Physiological Concentrations Of E2 Research Articles

Effect of estradiol replacement on hippocampal concentrations of estrogens in aged rhesus macaques maintained on an obesogenic diet

Replacement involving estrogens has proven efficacy at treating a wide range of disorders that develop with menopause or after surgical removal of the ovaries. Here, we tested whether an estradiol (E2) replacement paradigm that recapitulates physiological E2 levels in the circulation also recapitulates physiological E2 levels within the hippocampus. E2 was delivered continuously to old ovariectomized (OVX) rhesus macaques, maintained on a high-fat, high-sugar Western-style diet (WSD) for ∼30 months, via subcutaneous implants; this resulted in physiological concentrations of both estrone (E1) and E2 in the circulation (determined by LC-MS/MS). Surprisingly, however, hippocampal concentrations of E2 were markedly (P < 0.01) higher than in ovary-intact animals maintained on a regular chow diet. The data suggest that E2 replacement paradigms that appear to recapitulate physiological E2 concentrations in the circulation may produce hyper-physiological E2 levels within some brain areas, especially when individuals are maintained on a WSD.

Modulating Effects of Progesterone on Spontaneous Nocturnal and Ghrelin-Induced GH Secretion in Postmenopausal Women.

Oral administration of estradiol (E2) generally increases GH secretion in postmenopausal women. Oral administration of E2 is associated with a decrease in IGF-1, whereas parenteral or transdermally administered E2 may have no effect on GH. The effect of progesterone (P4) on GH secretion has rarely been studied. We hypothesized that moderately increased serum E2 levels stimulate GH and that P4 modulates E2-stimulated GH secretion. Four parallel groups of randomly assigned postmenopausal women (n = 40). Treatments were saline placebo and oral placebo, saline placebo and oral micronized P4 (3 × 200 mg/d IM), E2 (5 mg IM) and oral placebo, and E2 IM and oral micronized P4. Outcome measures were overnight GH secretion (10 hours), stimulated (ghrelin, 0.3 µg/kg IV bolus) GH secretion, and CT-estimated visceral fat. Intramuscular E2 administration did not alter nocturnal and ghrelin-stimulated GH secretion. Nocturnal GH secretion was not changed by P4 administration. However, P4 diminished ghrelin-stimulated pulsatile GH release with or without E2 (average, 7.20 ± 2.14 and 9.58 ± 1.97 µg/L/2 h, respectively; P = 0.045). Respective outcomes for mean GH concentrations and GH peak amplitudes were 0.97 ± 0.31 and 1.52 μg/L ± 0.29 (P = 0.025) and 2.76 ± 1.04 and 3.95 μg/L ± 0.90 (P = 0.031). Ghrelin-stimulated GH secretion correlated negatively with P4 concentration with or without correction for visceral fat area in the regression equation (R = 0.49, P = 0.04, β = -0.040 ± 0.016). Low-range physiological E2 concentrations do not affect spontaneous or ghrelin-stimulated pulsatile GH secretion. Conversely, P4 inhibits ghrelin-stimulated GH secretion in a concentration-dependent fashion. The mechanistic aspects and physiological significance of natural P4's regulation of ghrelin-evoked GH secretion require further study.

Moderate intensity continuous training reverses the detrimental effects of ovariectomy on RyR1 phosphorylation in rat skeletal muscle

