Mineralocorticoid Receptor Agonist Research Articles

The selective mineralocorticoid receptor modulator AZD9977 reveals differences in mineralocorticoid effects of aldosterone and fludrocortisone.

Introduction:AZD9977 is a novel mineralocorticoid receptor (MR) modulator, which in preclinical studies demonstrated organ protection without affecting aldosterone-regulated urinary electrolyte excretion. However, when tested in humans, using fludrocortisone as an MR agonist, AZD9977 exhibited similar effects on urinary Na+/K+ ratio as eplerenone. The aim of this study is to understand whether the contradictory results seen in rats and humans are due to the mineralocorticoid used.Materials and methods:Rats were treated with single doses of AZD9977 or eplerenone in combination with either aldosterone or fludrocortisone. Urine was collected for five to six hours and total amounts excreted Na+ and K+ were assessed.Results:AZD9977 dose-dependently increased urinary Na+/K+ ratio in rats when tested against fludrocortisone, but not when tested against aldosterone. Eplerenone dose-dependently increased urinary Na+/K+ ratio when tested against fludrocortisone as well as aldosterone.Conclusions:The data suggest that the contrasting effects of AZD9977 on urinary electrolyte excretion observed in rats and humans are due to the use of the synthetic mineralocorticoid fludrocortisone. Future clinical studies are required to confirm the reduced electrolyte effects of AZD9977 and the subsequent lower predicted hyperkalemia risk.

Evaluation of Heart Failure Medication Prescriptions using an Online Drug Discount Program

Introduction Drug discount card programs have emerged as an additional option for patients to offset the increasing costs of drugs. However, there is limited knowledge about online drug discount programs and their role on prescription medications. We evaluated the frequency of drug discount card utilization and estimated cost savings associated with heart failure (HF) medication prescriptions. Methods We conducted a retrospective study of all HF prescriptions filled through the NeedyMeds.org drug discount card program nationwide, from January 2009 to December 2016. We evaluated the frequency of drug discount card prescriptions (across pharmacy types, pharmacy location, by prescriber specialty and by drug class) and calculated cost savings (average per drug discount card and total program dollars saved) for entire study period and for each year (from 2009 to 2016). Results We identified 381,347 prescriptions (8.2% of all prescriptions) for medications that can be used for heart failure with drug discount cards (83.7% at national, 5.7% at regional and 9.8% at local pharmacies). Most prescriptions were filled at urban locations (89.1% in urban clusters, 7.6% in urbanized areas) and in ZIP-codes with lower median household income (65.5%). Angiotensin-converting enzyme inhibitors and selected angiotensin receptor blockers were the most prescribed drugs with discount cards (44.3%) followed by beta blockers (27.1%), diuretics (21.8%), and mineralocorticoid receptor agonists (3.5%). The prescriptions for newer drugs were limited (sacubitril-valsartan: 12; ivabradine: 27). The number of HF prescriptions with discount cards increased from 2577 in 2009 to 64,750 in 2016. Similar increases in the number of prescriptions were noted across all drug classes. A total of 224,049 prescriptions for HF medications (59% of the total) benefited from the program resulting in total savings of $4,739,204 with a median cost saving of $9.30 (41.5%) per prescription. Conclusion Drug discount prescription use increased during the study period, resulting in cost savings on HF prescription medications (approximately $9 in savings per prescription) compared to the original cost charged by pharmacies. Lack of use for newer drugs may suggest unwillingness of pharmaceutical company or pharmacy benefit manager to provide this option. Overall, while on-line drug assistance programs may reduce financial burden for generic drugs, pricing transparency remains obtuse.

Potential endocrine-disrupting effects of metals via interference with glucocorticoid and mineralocorticoid receptors

As a result of human activities, the pollution of metals is becoming ubiquitous in the environment. Among various toxicological mechanisms of action, metals have been considered as endocrine-disrupting chemicals (EDCs) through interference with steroid receptors. However, information regarding the potential endocrine disruption of metals on glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) is especially scarce. In this study, a total of 16 metals were assessed for their GR/MR activities using luciferase reporter gene assay. None of the tested metals exhibited GR or MR agonistic activity, but a total of 7 and 5 candidate metals showed obvious GR and MR antagonistic properties, respectively. All 7 GR antagonistic metals [ BaCl2, CoCl2, CuCl2, Pb(NO3)2, LiCl, SnCl2 and ZnCl2] inhibited glucocorticoid-responsive gene GILZ expression in J774A.1 cells. Further investigations indicated that the 5 MR antagonistic metals [ CdCl2, Pb(NO3)2, LiCl, MnCl2 and SnCl2] antagonized aldosterone-inhibited hepatocellular carcinoma cell proliferation. Among these metals, Pb(NO3)2, LiCl, and SnCl2 showed both anti-glucocorticoid and anti-mineralocorticoid activities. Comprehensive screening and evaluation of GR and MR antagonists and agonists among metals should be considered to better understand the ecological and health risks of metals.

