Cardiometabolic Effects of DOCA-Salt in Mice Depend on Ambient Temperature.

BACKGROUND: Mice prefer warmer environments than humans. For this reason, behavioral and physiological thermoregulatory responses are engaged by mice in response to a standard room temperature of 22 to 24 °C. Autonomic mechanisms mediating thermoregulatory responses overlap with mechanisms activated in hypertension, and, therefore, we hypothesized that housing at thermoneutral temperatures (TNs; 30 °C) would modify the cardiometabolic effects of deoxycorticosterone acetate (DOCA)-salt in mice. METHODS: The effects of DOCA-salt treatment upon ingestive behaviors, energy expenditure, blood pressure, heart rate (HR), and core temperature were assessed in C57BL/6J mice housed at room temperature or TN. RESULTS: Housing at TN reduced food intake, energy expenditure, blood pressure, and HR and attenuated HR responses to acute autonomic blockade by chlorisondamine. At room temperature, DOCA-salt caused expected increases in fluid intake, sodium retention in osmotically inactive pools, blood pressure, core temperature, and also caused expected decreases in fat-free mass, total body water, and HR. At TN, the effects of DOCA-salt upon fluid intake, fat gains, hydration, and core temperature were exaggerated, but effects on energy expenditure and HR were blunted. Effects of DOCA-salt upon blood pressure were similar for 3 weeks and exaggerated by TN housing in the fourth week. CONCLUSIONS: Ambient temperature robustly influences behavioral and physiological functions in mice, including metabolic and cardiovascular phenotype development in response to DOCA-salt treatment. Studying cardiometabolic responses of mice at optimal ambient temperatures promises to improve the translational relevance of rodent models.

Corticosteroid plasma kinetics and gonadal receptor gene expression during the reproductive cycle in female Eurasian Perch: Investigation of the roles of corticosteroids in vitellogenesis.

To improve the quality of reproduction in Eurasian perch, Perca fluviatilis L., which is a promising candidate for Eurasian freshwater aquaculture that is currently cultivated in recirculating aquaculture systems (RAS), investigating the hormones that mediate and affect reproduction in this species is indispensable. The literature defines a group of four major corticosteroids (11-deoxycorticosterone, 11-deoxycortisol, corticosterone and cortisol) that might mediate critical stages of reproduction in female perch. Unfortunately, neither the basic roles nor the kinetics of these four corticosteroids throughout the reproductive cycle of female perch have been well defined to date. In this study, we therefore elucidated the plasma kinetics of these four corticosteroids during the reproductive cycle of domesticated female perch while monitoring the expression of the different receptors and enzymes that mediate their production and possible functions. Additionally, we performed an in vitro experiment during late vitellogenesis to investigate the possible direct roles of these steroids during that stage. Our results revealed that these four corticosteroids were detectable throughout the reproductive cycle, and the levels of most of them (11-deoxycorticosterone, 11-deoxycortisol, and cortisol) fluctuated significantly depending on the stage of reproduction. 11-Deoxycorticosterone and 11-deoxycortisol exhibited their highest levels, 1.8 ng/ml and 58 ng/ml, respectively, at the beginning of the reproductive cycle. By the end of the reproductive cycle, 11-deoxycortisol and cortisol plasma levels exhibited a surge, reaching 58 ng/ml and 150 ng/ml, respectively. During the perch reproductive cycle, the corticosteroid receptor complex is not regulated only at the hormone level, as the expression levels of all corticosteroid receptor genes showed a progressive and similar decline. In vitro exposure of vitellogenic oocytes to some of these corticosteroids (11-deoxycorticosterone and 11-deoxycortisol) induced an increase in yolk globule diameter and a decrease in the density of yolk globules, which indicates the involvement of both of these hormones in yolk globule coalescence. Taken together, these results implicate corticosteroids in the reproductive cycle, although the related cellular mechanisms remain to be investigated.

Effect of 11-Deoxycorticosterone in the Transcriptomic Response to Stress in Rainbow Trout Skeletal Muscle.

