Non-genomic Effects Research Articles

Triiodothyronine Improves Contractile Function of Hypoxic/Reoxygenated Human Right Atrial Tissue

BackgroundIn patients with acute myocardial infarction, the one‐year mortality remains at 7‐15% despite successful interventional reperfusion. Thus, innovative treatment strategies beyond reperfusion are needed. In isolated perfused rat hearts, triiodothyronine (T3), when given during early reperfusion in a supraphysiological concentration, improves left ventricular function and reduces infarct size (Basic Res Cardiol 2021;116:27).AimTo investigate whether or not such acute, non‐genomic effects of T3 also protect human myocardium.MethodsWe isolated right atrial trabeculae from patients undergoing routine coronary artery bypass grafting and investigated the effect of T3 on contractile function during hypoxia/reoxygenation. Trabeculae (≥ 3 mm length, ≤ 1 mm diameter) were isolated in cardioplegic buffer and transferred to Tyrode buffer. Electrically initiated (1 Hz) developed force of contraction (mN/mm2) was recorded for 10 min at baseline (95% O2 – 5% CO2, glucose). Hypoxia/reoxygenation (60 min/30 min) was induced by changing the buffer gas supply and composition and by increasing the stimulation rate (95% N2 – 5% CO2, choline chloride, 3 Hz) and returning back to baseline. T3 (500 µg/l) or NaOH (as solvent control), respectively, was administered A) during hypoxia and reoxygenation (n=11 vs. n=10) or B) 15 min before and during reoxygenation (n=12 vs. n=14).ResultsAt baseline, before T3 or NaOH treatment, the contractile function of trabeculae was comparable (mN/mm2: 15.4±3.5, 13.8±4.4 in protocol A, and 14.5±4.1, 13.7±4.0 in protocol B, respectively). T3 improved the contractile function during reoxygenation, irrespectively of whether given continuously (A) or just before and during reoxygenation (B; see Figure).ConclusionT3 protects patient myocardium: contractile function is improved during reoxygenation in right atrial trabeculae. This bioassay will now permit an identification of causally involved cardioprotective signaling molecules of such non‐genomic protection in human tissue by T3.

Targeting Estrogen Non‐Genomic Signaling in a Three‐Dimensional (3D) in VitroModel of Inflammatory Breast Cancer

Inflammatory Breast Cancer (IBC) is the most aggressive form of breast cancer with a low overall 5‐year survival rate of ~30‐60%. To this date the molecular signature associated with IBC has not been fully characterized, which lead to a lack of effective targeted therapeutics, especially for those patients that account for the 20‐40% of triple‐negative (TNBC) IBC cases. Several studies established that molecular signature of IBC shows important differences from the other breast cancer subtypes, such as overexpression of ErbB receptors, RhoC GTPase and Cox‐2, E‐cadherin aberrant expression, and loss of WISP3, among others. However, these molecular alterations do not explain completely the aggressive and rapid IBC progression. Recently, several studies have shown that estrogen can exert non‐genomic effect in triple‐negative IBC and other TNBCs, mediated by the expression of alternate estrogen receptors, including ERα36 and GPR30. Estrogen non‐genomic signaling refers to the rapid action of these alternate estrogen receptors that upon estrogen binding activates protein‐kinase cascades in the cytoplasm. We hypothesize that estrogen can elicit a specific non‐genomic signaling cascade that promotes the acquisition of aggressive oncogenic phenotypes in IBC and that targeting this pathway can be an effective anticancer therapy for IBC. To test this hypothesis, we used pharmacological inhibitors of the Epidermal Growth Factor Receptor (EGFR) alone and GPR30 alone or in combination. The first approach was to determine if inhibiting estrogen non‐genomic signaling by targeting one of the alternate receptors, GPR30, affects cell proliferation in a 3D colony formation assay using Matrigel. Preliminary observations showed that after G15 treatment (GPR30 inhibitor), there was a slight reduction in cell proliferation compared to E2, but when combined with IRESSA (EGFR inhibitor) there was a further decreased on proliferation when compared to IRESSA alone. Ongoing experiments include the effects of Icaritin (modulator of ERα36) and knockdown of ERα36 using siRNA experiments to test the effects in cell proliferation, migration and invasion. Also, we are in the process of determining the half‐maximal inhibitory concentration (IC50) of G15, IRESSA and IRESSA + G15 by performing a dose response curve (Alamar Blue cell viability assay) of SUM149 cell line, to observe if there is an additive effect when combined the pharmacological inhibitors of EGFR and GPR30. This study will provide the foundation for further characterization of the role estrogen non‐genomic signaling in the progression of IBC and to test its potential as a therapeutic target.