Abstract 232: Medication Cost Savings Using an On-line Drug Discount Program

Objective: To evaluate the frequency of drug discount card utilization and to estimate cost savings associated with heart failure (HF) medication prescriptions. Methods: We conducted a retrospective study of all HF prescriptions filled through the NeedyMeds.org drug discount card program nationwide, from January 2009 to December 2016. We evaluated the frequency of drug discount card prescriptions (across pharmacy types, pharmacy location, by prescriber specialty and by drug class) and calculated cost savings (average per drug discount card and total program dollars saved) for entire study period and for each year (from 2009 to 2016). Findings: A total of 381,347 prescriptions for medications that can be used for HF with drug discount cards were identified during the study period (83.7% at national, 5.7% at regional and 9.8% at local pharmacies). Most prescriptions were filled at urban locations (89.1% in urban clusters, 7.6% in urbanized areas) and in ZIP-codes with lower median household income (65.5%). Angiotensin-converting enzyme inhibitors and selected angiotensin receptor blockers were the most prescribed drugs with discount cards (44.1%) followed by beta blockers (27.5%), diuretics (21.5%), and mineralocorticoid receptor agonists (3.9%). The number of HF prescriptions with drug discount cards increased from 2577 in 2009 to 64,750 in 2016. Increase in the number of prescriptions was also noted for all drug classes from 2009 to 2016. Overall 224,049 prescriptions for HF medications (59% of the total) benefited from the program resulting in total savings of $4,739,204 with a median cost saving of $9.30 (41.5%) per prescription. Conclusion: Use of a drug discount program resulted in cost savings on HF prescription medications (approximately $9 in savings per prescription) compared to the original cost charged by pharmacies. While these drug assistance programs may reduce financial burden, continued efforts should be made to improve adherence to medications and for better outcomes.

Chronically Increased Aldosterone In Vivo Attenuates Coronary Vasodilation to Adenosine

Adenosine‐induced coronary vasodilation is an important cardioprotective mechanism during cardiac hypoxia and ischemia and is utilized clinically for the determination of coronary flow reserve (CFR), an independent predictor of cardiac mortality. Impaired coronary vasodilation to adenosine is more common in diseases associated with inappropriate activation of the renin‐angiotensin‐aldosterone system (RAAS) and recent evidence indicates that systemic blockade of mineralocorticoid receptors (MR) improves CFR in patients with diabetes. The mechanisms underlying RAAS‐dependent attenuation of coronary adenosine‐induced vasodilation, however, remain unclear. Therefore, we hypothesized that chronic infusion of the MR agonist aldosterone would blunt coronary adenosine‐mediated vasodilation and that this would involve reduced adenosine type 2A (A2A) receptor‐dependent vasodilation. Studies were performed using 13–15 week old male C57BL/6J mice infused with either vehicle (95% EtOH in saline) or a subpressor dose of aldosterone (250 μg/kg/d) chronically for 4 weeks via osmotic minipump. After infusion, left ventricular coronary arteries were dissected and function was assessed by wire myography. Chronic infusion of aldosterone raised circulating aldosterone concentrations but did not increase heart weight. Aldosterone infusion blunted coronary vasodilation to adenosine, particularly within the range of adenosine concentrations relevant to those in vivo during cardiac hypoxia (1–10 μM), but did not reduce maximal adenosine‐induced vasodilation. Coronary vasodilation to the endothelium‐dependent vasodilator acetylcholine was also blunted by aldosterone infusion. Additional studies revealed that coronary adenosine‐induced vasodilation in the mouse is entirely dependent on activation of K+ channels and that selective activation of adenosine type 2B (A2B) receptors with BAY 60–6583 induces greater vasodilation that activation of A2A receptors with CGS‐21680. Chronic aldosterone infusion blunted coronary vasodilation in response to selective A2A, but not selective A2B, activation. RT‐PCR revealed no difference in aortic A2A and A2B gene expression. Taken together, our data demonstrate that chronic elevations of aldosterone blunt coronary vasodilation to adenosine via impaired A2A‐dependent vasodilation. This aldosterone‐dependent blunting of adenosine‐mediated vasodilation has implications for cardiac injury in response to hypoxia in states associated with inappropriate RAAS activation such as obesity and diabetes.Support or Funding InformationFunded by VHA BLR&D BX002030 (SBB) and a STRIDE Undergraduate Summer Research Fellowship (MK) supported by the American Physiological Society and NIH NHLBI R25 HL115473‐01.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Deletion of BMAL1 in Smooth Muscle Cells Protects Mice From Abdominal Aortic Aneurysms.

