Mineralocorticoid Receptor Gene Expression Research Articles

Alpha-synuclein-induced stress sensitivity renders the Parkinson’s disease brain susceptible to neurodegeneration

A link between chronic stress and Parkinson’s disease (PD) pathogenesis is emerging. Ample evidence demonstrates that the presynaptic neuronal protein alpha-synuclein (asyn) is closely tied to PD pathogenesis. However, it is not known whether stress system dysfunction is present in PD, if asyn is involved, and if, together, they contribute to neurodegeneration. To address these questions, we assess stress axis function in transgenic rats overexpressing full-length wildtype human asyn (asyn BAC rats) and perform multi-level stress and PD phenotyping following chronic corticosterone administration. Stress signaling, namely corticotropin-releasing factor, glucocorticoid and mineralocorticoid receptor gene expression, is also examined in post-mortem PD patient brains. Overexpression of human wildtype asyn leads to HPA axis dysregulation in rats, while chronic corticosterone administration significantly aggravates nigrostriatal degeneration, serine129 phosphorylated asyn (pS129) expression and neuroinflammation, leading to phenoconversion from a prodromal to an overt motor PD phenotype. Interestingly, chronic corticosterone in asyn BAC rats induces a robust, twofold increase in pS129 expression in the hypothalamus, the master regulator of the stress response, while the hippocampus, both a regulator and a target of the stress response, also demonstrates elevated pS129 asyn levels and altered markers of stress signalling. Finally, defective hippocampal stress signalling is mirrored in human PD brains and correlates with asyn expression levels. Taken together, our results link brain stress system dysregulation with asyn and provide evidence that elevated circulating glucocorticoids can contribute to asyn-induced neurodegeneration, ultimately triggering phenoconversion from prodromal to overt PD.

Cell-type specific effects of mineralocorticoid receptor gene expression suggest intercellular communication regulating fibrosis in skeletal muscle disease.

Introduction: Duchenne muscular dystrophy (DMD) is a fatal striated muscle degenerative disease. DMD is caused by loss of dystrophin protein, which results in sarcolemmal instability and cycles of myofiber degeneration and regeneration. Pathology is exacerbated by overactivation of infiltrating immune cells and fibroblasts, which leads to chronic inflammation and fibrosis. Mineralocorticoid receptors (MR), a type of nuclear steroid hormone receptors, are potential therapeutic targets for DMD. MR antagonists show clinical efficacy on DMD cardiomyopathy and preclinical efficacy on skeletal muscle in DMD models. Methods: We have previously generated myofiber and myeloid MR knockout mouse models to dissect cell-specific functions of MR within dystrophic muscles. Here, we compared skeletal muscle gene expression from both knockouts to further define cell-type specific signaling downstream from MR. Results: Myeloid MR knockout increased proinflammatory and profibrotic signaling, including numerous myofibroblast signature genes. Tenascin C was the most highly upregulated fibrotic gene in myeloid MR-knockout skeletal muscle and is a component of fibrosis in dystrophic skeletal muscle. Surprisingly, lysyl oxidase (Lox), canonically a collagen crosslinker, was increased in both MR knockouts, but did not localize to fibrotic regions of skeletal muscle. Lox localized within myofibers, including only a region of quadriceps muscles. Lysyl oxidase like 1 (Loxl1), another Lox family member, was increased only in myeloid MR knockout muscle and localized specifically to fibrotic regions. Discussion: This study suggests that MR signaling in the dystrophic muscle microenvironment involves communication between contributing cell types and modulates inflammatory and fibrotic pathways in muscle disease.

First Evidence of Mineralocorticoid Receptor Gene and Protein Expression in Rat and Human Thyroid Tissues and Cell Cultures.

