Rapid Nongenomic Effects Research Articles

G protein-coupled estrogen receptor 1 (GPER, GPR 30) in normal human endometrium and early pregnancy decidua

The recently identified trans-membrane G protein-coupled estrogen receptor 1 (GPER, GPR30) has been implicated in rapid non-genomic effects of estrogens. This focuses on expression and localization of GPER mRNA and protein in normal cyclic endometrium and early pregnancy decidua. Real-time PCR, western blotting, in situ hybridization and immuno-histochemistry were used. Endometrial expression of GPER mRNA was lower in the secretory phase than in the proliferative phase, and even lower in the decidua. The expression pattern was similar to that of ERα mRNA, but different from that of ERβ mRNA. Western blot detected GPER protein as a 54 kDa band in all endometrial and decidual samples. In contrast to the mRNA, GPER protein did not show cyclic variations. Apparently, a lower amount of mRNA is sufficient to maintain protein levels in the secretory phase. GPER mRNA was predominantly localized in the epithelium of mid- and late-proliferative phase endometrium, whereas expression in early proliferative and secretory glands could not be distinguished from the diffuse stromal signal, which was present throughout the cycle. Immuno-staining for GPER was stronger in glandular and luminal epithelium than in the stroma throughout the cycle. The cyclic variations of GPER mRNA obviously relate to strong epithelial expression in the proliferative phase, and the expression pattern suggests regulation by ovarian steroids. GPER protein is present in endometrial tissue throughout the cycle, and the epithelial localization suggests potential functions during sperm migration at mid-cycle, as well as decidualization and blastocyst implantation in the mid-secretory phase.

The nongenomic effects of progesterone in repressing iNOS activation through P38MAPK pathways in gonococci-infected polymorphonuclear leukocytes and the clinical significance

Progesterone has nongenomic effects on inducible nitric oxide synthase (iNOS), which is mediated by mitogen activated protein kinase (MAPK) pathways. This effect is supposed to have some potential association with asymptomatic gonococcal infections in women by immunological depression. In this study, polymorphonuclear leukocytes (PMNs) challenged by gonococci were used to study the nongenomic effects of progesterone. The activation of iNOS was assessed by measuring [(3)H] L-arginine converses to [(3)H] L-citrulline, and the activity of MAPK was detected by Western blot. It was found that the activity of iNOS and the yields of NO were enhanced significantly in gonococci-challenged PMNs compared with the controls (P<0.01). Progesterone could repress the activation of iNOS through P38MAPK pathway within PMNs (P<0.05), which could be blocked by SB203580 (P<0.01), but not by actinomycin D (P>0.05). It was also found subsequently that in the serum specimens collected from gonococci-infected but asymptomatic women, the progesterone level was higher than that in women with severe symptoms (P<0.01). Moreover, the yield of NO had an inverse correlation with progesterone. With these results it suggested that the rapid nongenomic effects of progesterone may inhibit iNOS activation and NO yields mediated by P38MAPK pathways, which were supposed to be concerned with asymptomatic women infected with gonococci.

Testosterone and Cholesterol Vasodilation of Rat Aorta Involves L-Type Calcium Channel Inhibition

Testosterone has rapid nongenomic vasodilator effects which could be involved in protective cardiovascular actions. Several authors suggested specific mechanisms to explain this effect, but this matter was not clarified yet. We studied the actions of testosterone and cholesterol on endothelium-denuded rat aorta and their effects on the L-type Ca2+ current (ICa,L) and potassium current (IK). Testosterone (1–100 μM) totally relaxed, in a rapid and concentration-dependent way, the aortic rings contracted by KCl or by (−)-Bay K8644 (BAY). Cholesterol also fully relaxed the contractions induced by KCl. None of the potassium channel antagonists tested (glibenclamide, tetraethylammonium and 4-aminopyridine) modified significantly the relaxant effect of testosterone. The antagonist of classic testosterone receptors, flutamide, did not modify the vasorelaxant effect of testosterone. Furthermore, testosterone and cholesterol inhibited either basal and BAY-stimulated ICa,L in A7r5 cells and they have no effects on IK. In summary, our results demonstrate that cholesterol and testosterone relax rat aorta by inhibiting LTCC. This effect of testosterone is not mediated by the classic hormone receptor or by potassium channel activation. These results suggest that the vasodilator mechanism of cholesterol and testosterone is the same.