Abdominal aortic aneurysm (AAA) has high mortality rate when ruptured, but currently, there is no proven pharmacological therapy for AAA because of our poor understanding of its pathogenesis. The current study explored a novel role of smooth muscle cell (SMC) BMAL1 (brain and muscle Arnt-like protein-1)-a transcription factor known to regulate circadian rhythm-in AAA development. SMC-selective deletion of BMAL1 potently protected mice from AAA induced by (1) MR (mineralocorticoid receptor) agonist deoxycorticosterone acetate or aldosterone plus high salt intake and (2) angiotensin II infusion in hypercholesterolemia mice. Aortic BMAL1 was upregulated by deoxycorticosterone acetate-salt, and deletion of BMAL1 in SMCs selectively upregulated TIMP4 (tissue inhibitor of metalloproteinase 4) and suppressed deoxycorticosterone acetate-salt-induced MMP (matrix metalloproteinase) activation and elastin breakages. Moreover, BMAL1 bound to the Timp4 promoter and suppressed Timp4 transcription. These results reveal an important, but previously unexplored, role of SMC BMAL1 in AAA. Moreover, these results identify TIMP4 as a novel target of BMAL1, which may mediate the AAA protective effect of SMC BMAL1 deletion.

Abstract 058: Deoxycorticosterone-induced Hypertension Caused by Fluconazole

Deoxycorticosterone (DOC), a steroid precursor of aldosterone, constitutes an endogenous mineralocorticoid receptor agonist. Exogenous DOC produces a well-established animal model of mineralocorticoid-induced salt-dependent hypertension (HTN), but, except for rare cases of congenital adrenal hyperplasia with pediatric HTN due to CYP11 β1 (11 β hydroxylase, P450c11 β ) deficiency, DOC is rarely considered a cause of clinical hypertension (HTN). We recently suspected DOC as the novel mechanism of severe low-renin HTN (blood pressure, BP 220/150 mmHg, plasma renin activity 0.21 ng/mL/hour) that developed de novo in a 47- year old African American man with previously normal BP (118/74) during treatment of disseminated coccidiodomycosis with high-dose fluconazole, which blocks P450 enzymes in the fungal cell wall. To determine if the fluconazole also inhibited human CYP11β1, the adrenal P450 enzyme that converts DOC to corticosterone, we performed a comprehensive adrenal steroid panel on the patient’s serum using mass spectrometry. During the HTN, his serum [DOC] was 24-times the upper limit of normal (354 vs. <15 ng/dL), while corticosterone was low and aldosterone was undetectable. Both the HTN and the high DOC were caused by fluconazole, because they normalized after treatment ended. We then proved that fluconazole causes dose-dependent inhibition of CYP11β1 in vitro by transfecting a mammalian (hamster V79) cell line with human CYP11 β 1 and incubating the cells with DOC. The observed IC50—10 μM— is certainly less than the plasma [fluconazole] during oral high-dose therapy. From these data, we conclude that high-dose fluconazole inhibits DOC conversion by human CYP11β1, causing in this adult patient an acquired form of severe DOC-induced HTN. Given the recent 10-fold increase in coccidiodomycosis cases in the United States and the marked propensity for disseminated disease in African American patients, BP should be monitored carefully during treatment with high-dose fluconazole. Additional case finding studies are warranted.

Aldosterone and Mineralocorticoid Receptors-Physiology and Pathophysiology.

