negative GC patients (n?=?40) and normal controls (n?=?40).

Results

There was a clear separation between GC patients and normal controls, and 33 differential metabolites were identified in the study. Moreover, GC patients were also well-classified according to LNM-positive or negative. Totally eight distinguishing metabolites were selected in the metabolic profiling of GC patients with LNM-positive or negative, suggesting the metabolic dysfunction in the process of LNM. According to further validation and analysis, especially BCAAs metabolism (leucine, isoleucine, valine), GSH and betaine may be as potential factors of diagnose and prognosis of GC patients with or without LNM.

Conclusion

To our knowledge, this is the first metabolomics study focusing on LNM of GC. The identified distinguishing metabolites showed a promising application on clinical diagnose and therapy prediction, and understanding the mechanism underlying the carcinogenesis, invasion and metastasis of GC.

     

Distinct responses of human granulosa lutein cells after hCG or LH stimulation in a spheroidal cell culture system

The growth and development of the corpus luteum (CL) is regulated by gonadotropic hormones. It is formed by granulosa cells (GCs), theca cells, and endothelial cells, and is the primary source of circulating progesterone. During early pregnancy only human chorionic gonadotropin (hCG) but not luteinizing hormone (LH) extends the life span of the CL, although hCG and LH interact with the same receptor and have similar actions on the CL. In this study a recently by our group established spheroidal GC culture assay served as a model of CL development on which we compared the actions of the gonadotropic hormones LH and hCG. To find out which signal pathways take part in the hormonal regulation of GC we stimulated GC-spheroids with modulators of protein kinases A and C dependent signaling cascades and determined their impact on sprout forming activity in GC. Our results indicate that PKA-dependent signaling pathways play a major role in mediating the hormonal-induced signaling cascades leading to sprouting in GC. Furthermore, this study strongly indicates that the different effects of hCG and LH in the maintenance of the CL may be reasoned in different signal transduction pathways triggered by hCG or LH.     

Glucocorticoid‐mediated modulation of morphological changes associated with aging in microglia

Microglia dynamically adapt their morphology and function during increasing age. However, the mechanisms behind these changes are to date poorly understood. Glucocorticoids (GCs) are long known and utilized for their immunomodulatory actions and endogenous GC levels are described to alter with advancing age. We here tested the hypothesis that age‐associated elevations in GC levels implicate microglia function and morphology. Our data indicate a decrease in microglial complexity and a concomitant increase in GC levels during aging. Interestingly, enhancing GC levels in young mice enhanced microglial ramifications, while the knockdown of the glucocorticoid receptor expression in old mice aggravated age‐associated microglial amoebification. These data suggest that GCs increase ramification of hippocampal microglia and may modulate age‐associated changes in microglial morphology.     

Involvement of bilitranslocase and beta-glucuronidase in the vascular protection by hydroxytyrosol and its glucuronide metabolites in oxidative stress conditions

Olive oil vascular benefits have been attributed to hydroxytyrosol (HT). However, HT biological actions are still debated because it is extensively metabolized into glucuronides (GCs). The aim of this study was to test HT and GC vasculoprotective effects and the underlying mechanisms using aorta rings from 8-week-old male Wistar rats. In the absence of oxidative stress, incubation with 100 μM HT or GC for 5 min did not exert any vasorelaxing effect and did not influence the vascular function. Conversely, in condition of oxidative stress [upon incubation with 500 μM tert-butylhydroperoxide (t-BHP) for 30 min], preincubation with HT or GC improved acetylcholine-induced vasorelaxation compared with untreated samples (no t-BHP). This protective effect was lost for GC, but not for HT, when a washing step (15 min) was introduced between preincubation with HT or GC and t-BHP addition, suggesting that only HT enters the cells. In agreement, bilitranslocase inhibition with 100 μM phenylmethanesulfonyl fluoride for 20 min reduced significantly HT, but not GC, effect on the vascular function upon stress induction. Moreover, GC protective effect (improvement of endothelium-dependent relaxation in response to acetylcholine) in oxidative stress conditions was reduced by preincubation of aorta rings with 300 μM D-saccharolactone to inhibit β-glucuronidase, which can deconjugate polyphenols. Finally, only HT was detected by high-pressure liquid chromatography in aorta rings incubated with GC and t-BHP. These results suggest that, in conditions of oxidative stress, GC can be deconjugated into HT that is transported through the cell membrane by bilitranslocase to protect vascular function.     