In aquaculture, many stressors can negatively affect growth in teleosts. It is believed that cortisol performs glucocorticoid and mineralocorticoid functions because teleosts do not synthesize aldosterone. However, recent data suggest that 11-deoxycorticosterone (DOC) released during stress events may be relevant to modulate the compensatory response. To understand how DOC modifies the skeletal muscle molecular response, we carried out a transcriptomic analysis. Rainbow trout (Oncorhynchus mykiss) were intraperitoneally treated with physiological doses of DOC in individuals pretreated with mifepristone (glucocorticoid receptor antagonist) or eplerenone (mineralocorticoid receptor antagonist). RNA was extracted from the skeletal muscles, and cDNA libraries were constructed from vehicle, DOC, mifepristone, mifepristone plus DOC, eplerenone, and eplerenone plus DOC groups. The RNA-seq analysis revealed 131 differentially expressed transcripts (DETs) induced by DOC with respect to the vehicle group, mainly associated with muscle contraction, sarcomere organization, and cell adhesion. In addition, a DOC versus mifepristone plus DOC analysis revealed 122 DETs related to muscle contraction, sarcomere organization, and skeletal muscle cell differentiation. In a DOC versus eplerenone plus DOC analysis, 133 DETs were associated with autophagosome assembly, circadian regulation of gene expression, and regulation of transcription from RNA pol II promoter. These analyses indicate that DOC has a relevant function in the stress response of skeletal muscles, whose action is differentially modulated by GR and MR and is complementary to cortisol.

Genetic Engineering of Lymphangiogenesis in Skin Does Not Affect Blood Pressure in Mouse Models of Salt-Sensitive Hypertension.

BACKGROUND: Recent studies have indicated that sodium storage is influenced by macrophages that secrete VEGF-C (vascular endothelial growth factor) during salt stress thus stimulating lymphangiogenesis, thereby acting as a buffer against increased blood pressure (BP). We aimed to explore the role of dermal lymphatics in BP and sodium homeostasis. Our hypothesis was that mice with reduced dermal lymphatic vessels were more prone to develop salt-sensitive hypertension, and that mice with hyperplastic vessels were protected. METHODS: Mice with either hypoplastic (Chy), absent (K14-VEGFR3 [vascular endothelial growth factor receptor 3]-Ig), or hyperplastic (K14-VEGF-C) dermal lymphatic vessels and littermate controls were given high-salt diet (4% NaCl in the chow), deoxycorticosterone acetate (DOCA)-salt diet and 1% saline to drink or nitric oxide blocker diet L-NG-nitro arginine methyl ester (followed by high salt diet). BP was measured by telemetric recording, and tissue sodium content by ion chromatography. RESULTS: In contrast to previous studies, high salt diet did not induce an increase in BP or sodium storage in any of the mouse strains investigated. DOCA-salt, on the other hand, gave an increase in BP in Chy and K14-VEGFR3-Ig not different from their corresponding WT controls. DOCA induced salt storage in skin and muscle, but to the same extent in mice with dysfunctional lymphatic vessels and WT controls. Lymph flow as assessed by tracer washout was not affected by the diet in any of the mouse strains. CONCLUSIONS: Our results suggest that dermal lymphatic vessels are not involved in salt storage or blood pressure regulation in these mouse models of salt-sensitive hypertension.

Na+ is shifted from the extracellular to the intracellular compartment and is not inactivated by glycosaminoglycans during high salt conditions in rats.

Recently, studies have emerged suggesting that the skin plays a role as major Na+ reservoir via regulation of the content of glycosaminoglycans and osmotic gradients. We investigated whether there were electrolyte gradients in skin and where Na+ could be stored to be inactivated from a fluid balance viewpoint. Na+ accumulation was induced in rats by a high salt diet (HSD) (8% NaCl and 1% saline to drink) or by implantation of a deoxycorticosterone acetate (DOCA) tablet (1% saline to drink) using rats on a low salt diet (LSD) (0.1% NaCl) on tap water as control. Na+ and K+ were assessed by ion chromatography in tissue eluates, and the extracellular volume by equilibration of 51 Cr-EDTA. By tangential sectioning of the skin, we found a low Na+ content and extracellular volume in epidermis, both parameters rising by ∼30% and 100%, respectively, in LSD and even more in HSD and DOCA when entering dermis. We found evidence for an extracellular Na+ gradient from epidermis to dermis shown by an estimated concentration in epidermis ∼2 and 4-5 times that of dermis in HSD and DOCA-salt. There was intracellular storage of Na+ in skin, muscle, and myocardium without a concomitant increase in hydration. Our data suggest that there is a hydration-dependent high interstitial fluid Na+ concentration that will contribute to the skin barrier and thus be a mechanism for limiting water loss. Salt stress results in intracellular storage of Na+ in exchange with K+ in skeletal muscle and myocardium that may have electromechanical consequences. KEY POINTS: Studies have suggested that Na+ can be retained or removed without commensurate water retention or loss, and that the skin plays a role as major Na+ reservoir via regulation of the content of glycosaminoglycans and osmotic gradients. In the present study, we investigated whether there were electrolyte gradients in skin and where Na+ could be stored to be inactivated from a fluid balance viewpoint. We used two common models for salt-sensitive hypertension: high salt and a deoxycorticosterone salt diet. We found a hydration-dependent high interstitial fluid Na+ concentration that will contribute to the skin barrier and thus be a mechanism for limiting water loss. There was intracellular Na+ storage in muscle and myocardium without a concomitant increase in hydration, comprising storage that may have electromechanical consequences in salt stress.