High 17β-Estradiol (E2) concentrations in isolated ventricular myocytes as well as a lack of ovarian hormones in cardiac muscle of ovariectomized (OVX) rodents has been shown to lead to arrhythmogenic effects by inducing post-translational modifications, including phosphorylation of the sarcoplasmic reticulum (SR) Ca2+ release channel ryanodine receptor-2 (RyR2). The effects of estrogens on the phosphorylation status of the RyR1 in skeletal muscle have not been investigated before. Furthermore, while high intensity exercise has been shown to increase RyR phosphorylation, there is no data on the effects of moderate intensity continuous training (MICT).The aims of the study were to investigate the effects of a 3-day treatment with low (1 nM, moderate (5 nM) and high (10 nM, 100 nM) E2 concentrations on RyR1 mRNA and protein expression and phosphorylation status (pRyRSer2844) in cultured C2C12 myotubes and to study the effects of OVX on RyR1 expression and phosphorylation in rat skeletal muscle in combination with 3 weeks of MICT.Treatment with low, physiological E2 concentrations reduced dihydropyridine receptor (DHPR) and RyR1 mRNA content in C2C12 myotubes compared to untreated control cells, whereas RyR1 protein phosphorylation (pRyRSer2844) was significantly increased after treatment with high, non-physiological E2 concentrations (p ≤ 0.05). RyR1 protein content (p ≤ 0.05) and pRyRSer2844 (p ≤ 0.05) were significantly elevated in skeletal muscle of OVX vs. sham-operated rats. Importantly, pRyRSer2844 levels were similar to sham-operated controls in OVX rats after MICT (OVX vs. OVX + MICT, p ≤ 0.05).Our results indicate, that one of the actions of estrogens is to alter skeletal muscle Ca2+ homeostasis by modulating the expression and phosphorylation of the RyR1 in skeletal muscle. Notably, regular MICT was able to counteract RyR1 phosphorylation in skeletal muscle of OVX rats.

Mas receptor is involved in the estrogen-receptor induced nitric oxide-dependent vasorelaxation

The Mas receptor is involved in the angiotensin (Ang)-(1–7) vasodilatory actions by increasing nitric oxide production (NO). We have previously demonstrated an increased production of Ang-(1–7) in human umbilical vein endothelial cells (HUVEC) exposed to estradiol (E2), suggesting a potential cross-talk between E2 and the Ang-(1–7)/Mas receptor axis. Here, we explored whether the vasoactive response and NO-related signalling exerted by E2 are influenced by Mas. HUVEC were exposed to 10nM E2 for 24h in the presence or absence of the selective Mas receptor antagonist A779, and the estrogen receptor (ER) antagonist ICI182780 (ICI). E2 increased Akt and endothelial nitric oxide synthase (eNOS) mRNA and protein expression, measured by RT-PCR and Western blot, respectively. Furthermore, E2 increased Akt activity (determined by the levels of phospho-Ser473) and eNOS activity (by the enhanced phosphorylation of Ser1177, the activated form), resulting in increased NO production, which was measured by the fluorescence probe DAF-2-FM. These signalling events were dependent on ER and Mas receptor activation, since they were abolished in the presence of ICI or A779. In ex-vivo functional experiments performed with a small-vessel myograph in isolated mesenteric vessels from wild-type mice pre-contracted with noradrenaline, the relaxant response to physiological concentrations of E2 was blocked by ICI and A779, to the same extent to that obtained in the vessels isolated from Mas-deficient. In conclusion, E2 induces NO production and vasodilation through mechanisms that require Mas receptor activation.

TCF7L2 involvement in estradiol- and progesterone-modulated islet and hepatic glucose homeostasis.

To evaluate the role of TCF7L2, a key regulator of glucose homeostasis, in estradiol (E2) and progesterone (P4)-modulated glucose metabolism, mouse insulinoma cells (MIN6) and human liver cancer cells (hepG2 and HUH7) were treated with physiological concentrations of E2 or P4 in the up- and down-regulation of TCF7L2. Insulin/proinsulin secretion was measured in MIN6 cells, while glucose uptake and production were evaluated in liver cancer cells. E2 increased insulin/proinsulin secretion under both basal and stimulated conditions, whereas P4 increased insulin/proinsulin secretion only under glucose-stimulated conditions. An antagonistic effect, possibly concentration-dependent, of E2 and P4 on the regulation of islet glucose metabolism was observed. After E2 or P4 treatment, secretion of insulin/proinsulin was positively correlated with TCF7L2 protein expression. When TCF7L2 was silenced, E2- or P4-promoted insulin/proinsulin secretion was significantly weakened. Under glucotoxicity conditions, overexpression of TCF7L2 increased insulin secretion and processing. In liver cancer cells, E2 or P4 exposure elevated TCF7L2 expression, enhanced the activity of insulin signaling (pAKT/pGSK), reduced PEPCK expression, subsequently increased insulin-stimulated glucose uptake, and decreased glucose production. Silencing TCF7L2 eliminated effects of E2 or P4. In conclusion, TCF7L2 regulates E2- or P4-modulated islet and hepatic glucose metabolism. The results have implications for glucose homeostasis in pregnancy.