The stress system and neuroplasticity

Event Abstract Back to Event The stress system and neuroplasticity Marian Joëls1* 1 University Medical Center Utrecht, Rudolf Magnus Institute, Netherlands Chronic stress (among other things) is associated with repeated exposure of the brain to high levels of corticosteroids, i.e. corticosterone in rodents. We have extensively studied the consequences of repeated exposure to high corticosteroid levels for the structure and function of the rodent brain. Chronic stress greatly affects neurogenesis, cell shape, synaptic strengthening and calcium influx of hippocampal cells. We also observed that the responsiveness to corticosterone alters when the organism has a history of chronic stress. For instance, corticosterone increases glutamate-mediated synaptic responses of dentate cells from chronically stressed but not from control animals. Hypothalamic circuitry is also affected by chronic stress exposure, i.e. we observed a reduced GABAergic projection to parvocellular neurons. Many of the effects due to chronic stress are readily reversible by temporary treatment with a glucocorticoid receptor antagonist. This suggests that effects of chronic stress critically depend on extensive activation of glucocorticoid receptors in the nucleus. Recently it has become evident, however, that corticosteroids also exert nongenomic effects, via mineralo- as well as glucocorticoid receptors, in the hippocampus and hypothalamus. Future experiments will need to examine if these rapid nongenomic effects of the stress hormone corticosterone are also changed by chronic stress. Conference: 2nd Selfish Brain Conference New research on the neurobiology of ingestive behaviour, 23554 Luebeck, Germany, 27 May - 28 May, 2010. Presentation Type: Oral Presentation Topic: Talks Citation: Joëls M (2010). The stress system and neuroplasticity. Front. Neurosci. Conference Abstract: 2nd Selfish Brain Conference New research on the neurobiology of ingestive behaviour. doi: 10.3389/conf.fnins.2010.08.00011 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 12 Apr 2010; Published Online: 12 Apr 2010. * Correspondence: Marian Joëls, University Medical Center Utrecht, Rudolf Magnus Institute, Utrecht, Netherlands, M.Joels@uva.nl Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Marian Joëls Google Marian Joëls Google Scholar Marian Joëls PubMed Marian Joëls Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Women rule

Female animals tolerate trauma and hemorrhage better than male animals AND Estrogen has rapid nongenomic effects that protect organs from damage and attenuate insult-induced inflammation MOREOVER The survival deficit from trauma and hemorrhage produced in ovariectomized female animals is repaired with administration of exogenous estrogen AND Women survive injury, sepsis, and trauma-hemorrhage-induced hypoxemia/reperfusion better than men THEREFORE Women rule ... in survival after trauma, thus, men would benefit from being more like women.

Nongenomic mechanisms of physiological estrogen-mediated dopamine efflux

BackgroundNeurological diseases and neuropsychiatric disorders that vary depending on female life stages suggest that sex hormones may influence the function of neurotransmitter regulatory machinery such as the dopamine transporter (DAT).ResultsIn this study we tested the rapid nongenomic effects of several physiological estrogens [estradiol (E2), estrone (E1), and estriol (E3)] on dopamine efflux via the DAT in a non-transfected, NGF-differentiated, rat pheochromocytoma (PC12) cell model that expresses membrane estrogen receptors (ERs) α, β, and GPR30. We examined kinase, ionic, and physical interaction mechanisms involved in estrogenic regulation of the DAT function. E2-mediated dopamine efflux is DAT-specific and not dependent on extracellular Ca2+-mediated exocytotic release from vesicular monoamine transporter vesicles (VMATs). Using kinase inhibitors we also showed that E2-mediated dopamine efflux is dependent on protein kinase C and MEK activation, but not on PI3K or protein kinase A. In plasma membrane there are ligand-independent associations of ERα and ERβ (but not GPR30) with DAT. Conditions which cause efflux (a 9 min 10-9 M E2 treatment) cause trafficking of ERα (stimulatory) to the plasma membrane and trafficking of ERβ (inhibitory) away from the plasma membrane. In contrast, E1 and E3 can inhibit efflux with a nonmonotonic dose pattern, and cause DAT to leave the plasma membrane.ConclusionSuch mechanisms explain how gender biases in some DAT-dependent diseases can occur.