Cardiometabolic effects of DOCA-salt in male C57BL/6J mice are variably dependent on sodium and nonsodium components of diet.

Hypertension characterized by low circulating renin activity accounts for roughly 25%-30% of primary hypertension in humans and can be modeled experimentally via deoxycorticosterone acetate (DOCA)-salt treatment. In this model, phenotypes develop in progressive phases, although the timelines and relative contributions of various mechanisms to phenotype development can be distinct between laboratories. To explore interactions among environmental influences such as diet formulation and dietary sodium (Na) content on phenotype development in the DOCA-salt paradigm, we examined an array of cardiometabolic endpoints in young adult male C57BL/6J mice during sham or DOCA-salt treatments when mice were maintained on several common, commercially available laboratory rodent "chow" diets including PicoLab 5L0D (0.39% Na), Envigo 7913 (0.31% Na), Envigo 2920x (0.15% Na), or a customized version of Envigo 2920x (0.4% Na). Energy balance (weight gain, food intake, digestive efficiency, and energy efficiency), fluid and electrolyte homeostasis (fluid intake, Na intake, fecal Na content, hydration, and fluid compartmentalization), renal functions (urine production rate, glomerular filtration rate, urine Na excretion, renal expression of renin, vasopressin receptors, aquaporin-2 and relationships among markers of vasopressin release, aquaporin-2 shedding, and urine osmolality), and blood pressure, all exhibited changes that were subject to interactions between diet and DOCA-salt. Interestingly, some of these phenotypes, including blood pressure and hydration, were dependent on nonsodium dietary components, as Na-matched diets resulted in distinct phenotype development. These findings provide a broad and robust illustration of an environment × treatment interaction that impacts the use and interpretation of a common rodent model of low-renin hypertension.

Aldosterone and dexamethasone activate African lungfish mineralocorticoid receptor: Increased activation after removal of the amino-terminal domain.

Aldosterone, the main physiological mineralocorticoid in humans and other terrestrial vertebrates, first appears in lungfish, which are lobe-finned fish that are forerunners of terrestrial vertebrates. Aldosterone activation of the MR regulates internal homeostasis of water, sodium and potassium, which was critical in the conquest of land by vertebrates. We studied transcriptional activation of the slender African lungfish MR by aldosterone, other corticosteroids and progesterone and find that aldosterone, 11-deoxycorticosterone, 11-deoxycortisol and progesterone have half-maximal responses (EC50 s) below 1 nM and are potential physiological mineralocorticoids. In contrast, EC50 s for corticosterone and cortisol were 23 nM and 66 nM, respectively. Unexpectedly, truncated lungfish MR, consisting of the DNA-binding, hinge and steroid-binding domains, had a stronger response to corticosteroids and progesterone than full-length lungfish MR, indicating that the N-terminal domain represses steroid activation of lungfish MR, unlike human MR in which the N-terminal domain contains an activation function. BLAST searches of GenBank did not retrieve a GR ortholog, leading us to test dexamethasone and triamcinolone for activation of lungfish MR. At 10 nM, both synthetic glucocorticoids are about 4-fold stronger than 10 nM aldosterone in activating full-length lungfish MR, leading us to propose that lungfish MR also functions as a GR.

ß-Arrestin-Biased Agonist Targeting the Brain AT1R (Angiotensin II Type 1 Receptor) Increases Aversion to Saline and Lowers Blood Pressure in Deoxycorticosterone Acetate-Salt Hypertension.

