Effects of Chronic Restraint Stress and 17-β-Estradiol Replacement on Oxidative Stress in the Spinal Cord of Ovariectomized Female Rats

Previous studies have shown sex-specific oxidative changes in spinal cord of rats submitted to chronic stress, which may be due to gonadal hormones. Here, we assessed total radical-trapping potential (TRAP), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and lipid peroxidation (evaluated by the TBARS test) in the spinal cord of ovariectomized (OVX) female rats. Female rats were subjected to OVX, and half of the animals received estradiol replacement. Animals were subdivided into controls and chronically stressed (for 40 days). Our findings demonstrate that chronic stress decreased TRAP, and increased SOD activity in spinal cord homogenates from ovariectomized female rats and had no effect on GPx activity. On the other hand, groups receiving 17β-estradiol replacement presented a decreased GPx activity, but no alteration in TRAP and in SOD activity. No differences in the TBARS test were found in any of the groups analyzed. In conclusion, our results support the idea that chronic stress induces an imbalance between SOD and GPx activities, additionally decreasing TRAP. Estradiol replacement did not reverse the effects of chronic stress, but induced a decrease in GPx activity. Therefore, estradiol replacement in ovariectomized chronically stressed rats could make the spinal cord more susceptible to oxidative injury.     

Heavy Metals Modulate Glutamatergic System in Human Platelets

Research strategies have been developed to characterize parameters in peripheral tissues that might easily be measured in humans as surrogate markers of damage, dysfunction or interactions involving neural targets of toxicants. The similarities between platelet and neuron may even be clinically important, as a number of biochemical markers show parallel changes in the central nervous system (CNS) and platelets. The purpose of our research was to investigate the effect of Hg²⁺, Pb²⁺ and Cd²⁺ on the [³H]-glutamate binding and [³H]-glutamate uptake in human platelets. The involvement of oxidative stress in the modulation of glutamatergic system induced by heavy metals was also investigated. The present study clearly demonstrates that Hg²⁺, Cd²⁺, and Pb²⁺ inhibited [³H]-glutamate uptake in human platelets. Hg²⁺ inhibited [³H]-glutamate binding, while Cd²⁺ and Pb²⁺ stimulated [³H]-glutamate binding in human platelets. Hg²⁺, Cd²⁺ and Pb²⁺ increased lipid peroxidation levels and reactive oxygen species (ROS) measurement in platelets. The present limited results could suggest that glutamatergic system may be used as a potential biomarker for neurotoxic action of heavy metals in humans.     

The Effects of Estradiol on Estrogen Receptor and Glutamate Transporter Expression in Organotypic Hippocampal Cultures Exposed to Oxygen--Glucose Deprivation

The molecular basis of estrogen-mediated neuroprotection against brain ischemia remains unclear. In the present study, we investigated changes in expression of estrogen receptors (ERs) α and β and excitatory amino acid transporters (EAAT) 1 and 2 in rat organotypic hippocampal slice cultures treated with estradiol and subsequently exposed to oxygen--glucose deprivation (OGD). Pretreatment with 17β-estradiol (10 nM) for 7 days protected the CA1 area of hippocampus against OGD (60 min), reducing cellular injury by 46% compared to the vehicle control group. Levels of ERα protein were significantly reduced by 20% after OGD in both vehicle- and estradiol-treated cultures, whereas ERβ was significantly up-regulated by 25% in the estradiol-treated cultures. In contrast, EAAT1 and EAAT2 levels were unchanged in response to estradiol treatment in this model of OGD. These findings suggest that estrogen-induced neuroprotection against ischemia might involve regulation of ERβ and, consequently, of the genes influenced by this receptor.Helena Cimarosti and Ross D. O’Shea, equal first authors.     