Dynamic changes in the tumor microenvironment under the effect of estradiol as a diagnostic tool and target for targeted cancer therapy

Activation of the estrogen receptor-α (ER-α) signaling pathway is a significant factor in the initiation of carcinogenesis in various types of tumors due to the genomic and non-genomic effects of estradiol in cancer cells. However, data on the expression of ER-α and aromatase on stromal and immune cells in the tumor microenvironment (TME) point to an additional mechanism by which estrogens increase tumor malignancy. There is growing evidence that TME can affect tumor immunity by increasing the immune response or reducing immunoreactivity.The important role of estrogen and the estrogen receptor signaling pathway in the response of the tumor microenvironment in cancer of various localizations, not only classical hormone-dependent cancers, has been proven. However, the clinical effectiveness of blocking the effect of estrogen on tumor growth has been primarily shown in cancer of the female reproductive system. At the same time, data on the significant role of TME in the development of endocrinotherapy resistance in breast cancer treatment are of great interest.Despite the possibilities of standard therapy, a more in-depth study on the role of various TME components in cancer evolution, creation of a micrometastatic niche, as well as in the response to therapy may result in development of new strategies for cancer treatment. It is also necessary to study the possibilities of overcoming the immunosuppressive effect of the estrogen receptor signaling pathway on TME in order to increase the survival rates in patients with hormone-dependent cancers, particularly, breast cancer.

Cardiac manifestations in hyperthyroidism.

Thyroid hormones have a fundamental impact on cardiac function that is mediated by genomic and nongenomic effects, alterations that condition physiological repercussions that lead to changes in frequency, contractility, rhythm and cardiac output as well as an increase in the incidence and prevalence of different cardiovascular diseases. This document presents an updated review of the implications that hyperthyroidism has in different cardiac conditions, including its importance in the evaluation of perioperative cardiovascular risk.

Pregnancy Achievement by Medical Assisted Reproduction Is Correlated to the G Protein-Coupled Receptor 30 mRNA Abundance in Human Spermatozoa

Estrogens, specifically 17β-estradiol (E2), play an important role in male health, including male fertility. The G protein-coupled receptor for estrogen 30 (GPR30) is essential for mediating the rapid non-genomic effects of E2 on a variety of testicular cells, including spermatozoa, although its molecular effects remain largely unknown. In this work, we hypothesized that the GPR30 mRNA abundance in spermatozoa could be correlated to sperm quality. Sperm GPR30 mRNA could also be carried into the oocyte, potentially impacting embryo development and the success of a pregnancy. For this study, 81 sperm samples were collected from couples seeking fertility treatment and undergoing medically assisted reproduction treatments (ART), following the World Health Organization guidelines. GPR30 mRNA abundance in spermatozoa was assessed with a quantitative polymerase chain reaction. The resulting data show that there is no correlation between the abundance of the GPR30 transcript with paternal BMI, age, or sperm quality parameters. Interestingly, we observed that higher levels of GPR30 mRNA abundance in spermatozoa were related to the achievement of biochemical pregnancy and clinical pregnancy (p < 0.05) by couples undergoing treatment. These results highlight the role of the sperm’s RNA cargo in offspring development, suggesting that spermatozoa mRNA content can influence ART success.

Rapid Nontranscriptional Effects of Calcifediol and Calcitriol.

Classically, a secosteroid hormone, vitamin D, has been implicated in calcium and phosphate homeostasis and has been associated with the pathogenesis of rickets and osteomalacia in patients with severe nutritional vitamin D deficiency. The spectrum of known vitamin D-mediated effects has been expanded in recent years. However, the mechanisms of how exactly this hormone elicits its biological function are still not fully understood. The interaction of this metabolite with the vitamin D receptor (VDR) and, subsequently, with the vitamin D-responsive element in the region of specific target genes leading to the transcription of genes whose protein products are involved in the traditional function of calcitriol (known as genomic actions). Moreover, in addition to these transcription-dependent mechanisms, it has been recognized that the biologically active form of vitamin D3, as well as its immediate precursor metabolite, calcifediol, initiate rapid, non-genomic actions through the membrane receptors that are bound as described for other steroid hormones. So far, among the best candidates responsible for mediating rapid membrane response to vitamin D metabolites are membrane-associated VDR (VDRm) and protein disulfide isomerase family A member 3 (Pdia3). The purpose of this paper is to provide an overview of the rapid, non-genomic effects of calcifediol and calcitriol, whose elucidation could improve the understanding of the vitamin D3 endocrine system. This will contribute to a better recognition of the physiological acute functions of vitamin D3, and it could lead to the identification of novel therapeutic targets able to modulate these actions.