Activation of central AT1Rs (angiotensin type 1 receptors) is required for the increased blood pressure, polydipsia, and salt intake in deoxycorticosterone acetate (DOCA)-salt hypertension. TRV120027 (TRV027) is an AT1R-biased agonist that selectively acts through ß-arrestin. We hypothesized that intracerebroventricular administration of TRV027 would ameliorate the effects of DOCA-salt. In a neuronal cell line, TRV027 induced AT1aR internalization through dynamin and clathrin-mediated endocytosis. We next evaluated the effect of chronic intracerebroventricular infusion of TRV027 on fluid intake. We measured the relative intake of water versus various saline solutions using a 2-bottle choice paradigm in mice subjected to DOCA with a concomitant intracerebroventricular infusion of either vehicle, TRV027, or losartan. Sham mice received intracerebroventricular vehicle without DOCA. TRV027 potentiated DOCA-induced water intake in the presence or absence of saline. TRV027 and losartan both increased the aversion for saline-an effect particularly pronounced for highly aversive saline solutions. Intracerebroventricular Ang (angiotensin) II, but not TRV027, increased water and saline intake in the absence of DOCA. In a separate cohort, blood pressure responses to acute intracerebroventricular injection of vehicle, TRV, or losartan were measured by radiotelemetry in mice with established DOCA-salt hypertension. Central administration of intracerebroventricular TRV027 or losartan each caused a significant and similar reduction of blood pressure and heart rate. We conclude that administration of TRV027 a selective ß-arrestin biased agonist directly into the brain increases aversion to saline and lowers blood pressure in a model of salt-sensitive hypertension. These data suggest that selective activation of AT1R ß-arrestin pathways may be exploitable therapeutically.

ELABELA attenuates deoxycorticosterone acetate/salt-induced hypertension and renal injury by inhibition of NADPH oxidase/ROS/NLRP3 inflammasome pathway.

ELABELA (ELA), a 32-residue hormone peptide abundantly expressed in adult kidneys, has been identified as a novel endogenous ligand for APJ/Apelin receptor. The aim of this study was to investigate the role of ELA in deoxycorticosterone acetate (DOCA)/salt-induced hypertension and further explore the underlying mechanism. In DOCA/salt-treated rats, the mRNA level of ELA greatly decreased in the renal medulla. Next, overexpression of ELA in the kidney was found to attenuate DOCA/salt-induced hypertension and renal injury, including lower blood pressure, reversed glomerular morphological damage, decreased blood urea nitrogen (BUN), and blocked the accumulation of fibrotic markers. Mechanistically, ELA overexpression inhibited renal nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and subsequent reactive oxygen species (ROS) production, thus resulted in the blockade of formation and activation of Nod-like receptor protein 3 (NLRP3) inflammasome. The inhibitory effects of ELA on Aldosterone-stimulated NADPH oxidase/ROS/NLRP3 inflammasome pathway were confirmed in the human renal tubular cells. Furthermore, our in vivo and in vitro results showed that the deficiency of the apelin receptor APJ did not influence the antihypertensive effect and blockage to NADPH oxidase/ROS/NLRP3 pathway of ELA. Moreover, in heterozygous ELA knockout mice (ELA+/-), the ELA deficiency remarkably accelerated the onset of DOCA/salt-induced hypertension. Our data demonstrate that ELA prevents DOCA/salt-induced hypertension by inhibiting NADPH oxidase/ROS/NLRP3 pathway in the kidney, which is APJ independent. Pharmacological targeting of ELA may serve as a novel therapeutic strategy for the treatment of hypertensive kidney disease.

Analysis of Modulation of the ρ1 GABAA Receptor by Combinations of Inhibitory and Potentiating Neurosteroids Reveals Shared and Distinct Binding Sites.

The ρ1 GABAA receptor is prominently expressed in the retina and is present at lower levels in several brain regions and other tissues. Although the ρ1 receptor is insensitive to many anesthetic drugs that modulate the heteromeric GABAA receptor, it maintains a rich and multifaceted steroid pharmacology. The receptor is negatively modulated by 5ß-reduced steroids, sulfated or carboxylated steroids, and ß-estradiol, whereas many 5α-reduced steroids potentiate the receptor. In this study, we analyzed modulation of the human ρ1 GABAA receptor by several neurosteroids, individually and in combination, in the framework of the coagonist concerted transition model. Experiments involving coapplication of two or more steroids revealed that the receptor contains at least three classes of distinct, nonoverlapping sites for steroids, one each for the inhibitory steroids pregnanolone (3α5ßP), 3α5ßP sulfate, and ß-estradiol. The site for 3α5ßP can accommodate the potentiating steroid 5αTHDOC. The findings are discussed with respect to receptor modulation by combinations of endogenous neurosteroids. SIGNIFICANCE STATEMENT: The study describes modulation of the ρ1 GABAA receptor by neurosteroids. The coagonist concerted transition model was used to determine overlap of binding sites for several inhibitory and potentiating steroids.