Bead rings at the endoplasmic reticulum-golgi complex boundary: morphological changes accompanying inihibition of intracellular transport of secretory proteins in arthropod fat body tissue

Golgi complex beads are 10-nm particles arranged in rings on the smooth surface of rough endoplasmic reticulum (ER) makind the forming face of the Golgi complex (GC). In arthropod cells they stain specifically with bismuth. Their morphology has been studied after treatment with reagents known to interfere with GC function. Inhibitors of oxidative phosphorylation (antimycin A, cyanide, and anoxia), but not an inhibitor of glycolysis (iodoacetate), both cause the bead rings to collapse and the GC saccules to round up, and inhibit transition vesicle (TV) formation. Cycloheximide blocks protein synthesis on ribosomes but does not stop TV formation or disrupt bead rings, even after prolonged treatment (6 h) to allow emptying of the rough ER cisternae. Thus the collapse of bead rings is not attributable to inhibition of protein synthesis, and the ring structure of beads does not require continued protein synthesis and secretion for its maintenance. Valinomycin has effects on the GC similar to those of antimycin A, but {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, monensin, and lasalocid do not affect bead ring structure or TV formation. These results are consistent with valinomycin’s secondarily uncoupling mitochondria, which collapses bead rings and prevents TV formation. Thus inhibitors of oxidative phosphorylation do not influence the beads through cation movement. Because mononsin and lasalocid block secretion at the level of the condensing vacuoles, bead rings are not influenced by blocks in secretion distal to them or by the backup of secretory material. These experiments are consistent with inhibitors of oxidative phosphorylation collapsing bead rings by decreasing intracellular ATP. The concomitant block to TV formation and the collapse of bead rings suggests that integrity of the bead rings is essential for the transport of secretory material from the rough ER to the GC.     

Glucocerebroside inhibits NADPH oxidase activation in cell-free system

We reported earlier that monocytes and macrophages from patients with type I Gaucher disease have a decreased capacity to generate superoxide anion (O(2)(-)) on stimulation with opsonized S. aureus or formyl-methionyl-leucyl-phenylalanine. In this study, various forms of the cell-free assay system were used to probe the hypothesis that glucocerebroside (GC) accumulating in Gaucher patients' phagocytes may interfere with the activation of NADPH oxidase. Xanthine/xanthine oxidase assay was applied to explore the possibility that GC may scavenge O(2)(-). We found that addition of GC to the crude, semirecombinant or fully purified cell-free systems inhibited activation of NADPH oxidase in a concentration-dependent manner. The inhibitory effect of GC could be overcome by increased concentrations of p47(phox) and p67(phox). In contrast, O(2)(-) generation was not decreased by GC added to the assembled, catalytically active enzyme complex. In the xanthine/xanthine oxidase system, GC had no effect on the generation of O(2)(-). These data indicate that assembly of the respiratory burst oxidase of phagocytic cells may be a possible target of the pathologic actions of GC.     

Bipolar manifestation of cataleptic reactions in rats

Selection of GC rats for the predisposition to cataleptic freezing has increased not only the frequency, intensity, and duration of freezing, but also the proportion of irritable or "nervous" rats with enhanced anxiety, defensive behavior with vocalization, jerky running, and jumpiness. An increased amplitude of the startle reflex is a correlate of this "nervousness." The results of the comparison of some behavioral characters in the nervous and freezing GC rats, as well as in F1 and F2 offspring from homogeneous crosses between nervous and freezing GC rats suggest that cataleptic freezing and nervousness are two poles of the same bipolar catatonic reaction. They have a common mechanism, with the alternative or preferential expression of one particular form of the reaction is determined by the external and internal environments or the set of modifier genes in the given individual.     

Ultrastructural Observation on Microtubules of the Generative Cell in Amaryllis Germinated Pollens and Pollen Tubes

The organization of microtubules (MTs) in the generative cell (GC) of germinated pollen and pollen tube in Amaryllis vittata Ait. has been studied with electron microscopy. At the beginning of pollen germination, the GC is long elliptic in shape, and is surrounded by its own membrane and also by that of the vegetative cell (VC) ,both of which appear undulated. In cross section, the GC appears roundish and has many lobes. The MT system of GC is mainly organized in bundles, but single MTs can also be observed. The MT bundles are generally located in the lobes, directly beneath the plasma membrane of the cell. These MT bundles orientate along the longitudinal axis of the cell. They are formed by aggregation of 5–6 MTs at least,more often about 30 MTs. In the bundles the MTs are often linked to each other by "cross-bridge". The single tubules in the eytopiasm distribute randomly in different orientations. When the GC has migrated into the pollen tube after germination ,it becomes elongated and has cytoplasmic extensions both in the anterior and posterior end of the cell. The organization of MTs of the GC in pollen tube is similar to that in the germinated pollen grain,but the number of MTs in a bundle often increases to 50–60. In the bundle the "cross-bridges" between the MTs which always link 3–5 MTs, are still seen clearly. Positional shift between the GC and Vegetative nucleus (VN) may take place during the growth of pollen tube. The physical association between GC and VN may be demonstrated some ultrastructural figures. It may be seen that irregular cytoplasmic extensions in the anterior end of the GC is always enclosed by the VN and the projections of the cytoplasmic extensions lie within enclaves of the VN. There are many MTs sheets in the lobes or extensions in the cytoplasm of the GC. Thus the present study demonstrates that MTs have an important role in maintaining the peculiar shape of the GC and the close association between GC and VN. However, it seems that the MTs are probably also engaged in the movement of the GC during pollen growth.