Role of Dexamethasone and Methylprednisolone Corticosteroids in Coronavirus Disease 2019 Hospitalized Patients: A Review.

The WHO announced coronavirus disease 2019 (COVID-19) as a pandemic disease globally on March 11, 2020, after it emerged in China. The emergence of COVID-19 has lasted over a year, and despite promising vaccine reports that have been produced, we still have a long way to go until such remedies are accessible to everyone. The immunomodulatory strategy has been kept at the top priority for the research agenda for COVID-19. Corticosteroids have been used to modulate the immune response in a wide range of diseases for the last 70 years. These drugs have been shown to avoid and reduce inflammation in tissues and the bloodstream through non-genomic and genomic effects. Now, the use of corticosteroids increased the chance of survival and relief by combating the viral strong inflammatory impacts and has moved to the forefront in the management of patients seeking supplemental oxygen. The goal of this review is to illuminate dexamethasone and methylprednisolone, i.e., in terms of their chemical and physical properties, role in COVID-19 patients suffering from pneumonia, the proposed mode of action in COVID-19, pharmacokinetics, pharmacodynamics, clinical outcomes in immunocompromised populations with COVID-19, interaction with other drugs, and contradiction to explore the trends and perspectives for future research. Literature was searched from scientific databases such as Science Direct, Wiley, Springer, PubMed, and books for the preparation of this review. The RECOVERY trial, a massive, multidisciplinary, randomized, and open-label trial, is mainly accountable for recommendations over the usage of corticosteroids in COVID-19 patients. The corticosteroids such as dexamethasone and methylprednisolone in the form of medication have anti-inflammatory, analgesic, and anti-allergic characteristics, including the ability to inhibit the immune system. These drugs are also recommended for treating symptoms of multiple ailments such as rheumatic and autoimmune diseases, leukemia, multiple myeloma, and Hodgkin’s and non-Hodgkin’s lymphoma along with other drugs. Toxicology studies proved them safe usually at low dosage via oral or other routes.

The effects of G protein-coupled receptor 30 (GPR30) on cardiac glucose metabolism in diabetic ovariectomized female rats.

Diabetic cardiometabolic disorders are characterized by significant changes in cardiac metabolism and are increased in postmenopausal women, which emphasize the role of 17β-estradiol (E2). Despite this, there are few safe and effective pharmacological treatments for these disorders. The role of G protein-coupled estrogen receptor (GPR30), which mediates the non-genomic effects of E2, is mostly unexplored. In this study, we used ovariectomy (menopausal model) and type 2 diabetic (T2D) rats' models to evaluate the preclinical action of G-1 (GPR30 agonist) against cardiometabolic disorders. T2D was induced by a high-fat diet and a low dose of streptozotocin. G-1 was administrated for six weeks after the establishment of T2D. We found that G-1 counteracts the effects of T2D and ovariectomy by increasing the body weight, reducing fasting blood sugar, heart weight, and heart weight to body weight ratio. Also, both ovariectomy and T2D led to decreases in the cardiac protein levels of hexokinase 2 (HK2) and GLUT4, while G-1-treated female rats reversed these changes and only increased HK2 protein level. In addition, T2D and ovariectomy increased glucose and glycogen content in the heart, but G-1 treatment significantly reduced them. In conclusion, our work demonstrates that G-1 as a selective GPR30 agonist is a viable therapeutic approach against T2D and cardiometabolic diseases in multiple preclinical female models.

Gaps in the knowledge of thyroid hormones and placental biology†.