Generalized Concentration Addition Model Predicts Glucocorticoid Activity Bioassay Responses to Environmentally Detected Receptor-Ligand Mixtures.

Many glucocorticoid receptor (GR) agonists have been detected in waste and surface waters domestically and around the world, but the way a mixture of these environmental compounds may elicit a total glucocorticoid activity response in water samples remains unknown. Therefore, we characterized 19 GR ligands using a CV1 cell line transcriptional activation assay applicable to water quality monitoring. Cells were treated with individual GR ligands, a fixed ratio mixture of full or partial agonists, or a nonequipotent mixture with full and partial agonists. Efficacy varied (48.09%-102.5%) and potency ranged over several orders of magnitude (1.278 × 10-10 to 3.93 × 10-8 M). Concentration addition (CA) and response addition (RA) mixtures models accurately predicted equipotent mixture responses of full agonists (r2 = 0.992 and 0.987, respectively). However, CA and RA models assume mixture compounds produce full agonist-like responses, and therefore they overestimated observed maximal efficacies for mixtures containing partial agonists. The generalized concentration addition (GCA) model mathematically permits < 100% maximal responses, and fell within the 95% confidence interval bands of mixture responses containing partial agonists. The GCA, but not CA and RA, model predictions of nonequipotent mixtures containing both full and partial agonists fell within the same statistical distribution as the observed values, reinforcing the practicality of the GCA model as the best overall model for predicting GR activation. Elucidating the mechanistic basis of GR activation by mixtures of previously detected environmental GR ligands will benefit the interpretation of environmental sample contents in future water quality monitoring studies.

The effects of estradiol and testosterone on renal tissues oxidative after central injection of angiotensin II in female doca - salt treated rats.

Background Although numerous studies have proven that estrogen (Est) has a protective effect on the development of hypertension, more research needs to be done to show its detailed mechanism in a variety of hypertension. The important role of active oxygen species in blood pressure is well defined. We examined whether or not sex hormones change the growth of reactive oxygen species (ROS) in kidneys after central microinjection of angiotensin II (Ang II). Materials and methods Female Wistar rats, 8 weeks old (200 ± 10 g) were used in this study. The animal groups were (1) Sham, (2) Ovariectomy (OVX), (3) Sham-Hypertension (Sham-Hyper), (4) OVX-Hypertension (OVX-Hyper), (5) Sham-Hyper-Est, (6) OVX-Hyper-Est ; (7) Sham-Hyper-Testosterone (Tst) and (8) OVX-Hyper-Tst. Solutions of 1% NaCl and 0.1 KCl were used and desoxycorticostrone (doca-salt) was injected (45 mg/kg) 3 times a week in Hypertension groups. Estradiol and Tst (2 mg/kg and 5 mg/kg ; daily; subcutaneously) for 4 weeks. Ang II (50 µM, 5 µL) was microinjected by intracerebroventricular ( i.c.v.) infusion and malondialdehyde (MDA) and thiol in the kidneys were measured. Results MDA in the kidneys was increased by Ang II and doca-salt treatments. Both estradiol and Tst decreased the kidney's MDA. The level of thiol was higher in Hyper groups and reversed after treatment with estradiol and Tst. Conclusions Our findings suggest that central effect of Ang II on blood pressure and kidney disease is accompanied with increased levels of oxidative stress in the kidneys. Indeed sex hormones change the ROS level in the kidneys after central microinjection of Ang II. .

Endogenous regulation of 11-deoxycorticosterone (DOC) and corticosteroid receptors (CRs) during rainbow trout early development and the effects of corticosteroids on hatching.