Targeted brain delivery of 17β-estradiol via nasally administered water soluble prodrugs

The utility of the nasal route for the systemic delivery of 17β-estradiol was studied using watersoluble prodrugs of 17β-estradiol. This delivery method was examined to determine if it will result in preferential delivery to the brain. Several alkyl prodrugs of 17β-estradiol were prepared and their physicochemical properties were determined. In vitro hydrolysis rate constants in buffer, rat plasma, and rat brain homogenate were determined by high-performance liquid chromatography. In vivo nasal experiments were carried out on rats. Levels of 17β-estradiol in plasma and cerebral spinal fluid (CSF) were determined with radioimunoassay using a gamma counter. The study revealed that the aqueous solubilities of the prodrugs were several orders of magnitude greater than 17β-estradiol with relatively fast in vitro conversion in rat plasma. Absorption was fast following nasal delivery of the prodrugs with high bioavailability. CSF 17β-estradiol concentration was higher following nasal delivery of the prodrugs compared to an equivalent intravenous dose. It was determined that water-soluble prodrugs of 17β-estradiol can be administered nasally. These prodrugs are capable of producing high levels of estradiol in the CSF and as a result may have a significant value in the treatment of Alzheimer's disease. Published: March 25, 2002.     

Naturally Occurring Compounds Affect Glutamatergic Neurotransmission in Rat Brain

Natural products, including those derived from plants, have largely contributed to the development of therapeutic drugs. Glutamate is the main excitatory neurotransmitter in the central nervous system and it is also considered a nociceptive neurotransmitter, by acting on peripheral nervous system. For this reason, in this study we investigated the effects of the hydroalcooholic extracts from Drymis winteri (polygodial and drimanial), Phyllanthus (rutin and quercetine), Jathopha elliptica (jatrophone), Hedyosmum brasiliense (13HDS), Ocotea suaveolens (Tormentic acid), Protium kleinii (αβ-amyrin), Citrus paradise (naringin), soybean (genistein) and Crataeva nurvala (lupeol), described as having antinociceptive effects, on glutamatergic transmission parameters, such as [³H]glutamate binding, [³H]glutamate uptake by synaptic vesicles and astrocyte cultures, and synaptosomal [³H]glutamate release. All the glutamatergic parameters were affected by one or more of these compounds. Specifically, drimanial and polygodial presented more broad and profound effects, requiring more investigation on their mechanisms. The putative central side effects of these compounds, via the glutamatergic system, are discussed.     

Steroidal and growth factor regulation of [3H]thymidine incorporation by cultured endosalpingeal cells of the bovine oviduct

Summary Cultured cells from the bovine endosalpinx were used to evaluate effects of estradiol-17β, progesterone, epidermal growth factor, and insulinlike growth factors I and II on [³H]thymidine incorporation. Cells were treated with hormones and growth factors when approximately 50% confluent. After 24 h, DNA synthesis was quantified by pulsing cells with [³H]thymidine for 12 h and determining uptake into DNA. Cells prepared by mechanical dispersal incorporated more [³H]thymidine than cells dispersed with collagenase. However, hormonal responses were the same for both types of cells. As compared to plastic, cells on a Matrigel substratum exhibited lower incorporation of [³H]thymidine and were unresponsive to hormones. Estradiol-17β increased [³H]thymidine incorporation slightly at 10⁻¹⁰ mol/liter and higher. Epidermal growth factor, insulinlike growth factor-I, and insulinlike growth factor-II also stimulated [³H]thymidine incorporation. Effects of insulinlike growth factor-I were greater for cells treated with estradiol-17β. In the absence of estradiol, progesterone inhibited [³H]thymidine incorporation at 1, 10, and 100 ng/ml. When estradiol-17β was present, progesterone stimulated [³H]thymidine incorporation at 1 ng/ml and reduced incorporation at 100 ng/ml. In conclusion, [³H]thymidine incorporation by cultured oviductal endosalpingeal cells can be regulated by ovarian steroids and growth factors. These molecules may represent signals through which the ovary, embryo, and oviduct regulate oviductal growth. Work conducted while on a sabbatical leave supported by the Deutsche Forschungsgemeinschaft.     