Thyroid hormones (THs) are required for the growth and development of the fetus, stimulating anabolism, and oxygen consumption from the early stages of pregnancy to the period of fetal differentiation close to delivery. Maternal changes in the hypothalamic-pituitary-thyroid axis are also well known. In contrast, several open questions remain regarding the relationships between the placenta and the maternal and fetal TH systems. The exact mechanism by which the placenta participates in regulating the TH concentration in the fetus and mother and the role of TH in the placenta are still poorly studied. In this review, we aim to summarize the available data in the area and highlight significant gaps in our understanding of the ontogeny and cell-specific localization of TH transporters, TH receptors, and TH metabolic enzymes in the placenta in both human and rodent models. Significant deficiencies also exist in the knowledge of the contribution of genomic and nongenomic effects of TH on the placenta and finally, how the placenta reacts during pregnancy when the mother has thyroid disease. By addressing these key knowledge gaps, improved pregnancy outcomes and management of women with thyroid alterations may be possible.

PAQR5 Expression Is Suppressed by TGFβ1 and Associated With a Poor Survival Outcome in Renal Clear Cell Carcinoma.

BackgroundRenal cell carcinoma (RCC) was sex-hormone responsive, and clinical trials using progesterone significantly reduced the incidence of distal metastasis after radical nephrectomy. Recently membrane-bound progesterone receptors (mPRs) were discovered to mediate the non-genomic effect of progesterone. Aberrant expressions of these mPRs were reported in human breast, ovarian, urinary bladder, brain, uterine, and prostate cancers. However, their expression profiles in RCC are yet to be assessed.MethodsMultiple datasets from RNA sequencing (RNA-seq), cDNA microarray, and proteomic analysis were used to compare gene expression between cancerous and normal kidney tissues. Immunohistochemistry was conducted to examine protein expression in kidney tissues. Promoter methylation levels were assessed for correlation analysis with gene expression.ResultsOf the seven membrane-bound progesterone receptor genes, the progestin and adipoQ receptor-5 (PAQR5) gene is predominantly expressed in normal kidney tissue but was significantly downregulated in RCC tissues. PAQR5 downregulation correlated with tumor stage, cancer grade, lymph node invasion, and distal metastasis only in clear cell RCC (ccRCC) tissues. PAQR5 downregulation was associated with an increased promoter DNA methylation and a poor survival outcome in ccRCC patients. In addition, PAQR5 expression inversely correlated with transforming growth factor beta-1 (TGFB1) expression, and TGFβ1 treatment significantly reduced PAQR5 gene expression.ConclusionPAQR5 is a novel prognostic biomarker in ccRCC and is negatively regulated by the TGFβ1 pathway.

G-Protein-Coupled Estrogen Receptor Enhances the Stemness of Triple-Negative Breast Cancer Cells and Promotes Malignant Characteristics

G-protein coupled estrogen receptor (GPER) is a transmembrane receptor that mediates non-genomic effects of estrogen. This study aimed to investigate the role of GPER in the stemness formation and malignancies in triple negative breast cancer (TNBC) cells. Spheroids of MDA-MB-468 cells were induced by mammosphere culture, and the proportion of the CD44+ /CD24−/low stem cell subpopulation was detected. Malignant characteristics, expression of GPER and stemness-related markers, and tumorigenesis in a xenograft assay were compared between the mammospheres and adherent cultured cells. The impacts of 17β-estradiol (E2) and the GPER-specific antagonist G15 were studied in in vitro assays. The proportion of the CD44+ /CD24−/low subpopulation was increased in the mammospheres of MDA-MB-468 cells, which also showed higher expression of GPER and stemness-related markers than adherent cultured cells. The abilities of spherical colonies to proliferate, invade, and form colonies in soft agar were enhanced. Spherical cells exhibited stronger tumorigenesis ability than adherent cells in the xenograft assay. E2 treatment enhanced tumorigenicity of both adherent and spherical cells. Spherical cells treated with E2 had stronger proliferation, invasion, and colony formation abilities than other groups. Pretreatment with G15 effectively blocked the stimulation by E2. In conclusion, the expression of GPER in TNBC cells is positively related to stemness and malignant features.

Cocoa flavonoids: a promising ingredient that potentiates vitamin d action in skeletal muscle cells

Rationale: Vitamin D (VitD) effects take place by binding to its receptor (VDR) which is a classical nuclear receptor responsible for genomic effects, but there is also a fraction of the VDR located in the muscle plasma membrane that is responsible for its non-genomic effects. In addition to the established effect of VitD in bone and mineral homeostasis, VitD insufficiency/deficiency is linked to a range of muscle disorders including frailty and muscle loss/dysfunction. Flavonoids decrease insulin resistance and have antioxidant and anti-inflammatory properties by activating Akt signaling pathways. Our study has aimed to evaluate, in VitD deficient conditions, if cocoa flavonoids (CF) are able to mediate the interacction between VitD and skeletal muscle glucose uptake by triggerring the modulation of its genomic and non-genomic signaling pathways. This could be useful in pathological deficient VitD situations, such as diabetes or sarcopenia where muscle function is compromised due to the onset of insulin resistance.