Clear evidence for a physiological role of the mineralocorticoid-like hormone 11-deoxycorticosterone (DOC) and the mineralocorticoid receptor (MR) in fish is still lacking. Efforts to demonstrate an osmoregulatory role for this hormone has so far not been conclusive, while a few scattered studies have indicated a role for DOC in development and reproduction. In this study, we investigate the onset of de novo DOC synthesis in parallel with endogenous corticosteroid receptor mRNA production from fertilization to the swim-up stage in rainbow trout. Whole egg DOC content decreased from fertilization until hatching followed by an increase to pre-fertilization levels just after hatching. Onset of de novo transcription of corticosteroid receptor mRNA's was observed shortly after the midblastula transition; initially glucocorticoid receptor 2 (GR2) followed by MR and then GR1. Non-invasive introduction of DOC or cortisol at fertilization resulted in altered corticosteroid receptor regulation and accelerated hatching date, suggesting a regulatory role in trout ontogenesis of both hormones through MR signaling pathway. The results presented in this study suggest a possible physiological role of the DOC-MR signaling pathway during fish ontogenesis, at fertilization and just after hatching.

Effect of the allopregnanolone and allotetrahydrodeoxycorticosteron on spike-wave discharges in the EEG of absence epilepsy rat models.

Absence epilepsy is a generalized nonconvulsive type of epilepsy that is characterized by spike-wave discharges (SWD) with a frequency of 2.5-4 Hz in the EEG. The activation of the GABAergic system in central nervous system suppresses convulsive seizures but exacerbates absence seizures. Endogenous neuroactive steroids such as 3α-hydroxy-5α-pregnan-20-one (3α,5α-THPROG; allopregnanolone) and 3α,21-dihydroxy-5α-pregnan-20-one (3α,5α-THDOC, allotetrahydrodeoxycorticosteron) are GABA-A receptor-positive allosteric modulators. Finasteride which is a 5α-reductase inhibitor can selectively block the synthesis of endogenous steroids. In this study, we compared the effects of endogenous steroids (THPROG and THDOC) on SWD by using finasteride-treated Wistar Albino Glaxo from Rijswijk (WAG/Rij) rats as a model of absence epilepsy. Wistar (WIS-THPROG and WIS-THDOC) and WAG/Rij (WAG-THPROG and WAG-THDOC) rats were divided into 4 groups (n = 8). After stereotactic surgical procedures, all rats were prepared for direct cortical EEG measurement. Following finasteride administration to each group, THPROG was administered to WIS-THPROG and WAG-THPROG groups, and THDOC to WIS-THDOC and WAG-THDOC groups intraperitoneally. While there was no any SWD activity detected in WIS-THPROG and WIS-THDOC groups, a significant increase in SWD count in WAG-THPROG (p = 0.012) and in WAG-THDOC (p = 0.012), and in SWD total duration in WAG-THPROG (p = 0.012) and WAG-THDOC groups (p = 0.011) were observed after steroid injection. No difference between the efficacy of THPROG and THDOC on absence seizures in WAG/Rij rats was observed.

Presynaptic Regulation of Tonic Inhibition by Neuromodulatory Transmitters in the Basal Amygdala.

Tonic inhibition mediated by ambient levels of GABA that activate extrasynaptic GABAA receptors emerges as an essential factor that tunes neuronal network excitability in vitro and shapes behavioral responses in vivo. To address the role of neuromodulatory transmitter systems on this type of inhibition, we employed patch clamp recordings in mouse amygdala slice preparations. Our results show that the current amplitude of tonic inhibition (Itonic) in projection neurons of the basal amygdala (BA) is increased by preincubation with the neurosteroid THDOC, while the benzodiazepine diazepam is ineffective. This suggests involvement of THDOC sensitive δ subunit containing GABAA receptors in mediating tonic inhibition. Moreover, we provide evidence that the neuromodulatory transmitters NE, 5HT, and ACh strongly enhance spontaneous IPSCs as well as Itonic in the BA. As the increase in frequency, amplitude, and charge of sIPSCs by these neuromodulatory transmitters strongly correlated with the amplitude of Itonic, we conclude that spill-over of synaptic GABA leads to activation of Itonic and thereby to dampening of amygdala excitability. Since local injection of THDOC, as a positive modulator of tonic inhibition, into the BA interfered with the expression of contextual fear memory, our results point to a prominent role of Itonic in fear learning.

Physiological and proteomic responses to corticosteroid treatments in Eurasian perch, Perca fluviatilis: Investigation of immune-related parameters.