Estradiol, acting through ERα, induces endothelial non-classic renin-angiotensin system increasing angiotensin 1–7 production

Intracellular renin-angiotensin system (RAS) can operate independently of the circulating RAS. Estrogens provide protective effects by modulating the RAS. Our aim was to investigate the effect of estradiol (E2) on angiotensin converting enzymes (ACE) 1 and ACE2 expression and activities in human endothelial cells (HUVEC), and the role of estrogen receptors (ER). The results confirmed the presence of active intracellular RAS in HUVEC. Physiological concentrations of E2 induced a concentration-dependent increase of ACE1 and ACE2 mRNA expression and ACE1, but not ACE2, protein levels. ACE1 and ACE2 enzymatic activities were also induced with E2. These effects were mediated through ERα activation, since ER antagonists ICI 182780 and MPP completely abolished the effect of E2. Moreover, the ERα agonist PPT mirrored the E2 effects on ACE1 and ACE2 protein expression and activity. Exposure of endothelial cells to E2 significantly increased Ang-(1–7) production. In conclusion, E2 increases Ang-(1–7) production, through ERα, involving increased ACE1 and ACE2 mRNA expression and activity and ACE1 protein levels.

Estrogen reduces mechanical injury-related cell death and proteoglycan degradation in mature articular cartilage independent of the presence of the superficial zone tissue

To study the effect of 17β-estradiol (E2) and the superficial zone (SFZ) on cell death and proteoglycan degradation in articular cartilage after a single injurious compression invitro. Cartilage explants from the femoropatellar groove of 2 year old cows with or without the SFZ were cultured serum-free with physiological concentrations of E2 and injured by an unconfined single load compression (strain 50%, velocity 2mm/s). After 96hcell death was measured histomorphometrically (nuclear blebbing (NB) and TUNEL staining) and release of glycosaminoglycans (GAG) by DMMB assay. Injurious compression increased significantly the number of cells with NB and TUNEL staining and release of GAG. Physiological concentrations of E2 prevented the injury-related cell death and reduced the GAG release significantly in a receptor-mediated manner (shown by co-stimulation with the antiestrogen fulvestrant/faslodex/ICI-182,780). The presence of the SFZ did not alter the NB response to either the mechanical injury or E2, but reduced the overall release of GAG significantly. E2 prevents injury-related cell death and GAG release, and might be useful for the development of treatment options for either cartilage-related sports injuries or osteoarthritis (OA). The SFZ does not seem to play an important role in (1) the E2-related tissue response and (2) the mechanically-induced cell death in deeper regions of the explants and GAG release. The latter might be related to the unconfined nature of the injury model.

17β-Estradiol suppresses MHC class I chain-related B gene expression via an intact GC box

Major histocompatibility complex class I chain-related B (MICB) is a membrane-bound glycoprotein involved in both innate and adaptive immunity through its interaction with NKG2D receptors present on γδ T, αβ CD8(+) T, and natural killer cells. Factors known to upregulate MICB expression include heat shock, viral or bacterial infection, and tumorigenesis, and here, we explored the effect of 17β-estradiol (E2) on MICB regulation. Physiological concentrations of E2 were found to suppress MICB mRNA and surface protein levels and this effect was antagonized by the antiestrogen ICI 182780. The inhibitory effect of E2 was also observed for other NKG2D ligands, MICA and ULBPs. Evaluation of promoter fragments from the common MICB*00502 allele revealed that inhibition of transcription by E2 required the GC box at -87. The electrophoretic mobility shift assay and supershift analysis established the presence of SP1, SP3, or estrogen receptor α recognition sites within the MICB promoter sequence and interaction of these factors in situ was confirmed by chromatin immunoprecipitation. We conclude that E2 upon forming a complex with its cognate receptor suppresses MICB expression through binding with SP1/SP3 sites within the MICB promoter GC box. These results suggest that the partial benefit of 17β-estradiol on autoimmune diseases may be mediated by reducing the immune NKG2D ligands like MICB.