A comparative study of the effect of 17β-estradiol and estriol on peripheral pain behavior in rats

Although estradiol has been reported to influence pain sensitivity, the role of estriol (an estradiol metabolite and another widely used female sex hormone) remains unclear. In this study, pain behavior tests, whole-cell patch clamp recording and Western blotting were used to determine whether estriol plays a role in pain signal transduction and transmission. Either systemic or local administration of 17β-estradiol produced a significant rise of mechanical pain threshold, while estriol lacked this effect in normal and ovariectomized (OVX) rats following estriol replacement. Local administration of 17β-estradiol or estriol significantly decreased ATP-induced spontaneous hind-paw withdrawal duration (PWD), which was blocked by an estrogen receptor antagonist, ICI 182, 780. However, systemic application of estriol in normal or OVX rats lacked this similar effect. In cultured dorsal root ganglion neurons, estriol attenuated α,β-methylene ATP-induced transient currents which were blocked by ICI 182, 780. In complete Freund's adjuvant treated (CFA) rats, systemic application of 17β-estradiol or estriol decreased the mechanical pain threshold significantly, but did not change the inflammatory process. Similar effects were observed after estriol replacement in OVX rats. The expression of c-fos in lumbosacral spinal cord dorsal horn (SCDH) was increased significantly by administration of 17β-estradiol but not estriol, and not by estriol replacement in OVX rats. These results suggest that 17β-estradiol but not estriol plays an anti-hyperalgesic role in physiological pain. However, both peripheral 17β-estradiol and estriol play anti-hyperalgesic roles in ATP-induced inflammatory pain. Systemic application of estriol as well as 17β-estradiol plays hyperalgesic roles in CFA-induced chronic pain.     

Estrogen regulation and ion dependence of taurine uptake by MCF-7 human breast cancer cells

It has been reported that estrogen receptor-positive MCF-7 cells express TauT, a Na⁺-dependent taurine transporter. However, there is a paucity of information relating to the characteristics of taurine transport in this human breast cancer cell line. Therefore, we have examined the characteristics and regulation of taurine uptake by MCF-7 cells. Taurine uptake by MCF-7 cells showed an absolute dependence upon extracellular Na⁺. Although taurine uptake was reduced in Cl^- free medium a significant portion of taurine uptake persisted in the presence of NO₃ ^-. Taurine uptake by MCF-7 cells was inhibited by extracellular β-alanine but not by L-alanine or L-leucine. 17β-estadiol increased taurine uptake by MCF-7 cells: the V_max of influx was increased without affecting the K_m. The effect of 17β-estradiol on taurine uptake by MCF-7 cells was dependent upon the presence of extracellular Na⁺. In contrast, 17β-estradiol had no significant effect on the kinetic parameters of taurine uptake by estrogen receptor-negative MDA-MB-231 cells. It appears that estrogen regulates taurine uptake by MCF-7 cells via TauT. In addition, Na⁺-dependent taurine uptake may not be strictly dependent upon extracellular Cl^-.     

Estradiol Attenuates Tau Hyperphosphorylation Induced by Upregulation of Protein Kinase-A

Protein kinase A (PKA) plays a crucial role in tau hyperphosphorylation, an early event of Alzheimer disease (AD), and 17β-estradiol replacement in aging women forestalls the onset of AD. However, the role of estradiol in PKA-induced tau hyperphosphorylation is not known. Here, we investigated the effect of 17β-estradiol on cAMP/PKA activity and the PKA-induced tau hyperphosphorylation in HEK293 cells stably expressing tau441. We found that 17β-estradiol effectively attenuated forskolin-induced overactivation of PKA and elevation of cAMP, and thus prevented tau from hyperphosphorylation. These data provide the first evidence that 17β-estradiol can inhibit PKA overactivation and the PKA-induced tau hyperphosphorylation, implying a preventive role of 17β-estradiol in AD-like tau pathology. Xin-An Liu and Ling-Qiang Zhu contributed equally to this work.     

Analysis of a Vesicular Glutamate Transporter (VGLUT2) Supports a Cell-leakage Mode in Addition to Vesicular Packaging