Aldosterone (Aldo) exerts its action through binding with the mineralocorticoid receptor (MR). Clinically, a link between primary aldosteronism (PA) and thyroid diseases has been hypothesised. However, the presence and activity of MR on the thyroid have not yet been demonstrated. We investigated the gene/protein expression and activation of MR in primary thyroid cell cultures (normal rat thyroid [FRTL-5] and human papillary thyroid cancer [PTC] cell lines, BCPAP and K1) through qRT-PCR analysis, immunofluorescence, and confocal microscopy. We also studied the effects of Aldo on thyroid-specific and inflammation genes in vitro. Paired human normal and neoplastic thyroid tissues were also studied. We demonstrated both gene and protein expression and activation of MR in normal rat thyroid and human PTC lines. Incubation with Aldo induced an acute increase in IL-6 expression in both the FRTL-5 and BCPAP lines, which was antagonised by spironolactone, and an acute and late upregulation of thyroid-specific genes in FRTL-5. MR was also expressed at both gene and protein levels in normal human thyroid tissues and in PTC, with a progressive decline during neoplastic tumourigenesis, particularly in more aggressive histotypes. We present the first evidence of MR gene and protein expression in both normal and pathological thyroid cells and tissues. We have shown that MR is present and functionally activated in thyroid tissue. Binding of Aldo to MR induces the expression of inflammatory and thyroid-specific genes, and the thyroid may thus be considered a novel mineralocorticoid target tissue.

Is chronic stress a causal mechanism for small mammal population cycles? Reconciling the evidence.

Chronic stress has long been hypothesized to play a role in driving population cycles. Christian (1950) hypothesized that high population density results in chronic stress and mass "die-offs" in small mammal populations. Updated variations of this hypothesis propose that chronic stress at high population density may reduce fitness, reproduction, or program aspects of phenotype, driving population declines. We tested the effect of density on the stress axis in meadow voles (Microtus pennsylvanicus) by manipulating population density in field enclosures over three years. Using fecal corticosterone metabolites as a non-invasive measure of glucocorticoid (GC) concentrations, we found that density alone was not associated with GC differences. However, we found that the seasonal relationship of GC levels differed by density treatment, with high-density populations having elevated GC levels early in the breeding season and decreasing towards late summer. We additionally tested hippocampal glucocorticoid receptor and mineralocorticoid receptor gene expression in juvenile voles born at different densities, with the hypothesis that high density may reduce receptor expression, altering negative feedback of the stress axis. We found that females had marginally higher glucocorticoid receptor expression at high density, no effect in males, and no detectable effect of density on mineralocorticoid receptor expression in either sex. Hence, we found no evidence that high density directly impairs negative feedback in the hippocampus, butrather female offspring may be better equipped for negative feedback. We compare our findings with prior studies to attempt to disentangle the complicated relationship between density, seasonality, sex, reproduction and the stress axis.

Mechanism by Which Inflammation and Oxidative Stress Induce Mineralocorticoid Receptor Gene Expression in Aging Vascular Smooth Muscle Cells.

Vascular MR (mineralocorticoid receptor) expression increases with age driving aging-associated vascular stiffness and hypertension. MR has two isoforms (1α and 1β) with distinct 5'-untranslated and promoter sequences (P1 and P2), but the gene regulatory mechanisms remain unknown. We investigated mechanisms driving MR gene transcriptional regulation in aging human smooth muscle cells (SMC). MR was quantified in aortic tissue and primary human aortic SMC (HASMC) comparing adult and aged donors and adult HASMC treated with H2O2, to induce aging. Predicted transcription factor (TF) binding sites in the MR gene were validated using chromatin immunoprecipitations and reporter assays. The impact of TF inhibitors on MR isoforms and fibrosis target gene expression was examined. Expression of both MR mRNA isoforms increased with donor age or H2O2 treatment in HASMCs. HIF1α (hypoxia-inducible factor) and the inflammatory TF NFκB (nuclear factor kappa B) both increased with age in HASMCs and are predicted to bind MR promoters. H2O2 induced HIF1α and NFκB expression and DNA binding of HIF1α to the MR P1 promoter and of NFκB to both MR promoters in HASMCs. HIF1α inhibition decreased MR-1α isoform expression while NFκB inhibition decreased both MR isoforms. HIF1α, NFκB, and MR inhibition decreased the expression of a SMC-MR target gene implicated in vascular fibrosis. In human aortic tissues, expression of HIF1α and NFκB each positively correlated with donor age and MR expression (P<0.0001). These data implicate the inflammatory TF, NFκB, and oxidative stress-induced TF, HIF1α, in regulating SMC MR transcription in aging HASMCs, which drives aging-related vascular stiffness and cardiovascular disease.

Tight junction proteins in the small intestine and prefrontal cortex of female rats exposed to stress of chronic isolation starting early in life.