17β-estradiol modulates mitochondrial Ca2+ flux in rat caudate nucleus and brain stem

The aim of this study was to examine the rapid non-genomic effect of 17β-estradiol (E2) on Ca2+ transport in mitochondria isolated from the nerve terminals (synaptosomes) of caudate nuclei (NC) and brain stems (BS) of ovariectomised female rats. In physiological conditions no effect of E2 on Ca2+ influx into synaptosomal mitochondria through ruthenium red (RR)-sensitive uniporter was observed. However, in the presence of uncoupling agent carbonyl cyanide4-(trifluoromethoxy)phenylhydrazone (FCCP) (1μmol/l), pre-treatment with 0.5nmol/l E2 protected mitochondrial membrane potential and consequently increased Ca2+ influx (2.3-fold in NC and 3.1-fold in BS). At the same time, 0.5nmol/l E2 by increasing the affinity of mitochondrial Na+/Ca2+ exchanger for Na+ inhibited mitochondrial Ca2+ efflux in NC and BS by about 40%. Also, the specific binding of physiological E2 concentrations (0.1–10nmol/l) to isolated synaptosomal mitochondria was detected. Using membrane impermeable E2 bound to bovine serum albumin and selective inhibitor of mitochondrial Na+/Ca2+ exchanger, we obtained that E2’s action on mitochondrial Ca2+ efflux at least partially is due to the direct effects on the mitochondrial membrane and/or Na+/Ca2+ exchanger located in inner mitochondrial membrane. Our results implicate E2 as a modulator of Ca2+ concentration in mitochondrial matrix, and ultimately in the cytosol. Given the vital role of Ca2+ in regulation of total nerve cells activity, especially energy metabolism, neurotransmission and directing the cells toward survival or cell death, the effects on mitochondrial Ca2+ transport could be one of the important modes of E2 neuromodulatory action independent of the genome.

The Effects of 17β-estradiol on Mitochondrial Biogenesis and Function in Breast Cancer Cell Lines are Dependent on the ERα/ERβ Ratio

Background/Aims: 17β-estradiol (E2) is a risk factor for the development of breast cancer, and cause tumorigenesis in epithelial breast cells. Moreover, E2 has distinct effects on different tissues that are attributed to the presence of two estrogen receptor isoforms, ERα and ERβ. Methods: The effect of E2 on mitochondrial biogenesis and function was investigated in two breast cancer cell lines with different estrogen receptor ratios, MCF-7 (high ERα/ERβ ratio) and T47D (low ERα/ERβ ratio) cell lines treated with physiological concentrations of E2 (1 nM). Results: Mitochondria of the MCF-7 cell line showed an increase in proliferation but a decrease in functionality, while the T47D cell line, with low ERα/ERβ ratio, maintained functionality with fewer mitochondria. Conclusion: Our results suggest that ERs endowment and its subtypes relation have an effect on treatment response and could contribute new ideas about mitochondria and ERs in breast cancer, as well as new indicators to the disease progression.

Physiological levels of soy isoflavones reduce proliferation and promote apoptosis in the ERβ-positive breast cancer cell line MDA-MB-231