Aldosterone is a uniquely terrestrial hormone, first appearing in lungfish, which have both gills and lungs. Mineralocorticoid receptors (MRs), on the other hand, evolved much earlier, and are found in cartilaginous and bony fish, presumptive ligand cortisol. MRs have equivalent high affinity for aldosterone, progesterone, and cortisol; in epithelia, despite much higher cortisol circulating levels, aldosterone selectively activates MRs by co-expression of the enzyme 11β-hydroxysteroid dehydrogenase, Type 11. In tissues in which the enzyme is not expressed, MRs are overwhelmingly occupied but not activated by cortisol, which normally thus acts as an MR antagonist; in tissue damage, however, cortisol mimics aldosterone and acts as an MR agonist. The risk profile for primary aldosteronism (PA) is much higher than that in age-, sex-, and blood pressure-matched essential hypertensives. High levels of aldosterone per se are not the problem: in chronic sodium deficiency, as seen in the monsoon season in the highlands of New Guinea, plasma aldosterone levels are extraordinarily high, but cause neither hypertension nor cardiovascular damage. Such damage occurs when aldosterone levels are out of the normal feedback control, and are inappropriately elevated for the salt status of the individual (or experimental animal). The question thus remains of how excess salt can synergize with elevated aldosterone levels to produce deleterious cardiovascular effects. One possible mechanism is through the agency of the elusive ouabain-like factors (OLFs). Such factors are secreted from the adrenal in response to ACTH (adrenalocortical tropic hormone), to angiotensin via AT2R, and—the polar opposite of aldosterone—to sodium loading. They act on blood vessels to cause vasoconstriction and thus elevate blood pressure to dump excess sodium through pressure natriuresis. Their levels are chronically elevated in PA in response to the continually elevated sodium status, and they thus act to constrict coronary and systemic arteries. In the context of the elevated blood volume and total body sodium in a PA patient, this raises blood pressure and acts as the proximate cause of cardiovascular damage. If this is the case, it would appear to offer new insights into therapy for PA. One would be the use of digibindin, or its more recent successors as antagonists of OLFs acting on Na/K ATPase at the vessel wall. A second would be to routinely combine a low dose MR antagonist, an ENaC inhibitor, and sodium restriction as first-line therapy for bilateral aldosterone overproduction. Finally, for unilateral cases post-surgery, there is good reason to include low-dose MRs in drug therapy if required, given the ability of cortisol in damaged blood vessels to mimic aldosterone vasoconstrictor action.

Abstract 481: Pharmacological Inhibition of Calpain Attenuates Mineralocorticoid Receptor Agonist and High Salt-Induced Abdominal Aortic Aneurysm Incidence and Aortic Rupture in Mice

Background and Objective: Recently, we demonstrated that pharmacological and genetic inhibition of calpains, a class of calcium-activated neutral cysteine proteases, attenuated angiotensin II (AngII)-induced abdominal aortic aneurysms (AAAs) in mice. A newly identified non-invasive model of aneurysm has been developed in mice using deoxycorticosterone acetate (DOCA) coated pellets and oral administration of high salt. It is interesting to note that in this model, systemic administration of renin angiotensin inhibitors had no effect on aneurysm formation. The purpose of this study was to test whether pharmacological inhibition of calpain would influence AAA formation induced by mineralocorticoid receptor agonism and high salt in mice. Methods and Results: To determine whether calpain inhibition influenced DOCA and high salt-induced AAAs, 9 month old male C57BL/6 mice were administered calpain inhibitor, BDA-410 (30 mg/kg/day) or vehicle (n=10 per group) by oral gavage 1 week before DOCA pellet implantation and throughout the subsequent 21 days. Mice were implanted with DOCA pellets (50mg/ mouse for 21 days) and maintained on high salt (0.9% NaCl and 0.2% KCl) water. Calpain inhibition significantly suppressed AAA incidence (defined as increase in aortic diameter or AAA rupture; Vehicle = 60% vs BDA = 20%; P<0.05) compared to the vehicle group. Calpain inhibition also significantly protected DOCA + high salt-induced aortic rupture (Vehicle = 6/10 vs BDA = 1/10; P<0.05) in mice. Conclusion: These data demonstrate that calpain activation plays a critical role in not only AngII-induced, but also aldosterone pathways accelerated AAA formation in mice.

Gene expression effects of glucocorticoid and mineralocorticoid receptor agonists and antagonists on normal human skeletal muscle.