Simultaneous evaluation of barrier protein expression in the gut and the brain and their modulation under stress conditions have not been studied before now. As the permeability and function of the gut and blood-brain barrier are different and both express the MRs, we hypothesized that stress of post-weaning social isolation induces changes in tight junction protein expression in the gut which are (1) independent of changes in the brain and (2) are mediated via the mineralocorticoid receptor (MR). First, using UPLC-MS/MS we have successfully validated and selected a dose (1.2mg/rat/day) of the MR antagonist spironolactone to treat female rats exposed to stress of chronic isolation or control conditions from postnatal day 21 for 9weeks. Isolation stress caused an enhancement of gene expression of occludin and ZO-1 and a decrease in claudin-5 and MR expression in both the small intestine and prefrontal cortex. Isolation stress failed to decrease claudin-5 (small intestine) and MR (prefrontal cortex) gene expression in spironolactone-treated rats. MR blockade resulted in a decrease in claudin-15 expression in the small intestine. Anxiogenic effect of chronic stress, measured in elevated plus-maze test, was partly prevented by spironolactone treatment. Claudins, the main regulators of intestinal barrier permeability responded to chronic stress of social isolation and/or simultaneous blockade of MR in female rats by alterations independent of changes in the brain cortex. The results suggest a physiological role of MR in the control of claudin expression in the small intestine, but not in the brain cortex.

Steroidal and non-steroidal mineralocorticoid receptor antagonists in cardiorenal medicine.

This review covers the last 80 years of remarkable progress in the development of mineralocorticoid receptor (MR) antagonists (MRAs) from synthesis of the first mineralocorticoid to trials of nonsteroidal MRAs. The MR is a nuclear receptor expressed in many tissues/cell types including the kidney, heart, immune cells, and fibroblasts. The MR directly affects target gene expression—primarily fluid, electrolyte and haemodynamic homeostasis, and also, but less appreciated, tissue remodelling. Pathophysiological overactivation of the MR leads to inflammation and fibrosis in cardiorenal disease. We discuss the mechanisms of action of nonsteroidal MRAs and how they differ from steroidal MRAs. Nonsteroidal MRAs have demonstrated important differences in their distribution, binding mode to the MR and subsequent gene expression. For example, the novel nonsteroidal MRA finerenone has a balanced distribution between the heart and kidney compared with spironolactone, which is preferentially concentrated in the kidneys. Compared with eplerenone, equinatriuretic doses of finerenone show more potent anti-inflammatory and anti-fibrotic effects on the kidney in rodent models. Overall, nonsteroidal MRAs appear to demonstrate a better benefit–risk ratio than steroidal MRAs, where risk is measured as the propensity for hyperkalaemia. Among patients with Type 2 diabetes, several Phase II studies of finerenone show promising results, supporting benefits on the heart and kidneys. Furthermore, finerenone significantly reduced the combined primary endpoint (chronic kidney disease progression, kidney failure, or kidney death) vs. placebo when added to the standard of care in a large Phase III trial.

Prenatal urban traffic noise exposure impairs spatial learning and memory and reduces glucocorticoid receptor expression in the hippocampus of male rat offspring.

Exposure to noise stress during early life may permanently affect the structure and function of the central nervous system. The aim of this study was to evaluate the effects of prenatal exposure to urban traffic noise on the spatial learning and memory of the rats' offspring and the expression of glucocorticoid receptors (GRs) in their hippocampi. Three g\roups of pregnant rats were exposed to recorded urban traffic noise for 1, 2 or 4 h/day during the last week of pregnancy. At the age of 45 days, their male offspring were introduced to the Morris water maze (MWM) for assessment of spatial learning and memory. The corticosterone levels were measured in the offspring's sera by radioimmunoassay, and the relative expression of glucocorticoid and mineralocorticoid receptors (MRs) in their hippocampi was evaluated via RT-PCR. Facing urban traffic noise for 2 and 4 h/day during the third trimester of pregnancy caused the offspring to spend more time and to travel a larger distance than the controls to find the target platform. Analogously, these two groups were inferior to their control counterparts in the probe test. Also, prenatal noise stress elevated the corticosterone concentration in the sera of the rats' offspring and dose-dependently decreased the relative expression of the mRNA of both GRs and MRs in their hippocampi. Urban traffic noise exposure during the last trimester of pregnancy impairs spatial learning and memory of rat offspring and reduces GRs and MRs gene expression in the hippocampus.