Estradiol-17β Inhibits Gonadotropin-Releasing Hormone-Induced Ca2+ in Gonadotropes to Regulate Negative Feedback on Luteinizing Hormone Release

In pituitary gonadotropes, estrogens have biphasic actions to cause an initial negative feedback followed by a positive feedback on LH secretion, but the mechanisms involved are not clearly understood. To investigate the feedback effects of estrogen, we used mixed ovine pituitary cell cultures (48-72 h), which were treated with 10(-9) M estradiol-17beta (E(2)) or vehicle followed by a pulse of 10(-9) M GnRH. Medium was collected for LH assay and cells extracted to determine activation of MAPK (phosphorylated ERK-1/2). E(2) treatment for 5 min reduced GnRH-induced LH release and caused phosphorylation of ERK-1/2. E(2) alone also caused phosphorylation of ERK-1/2, similar to the response evoked by GnRH alone. GnRH increased cytoplasmic intracellular free calcium concentration ([Ca(2+)](i)) and this was abolished by 2 min pretreatment with E(2) or E-bovine serum albumen conjugate. Blockade of Ca(2+) channels with nifedipine had no effect on the initial peak of GnRH-induced increase in [Ca(2+)](i) but reduced its duration by 27 +/- 6%. Depletion of intracellular Ca(2+) stores with thapsigargin prevented GnRH-induced increase in [Ca(2+)](i). Thapsigargin (10(-7) M) or nifedipine (10(-5) M) pretreatment (15 min) of cells lowered GnRH-induced LH secretion by 30 +/- 6 and 50% +/- 4%, respectively. We conclude that inhibition of the GnRH-induced increase in [Ca(2+)](i) in gonadotropes by E(2) is a likely mechanism for the negative feedback effect of E(2) on LH secretion involving a rapid nongenomic effect of E(2). Activation of the MAPK pathway by E(2) may be the mechanism for the time-delayed positive feedback effect on LH secretion at the level of the gonadotrope.

Nongenomic uterine relaxing effect of RU 486 (mifepristone) prior to its antiprogesterone activity in the human pregnancy

The antiprogestin mifepristone (RU 486) is used for termination of pregnancy, as RU 486 blocks the quiescent action of progesterone, increases uterine contractility, sensitizes the myometrium to prostaglandins, and elicits cervical ripening. Since RU 486 represents a class of compound that is structurally related to steroid hormones, some of which possess a nongenomic uterine relaxing effect, we investigated the potential nongenomic relaxing action of RU 486 on the human pregnant myometrium. Myometrial tissues were obtained from pregnant women undergoing elective cesarean section at term and were isometrically recorded. RU 486 caused relaxation on spontaneous contractility and high potassium-induced contractions with lower relaxing efficacy than progesterone. The progesterone receptor-blocking activity of RU 486 did not antagonize the uterine relaxation of progesterone. Moreover, contractions induced by oxytocin or different prostaglandins (PGF2α, PGE2, and a prostaglandin analogue, misoprostol) were inhibited rather than increased by RU 486. RU 486 induced a rapid and reversible relaxing effect, which was unaffected by inhibitors of protein synthesis and transcription, implying that RU 486 acts through a nongenomic mechanism. This study reveals that RU 486: (i) reduced high potassium-induced contraction and prevented calcium-induced contraction in depolarized tissue; and (ii) relaxed the oxytocin- and prostaglandin-induced contractions, indicating a blockade of voltage- and receptor-operated calcium channels by RU 486. These data show that this antiprogestin may induce a rapid nongenomic antiuterotonic effect prior to its antiprogesterone action.

Androgen receptor's destiny in mammalian oocytes: a new hypothesis

Unlike the well-established roles of androgen and androgen receptor (AR) in males, the functions of this steroid and its receptor in the ovary are still unclear. For decades, androgen and AR have long been considered to play a negative (at least not a positive) role in mammalian oocyte maturation. However, recent studies by us and others showed their positive influence in promoting meiotic maturation. On the other hand, rapid non-genomic effects of androgens have been observed and are now generally accepted as contributing to the physiological effects of the steroids and their related receptors in somatic cells, and this has stimulated us to explore the complex roles of AR in the ovary. Based on the classic dogma and new findings, we collected evidence to propose that the expression of AR shifts from the oocytes to the theca cells and finally disappears in the oocytes during evolution. It is suggested that the non-genomic pathway involving androgen and AR in the mammalian oocytes, unlike somatic cells, cells will undergo elimination. The function of androgen and AR in promoting meiotic maturation may have been replaced gradually by gonadotrophins. Moreover, a possible relationship between AR and polycystic ovary syndrome is also discussed, which might provide a clue for the pathology of the disease.