Mineralocorticoid and glucocorticoid receptors are closely related steroid hormone receptors that regulate gene expression through many of the same hormone response elements. However, their transcriptional activities and effects in skeletal muscles are largely unknown. We recently identified mineralocorticoid receptors (MR) in skeletal muscles after finding that combined treatment with the angiotensin-converting enzyme inhibitor lisinopril and MR antagonist spironolactone was therapeutic in Duchenne muscular dystrophy mouse models. The glucocorticoid receptor (GR) agonist prednisolone is the current standard-of-care treatment for Duchenne muscular dystrophy because it prolongs ambulation, likely due to its anti-inflammatory effects. However, data on whether glucocorticoids have a beneficial or detrimental direct effect on skeletal muscle are controversial. Here, we begin to define the gene expression profiles in normal differentiated human skeletal muscle myotubes treated with MR and GR agonists and antagonists. The MR agonist aldosterone and GR agonist prednisolone had highly overlapping gene expression profiles, supporting the notion that prednisolone acts as both a GR and MR agonist that may have detrimental effects on skeletal muscles. Co-incubations with aldosterone plus either nonspecific or selective MR antagonists, spironolactone or eplerenone, resulted in similar numbers of gene expression changes, suggesting that both drugs can block MR activation to a similar extent. Eplerenone treatment alone decreased a number of important muscle-specific genes. This information may be used to develop biomarkers to monitor clinical efficacy of MR antagonists or GR agonists in muscular dystrophy, develop a temporally coordinated treatment with both drugs, or identify novel therapeutics with more specific downstream targets.

Mineralocorticoid Receptor Agonist as a Predictor of Treatment Outcome in Depression with and without Early Life Stress

Mineralocorticoid Receptor Agonist as a Predictor of Treatment Outcome in Depression with and without Early Life Stress

Activation of the Mineralocorticoid Receptor Stimulates Protein Disulfide Isomerase: Role in Sickle Cell Disease

Activation of the mineralocorticoid receptor (MR), a critical component of the Renin-Angiotensin-Aldosterone (ALDO)-System (RAAS), has been shown to play an important role in inflammatory and vascular endothelial responses in addition to its well-described effects on sodium and water homeostasis. Activationof endothelial cells leads to, among other factors, increased endothelin-1 (ET-1) and protein disulfide isomerase (PDI) release. PDI and ET-1 contribute to vascular inflammation and are increased in patients with SCD and sickle transgenic mouse models. The MR is a member of the steroid family of nuclear receptors and transcription factors that upon activation binds to hormone response elements of edn1, the gene for ET-1, leading to increased ET-1 expression.In vivo, blockade of MR has been shown to reduce circulating ET-1 levels and ET-1 mRNA expression. However, the role of MR in SCD is unclear. We hypothesized that MR blockade in sickle transgenic mice would reduce PDI activity and improve hematological parameters and inflammation. We first studied EA.hy926 (EA) cells, a human endothelial cell line that expresses MR. We incubated EA cells with ALDO (10-8 M), an MR agonist, for 24 hr and observed a rise in PDI mRNA levels by qRT-PCR (P<0.01, n=5), an event that was blocked by pre-incubation of EA cells with 1 μM canrenoic acid (CA), an MR antagonist (P<0.05, n=5). We then measured PDI activity in the supernatant of ALDO-stimulated EA cells using a Di-E-GSSH fluorescent marker and observed a rise in PDI activity following ALDO (10-8 M) when compared to vehicle treatment (P<0.05; n=5). To test the in vivo effects of MR activation, we studied Berkeley Sickle Transgenic (BERK) mice that were randomized to receive either normal rodent chow or chow containing eplerenone (156 mg/kg per day), an MR antagonist (MRA), for 14 days. We observed significantly lower plasma PDI activity in mice treated with MRA than those on regular chow (63.7 ± 8.7 control diet to 47.9 ± 2.4 eplerenone, Relative Fluorescence Units (RFU); P<0.005, n=6 and 9, respectively). Treatment with MRA was associated with reduced plasma ET-1 and myeloperoxidase (MPO) levels in BERK mice. We also studied RBC Gardos channel activity in these mice and observed a significant reduction in clotrimazole-sensitive K+ efflux following MR blockade (2.49±0.5 control and 1.37±0.3 mmol/1013 cells x hr; P<0.04 n= 5 and 7 respectively). Consistent with these results, MR blockade was associated with increases in both erythrocyte MCV (41.3±2.5 vs 47.4±1.1 fL, P<0.03, n=7) and reticulocyte MCV (53.6.3±2.8 vs 60.1±0.6 fL, P<0.02, n=7). We also studied gene expression by qRT-PCR in heart tissue from these mice and observed that MR blockade reduced mRNA expression of: ET-1 (0.654 ± 0.233, P<0.05, n=5 and n=7); PDI (0.546 ± 0.063, P<0.01, n=5 and n=7); and Tumor Necrosis Factor Receptor Superfamily Member 1A mRNA (0.464 ± 0.061, P<0.01, n=5 and n=7). Thus, our results suggest a novel role for RAAS and, in particular, MR activation in SCD. DisclosuresNo relevant conflicts of interest to declare.