VGLUT2 is one of three vesicular glutamate transporters that play crucial roles in glutamatergic excitatory neurotransmission. We explored the functional properties of the rat VGLUT2 by heterologous expression of VGLUT2 in Xenopus oocytes. Immunocytochemical analysis indicated that most VGLUT2 protein was expressed in intracellular compartments but that some expression occurred also on the plasma membrane. Functional analysis revealed VGLUT2 to be active in two independent modes, namely, uptake into intracellular organelles and efflux at the plasma membrane. VGLUT-specific transport was identified based on the strong preference for glutamate over aspartate—in contrast to plasma-membrane or mitochondrial glutamate transporters—and sensitivity to known VGLUT blockers. VGLUT2 expression in oocytes (1) stimulated the influx of l-[³H]glutamate, but not d-[³H]aspartate, into digitonin-permeabilized oocytes and (2) stimulated efflux of l-glutamate, but not l-aspartate, from intact oocytes preinjected with ³H-labeled amino acids. In the latter assay, cellular efflux of glutamate (which was blocked by rose bengal and trypan blue) may be analogous to vesicular packaging of glutamate. Our data are consistent with VGLUT2-mediated H⁺/l-glutamate antiport, but not antiport with chloride. Expression of mammalian VGLUT1 and VGLUT3 also stimulated l-[³H]glutamate efflux from Xenopus oocytes, suggesting that this phenomenon is a general feature of vesicular glutamate transporters. Our findings support the idea that vesicular glutamate transporters, when transiently expressed on the neuronal plasma membrane, may mediate Ca²⁺-independent glutamate leakage in addition to their traditional role of packaging glutamate into synaptic vesicles for Ca²⁺-dependent exocytosis. Special issue article in honor of Dr. Frode Fonnum.     

Estradiol stimulates Akt, AMP-activated protein kinase (AMPK) and TBC1D1/4, but not glucose uptake in rat soleus

Post-menopausal women exhibit decreases in circulating estrogen levels and whole body insulin sensitivity, suggesting that estrogen regulates skeletal muscle glucose disposal. Thus, we assessed whether estrogen stimulates glucose uptake or enhances insulin sensitivity in skeletal muscle. Ex vivo muscle stimulation with 17β-estradiol (10 nM) resulted in a rapid (?10 min) increase in the phosphorylation of Akt, AMP-activated protein kinase (AMPK), and TBC1D1/4, key signaling proteins that regulate glucose uptake in muscle. Treatment with the estrogen receptor antagonist, ICI 182,780, only partly inhibited signaling, suggesting both an estrogen receptor-dependent and independent mechanism of estradiol action. 17β-Estradiol did not stimulate ex vivo muscle [³H]-2-deoxyglucose uptake or enhance insulin-induced glucose uptake, demonstrating discordance between the estradiol-induced stimulation of signaling proteins and muscle glucose uptake. This study is the first to demonstrate that estradiol stimulates Akt, AMPK, and TBC1D1/4 in intact skeletal muscle, but surprisingly, estradiol does not stimulate muscle glucose uptake.     

Effects of ammonia onl-glutamate uptake in cultured astrocytes

The effect of ammonia onl-glutamate (L-GLU) uptake was examined in cultured astrocytes. Acute ammonia treatment (5–10 mM) enhanced L-[³H]GLU uptake by 20–42% by increasing the V_max; this persisted for 2 days and then started to decline. Ammonia, however, did not affect the uptake ofd-[³H]aspartate (D-ASP), a non-metabolizable analog of L-GLU, that uses the same transport carrier as L-GLU. Also, L-GLU uptake was not affected during the first 2 min of the assay. Thus, ammonia did not have an acute effect on L-GLU transport (translocation); rather, ammonia enhanced the accumulation or “trapping” of L-GLU or its by-products. Chronic ammonia treatment, on the other hand, inhibited L-GLU transport in astrocytes by ∼30–45% and this was due to a decrease in V_max, suggesting that the number of L-GLU transporters was decreased. This inhibitory effect was observed after 1 day of treatment and persisted for at least 7 days. The inhibition of L-GLU transport was partially reversible following removal of ammonia. The effects of ammonia on L-GLU transport and uptake may explain the abnormal L-GLU neurotransmission observed in hyperammonemia/hepatic encephalopathy, and the brain swelling associated with fulminant hepatic failure.     