The comparative effects of cortisol and 11-deoxycorticosterone (DOC), two major corticosteroids in fish, have yet received little attention in teleosts. We evaluated the proteomic and immune responses of Eurasian perch to chronic corticosteroid treatments. We implanted immature perch with cortisol (80mg/kg) or DOC (4mg/kg) and measured the proportions of blood leucocytes, immune indices in the plasma, spleen and liver (complement and lysozyme activity, total immunoglobulin and immune gene expression in the tissues) and differential proteome expression (corticosteroid versus control) in the liver and the spleen on days 2, 4 and 14 post-treatment. Implantation of cortisol decreased the ratio of blood leucocytes and depressed Ig levels in both organs while DOC modulated the proportion of leucocyte sub-populations (increase in lymphocytes and decrease in granulocytes). In contrast, the innate humoral immunity was not strongly influenced by any of corticosteroid implants. The only immune parameter that was significantly affected was lysozyme, after DOC treatment. A number of proteins were differentially regulated by these hormones and some were identified in the liver (21 for cortisol and 8 for DOC) and in the spleen (10 for cortisol and 10 for DOC). None of the proteins was directly linked to immunity, except the natural killer enhancing factor, which was repressed by cortisol in the spleen. Our results also confirm that the proteins involved in energetic and glucose metabolism are affected by corticosteroids. Furthermore, these corticosteroids differently regulate immune status in Eurasian perch and they primarily impact leucocytes, as opposed to innate immune function.

DOCA/salt hypertension alters Period1 and orexin-related gene expression in the medulla and hypothalamus of male rats: Diurnal influences.

In peripheral tissues, aldosterone alters expression of multiple genes, including the clock gene Period 1 (Per1), 11 beta-hydroxysteroid dehydrogenase-2 (11-HSD2), and α-ENAC, the epithelial sodium channel subunit. We evaluated the impact of chronic aldosterone exposure (DOCA) and salt intake on nocturnal changes in gene expression in the male Sprague Dawley rat brain. Additionally, genes associated with the orexin (ORX) system were also evaluated based on the role of this neuropeptide in arousal, feeding and hypertension and an interconnection with Per1 expression. DOCA/salt treatment increased saline intake primarily at night, elevated arterial pressure and lowered heart rate. In the medulla oblongata, DOCA/salt upregulated Per1, 11-HSD2, and α-ENAC expression independent of time of day, but did not change ORX receptor type 1 (ORX-R1) or type 2 (ORX-R2) expression. ORX-R1, and ORX-R2 expression in the medulla did however correlate with Per1 expression following DOCA/salt treatment but not in controls. In the hypothalamus, DOCA/salt treatment upregulated Per1, ORX-A, and ORX-R2 expression, in general, and Per1 and ORX-A expression at night. ORX-A, ORX-R1 and ORX-R2 expression in the hypothalamus correlated with Per1 expression following DOCA/salt but not in controls. These findings demonstrate for the first time that DOCA/salt hypertension modulates ORX gene expression in the brain and suggest that changes in expression in the ORX system may occur directly or indirectly via aldosterone-induced changes in Per1 expression. Our findings also build on emerging evidence that monitoring gene expression during both the day and night is critical to understanding the role of specific genes in hypertension.

Desoxycorticosterone Pivalate Duration of Action and Individualized Dosing Intervals in Dogs with Primary Hypoadrenocorticism.

BACKGROUND: Clinicians alter dosing for desoxycorticosterone pivalate (DOCP) to mitigate costs, but this practice has not been critically evaluated in a prospective clinical trial. HYPOTHESIS/OBJECTIVES: The duration of action of DOCP is longer than 30 days in dogs with primary hypoadrenocorticism (PH). ANIMALS: A total of 53 client-owned dogs with PH. Twenty-four dogs with newly diagnosed PH (Group 1) and 29 dogs with treated PH (Group 2). METHODS: Prospective, multicenter, clinical trial. For phase I, DOCP was administered and plasma sodium and potassium concentrations were measured until the dog developed hyponatremia or hyperkalemia at a planned evaluation, or displayed clinical signs with plasma electrolyte concentrations outside of the reference interval independent of a planned evaluation, thus defining DOCP duration of action. Plasma electrolyte concentrations then were assessed at the end of the individualized dosing interval (IDI; i.e., DOCP duration of action minus 7 days, phase II and at least 3 months after concluding phase II, phase III). RESULTS: The duration of action of DOCP in dogs in phase I with naïve PH (n = 24) ranged from 32 to 94 days (median, 62 days; 95% confidence interval [CI], 57, 65) and previously treated PH (n = 29) from 41 to 124 days (median, 67 days; CI, 56, 72). Overall, the final DOCP dosing interval for all dogs that completed phase II (n = 36) ranged from 38 days to 90 days (median, 58 days; CI, 53, 61). No dog that completed phase III (n = 15) required reduction in the IDI. The DOCP duration of action, independent of group, was not significantly associated with several baseline variables. The median drug cost reduction using IDI was approximately 57.5% per year. CONCLUSION AND CLINICAL IMPORTANCE: The duration of action of DOCP in dogs with PH is >30 days, and plasma sodium and potassium concentrations can be maintained with an IDI >30 days long term.