Cardiac hypertrophy and thyroid hormone signaling

Thyroid hormone exerts a large number of influences on the cardiovascular system. Increased thyroid hormone action increases the force and speed of systolic contraction and the speed of diastolic relaxation and these are largely beneficial effects. Furthermore, thyroid hormone has marked electrophysiological effects increasing heart rate and the propensity for atrial fibrillation and these effects are largely mal-adaptive. In addition, thyroid hormone markedly increases cardiac angiogenesis and decreases vascular tone. These multiple thyroid hormone effects are largely mediated by the action of nuclear based thyroid hormone receptors (TR) the thyroid hormone receptor alpha and beta. TRα is the predominant isoform in the heart. Rapid nongenomic thyroid hormone effects also occur, which can be clearly demonstrated in ex-vivo experiments. Some of the most marked thyroid hormone effects in cardiac myocytes involve influences on calcium flux, with thyroid hormone promoting expression of the gene encoding the calcium pump of the sarcoplasmic reticulum (SERCa2). In contrast, in hypothyroid animals phospholamban levels, which inhibit the SERCa2 pump, are increased. In addition, marked effects are exerted on the calcium channel of the sarcoplasmic reticulum the ryanodine channel. Related to myofibrillar proteins, myosin heavy chain alpha is increased by T3 and MHC beta is decreased. Complex and interesting interactions occur between cardiac hypertrophy induced by excess thyroid hormone action and cardiac hypertrophy occurring with heart failure. The thyroid hormone mediated cardiac hypertrophy in its initial phases presents a physiological hypertrophy with increases in SERCa2 levels and decreased expression of MHC beta. In contrast, pressure overload induced heart failure leads to a “pathological” cardiac hypertrophy which is largely mediated by activation of the calcineurin system and the MAPkinases signaling system. Recent evidence indicates that heart failure can lead to a downregulation of the thyroid hormone signaling system in the heart. In the failing heart, decreases of thyroid hormone receptor levels occur. In addition, serum levels of T4 and T3 are decreased with heart failure in the frame of the non-thyroidal illness syndrome. The decrease in T3 serves as an indicator for a bad prognosis in the heart failure patient being linked to increased mortality. In animal models, it can be shown that in pressure overload-induced cardiac hypertrophy a decrease of thyroid hormone receptor levels occurs. Cardiac function can be improved by increasing expression of thyroid hormone receptors mediated by adeno-associated virus based gene transfer. The failing heart may develop a “hypothyroid” status contributing to diminished cardiac contractile function.

Alkylphenol Xenoestrogens with Varying Carbon Chain Lengths Differentially and Potently Activate Signaling and Functional Responses in GH 3 /B 6 /F10 Somatomammotropes

BackgroundAlkylphenols varying in their side-chain lengths [ethyl-, propyl-, octyl-, and nonylphenol (EP, PP, OP, and NP, respectively)] and bisphenol A (BPA) represent a large group of structurally related xenoestrogens that have endocrine-disruptive effects. Their rapid nongenomic effects that depend on structure for cell signaling and resulting functions are unknown.ObjectivesWe compared nongenomic estrogenic activities of alkylphenols with BPA and 17β-estradiol (E2) in membrane estrogen receptor-α–enriched GH3/B6/F10 pituitary tumor cells. These actions included calcium (Ca) signaling, prolactin (PRL) release, extracellular-regulated kinase (ERK) phosphorylation, and cell proliferation.MethodsWe imaged Ca using fura-2, measured PRL release via radioimmunoassay, detected ERK phosphorylation by fixed cell immunoassay, and estimated cell number using the crystal violet assay.ResultsAll compounds caused increases in Ca oscillation frequency and intracellular Ca volume at 100 fM to 1 nM concentrations, although long-chain alkylphenols were most effective. All estrogens caused rapid PRL release at concentrations as low as 1 fM to 10 pM; the potency of EP, PP, and NP exceeded that of E2. All compounds at 1 nM produced similar increases in ERK phosphorylation, causing rapid peaks at 2.5–5 min, followed by inactivation and additional 60-min peaks (except for BPA). Dose–response patterns of ERK activation at 5 min were similar for E2, BPA, and PP, whereas EP caused larger effects. Only E2 and NP increased cell number. Some rapid estrogenic responses showed correlations with the hydrophobicity of estrogenic molecules; the more hydrophobic OP and NP were superior at Ca and cell proliferation responses, whereas the less hydrophobic EP and PP were better at ERK activations.ConclusionsAlkylphenols are potent estrogens in evoking these nongenomic responses contributing to complex functions; their hydrophobicity can largely predict these behaviors.