Effects of mineralocorticoid-receptor stimulation on risk taking behavior in young healthy men and women

Risk taking is influenced by stress, with riskier decisions after exposure to an acute stressor and consecutively elevated cortisol levels. In the brain, cortisol acts on two receptors with different functional profiles: the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR).In the current study we investigated the effect of MR stimulation on risk taking behavior in 80 young healthy participants (40 women, mean age=23.9). We administered 0.4mg fludrocortisone, a MR agonist, in a between-subjects, placebo controlled design. Subsequently, participants conducted an established risk taking paradigm, the Balloon-Analogue-Risk-Task (BART). We also used two questionnaires to assess risk taking and decision behavior as trait measures.We found a treatment effect with riskier decisions in the fludrocortisone group. Furthermore, we found a sex effect with more risk taking in men. There was no statistically significant interaction between both factors.Our results indicate that acute MR stimulation leads to riskier decisions in women and men. Our findings argue for an important role of the MR in decision making processes.

Corticosteroid and progesterone transactivation of mineralocorticoid receptors from Amur sturgeon and tropical gar.

The response to a panel of steroids by the mineralocorticoid receptor (MR) from Amur sturgeon and tropical gar, two basal ray-finned fish, expressed in HEK293 cells was investigated. Half-maximal responses (EC50s) for transcriptional activation of sturgeon MR by 11-deoxycorticosterone, corticosterone, 11-deoxycortisol, cortisol and aldosterone, and progesterone (Prog) were between 13 and 150 pM. For gar MR, EC50s were between 8 and 55 pM. Such low EC50s support physiological regulation by these steroids of the MR in sturgeon and gar. Companion studies with human and zebrafish MRs found higher EC50s compared with EC50s for sturgeon and gar MRs, with EC50s for zebrafish MR closer to gar and sturgeon MRs than was human MR. For zebrafish MR, EC50s were between 75 and 740 pM; for human MR, EC50s were between 65 pM and 2 nM. In addition to Prog, spironolactone (spiron) and 19nor-progesterone (19norP) were agonists for all three fish MRs, in contrast with their antagonist activity for human MR, which is hypothesized to involve serine-810 in human MR because all three steroids are agonists for a mutant human Ser810Leu-MR. Paradoxically, sturgeon, gar, and zebrafish MRs contain a serine corresponding to serine-810 in human MR. Our data suggest alternative mechanism(s) for Prog, spiron, and 19norP as MR agonists in these three ray-finned fishes and the need for caution in applying data for Prog signaling in zebrafish to human physiology.

Mineralocorticoid receptor stimulation effects on spatial memory in healthy young adults: A study using the virtual Morris Water Maze task

ObjectivesStress hormones such as cortisol are known to influence a wide range of cognitive functions, including hippocampal based spatial memory. In the brain, cortisol acts via two different receptors: the glucocorticoid (GR) and the mineralocorticoid receptor (MR). As the MR has a high density in the hippocampus, we examined the effects of pharmacological MR stimulation on spatial memory. MethodsEighty healthy participants (40 women, 40 men, mean age=23.9years±SD=3.3) completed the virtual Morris Water Maze (vMWM) task to test spatial encoding and spatial memory retrieval after receiving 0.4mg fludrocortisone, a MR agonist, or placebo. ResultsThere was no effect of MR stimulation on spatial encoding during the vMWM task. However, participants who received fludrocortisone exhibited improved spatial memory retrieval performance. There was neither a main effect of sex nor a sex-by-treatment interaction. ConclusionIn young healthy participants, MR stimulation improved hippocampal based spatial memory retrieval in a virtual Morris Water Maze task. Our study not only confirms the importance of MR function in spatial memory, but suggests beneficial effects of acute MR stimulation on spatial memory retrieval in humans.

Stress and Depression: a Crucial Role of the Mineralocorticoid Receptor.