Acute stress in adolescence vs early adulthood following selective deletion of dysbindin-1A: Effects on anxiety, cognition and other schizophrenia-related phenotypes

As exposure to stress has been linked to the onset and maintenance of psychotic illness, its pathogenesis may involve environmental stressors interacting with genetic vulnerability. To establish whether acute stress interacts with a targeted mutation of the gene encoding the neurodevelopmental factor dystrobrevin-binding protein 1 (DTNBP1), resulting in a specific loss of the isoform dysbindin-1A, to influence schizophrenia-relevant phenotypes in mice during adolescence and adulthood. Male and female mice with a heterozygous or homozygous deletion of DTNBP1 were assessed in the open field test following acute restraint stress in adolescence (Day 35) and young adulthood (Day 60-70). Effects of acute restraint stress on memory retention in the novel object recognition test was also assessed in adulthood. Baseline corticosterone was measured in serum samples and, brain-derived neurotrophic factor (BDNF), glucocorticoid and mineralocorticoid receptor gene expression levels were measured in the hippocampus of adult mice. In the open field, deletion of dysbindin-1A induced hyperactivity and attenuated the action of stress to reduce hyperactivity in adolescence but not in adulthood; in females deletion of dysbindin-1A attenuated the effect of acute stress to increase anxiety-related behaviour in adolescence but not in adulthood. In the novel object recognition test, deletion of dysbindin-1A impaired memory and also revealed an increase in anxiety-related behaviour and a decrease in hippocampal BDNF gene expression in males. These data suggest that deletion of dysbindin-1A influences behaviours related to schizophrenia and anxiety more robustly in adolescence than in adulthood and that dysbindin-1A influences stress-related responses in a sex-dependent manner.

Eplerenone attenuates pathological pulmonary vascular rather than right ventricular remodeling in pulmonary arterial hypertension

BackgroundAldosterone is a mineralocorticoid hormone critically involved in arterial blood pressure regulation. Although pharmacological aldosterone antagonism reduces mortality and morbidity among patients with severe left-sided heart failure, the contribution of aldosterone to the pathobiology of pulmonary arterial hypertension (PAH) and right ventricular (RV) heart failure is not fully understood.MethodsThe effects of Eplerenone (0.1% Inspra® mixed in chow) on pulmonary vascular and RV remodeling were evaluated in mice with pulmonary hypertension (PH) caused by Sugen5416 injection with concomitant chronic hypoxia (SuHx) and in a second animal model with established RV dysfunction independent from lung remodeling through surgical pulmonary artery banding.ResultsPreventive Eplerenone administration attenuated the development of PH and pathological remodeling of pulmonary arterioles. Therapeutic aldosterone antagonism – starting when RV dysfunction was established - normalized mineralocorticoid receptor gene expression in the right ventricle without direct effects on either RV structure (Cardiomyocyte hypertrophy, Fibrosis) or function (assessed by non-invasive echocardiography along with intra-cardiac pressure volume measurements), but significantly lowered systemic blood pressure.ConclusionsOur data indicate that aldosterone antagonism with Eplerenone attenuates pulmonary vascular rather than RV remodeling in PAH.

Ovine hippocampal mRNA expression in offspring from dams supplemented with fishmeal and stress challenged in late pregnancy with endotoxin.

Previous research has shown that adulthood disease can be attributed to stress events that occur during gestation. The objective of the present study was to determine whether maternal stress during late pregnancy, using a bacterial endotoxemia model, causes changes in hippocampal mRNA expression of candidate genes related to hypothalamic-pituitary-adrenal axis (HPAA) regulation in sheep. This study also sought to investigate whether maternal diets supplemented with fishmeal (FM) rich in omega-3 polyunsaturated fatty acids (PUFAs) offer protection to the fetus when subjected to maternal endotoxin stress. Using RT-qPCR, relative mRNA expression was assessed in both fetal lambs and 6-month-old lambs from dams supplemented with soybean meal (SM) or FM and challenged with either endotoxin or saline. From this it was found that fetal mineralocorticoid receptor (MR) gene expression had a tendency to be altered during endotoxin challenge, however, in the 6-month-old offspring, MR and glucocorticoid receptor (GR) gene expression were differently altered across treatment groups. These results suggest that gene regulation within the hippocampus is altered into adulthood by maternal endotoxin stress and that dietary supplementation affects outcome.