Effect of aldosterone on the regulation of the volume of principal cells of rat cortical collecting duct epithelium in early postnatal development

A rapid nongenomic regulatory effect of aldosterone on the volume of principal cells in the cortical segment of cortical collecting ducts (CCDs) of rat kidney was studied using the fluorescent dye calcein under hypoosmotic shock conditions (280‐140 mOsm/kg). In the presence of aldosterone (10 nM), the amplitude of cell volume growth significantly decreased ( p < 0.05) and the cell volume recovery time increased twice both in adult and ten-day-old animals. Age differences were detected when the time required for the cell volume recovery under hypoosmotic conditions was determined: in the adult animals, the cell volume recovered much more rapidly (by an order of magnitude) than in the ten-day-old animals both in the control and in the presence of aldosterone ( 0.09 ± 0.02 vs 2.8 ± 0.3 and 0.21 ± 0.03 vs 4.4 ± 0.5 , respectively, p < 0.001). This was the first study to demonstrate the involvement of aldosterone in the regulation of amplitude and dynamics of changes in the volume of CCD principal cells in early postnatal ontogeny. One of the key aspects of development of the osmoregulatory function of the kidney is the development of the molecular mechanisms of regulation of sodium transport in nephron epithelial cells by aldosterone. It is known that, in early ontogeny, kidneys of altricial animals including rats are insensitive to the antinatriuretic effect of aldosterone. Regulation of sodium transport in postnatal ontogeny of the rat is detected starting from the tenth day of life, when the transition from milk feeding to independent feeding is ended. The molecular mechanisms underlying the development of hormonal sensitivity of target cells of an immature kidney have been studied insufficiently well. It is known that the main proteins involved in the transmembrane sodium transport in the principal cells of the CCDs of rat nephron are the proteins of the apical epithelial of the sodium channel (ENaC) and the basolateral sodium pump ( Na + ,a + -ATPase) [6, 7]. The intracellular sodium concentration ([ ]) in principal cells is primarily determined by the dynamic equilibrium between the sodium influx through ENaC and its efflux form the cell into the intersticium against the concentration gradient by ATP-driven Na + ,a + -ATPase. Earlier, we showed that the expression of the genes encoding these proteins is suppressed and the intracellular sodium concentration in CCD principal cells is decreased in kidneys of tenday-old animals [1, 4]. At the same time, we discovered that aldosterone had a rapid nongenomic effect expressed in maintaining an increased [ ] in CCD principal cells at a low (14 mM) sodium concentration in the ambient medium both in adult and ten-day-old rats [4]. These data led us to assume that, along with the well-known mineralocorticoid effect, aldosterone is also involved in the regulation of the volume of CCD cells in rat nephron in hypoosmotic medium and that this mechanism functions even in the preweaning period. However, the rapid nongenomic effects of aldosterone on the regulation of the volume of CCD principal cells have been studied insufficiently comprehensively. In view of above, the goal of this study was to investigate the rapid nongenomic effect of aldosterone at a physiological concentration on the volume of CCD principal cells in kidneys of ten-day-old and adult rats under hypoosmotic shock conditions. Experiments were performed with CCDs of ten- and sixty-day-old Wistar rats, which were obtained by microdissection. The changes in the cell volume were assessed by the changes in the fluorescence intensity of the intracellular dye calcein and expressed in relative units. Fluorescence intensity was measured in the cells located at the open end of a CCD fragment; the basolateral and apical surfaces of these cells are in contact with the ambient solution. Continuous fluorescence recording during cell volume changes in response to osmotic shock was performed in our modification [11]. Renal tubule fragments contained in 100 µ l of suspension in aOa culture medium were loaded on a polylysinecoated slide and loaded with the fluorescent dye calcein

Cell-specific signal transduction pathways regulating Na+-K+-ATPase. Focus on “Short-term effects of thyroid hormones on the Na+-K+-ATPase activity of chick embryo hepatocytes during development: focus on signal transduction”

the na+-k+-atpase is a complex integral membrane protein that carries out active transport of sodium and potassium across the cell plasma membrane, maintaining the ionic gradients. Each subunit has multiple isozymes that are expressed in tissue and developmental specific fashion. In addition to