In Vitro Non-Genomic Effects of Calcifediol on Human Preosteoblastic Cells.

Several recent studies have demonstrated that the direct precursor of vitamin D3, the calcifediol [25(OH)D3], through the binding to the nuclear vitamin D receptor (VDR), is able to regulate the expression of many genes involved in several cellular processes. Considering that itself may function as a VDR ligand, although with a lower affinity, respect than the active form of vitamin D, we have assumed that 25(OH)D3 by binding the VDR could have a vitamin’s D3 activity such as activating non-genomic pathways, and in particular we selected mesenchymal stem cells derived from human adipose tissue (hADMSCs) for the in vitro assessment of the intracellular Ca2+ mobilization in response to 25(OH)D3. Our result reveals the ability of 25(OH)D3 to activate rapid, non-genomic pathways, such as an increase of intracellular Ca2+ levels, similar to what observed with the biologically active form of vitamin D3. hADMSCs loaded with Fluo-4 AM exhibited a rapid and sustained increase in intracellular Ca2+ concentration as a result of exposure to 10−5 M of 25(OH)D3. In this work, we show for the first time the in vitro ability of 25(OH)D3 to induce a rapid increase of intracellular Ca2+ levels in hADMSCs. These findings represent an important step to better understand the non-genomic effects of vitamin D3 and its role in endocrine system.

Integrative Analysis of Gut Microbiota and Fecal Metabolites in Rats after Prednisone Treatment.

ABSTRACTPrednisone (PRED) is a synthetic glucocorticoid (GC) widely used in immune-mediated diseases for its immunosuppressive and anti-inflammatory properties. The effects of GC are achieved by genomic and nongenomic mechanisms. However, the nongenomic effects are largely unknown. Thus, we aimed to investigate how long-term prednisone therapy changes the composition of the gut microbiota and fecal metabolites in rats. Male Sprague-Dawley rats were randomly assigned to a control (CON) group and a PRED group, which received prednisone treatment daily for 6 weeks by gavage. The V3 to V4 regions of bacterial 16S rRNA genes were amplified and sequenced after the total bacterial DNA was extracted from fecal samples. The alpha and beta diversities were calculated. The compositional alteration of the gut microbiota at different taxonomic levels was analyzed using the Metastats method. Meanwhile, the fecal metabolites were quantitated in an ultra-performance liquid chromatography system. Similar microbial richness and diversity between the CON and PRED groups were indicated by the alpha diversity results. The gut microbial communities differed significantly between two groups. The relative abundances of the genera Eisenbergiella, Alistipes, and Clostridium XIVb decreased, whereas that of Anaerobacterium increased significantly in rats after the 6-week prednisone treatment. In total, 11 downregulated and 10 upregulated fecal metabolites were identified. Differential fecal metabolites were enriched in the pathways, including phenylalanine metabolism, butanoate metabolism, and propanoate metabolism. The lowered production of short-chain fatty acids was associated with the decreased relative abundance of the genera Alistipes and Clostridium XIVb and increased abundance of the genus Anaerobacterium. The composition of the gut microbiota and fecal metabolites was changed after long-term prednisone treatment. This may help us to understand the pharmacology of prednisone.IMPORTANCE Prednisone is widely used in chronic glomerular diseases, immunological disorders, and rheumatic diseases for its anti-inflammatory and immunosuppressive properties. It is a synthetic glucocorticoid (GC) that shows therapeutic effects after conversion to prednisolone by the liver. Prolonged GC therapy causes anti-inflammatory effects; it also results in a variety of adverse events, including obesity, hypertension, psychiatric symptoms, and dyslipidemia. The therapeutic effects and adverse events of GCs may be associated with changes in the gut microbiota, as the host might be affected by the metabolites generated by the altered gut microbes. Thus, we investigated how long-term prednisone therapy changed the composition of the gut microbiota and fecal metabolites in rats. This study may shed new light on the pharmacology of prednisone.

Smoothelin-Like Protein 1 Regulates Development and Metabolic Transformation of Skeletal Muscle in Hyperthyroidism.