Prenatal corticosterone altered glucocorticoid receptor and glucocorticoid metabolic enzyme gene expression in chicken ovary

Abstract Objective The acute stress response is an adaptive physiological mechanism which allows an organism to respond and survive deleterious stimuli in the surrounding environment. In mammals, prenatal glucocorticoids exposure (GCs) reprograms offspring phenotype and reproductive performance. In the present study, we investigated potential prenatal GC exposure on the glucocorticoid receptor (GR), mineralocorticoid receptor (MR) and GC metabolic genes mRNA expression in the ovary of chickens. Methods We injected low (0.2 μg) and high (1.0 μg) doses of corticosterone (CORT) in ovo before incubation and measured the changes in GCs metabolic enzymes genes in ovarian follicles 1 (F1), F2 and F3 post hatching. Results The high dose CORT treatment significantly ( P P in ovo injection of high dose CORT significantly ( P Conclusions Prenatal CORT exposure modified GR, MR and GC metabolic enzymes gene expression in ovarian follicles, thus it may reprogram reproductive function.

AMPK as a therapeutic target for metabolic disorders: interactions with the renin–angiotensin–aldosterone system in adipocytes

BackgroundUpregulation of the renin-angiotensin-aldosterone system (RAAS) in adipocytes has been implicated in low-grade adipose inflammation, obesity, the metabolic syndrome, and insulin resistance. These adverse effects can be counter-regulated by AMP-activated protein kinase (AMPK), which has anti-inflammatory actions. We investigated the association between AMPK and the RAAS in human adipocytes to determine whether AMPK activation ameliorates injurious effects of angiotensin II and aldosterone. MethodsHuman liposarcoma-derived SW872 cells were differentiated into adipocytes and then stimulated in the presence or absence of the AMPK activators AICAR (1 mM, 8–24 h) or A769662 (100–300 μM, 8–24 h), angiotensin II (100 nM, 5 min), angiotensin II type 2 receptor inhibitor PD123319, aldosterone (1 μM, 6h), and the proinflammatory cytokines tumour necrosis factor (TNF) α and interleukin (IL) 1β (10 ng/mL, 6 h). RT-PCR, immunoblotting, and ELISA were performed to evaluate expression and activation of the RAAS and proinflammatory signalling pathways. FindingsAngiotensinogen (AGT), angiotensin II type 2 receptor (AGTR2), and mineralocorticoid receptor (MR) expression increased during adipocyte differentiation; angiotensin II type 1 receptor (AGTR1) was undetectable. AICAR decreased AGT, angiotensin I converting enzyme (ACE), and MR gene expression, and chemokines MCP-1, CXCL-1, and CXCL-10. A769662 also decreased MR expression yet increased aldosterone secretion and levels of downstream MR target serum and glucocorticoid regulated kinase (SGK-1). Conversely, aldosterone stimulation decreased SGK1 gene expression and attenuated proinflammatory effects of TNFα and IL1β on IL6 gene expression. Angiotensin II stimulated AMPK, effects that were blocked by PD123319. InterpretationOur findings suggest an interaction between AMPK and the RAAS in adipocytes, where AMPK downregulates the local RAAS and attenuates inflammatory responses. These processes are associated with activation of the angiotensin II type 2 receptor, which is the protective axis of angiotensin II signalling. AMPK might be an important modulator of RAAS signalling in adipocytes and could be an attractive target to ameliorate adipose inflammation in conditions associated with upregulation of the RAAS. FundingDiabetes UK, Sir George Alberti Research Training Fellowship.

Crosstalk between peroxisome proliferator-activated receptor-γ and mineralcorticoid receptor in TNF-α activated renal tubular cell.