Combining Established and Novel Immunomodulators: Synergistic Inhibition of Major T Cell Effector Functions by Dexamethasone and Dasatinib

Introduction. An increasing body of evidence suggests that in addition to their molecular targeted antitumor activity tyrosine kinase inhibitors (TKIs) exert immunomodulatory effects on T cells. Others (Blake et al 2008; Schade et al 2008) and we (Weichsel et al 2008) have recently shown that dasatinib (a multitargeting TKI) may lead to a complete inhibition of TCR dependent T cell effector functions via the SRC kinases LCK and FYN. Because TKIs act as competitive enzyme inhibitors with short half life, they permit better side effect control in the clinic. We investigated the impact of combining clinically relevant doses of dasatinib (1–100nM) with the established immunosuppressant dexamethasone (1–1000nM) on T cells.Materials and Methods. Purified human CD3+ T cells from healthy blood donors were studied directly ex vivo. Functional outcomes assessed included cytokine production (IL-2), activation (CD69 upregulation), proliferation (CFSE dilution) and apoptosis/necrosis induction. EBV or CMV specific proliferation of antigen specific CD8+ T cells were evaluated applying tetramer technology. To distinguish between rapid non-genomic and genomic effects all assessments compared the impact of pre-treating T-cells with dexamethasone for 1 vs. 24h.Results and Discussion. Complete inhibition of proliferation and activation occurred with dasatinib alone at levels of 50nM and above, whereas application of dexamethasone did not lead to a complete inhibition even at doses up to 1000nM. Dose-dependent inhibition of with the monoclonal CD3 antibody OKT3 induced T cell activation and proliferation was observed with the combination of dasatinib and dexamethasone. Strongest synergistic inhibitory effects of the drug combination were observed for OKT3 induced cytotoxic CD8+ T cell proliferation (mean±SEM given: OKT3 induced 63±5% proliferating T cells after 4 days incubation, OKT3 + 10nM dexamethasone 44±5%, OKT3 + 10nM dasatinib 39±10%, OKT3 + combination 16±4%; n=5; p<0.05). A significant inhibition of OKT3 induced up-regulation of the activation marker CD69 was demonstrated with the combination but not for dexamethasone alone (n=5). Our previously published data on dasatinib alone also showed inhibition of OKT3 induced up-regulation of CD69 expression, and this was potentiated in combination with dexamethasone. The pre-treatment time did not influence the dexamethasone effect except for increased reduction of IL-2 production after 24h vs. 1h pre-incubation. Overall, helper CD4+ T cells were more sensitive to the inhibitory effects of the drug combination regarding activation and proliferation than cytotoxic CD8+ T cells. Of note, synergistic effects occurred primarily in the different memory CD4 and CD8 T cell subsets but not in the naïve CD4 and CD8 T cells (e.g. for CD8+CD45RO+CD27+ memory T cells mean±SEM given for percentage of proliferating cells after 4 days: OKT3 92±1%, OKT3 + 100nM dexamethasone 77±2%, OKT3 + 10nM dasatinib 69±15%, and OKT3 + combination 27±11%; n=5; p<0.05). IL-2 production in purified T cells was significantly reduced (p<0.05) in a dose dependent nature for both dasatinib and dexamethasone compared to the OKT3 stimulated condition, either alone or in combination (n=5). Similarly, activation induced cell death (AICD) was significant reduced when the two drugs were combined, whereas no synergistic effects were observed regarding necrosis inhibition (n=5; p<0.05). In contrast, initial results suggested that dexamethasone did not inhibit clinically relevant EBV or CMV antigen specific CD8+ memory T cell proliferation when used alone and did not show synergistic effects with dasatinib (n=3). This may be due to a reduced sensitivity of the specific viral memory subsets towards dexamethasone.Outlook and Conclusion. As viral reactivation especially with CMV is a major cause for morbidity after allogeneic stem cell transplantation, our results warrant further studies in vitro and in vivo. With an indication that each drug, when combined could be used at reduced dose, this research may then pave the way for synergistic uses of TKIs and glucocorticosteroids in treatment of graft versus host disease or autoimmune diseases potentially without increased risk of infections.