Dietary soy, particularly the high levels consumed as part of traditional Eastern-Asian diets, may be protective against certain subtypes of breast cancer(1). This effect is partly related its high isoflavone phytoestrogen content (genistein and daidzein). However much in vitro evidence suggests a contradictory, growth promoting impact of physiological (serum) levels of isoflavones in oestrogen receptor alpha positive (ERa+) breast cancer cell lines(2). This has led to the contraindication of isoflavone supplement use in post menopausal breast cancer survivors. The inconsistency between epidemiological and in vitro evidence may relate to the relative expression levels of ERa and its counterpart ERb. Unlike in cell lines predominantly expressing ERa, the proliferation of ERb-dominant cell lines can be inhibited by concentrations of genistein as low as 1 nM(3,4). This information is relevant as the ERa - /b+ subgroup represent approximately 18% of all breast tumours(5) but the beta-receptor is not routinely tested for upon diagnosis. We assessed the impact of physiologically relevant concentrations (serum levels achievable through diet: 0.01nM to 31.6 mM) of the soy isoflavones genistein and daidzein on proliferation (MTT assay) and apoptosis with the Annexin V-Cy32 Apoptosis Detection Kit and DAPI staining/Nuclear Area Factor (NAF) calculation in the ERa - /b+ breast cancer cell line MDA-MB-231. Low doses of genistein or daidzein (‡ 0.01 nM) inhibited MDA-MB-231 proliferation by around 20%, although this frequently failed to achieve statistical significance. The highest concentrations used (31.6 mM) resulted in a dramatic decrease in percent proliferation compared with a vehicle only control (meanSD : 48.512.6, p = 0.004 and 48.413.1, p<0.001 for genistein and daidzein respectively; n = 3). 17b-oestradiol (E2; the major circulating oestrogen) also slightly reduced proliferation at its pre- and post-menopausal serum concentrations (1nM and 1 pM respectively). This reduction in proliferation was associated with an observed increase in cell death at 10 and 31.6 mM genistein and daidzein, which was at least partly explained by an increase in the percentage of apoptotic cells. While the combination of E2 and isoflavones failed to show any synergistic growth inhibitory, or pro-apoptotic effects, some inhibition of proliferation was still documented. This implies that the growth inhibitory effects of genistein, daidzein and E2 may not be regulated by the same mechanisms. However, more importantly, the apoptotic cell death observed with soy isoflavone treatment was not abrogated by the addition of physiological E2 concentrations. Overall, our data suggests that at concentrations achievable through the diet (£ 10 mM) soy isoflavones may be potentially beneficial as chemotherapeutic agents against ERa - /b+ breast cancers, through their ability to reduce proliferation and promote apoptosis, even in the presence of physiological E2 levels. Routine determination of the expression levels of ERb in addition to ERa in breast cancer would be an invaluable biomarker to indicate the potential efficacy of this cheap and readily available treatment, and indicates a possible pharmacological target. 1. Wu AH, Yu MC, Tseng CC, Pike MC (2008) Brit J Cancer 98:9–14. 2. Hwang CS, Kwak HS, Lim HJ, et al. (2006) J Steroid Biochem Mol Bio 101:246–53. 3. Rajah TT, Du N, Drews N, Cohn R (2009) Pharmacology 84:68–73. 4. Kang X, Jin S, Zhang Q (2009) J Food Sci 74:H237–H242. 5. Skliris GP, Leygue E, Watson PH, Murphy LC (2008) J Steroid Biochem Mol Bio 109:1–10.

17β-Estradiol induces nongenomic effects in renal intercalated cells through G protein-coupled estrogen receptor 1

Steroid hormones such as 17β-estradiol (E2) are known to modulate ion transporter expression in the kidney through classic intracellular receptors. Steroid hormones are also known to cause rapid nongenomic responses in a variety of nonrenal tissues. However, little is known about renal short-term effects of steroid hormones. Here, we studied the acute actions of E2 on intracellular Ca(2+) signaling in isolated distal convoluted tubules (DCT2), connecting tubules (CNT), and initial cortical collecting ducts (iCCD) by fluo 4 fluorometry. Physiological concentrations of E2 induced transient increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) in a subpopulation of cells. The [Ca(2+)](i) increases required extracellular Ca(2+) and were inhibited by Gd(3+). Strikingly, the classic E2 receptor antagonist ICI 182,780 also increased [Ca(2+)](i), which is inconsistent with the activation of classic E2 receptors. G protein-coupled estrogen receptor 1 (GPER1 or GPR30) was detected in microdissected DCT2/CNT/iCCD by RT-PCR. Stimulation with the specific GPER1 agonist G-1 induced similar [Ca(2+)](i) increases as E2, and in tubules from GPER1 knockout mice, E2, G-1, and ICI 182,780 failed to induce [Ca(2+)](i) elevations. The intercalated cells showed both E2-induced concanamycin-sensitive H(+)-ATPase activity by BCECF fluorometry and the E2-mediated [Ca(2+)](i) increment. We propose that E2 via GPER1 evokes [Ca(2+)](i) transients and increases H(+)-ATPase activity in intercalated cells in mouse DCT2/CNT/iCCD.