In our previous study, we observed the crosstalk between peroxisome proliferator-activated receptor-γ (PPAR-γ) and angiotensin II in activated renal tubular cells. The present study is aimed to further explore the crosstalk between PPAR-γ and mineralocorticoid receptor (MR) in tumor necrosis factor (TNF)-α activated renal tubular cells. Human proximal renal tubular epithelial cells HK-2 were cultured with the pre-treatment of PPAR-γ agonist, pioglitazone (5 μM), MR antagonist, eplerenone (5 μM), or their combined treatment, followed by activation with TNF-α (20 ng/ml). In the parallel experiment, PPAR-γ inhibitor GW9662 (25 µM) was used to study the independence of PPAR-γ. Gene expression and protein synthesis of intercellular adhesion molecule-1 (ICAM-1), interleukin-6 (IL-6), MR and PPAR-γ were measured by RT-PCR, ELISA and Western blot, respectively; nuclear factor κB (NF-κB) nuclear translocation activity in the nucleus was examined by EMSA assay. TNF-α effectively activated HK-2 cells by up-regulating gene expression and protein synthesis of ICAM-1, IL-6 and MR and down-regulating PPAR-γ in a dose-dependent manner. TNF-α also significantly induced NF-κB nuclear translocation in HK-2 cells. Dual treatment of pioglitazone and eplerenone demonstrated synergistic effect on reducing ICAM-1 and IL-6 expression and alleviating NF-κB activation when compared with their monotherapies in TNF-α activated renal tubular cells. PPAR-γ antagonist, GW9662, significantly attenuated protective effect on ICAM-1, IL-6 and PPAR-γ expression by pioglitazone, eplerenone and their combined treatment. Our data suggest that pioglitazone, in a PPAR-γ-dependent manner, trans-represses MR signaling by suppressing NF-κB activation. MR antagonist also restored PPAR-γ expression. Dual treatment of pioglitazone and eplerenone present better efficacy in attenuating excessive inflammatory response in activated renal tubular cells under stimulation of TNF-α than single treatment.

The Expression of RAC1 and Mineralocorticoid Pathway-Dependent Genes are Associated With Different Responses to Salt Intake.

Rac1 upregulation has been implicated in salt-sensitive hypertension as a modulator of mineralocorticoid receptor (MR) activity. Rac1 could affect the expression of oxidative stress markers, such as hemoxigenase-1 (HO-1) or nuclear factor-B (NF-κB), and the expression of neutrophil gelatinase-associated lipocalin (NGAL), a cytokine upregulated upon MR activation. We evaluated RAC1 expression in relation of high salt intake and association with MR, NGAL, HO-1, and NF-κB expression, mineralo- and glucocorticoids levels, and inflammatory parameters. We studied 147 adult subjects. A food survey identified the dietary sodium (Na) intake. RAC1 expression was considered high or low according to the value found in normotensive subjects with low salt intake. We determined the gene expression of RAC1, MR, NGAL, HO-1, NF-κB, and 18S, isolated from peripheral leukocytes. We measured aldosterone, cortisol, sodium, potassium excretion, metalloproteinase (MMP9 y MMP2), and C-reactive protein. We identified 126 subjects with high Na-intake, 18 subjects had high, and 108 low-RAC1 expression. The subjects with high-RAC1 expression showed a significant increase in MR (P = 0.0002), NGAL (P < 0.0001) HO-1 (P = 0.0004), and NF-κB (P < 0.0001) gene expression. We demonstrated an association between RAC1 expression and MR (R sp 0.64; P < 0.0001), NGAL (R sp 0.48; P < 0.0001), HO-1 (R sp 0.53; P < 0.0001), and NF-κB (R sp0.52; P < 0.0001). We did not identify any association between RAC1 and clinical or biochemical variables. RAC1 expression was associated with an increase in MR, NGAL, NF-κB, and HO-1 expression, suggesting that RAC1 could be a mediator of cardiovascular damage induced by sodium, and may also useful to identify subjects with different responses to salt intake.

Spironolactone improves endothelial dysfunction in streptozotocin-induced diabetic rats.