Rapid non-genomic regulation of Ca2+ signals and insulin secretion by PPARα ligands in mouse pancreatic islets of Langerhans

PPAR alpha is a ligand-activated transcription factor belonging to the nuclear receptor superfamily. PPAR alpha is involved in the regulation of in vivo triglyceride levels, presumably through its effects on fatty acid and lipoprotein metabolism. Some nuclear receptors have been involved in rapid effects mediated by non-genomic mechanisms. In this paper, we report the rapid non-genomic effects of PPAR alpha ligands on the intracellular calcium concentration ([Ca2+]i), mitochondrial function, reactive oxygen species (ROS) generation, and secretion of insulin in freshly isolated mouse islets of Langerhans. The hypolipidemic fibrate PPAR alpha agonist WY-14 643 decreased the glucose-induced calcium oscillations in intact islets. This effect was mimicked by the synthetic agonist GW7647 and the endogenous agonist oleylethanolamide. The WY-14 643 action was rapid in onset (5 min) and was still produced in the presence of protein and mRNA synthesis inhibitors, cycloheximide, and actinomycin-d. This suggests that it is independent of gene transcription. In addition, WY-14 623 impaired mitochondrial function, increased ROS formation and decreased insulin release. PPAR alpha is present in beta-cells, mainly in the cytosol and nucleus, with a small subpopulation localized in the plasma membrane. However, the presence of the PPAR alpha ligand effects in mice bearing a disrupted Ppar alpha gene raises the possibility that the rapid effects of the agonists in pancreatic beta-cells are independent of the receptor. We conclude that PPAR alpha agonists produce a decrease in glucose-induced [Ca2+]i signals and insulin secretion in beta-cells through a rapid, non-genomic mechanism.

Progestin Receptor-Mediated Reduction of Anxiety-Like Behavior in Male Rats

BackgroundIt is well known progesterone can have anxiolytic-like effects in animals in a number of different behavioral testing paradigms. Although progesterone is known to influence physiology and behavior by binding to classical intracellular progestin receptors, progesterone's anxiety reducing effects have solely been attributed to its rapid non-genomic effects at the GABAA receptor. This modulation occurs following the bioconversion of progesterone to allopregnanolone. Seemingly paradoxical results from some studies suggested that the function of progesterone to reduce anxiety-like behavior may not be entirely clear; therefore, we hypothesized that progesterone might also act upon progestin receptors to regulate anxiety.Methodology/Principal FindingsTo test this, we examined the anxiolytic-like effects of progesterone in male rats using the elevated plus maze, a validated test of anxiety, and the light/dark chamber in the presence or absence of a progestin receptor antagonist, RU 486. Here we present evidence suggesting that the anxiolytic-like effects of progesterone in male rats can be mediated, in part, by progestin receptors, as these effects are blocked by prior treatment with a progestin receptor antagonist.Conclusion/SignificanceThis indicates that progesterone can act upon progestin receptors to regulate anxiety-like behavior in the male rat brain.

Effect of aldosterone on kinetics of intracellular sodium in cortical portion of collecting ducts in rat kidney

We studied the effect of protein kinase C inhibitor RO-31-8220 (10(-7) M) on rapid nongenomic effect of aldosterone in cells of isolated segment of distal region of collecting duct in rat kidney. Experiments with fluorescent dye Na-Green showed that the inhibitor abolished the modulating effect of aldosterone (10 nM) on intracellular sodium concentration at external sodium concentration of 14 mM. Aldosterone decreased by half the initial rate of the changes in internal sodium concentration in both 10-day and mature rats (p<0.05). Similarly to sodium channel blocker amiloride (10(-5) M), RO-31-8220 abolished rapid nongenomic effect of aldosterone on the rate of the changes in internal sodium concentration.