Inhibition of mitochondrial Na+-dependent Ca2+ efflux by 17β-estradiol in the rat hippocampus

Our results, as well as those of others, have indicated that 17β-estradiol (E2) exerts its nongenomic effects in neuronal cells by affecting plasma membrane Ca(2+) flux. In neuronal cells mitochondria possess Ca(2+) buffering properties as they both sequester and release Ca(2+). The goal of this study was to examine the rapid non-genomic effect of E2 on mitochondrial Ca(2+) transport in hippocampal synaptosomes from ovariectomised rats. In addition, we aimed to determine if, and to what extent, E2 receptors participated in mitochondrial Ca(2+) transport modulation by E2 in vitro. E2-specific binding and Ca(2+) transport was monitored. At physiological E2 concentrations (0.1-1.5 nmol/L), specific E2 binding to mitochondria isolated from hippocampal synaptosomes was detected with a B(max.) and K(m) of 37.6±2.6 fmol/mg protein and 0.69±0.14 nmol/L of free E2, respectively. The main mitochondrial Ca(2+) influx mechanism is the Ruthenium Red-sensitive uniporter driven by mitochondrial membrane potential. Despite no effect of E2 on Ca(2+) influx, a physiological E2 concentration (0.5 nmol/L) protected mitochondrial membrane potential and consequently Ca(2+) influx from the uncoupling agent carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (1 μmol/L). In neuronal cells the predominant mitochondrial Ca(2+) efflux mechanism is the Na(+)/Ca(2+) exchanger. E2 caused Ca(2+) efflux inhibition (by 46%) coupled with increased affinity of the Na(+)/Ca(2+) exchanger for Na(+). Using E2 receptor (ERα and ERβ) antagonists and agonists, we confirmed ERβ's involvement in E2-induced mitochondrial membrane potential protection as well as Ca(2+) efflux inhibition. In summary, our results indicate that the non-genomic neuromodulatory role of E2 in rat hippocampus is achieved by affecting mitochondrial Ca(2+) transport via, in part, mitochondrial ERβ.

Mechanisms of estrogen's effects on calcium signaling in vascular endothelial cells

17β‐estradiol (E2) exerts many cardioprotective effects via actions on endothelial cells (ECs). It has been observed that E2 increases Ca2+ signals produced in ECs in response to agonists. However, the underlying mechanisms are unclear. Here we have examined the main components of Ca2+ signaling in primary ECs in response to long‐term exposure to physiological concentrations of E2. E2 treatment for 48 hrs did not affect thapsigargin‐induced internal Ca2+ store release or store‐operated Ca2+ entry. However, we observed a 35% reduction in the activity of the calmodulin (CaM)‐dependent plasma membrane Ca2+‐ATPase (PMCA), the main Ca2+ efflux mechanism in ECs. In contrast, calmodulin (CaM) binding to the PMCA is increased by 50%, while total PMCA expression is not affected. Inhibition of pp60src‐mediated tyrosine phosphorylation of PMCA reveals the effects of E2‐induced increase in CaM binding to the PMCA, demonstrating a 30% increase in PMCA activity in E2‐treated cells. Our results suggest that via pp60src‐dependent phosphorylation of PMCA, estrogen decreases Ca2+ efflux, which provides an explanation for the enhanced Ca2+ signals in ECs exposed to E2.

Rapid actions of oestrogen on gonadotropin‐releasing hormone neurons; from fantasy to physiology?

Oestradiol (E2) exerts critical homeostatic feedback effects upon gonadotropin-releasing hormone (GnRH) neurons to maintain fertility. In the female, E2 has both negative and positive feedback actions to suppress and stimulate GnRH neuron activity at different times of the ovarian cycle. This review summarizes reported rapid E2 effects on native embryonic and adult GnRH neurons and attempts to put them into a physiological perspective. Oestrogen has been shown to rapidly modulate multiple processes in embryonic and adult GnRH neurons including intracellular calcium levels, electrical activity and specific second messenger pathways, as well as GnRH secretion itself. Evaluation of in vivo data suggests that there is no essential role for rapid E2 actions in the positive feedback mechanism but that they may comprise part of the negative feedback pathway. Adult GnRH neurons are only likely to be exposed to E2 from the gonads via the circulation with appropriate physiological E2 concentrations in the rodent being 10-50 pM for negative feedback ranging up to 400 pM for positive feedback. Although most studies to date have examined the effects of supraphysiological E2 levels on GnRH neurons, there is accumulating evidence that rapid E2 actions may have a physiological role in suppressing GnRH neuron activity.