Endothelial dysfunction is a critical initiator for developing diabetic vascular complications. Substantial clinical and experimental evidence suggests that aldosterone plays a crucial role in its pathogenesis. The present study aimed to investigate the effect of the mineralocorticoid receptor (MR) blocker, spironolactone, on diabetes-associated endothelial dysfunction and address the underlying mechanism(s) involved in this setting. Diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ) to rats and spironolactone was orally administered (50mg/kg/day). Our results showed a marked increase in aortic malondialdehyde (MDA) level and upregulation of the catalytic NADPH oxidase subunit, NOX2 gene expression alongside reducing catalase enzyme capacity, and the serum nitric oxide (NO) bioavailability in diabetic rats. This was associated with a significant reduction in endothelial nitric oxide synthase (eNOS) immunoreactivity and gene expression in diabetic aorta. The transforming growth factor-β (TGF-β) protein and the MR gene expression levels were significantly increased in the diabetic rat aorta. Moreover, the diabetic aorta showed a marked impairment in acetylcholine-mediated endothelium-dependent relaxation. Additionally, spironolactone significantly inhibited the elevated MDA, TGF-β, NOX2, and MR levels alongside correcting the dysregulated eNOS expression and the defective antioxidant function as well as NO bioavailability. Spironolactone markedly reversed the impaired endothelial function in the diabetic aorta. Collectively, our study demonstrates that spironolactone ameliorated the vascular dysfunction of diabetic aorta, at least partially via its anti-inflammatory and anti-oxidative effects alongside correcting the dysregulated eNOS and TGF-β expression. Thus, blockade of MR may represent a useful therapeutic approach against diabetic vasculopathy.

Expression of mineralocorticoid and glucocorticoid receptors in preautonomic neurons of the rat paraventricular nucleus.

Activation of mineralocorticoid receptors (MR) of the hypothalamic paraventricular nucleus (PVN) increases sympathetic excitation. To determine whether MR and glucocorticoid receptors (GR) are expressed in preautonomic neurons of the PVN and how they relate to endogenous aldosterone levels in healthy rats, retrograde tracer was injected into the intermediolateral cell column at T4 to identify preautonomic neurons in the PVN. Expression of MR, GR, 11-β hydroxysteroid dehydrogenase1 and 2 (11β-HSD1, 2), and hexose-6-phosphate dehydrogenase (H6PD) required for 11β-HSD1 reductase activity was assessed by immunohistochemistry. RT-PCR and Western blot analysis were used to determine MR gene and protein expression. Most preautonomic neurons were in the caudal mediocellular region of PVN, and most expressed MR; none expressed GR. 11β-HSD1, but not 11β-HSD2 nor H6PD immunoreactivity, was detected in the PVN. In rats with chronic low or high sodium intakes, the low-sodium diet was associated with significantly higher plasma aldosterone, MR mRNA and protein expression, and c-Fos immunoreactivity within labeled preautonomic neurons. Plasma corticosterone and sodium and expression of tonicity-responsive enhancer binding protein in the PVN did not differ between groups, suggesting osmotic adaptation to the altered sodium intake. These results suggest that MR within preautonomic neurons in the PVN directly participate in the regulation of sympathetic nervous system drive, and aldosterone may be a relevant ligand for MR in preautonomic neurons of the PVN under physiological conditions. Dehydrogenase activity of 11β-HSD1 occurs in the absence of H6PD, which regenerates NADP(+) from NADPH and may increase MR gene expression under physiological conditions.

Neonatal exposure to low dose corticosterone persistently modulates hippocampal mineralocorticoid receptor expression and improves locomotor/exploratory behaviour in a mouse model of Rett syndrome

Rett syndrome (RTT) is a pervasive neurodevelopmental disorder, primarily affecting girls. RTT causes a wide variety of debilitating symptoms and no cure currently exists. Mouse models bearing mutations in the Mecp2 gene recapitulate most physiological and behavioural RTT-related abnormalities. Stimulating neonatal environments (e.g. brief maternal separations or maternal low-dose corticosterone supplementation) reduce stress and fear responses at adulthood. The present study investigated whether impacting early in development the hypothalamic-pituitary-adrenal axis, by exposing Mecp2-308 mutant pups to a low dose of corticosterone (50 µg/ml, during the 1st week of life) may contrast RTT-related abnormalities in neuroendocrine regulation and behavioural adaptation at adulthood. In line with previous reports, when fully symptomatic, MeCP2-308 mice showed a reduction in the regular nocturnal hyperactivity in the home-cage and increased anxiety-like behaviours and plasma corticosterone (CORT) levels in response to restraint stress. An abnormal elevation in mRNA levels of mineralocorticoid receptors (MR) and BDNF gene was also evident in the hippocampus of fully symptomatic mutant mice. Neonatal CORT modulated MR gene expression and behavioural reactivity towards a novel object, also restoring wt-like levels of locomotor/exploratory behaviour in mutant mice. Enhanced sensitivity to the neonatal treatment (in terms of increase in GR and MR mRNA levels), was also evident in the hippocampus of MeCP2-308 mice compared to wt littermates. Present results corroborate the hypothesis that targeting the glucocorticoid system may prove valid in contrasting at least some of the RTT-related symptoms and provide evidence that pharmacological interventions during critical early time windows can persistently improve the behavioural phenotype of RTT mice. Current data also support the emerging role played by Mecp2 in mediating the epigenetic programming induced by early life events and indicate that, in the absence of functional MeCP2, programming of the central nervous system in response to early environmental stimuli is abnormally regulated.This article is part of the Special Issue entitled ‘Neurodevelopmental Disorders’.