Regulating Estrogen Receptor’s Cytoplasmic Partners

The estrogen (E 2 ) receptor is best known for its actions as a transcriptional activator, but evidence continues to accumulate for rapid nongenomic effects of estrogen receptors outside the nucleus as well. Le Romancer et al . provide evidence that interaction of estrogen receptor alpha (Erα) with phosphoinositide 3-kinase (PI3K) and the tyrosine kinase Src depends on covalent modification of ERα by arginine methylation. The arginine methyltransferase PRMT1 (protein arginine methyltransferase 1) is known to associate with Erα and to regulate its transcriptional activity. Le Romancer et al . developed an antibody that appears to specifically recognize ERα that is dimethylated on R260. Immunoprecipitation of ERα from the human breast cancer cell line MCF-7 with this antibody showed that methylation of the receptor was increased within 5 min of treatment of cells with E 2 and that the methylated form of the receptor was found exclusively in the cytoplasm (although the bulk of ERα was localized in the nucleus). Methylation of ERα was correlated with its association with Src and the p85 subunit of PI3K. Depletion of PRMT1 from cells by treating them with siRNA inhibited the formation of the protein complex. Examination of tyrosine phosphorylated proteins associated with the activated receptor identified focal adhesion kinase as a new component in the complex. In mouse NIH 3T3 cells transfected with wild-type ERα, phosphorylation of the kinase Akt was evident within 5 min of E 2 treatment, but such regulation of Akt was diminished in cells expressing a mutant form of ERα in which the site of methylation was modified to prevent methylation. About half of the invasive human breast cancer samples tested showed abnormally high amounts of ERα methylation, but it remains to be determined how this relates to tumor phenotypes and dependence of tumor growth on E 2 . M. Le Romancer, I. Treilleux, N. Leconte, Y. Robin-Lespinasse, S. Sentis, K. Bouchekioua-Bouzaghou, S. Goddard, S. Gobert-Gosse, L. Corbo, Regulation of estrogen rapid signaling through arginine methylation by PRMT1. Mol . Cell 31 , 212-221 (2008). [PubMed]

Testosterone impairs insulin sensitivity in 3T3-L1 adipocytes and the related nongenomic mechanisms

To investigate the influence of rapid nongenomic effect of androgen on the insulin sensitivity of mature adipocytes and the molecular mechanism thereof. Fetal mice [corrected] preadipocytes of the line 3T3-L1 were cultured to develop into mature adipocytes. 3T3-L1 adipocytes were pretreated with testosterone of the concentration of 10-9 to approximately 10(-5) mol/L for a short-time 0-30 minutes) or a long-time (24 hours). Insulin (Ins) 200 microl at the concentration 100 micromol/L and 2-deoxy [3H] glucose were added to examine the glucose uptake. Phosphorylation and protein expression of Ins receptor (InsR) and its downstream signaling molecules (Akt and GSK3beta) were analyzed by Western blotting. The Ins-stimulated glucose uptake after the pretreatment of testosterone for 30 min and 24 h decreased gradually in response to the increasing of the concentration of testosterone with the nadir both at the testosterone concentration of 10(-5) mol/L (both P < 0.05). The phosphorylation levels of InsR, Akt, and GSK3Pbeta were significantly down-regulated by adding of testosterone at the concentration of 10(-6) mol/L for 3-30 minutes (all P < 0.05) or by adding of testosterone at the concentrations of 10(-7) - 10(-6) mol/L for 24 hours (all P < 0.05). The protein expression levels of InsR, Akt, and GSK3Pbeta, however, were not significantly affected by testosterone treatment. Rapid nongenomic effect of androgen may contribute to the insulin resistance in adipocytes.

Estradiol, a key endocrine signal in the sexual differentiation and activation of reproductive behavior in quail

In Japanese quail, estrogen's effects on sexual behavior can be divided into three classes based on the underlying mechanisms and time-course of action and release. During embryonic life, the embryonic ovary secretes large amounts of estrogens. In contrast to what is observed in mammals where sexual differentiation essentially proceeds via masculinization of the males, in quail, females are demasculinized by their endogenous ovarian estrogens, an effect that can be blocked by injection of an aromatase inhibitor and mimicked in male embryos by an injection of estradiol. In adulthood, testosterone secreted by the testes is converted into estrogens by the preoptic aromatase. Locally produced estrogens activate male sexual behavior largely through the activation of estrogen receptors resulting in the transcription of a variety of genes, including brain aromatase (genomic effect). Both changes in estrogen production and action are observed within latencies ranging from a few hours to a few days, and are completely reversible. Additionally, brain aromatase activity can be modulated within minutes by calcium-dependent phosphorylations, triggered by variations in glutamatergic neurotransmission. These rapid changes in aromatase activity affect with relatively short latencies (10-15 min) the expression of male sexual behavior in quail and also in mice. Overall, the effects of estrogens on sexual behavior can thus be categorized into three classes: organizational (irreversible genomic action during ontogeny), activational (reversible genomic action during adulthood) and rapid nongenomic effects. Rapid and slower changes in brain aromatase activity match well with the two modes of estrogen action on behavior and provide temporal variations in the estrogens' bioavailability that should be able to support the entire range of established effects for this steroid.