Deoxycorticosterone/Salt-Mediated Cardiac Inflammation and Fibrosis Are Dependent on Functional CLOCK Signaling in Male Mice.

Activation of the mineralocorticoid receptor (MR) promotes inflammation, fibrosis, and hypertension. Clinical and experimental studies show that MR antagonists have significant therapeutic benefit for all-cause heart failure; however, blockade of renal MRs limits their widespread use. Identification of key downstream signaling mechanisms for the MR in the cardiovascular system may enable development of targeted MR antagonists with selectivity for pathological MR signaling and lower impact on physiological renal electrolyte handling. One candidate pathway is the circadian clock, the dysregulation of which is associated with cardiovascular diseases. We have previously shown that the circadian gene Per2 is dysregulated in hearts with selective deletion of cardiomyocyte MR. We therefore investigated MR-mediated cardiac inflammation and fibrosis in mice that lack normal regulation and oscillation of the circadian clock in peripheral tissues, that is, CLOCKΔ19 mutant mice. The characteristic cardiac inflammatory/fibrotic response to a deoxycorticosterone (DOC)/salt for 8 weeks was significantly blunted in CLOCKΔ19 mice when compared with wild-type mice, despite a modest increase at "baseline" for fibrosis and macrophage number in CLOCKΔ19 mice. In contrast, cardiac hypertrophy in response to DOC/salt was significantly greater in CLOCKΔ19 vs wild-type mice. Markers for renal inflammation and fibrosis were similarly attenuated in the CLOCKΔ19 mice given DOC/salt. Moreover, increased CLOCK expression in H9c2 cardiac cells enhanced MR-mediated transactivation of Per1, suggesting cooperative signaling between these transcription factors. This study demonstrates that the full development of MR-mediated cardiac inflammation and fibrosis is dependent on intact signaling by the circadian protein CLOCK.

Low-dose sustained-release deoxycorticosterone acetate-induced hypertension in Bama miniature pigs for renal sympathetic nerve denervation.

Recently, the effectiveness of renal sympathetic nerve denervation for treatment of hypertension has been doubted after SYMPLICITY HTN-3 trial. An ideal animal model is still unavailable for preclinical study about catheter-based renal sympathetic nerve denervation for treatment of hypertension. Traditional high-dose deoxycorticosterone acetate (DOCA)-induced hypertension pig model has some problems due to extensive end-organ damage. Based on the similarity in the anatomic characteristics of renal artery between pigs and humans, this study was undertaken to establish a low-dose sustained-release DOCA-induced hypertension model in pigs. A total of 14 pigs were subcutaneously implanted with low-dose DOCA in the abdomen and cannulated from the femoral artery for the measurement of blood pressure (BP). Plasma angiotensin I (Ang I), angiotensin II (Ang II), plasma renin activity (PRA), aldosterone (Ald), creatinine, epinephrine, and norepinephrine (NE) were determined before and after treatments. The kidneys were collected and processed for hematoxylin and eosin staining, Masson-Goldner trichromic, and periodic acid Schiff staining. Ten pigs survived for 1 month. Mean BP significantly increased after 2-week treatment (P < .001). The plasma Ang I, Ang II, PRA, and Ald significantly decreased (Ang I: 6.92 ± 6.06 vs. 2.22 ± 3.08, P = .002; Ang II: 768.85 ± 525.8 vs. 213.76 ± 148.63, P = .003; PRA: 1.68 ± 1.67 vs. 0.29 ± 0.39, P = .008; Ald: 0.37 ± 0.12 vs. 0.25 ± 0.09, P < .001), but norepinephrine significantly increased (7.59 ± 4.57 vs. 16.96 ± 10.38, P = .021). Plasma creatinine remained unchanged. Hisotological examination showed mild damage to the kidney. Low-dose sustained-release DOCA is able to induce hypertension in pigs. A femoral catheter is not only helpful for monitoring BP, but can be used to quickly exchange the renal sympathetic nerve denervation equipment.