Lack of elevations in glucocorticoids correlates with dysphoria-like behavior after repeated social defeat

Activity of the hypothalamic–pituitary–adrenocortical (HPA) axis is often abnormal in depression and could hold clues for better treatment of this debilitating disease. However, it has been difficult to use HPA activity as a depression biomarker because both HPA hyperactivity and HPA hypoactivity have been reported in depression. Melancholic depression has typically been associated with HPA hyperactivity, while atypical depression has been linked with HPA hypoactivity. Many animal models of chronic stress recapitulate behavioral aberrations and elevated HPA activity that could represent a model for melancholic depression. However, there are no animal models that could be used to elucidate the etiology or treatment of atypical depression. We have used repeated social defeat in mice to test the hypothesis that this chronic stress would induce dysphoria-like behavior associated with HPA hypoactivity in a subset of subjects. Intruder mice were placed in the home cage of an aggressive resident mouse for 5min/d for 30days. The majority of intruder mice had elevated basal plasma corticosterone (High Morning Corticosterone, or HMC) and adrenal 11β hydroxylase mRNA levels relative to control mice that were handled daily. However, a subset of intruder mice (Low Morning Corticosterone; LMC) exhibited basal plasma corticosterone and 11β hydroxylase mRNA levels that were indistinguishable from control levels. Significant changes in emotional behavior only occurred in LMC mice, which exhibited anxiety-like increases in activity and defecation during tail suspension and anhedonia-like decreases in sucrose preference. Relative to HMC mice, LMC mice also showed increases in gene expression of mineralocorticoid receptor in CA2 hippocampus, consistent with the possibility that HPA activity in this group is constrained by increased sensitivity to glucocorticoid negative feedback. LMC mice also exhibited increased c-fos gene expression compared to HMC mice in the paraventricular hypothalamus and lateral septum suggesting that central pathways fail to habituate to chronic stress even though adrenocortical activity is not stimulated. We conclude that LMC mice showed adrenocortical hyporesponsiveness, which in combination with the behavioral abnormalities in this group may represent a model for the HPA hypoactivity associated with atypical depression.

Age‐related changes in corticosteroid receptor expression and monoamine neurotransmitter concentrations in various brain regions of postnatal pigs

Negative early life experience may be associated with altered functioning of stress-related systems and may increase vulnerability to diseases later in life. Corticosteroids are important mediators of homeostasis and stress and exert their effects via two receptors, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR), and through the glucocorticoid-metabolizing enzymes 11β-hydroxysteroid dehydrogenase (11β-HSD) types 1 and 2 in a brain-region-specific manner. However, relatively little is known about the postnatal ontogeny of these receptors and enzymes in the central nervous system. Here we describe, for the first time, the postnatal ontogeny of central GR, MR, 11β-HSD1, and 11β-HSD2 gene expression and monoamine levels in stress-related brain regions of domestic pigs at 7, 21, and 35 days of age. During the postnatal period, there was an increase in GR, MR, and 11β-HSD1 mRNA expression in the pituitary and prefrontal cortex and an increase in MR mRNA expression in the hippocampus. We also demonstrated age-dependent changes in levels of noradrenaline and dopamine and their metabolites in the locus coeruleus, with the highest concentrations on day 7 compared with days 21 and 35. In conclusion, the dynamic changes in corticosteroid receptors and monoamines during neural development of postnatal pigs may represent periods of sensitivity to environmental stress that are comparable to some extent with those that are observed in primates and humans. Thus, these findings support the use of the domestic pig as an alternative animal model for humans in stress research.