Hyperthyroidism triggers a glycolytic shift in skeletal muscle (SKM) by altering the expression of metabolic proteins, which is often accompanied by peripheral insulin resistance. Our previous results show that smoothelin-like protein 1 (SMTNL1), a transcriptional co-regulator, promotes insulin sensitivity in SKM. Our aim was to elucidate the role of SMTNL1 in SKM under physiological and pathological 3,3′,5-Triiodo-L-thyronine (T3) concentrations. Human hyper- and euthyroid SKM biopsies were used for microarray analysis and proteome profiler arrays. Expression of genes related to energy production, nucleic acid- and lipid metabolism was changed significantly in hyperthyroid samples. The phosphorylation levels and activity of AMPKα2 and JNK were increased by 15% and 23%, respectively, in the hyperthyroid samples compared to control. Moreover, SMTNL1 expression showed a 6-fold decrease in the hyperthyroid samples and in T3-treated C2C12 cells. Physiological and supraphysiological concentrations of T3 were applied on differentiated C2C12 cells upon SMTNL1 overexpression to assess the activity and expression level of the elements of thyroid hormone signaling, insulin signaling and glucose metabolism. Our results demonstrate that SMTNL1 selectively regulated TRα expression. Overexpression of SMTNL1 induced insulin sensitivity through the inhibition of JNK activity by 40% and hampered the non-genomic effects of T3 by decreasing the activity of ERK1/2 through PKCδ. SMTNL1 overexpression reduced IRS1 Ser307 and Ser612 phosphorylation by 52% and 53%, respectively, in hyperthyroid model to restore the normal responsiveness of glucose transport to insulin. SMTNL1 regulated glucose phosphorylation and balances glycolysis and glycogen synthesis via the downregulation of hexokinase II by 1.3-fold. Additionally, mitochondrial respiration and glycolysis were measured by SeaHorse analysis to determine cellular metabolic function/phenotype of our model system in real-time. T3 overload strongly increased the rate of acidification and a shift to glycolysis, while SMTNL1 overexpression antagonizes the T3 effects. These lines of evidence suggest that SMTNL1 potentially prevents hyperthyroidism-induced changes in SKM, and it holds great promise as a novel therapeutic target in insulin resistance.

The role of glucocorticoids in the treatment of acute anaphylactic reaction

Food allergy is a growing health problem, which is particularly common among the youngest children. Anaphylaxis, which is defined as a sudden-onset and potentially fatal response to an allergen, is an indication for urgent treatment. Although intramuscular epinephrine is the treatment of choice, all therapeutic algorithms also recommend glucocorticoids. They play an important role in reducing the risk of late allergic reaction, and, due to their non-genomic effects, are also increasingly often mentioned in the context of early response to shock. This effect is directly proportional to the dose of the drug, and a reduced duration of the symptoms of anaphylactic shock is achieved with the use of high doses of glucocorticoids. The paper presents a case of a 3-month-old girl with an anaphylactic reaction after consuming a modified milk preparation. After systemic administration of glucocorticoids, a satisfactory therapeutic effect was observed in the child.

Pregnant women with more seizures have lower allopregnanolone concentrations

Neuroactive steroids have rapid, nongenomic effects on neuronal excitability. The effects in humans are less clear. We compared seizure control and concentrations of neuroactive steroids, known to influence neuroexcitability in animal studies, in pregnant women. Participants were prospectively followed throughout pregnancy with seizure-medication diaries and blood samples, assayed for steroid concentrations with gas chromatography-mass spectrometry. Baseline seizure frequency was calculated for the preconception year, and it was determined if seizure frequency was increased in each trimester. The Wilcoxon rank-sum test was used to compare neuroactive steroid concentrations in between the group with increased frequency to the group without, as calculated for the respective trimester, with the Holm-Bonferroni method to correct for multiple comparisons.Among eighty-three pregnancies included, twenty-eight had increased seizure frequency during at least one trimester (15, 18 and 10, respectively) compared to preconception seizure frequency. Allopregnanolone concentrations were lower in the 3rd trimester (p < 0.001), with a similar trend in the 1st (p = 0.08), for pregnancies with increased compared to those with stable seizure frequency. Other neuroactive steroid concentrations were similar. Our findings suggest that lower allopregnanolone concentrations are associated with increased seizure frequency during pregnancy. Validation of these finding in a larger cohort has potential important clinical applications.

Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons.

Gonadal steroid 17β-estradiol (E2) exerts rapid, non-genomic effects on neurons and strictly regulates learning and memory through altering glutamatergic neurotransmission and synaptic plasticity. However, its non-genomic effects on AMPARs are not well understood. Here, we analyzed the rapid effect of E2 on AMPARs using single-molecule tracking and super-resolution imaging techniques. We found that E2 rapidly decreased the surface movement of AMPAR via membrane G protein-coupled estrogen receptor 1 (GPER1) in neurites in a dose-dependent manner. The cortical actin network played a pivotal role in the GPER1 mediated effects of E2 on the surface mobility of AMPAR. E2 also decreased the surface movement of AMPAR both in synaptic and extrasynaptic regions on neurites and increased the synaptic dwell time of AMPARs. Our results provide evidence for understanding E2 action on neuronal plasticity and glutamatergic neurotransmission at the molecular level.

Thyroxine Induces Acute Relaxation of Rat Skeletal Muscle Arteries via Integrin αvβ3, ERK1/2 and Integrin-Linked Kinase.

Aim: Hyperthyroidism is associated with a decreased peripheral vascular resistance, which could be caused by the vasodilator genomic or non-genomic effects of thyroid hormones (TH). Non-genomic, or acute, effects develop within several minutes and involve a wide tissue-specific spectrum of molecular pathways poorly studied in vasculature. We aimed to investigate the mechanisms of acute effects of TH on rat skeletal muscle arteries.Methods: Sural arteries from male Wistar rats were used for isometric force recording (wire myography) and phosphorylated protein content measurement (Western blotting).Results: Both triiodothyronine (T3) and thyroxine (T4) reduced contractile response of sural arteries to α1-adrenoceptor agonist methoxamine. The effect of T4 was more prominent than T3 and not affected by iopanoic acid, an inhibitor of deiodinase 2. Endothelium denudation abolished the effect of T3, but not T4. Integrin αvβ3 inhibitor tetrac abolished the effect of T4 in endothelium-denuded arteries. T4 weakened methoxamine-induced elevation of phospho-MLC2 (Ser19) content in arterial samples. The effect of T4 in endothelium-denuded arteries was abolished by inhibiting ERK1/2 activation with U0126 as well as by ILK inhibitor Cpd22 but persisted in the presence of Src- or Rho-kinase inhibitors (PP2 and Y27632, respectively).Conclusion: Acute non-genomic relaxation of sural arteries induced by T3 is endothelium-dependent and that induced by T4 is endothelium-independent. The effect of T4 on α1-adrenergic contraction is stronger compared to T3 and involves the suppression of extracellular matrix signaling via integrin αvβ3, ERK1/2 and ILK with subsequent decrease of MLC2 (Ser19) phosphorylation.

Brightened body coloration in female guppies (Poecilia reticulata) serves as an in vivo biomarker for environmental androgens: The example of 17β-trenbolone

In vivo testing systems for environmental androgens are scarce. The aim of this study was to evaluate the potential of male-specific brightened body coloration in female guppies (Poecilia reticulata) to serve as an in vivo biomarker of environmental androgens using 17β-trenbolone as an example. The high bioaccumulation of 17β-trenbolone in the skin of female guppies suggests that it is a potential target tissue of environmental androgens. The coloration index, pigment cell ultrastructure, pigment levels, sexual attractiveness, and reproductive capability of female guppies were analyzed following 28 days of exposure to 20 ng/L, 200 ng/L, and 2000 ng/L 17β-trenbolone. Increases in the coloration index caused by 17β-trenbolone exposure were attributable to increased pteridine and melanin levels. Decreases in the sexual attractiveness, number of offspring, and survival rate of offspring suggested that the changes in body coloration translated into adverse outcomes. Finally, mRNA sequencing indicated that 17β-trenbolone increased pteridine levels by activating genomic effects of androgen receptor on xanthine dehydrogenase and increased melanin levels by exerting non-genomic effects targeting microphthalmia-associated transcription factor, tyrosinase, and tyrosinase-related protein 1 that were mediated by mitogen-activated protein kinase and calcium signaling pathways. We have derived a robust adverse outcome pathway of environmental androgens, and our findings suggest that indicators at different biological levels related to brightened body coloration in female guppies can serve as less-invasive or noninvasive in vivo biomarkers of short-term exposure to environmental androgens.