Cortisol and corticosterone act on the appraisal process, which comprises the selection of an appropriate coping style and the encoding of the experience for storage in the memory. This action exerted by the stress hormones is mediated by mineralocorticoid receptors (MRs), which are expressed abundantly in the limbic circuitry, particularly in the hippocampus. Limbic MR is down-regulated by chronic stress and during depression but induced by antidepressants. Increased MR activity inhibits hypothalamic-pituitary-adrenal axis activity, promotes slow wave sleep, reduces anxiety and switches circuit connectivity to support coping. Cortisol and emotion-cognition are affected by MR gene haplotypes based on rs5522 and rs2070951. Haplotype 1 (GA) moderates the effects of (early) life stressors, reproductive cycle and oral contraceptives. MR haplotype 2 (CA) is a gain of function variant that protects females against depression by association with an optimistic, resilient phenotype. Activation of MR therefore may offer a target for alleviating depression and cognitive dysfunction. Accordingly, the MR agonist fludrocortisone was found to enhance the efficacy of antidepressants and to improve memory and executive functions in young depressed patients. In conclusion, CORT coordinates via MR the networks underlying how an individual copes with stress, and this action is complemented by the widely distributed lower affinity glucocorticoid receptor (GR) involved in the subsequent management of stress adaptation. In this MR:GR regulation, the MR is an important target for promoting resilience.

Selective attention to emotional cues and emotion recognition in healthy subjects: the role of mineralocorticoid receptor stimulation.

Selective attention toward emotional cues and emotion recognition of facial expressions are important aspects of social cognition. Stress modulates social cognition through cortisol, which acts on glucocorticoid (GR) and mineralocorticoid receptors (MR) in the brain. We examined the role of MR activation on attentional bias toward emotional cues and on emotion recognition. We included 40 healthy young women and 40 healthy young men (mean age 23.9 ± 3.3), who either received 0.4mg of the MR agonist fludrocortisone or placebo. A dot-probe paradigm was used to test for attentional biases toward emotional cues (happy and sad faces). Moreover, we used a facial emotion recognition task to investigate the ability to recognize emotional valence (anger and sadness) from facial expression in four graded categories of emotional intensity (20, 30, 40, and 80%). In the emotional dot-probe task, we found a main effect of treatment and a treatment × valence interaction. Post hoc analyses revealed an attentional bias away from sad faces after placebo intake and a shift in selective attention toward sad faces compared to placebo. We found no attentional bias toward happy faces after fludrocortisone or placebo intake. In the facial emotion recognition task, there was no main effect of treatment. MR stimulation seems to be important in modulating quick, automatic emotional processing, i.e., a shift in selective attention toward negative emotional cues. Our results confirm and extend previous findings of MR function. However, we did not find an effect of MR stimulation on emotion recognition.

The Mineralocorticoid Agonist Fludrocortisone Promotes Survival and Proliferation of Adult Hippocampal Progenitors.

Glucocorticoid receptor (GR) activation has been shown to reduce adult hippocampal progenitor cell proliferation and neurogenesis. By contrast, mineralocorticoid receptor (MR) signaling is associated with neuronal survival in the dentate gyrus of the hippocampus, and impairment of hippocampal MR has been linked to pathological conditions, such as depression or neurodegenerative disorders. Here, we aimed to further clarify the protective role of MR in adult hippocampal neurons by studying the survival and proliferative effects of the highly potent MR agonist fludrocortisone (Fludro) in adult rat hippocampal progenitor cells (AHPs), along with the associated signaling mechanisms. Fludro, which upregulated MR but not GR expression, increased survival and proliferation and prevented apoptosis in AHPs cultured in growth factor-deprived medium. These effects were blunted by the MR antagonist spironolactone and by high doses of the GR agonist dexamethasone. Moreover, they involved signaling through cAMP/protein kinase A (PKA)/cAMP response element-binding protein, phosphoinositide 3-kinase (PI3K)/Akt and its downstream targets glycogen synthase kinase-3β (GSK-3β) and mammalian target of rapamycin. Furthermore, Fludro attenuated the detrimental effects of amyloid-β peptide 1–42 (Aβ1–42) on cell survival, proliferation, and apoptosis in AHPs, and increased the phosphorylation of both PI3K/Akt and GSK-3β, which was reduced by Aβ1–42. Finally, Fludro blocked Aβ1–42-induced hyperphosphorylation of Tau protein, which is a main feature of Alzheimer’s disease. Overall, these results are the first to show the protective and proliferative role of Fludro in AHPs, suggesting the potential therapeutic importance of targeting MR for increasing hippocampal neurogenesis and for treating neurodegenerative diseases.

Interactions between inflammatory mediators and corticosteroids regulate transcription of genes within the Kynurenine Pathway in the mouse hippocampus.