The Effects of Cycling Levels of 17β-Estradiol and Progesterone on the Magnitude of Temporomandibular Joint-Induced Nociception

A greater incidence of temporomandibular joint (TMJ) pain is reported in females, suggesting that gonadal hormones may play a role in this condition. However, the exact roles of 17beta-estradiol (E2) and progesterone (P4) in TMJ pain are not completely known. Two experiments were performed to determine the separate roles of E2 and P4 in TMJ nociception at various stages of the estrous cycle. Ovariectomized (OVX) rats were cycled with physiological concentrations of E2 or P4. The E2-cycled rats then received bilateral TMJ injections of saline (SAL) or complete Freund's adjuvant (CFA) on the morning of diestrus-2 (low E2 condition) or proestrus (high E2 condition). As a control, OVX rats (no ovarian E2 and no replacement) were injected with SAL or CFA. The TMJ nociception was measured using a validated novel method in which an increase in meal duration directly correlated to the intensity of deep TMJ nociception. In the E2 experiment, CFA injection, but not SAL, increased TMJ nociception in the OVX group, but the effect was less pronounced in diestrus-2 and even less in proestrus. In the P4 experiment, the rats receiving TMJ CFA in diestrus-2 (end of minor P4 surge) did not show an increase in TMJ nociception, whereas the rats injected in proestrus (major P4 surge), estrus (low P4), and metestrus (low P4) had similar increases in TMJ nociception. The hormones' concentration did not affect TMJ IL-1beta, IL-6, C-C motif ligand 20, or C-X-C motif ligand 2 or the trigeminal ganglia calcitonin gene-related peptide. The high physiological concentrations of E2 observed at proestrus and the low P4 concentrations observed at diestrus-2 attenuated or eliminated CFA-induced TMJ nociception. The results suggest that the cyclic estrous cycle concentrations of E2 and P4 can influence CFA-induced TMJ nociception in the rat.

Acute effects of oestrogen on the guinea pig and human IKr channels and drug‐induced prolongation of cardiac repolarization

Female gender is a risk factor for drug-induced arrhythmias associated with QT prolongation, which results mostly from blockade of the human ether-a-go-go-related gene (hERG) channel. Some clinical evidence suggests that oestrogen is a determinant of the gender-differences in drug-induced QT prolongation and baseline QT(C) intervals. Although the chronic effects of oestrogen have been studied, it remains unclear whether the gender differences are due entirely to transcriptional regulations through oestrogen receptors. We therefore investigated acute effects of the most bioactive oestrogen, 17beta-oestradiol (E2) at its physiological concentrations on cardiac repolarization and drug-sensitivity of the hERG (I(Kr)) channel in Langendorff-perfused guinea pig hearts, patch-clamped guinea pig cardiomyocytes and culture cells over-expressing hERG. We found that physiological concentrations of E2 partially suppressed I(Kr) in a receptor-independent manner. E2-induced modification of voltage-dependence causes partial suppression of hERG currents. Mutagenesis studies showed that a common drug-binding residue at the inner pore cavity was critical for the effects of E2 on the hERG channel. Furthermore, E2 enhanced both hERG suppression and QT(C) prolongation by its blocker, E4031. The lack of effects of testosterone at its physiological concentrations on both of hERG currents and E4031-sensitivity of the hERG channel implicates the critical role of aromatic centroid present in E2 but not in testosterone. Our data indicate that E2 acutely affects the hERG channel gating and the E4031-induced QT(C) prolongation, and may provide a novel mechanism for the higher susceptibility to drug-induced arrhythmia in women.

Male and female equality: still far from goal

Male and female equality: still far from goal

Regulation of the Hepatic Transcriptome by Estradiol and Progesterone in Cows.