BackgroundIncreased tryptophan metabolism towards the production of kynurenine via indoleamine/tryptophan-2,3-dioxygenases (DOs: Ido1, Ido2, and Tdo2) is strongly associated with the prevalence of major depressive disorder in patients and the induction of depression-like behaviors in animal models. Several studies have suggested that activation of the immune system or elevated corticosteroids drive DO expression; however, mechanisms linking cytokines, corticosteroids, and DOs to psychiatric diseases remain unclear. Various attempts have been made to correlate DO gene expression within the brain to behavior, but disparate results have been obtained. We believe that discrepancies arise as a result of the under-recognized existence of multiple mRNA transcripts for each DO. Unfortunately, there are no reports regarding how the multiple transcripts are distributed or regulated. Here, we used organotypic hippocampal slice cultures (OHSCs) to directly test the ability of inflammatory and stress mediators to differentially regulate DO transcripts.MethodsOHSCs were treated with pro-inflammatory mediators (interferon-gamma (IFNγ), lipopolysaccharide (LPS), and polyinosine-polycytidylic acid (pI:C)) with or without corticosteroids (dexamethasone (Dex: glucocorticoid receptor (GR) agonist), aldosterone (Aldo: mineralocorticoid receptor (MR) agonist), or corticosterone (Cort: GR/MR agonist)).ResultsIFNγ induced Ido1-full length (FL) and Ido1-variant (v) expression, and surprisingly, Dex, Cort, and Aldo interacted with IFNγ to further elevate expression of Ido1, importantly, in a transcript dependent manner. IFNγ, LPS, and pI:C increased expression of Ido2-v1 and Ido2-v3 transcripts, whereas only IFNγ increased expression of Ido2-v2. Overall Ido2 transcripts were relatively unaffected by GR or MR activation. Naïve mouse brain expresses multiple Tdo2 transcripts. Dex and Cort induced expression of only one of the three Tdo2 transcripts (Tdo2-FL) in OHSCs. ConclusionsThese results establish that multiple transcripts for all three DOs are expressed within the mouse hippocampus, under the control of distinct regulatory pathways. These data identify a previously unrecognized interaction between corticosteroid receptor activation and inflammatory signals on DO gene expression, which suggest that corticosteroids act to differentially enhance gene expression of Ido1, Ido2, and Tdo2.

Abstract 501: A Pivotal Role of Smooth Muscle Bmal1 in Mineralocorticoid Receptor Agonist plus Salt Induced Mouse Aortic Aneurysm

Rationale: Bmal1 is a basic helix loop helix (bHLH) transcription factor and is an indispensable core clock gene. Bmal1 is expressed in vascular smooth muscle and has been implicated in atherosclerosis and vascular remodeling. However, it is unknown whether vascular smooth muscle Bmal1 is involved in aortic aneurysm. We use a smooth muscle selective Bmal1 knockout (SM-Bmal1 -/- ) mouse and mineralocorticoid receptor agonists plus salt induced aortic aneurysm mouse model to address this specific question. Approach and Results: Aortic Aneurysm was induced in SM-Bmal1 -/- and littermate controls by subcutaneously implanting a 50mg DOCA (deoxycorticosterone acetate) pellet for 21 days or mini-pump with 200μg/Kg/day aldosterone for 28 days and drinking 0.9% saline solution. The abdominal aortic aneurysm incidence was dramatically decreased from control 30.7% (4-month-old) and 68.0% (8-month-old) to 4.0% (p&lt;0.05) and 0.0% (p&lt;0.001) in SM-Bmal1 -/- mice respectively. This was associated with a significant decrease in the aortic inner and external diameters. While SM-Bmal1 deletion did not alter the plasma sodium concentration, it significantly decreased the elastin breakage in aorta. In situ zymography assay demonstrated a significantly suppressed gelatinase activity in SM-Bmal1 -/- mouse aorta when the metalloproteinases (MMPs) were complexed with their tissue inhibitors (TIMPs). Gel zymography showed no decrease in MMP2/9 activities in the SM-Bmal1-KO mouse aorta, real-time PCR analysis showed TIMP4 mRNA and immunohistochemistry analysis showed TIMP4 protein were selectively up-regulated among the four TIMP isoforms. Mouse TIMP4 promoter contains 7 E-boxes, ChIP assay of the mouse aorta tissues showed 4 of them bound to Bmal1. Further TIMP4 promoter luciferase activity analysis demonstrated TIMP4 promoter activity was dramatically activated by the deletion of Bmal1. Conclusions: Deletion of Bmal1 from smooth muscle protects mice from mineralocorticoid receptor agonist plus salt induced aortic aneurysm at least in part via up-regulating TIMP4.