The steroid hormones, estradiol (E2) and progesterone (P4), regulate reproductive function and gene expression in many tissues. These steroids are primarily metabolized in the liver where they can also regulate gene expression. Previous studies in a variety of tissues have reported antagonism by P4 of E2 actions; although in some circumstances E2 and P4 act synergistically. The study was designed to evaluate the effects of E2 and/or P4 on gene expression of the bovine liver using the microarray technique. Methods: A 4X4 Latin Square design was used with ovariectomized Holstein cows randomly assigned to 4 treatment groups (n≡4): 1) No hormone supplementation; 2) E2 treatment (ear implant delivering physiological E2 concentrations); 3) P4 treatment (2 intravaginal progesterone-releasing devices producing ≈ 3.5 ng/ml); 4) E2+P4. After 14 d of treatment, liver biopsies were collected and frozen. To avoid carry-over effects, there was a 28 d interval between periods. The mRNA concentrations in the liver biopsies were evaluated on bovine-specific arrays from Affymetrix (Bovine GeneChip Genome Array) that are claimed to represent over 23,000 transcripts (trp). Signal values on perfect-match and mismatch probes of each trp were combined into expression indexes using the MAS5.0 algorithm, and the Quantile procedure was used to normalize the data. The MIXED procedure of SAS was used to test for the main effects of E2, P4 and their interaction, followed by an analysis of overrepresented categories of differentially expressed genes using the DAVID functional annotation clustering analysis. Results: Treatment with E2 caused significant differential expression of 2841 trp: 1208 upregulated, 1633 downregulated. E2-induced upregulation was inhibited by P4 for 87 (7.2%) of these trp; whereas, E2-induced downregulation was inhibited by P4 for 203 trp (12.4%). Treatment with P4 caused significant differential expression of 2512 trp: 1100 upregulated, 1415 downregulated. P4-induced upregulation or downregulation was reversed by E2 for almost none of the trp (3 or 5 trp). Interestingly, most of the trp were upregulated by both P4 and E2 (683 trp) or downregulated by both E2 and P4 (759 trp). For some of these genes there was a synergistic effect when E2+P4 were combined (193 [13.6%] of downregulated trp; 61 [5.5%] of upregulated trp). Downregulated trp were surprisingly enriched with genes encoding SRC homology-3 domain proteins for all treatments. Those downregulated trp were also enriched in intracellular protein transport and calcium-binding proteins. In addition, E2-down-regulated trp were enriched in general receptor, signaling, and transport functions; whereas, P4-downregulated trp were clustered in EGF-related pathways. For all treatments, upregulated trp were enriched in proteins related to mitochondria processes (electron transport, ATP, cellular metabolism), chaperone and ubiquitin-protein ligase activity and NAD/oxidoreductase. Conclusions: Treatment for 14d with E2, P4, or E2+P4 regulated numerous hepatic genes with more downregulated than upregulated trp. There was some antagonism of E2-regulated genes by P4 but not P4-regulated genes by E2. One of the most remarkable findings was the overlap between the genes regulated by E2 and P4 with >50% of the genes found to be regulated by both steroids. This finding explains the similarities in the clustering results. Supported by CAPES fellowship-Brazil and Pfizer Global Academic Alliance.

An estrogen‐induced decrease in voltage‐gated calcium channel expression attenuates coronary artery contractility

Estrogen (E2) can pharmacologically inhibit Ca2+ influx into vascular smooth muscle cells. However, little is known about the ability of E2 to alter vascular Ca2+ channel expression. The goal of our study was to determine if a physiological E2 concentration can decrease the expression of voltage‐gated calcium channels (VGCC) in small coronary arteries. Right coronary arteries were obtained from hearts of reproductively cycling sows. The distal end of the artery was cut into 4 mm rings or longitudinal strips and incubated for 24 hours in E2 (1 nM) or its ethanol solvent. Western blots using protein isolated from the arterial strips demonstrated a decreased expression of the pore‐forming α1C subunit of the VGCC in E2‐treated arteries. In parallel studies, isometric tension recording revealed that E2‐treated arterial rings contracted significantly less to 30 mM KCl. However, E2 and ethanol‐treated rings preconstricted with 60 mM KCl showed similar relaxation responses to E2 (1 nM to 1 μM). These findings suggest that physiological concentrations of E2 down‐regulate VGCC, thereby providing a potentially novel mechanism for limiting coronary vasoconstriction. In the same arteries, E2‐dependent relaxation is intact. By limiting the expression of VGCC, E2 may promote coronary blood flow and potentially lower the incidence of coronary events in pre‐menopausal women. Support: NCRR of the NIH, Grant #P20 RR‐16460