<Emphasis Type="Italic">In vivo</Emphasis> Quinolinic Acid Increases Synaptosomal Glutamate Release in Rats: Reversal by Guanosine

Glutamate, the main excitatory neurotransmitter in the mammalian central nervous system (CNS), plays important role in brain physiological and pathological events. Quinolinic acid (QA) is a glutamatergic agent that induces seizures and is involved in the etiology of epilepsy. Guanine-based purines (GBPs) (guanosine and GMP) have been shown to exert neuroprotective effects against glutamatergic excitotoxic events. In this study, the influence of QA and GBPs on synaptosomal glutamate release and uptake in rats was investigated. We had previously demonstrated that QA “in vitro” stimulates synaptosomal L-[³H]glutamate release. In this work, we show that i.c.v. QA administration induced seizures in rats and was able to stimulate synaptosomal L-[³H]glutamate release. This in vivo neurochemical effect was prevented by i.p. guanosine only when this nucleoside prevented QA-induced seizures. I.c.v. QA did not affect synaptosomal L-[³H]glutamate uptake. These data provided new evidence on the role of QA and GBPs on glutamatergic system in rat brain.     

NMR analysis reveals 17β-estradiol induced conformational change in ERβ ligand binding domain expressed in <Emphasis Type="Italic">E. coli</Emphasis>

Nuclear magnetic resonance (NMR) spectroscopy is a useful biophysical technique to study the ligand–protein interaction. In this report, we have used bacterially produced ERβ and its domains for studying the functional analysis of ligand–protein interaction. Briefly, ERβ and its transactivation domain (TAD) and ligand binding domain (LBD) were subcloned and overexpressed using a prokaryotic expression system. The recombinant proteins were purified using Ni⁺²-IDA affinity chromatography and analyzed by NMR. Purified ERβ and TAD show similar conformation in the absence or presence of 17β-estradiol. However, LBD shows altered conformation in the presence of 17β-estradiol. These findings suggest that ERβ produced in bacteria exhibits a conformation such that its LBD remains masked and consequently it binds less to 17β-estradiol. Such study may help to develop the therapeutic approaches for controlling the estradiol-mediated gene expression in hormone dependent diseases.     

Estradiol-17β stimulates proliferation of uterine epithelial cells cultured with stromal cells but not cultured separately

Summary There is indirect evidence that the in vivo proliferative response of rodent uterine epithelium to estrogen requires interaction with the underlying stroma in pre- and post-pubescent animals. To examine this potential requirement directly, the proliferative response of epithelium to 17β-estradiol in the presence or absence of stroma was measured in vitro. Uterine epithelial and stromal cells were isolated separately from immature or adult mice, and were maintained as monocultures or cocultures in defined, serum-free medium with or without 8 × 10⁻⁹ M 17β-estradiol. Incorporation of bromodeoxyuridine into the DNA was determined by immunolabeling to assay proliferation in individual cells. Cell morphology and immunolabeling of cytokeratin were used to distinguish epithelial from stromal cells. Treatment of cocultures with 17β-estradiol for 24 h increased the proliferation of epithelial cells relative to controls approximately threefold, whereas, in monocultures of epithelial or stromal cells 17β-estradiol decreased the number of bromodeoxyuridine-incorporating cells by approximately half. Furthermore, cell contact between epithelial and stromal cells was important for the effects of 17β-estradiol on cells in cocultures. Approximately three quarters of the 17β-estradiol-induced proliferation of epithelial cells in cocultures was produced by epithelial cells within colonies that were also contacting stromal cells. These results are consistent with the hypothesis that stromal cells mediate the estrogenic proliferative response, and provide evidence that this mediation involves cell contact or stroma-mediated changes in the microenvironment immediately around the epithelial cell.     

Comparable Attenuation of Aβ<Subscript>25–35</Subscript>-Induced Neurotoxicity by Quercitrin and 17β-Estradiol in Cultured Rat Hippocampal Neurons

In the present work, potential protective effects of quercitrin (a phytoestrogen) on Aβ-induced neurotoxicity in cultured rat hippocampal neurons were investigated in comparison with 17β-estradiol. Cell viability, oxidative status, and antioxidative potentials were used as comparative parameters. Co-exposure of cultured neurons to Aβ_25–35 with either quercitrin or 17β-estradiol (50–100 μM) for 72 h attenuated Aβ_25–35-induced neurotoxicity and lipid peroxidation, but not Aβ_25–35-induced ROS accumulation. However, only 17β-estradiol counteracted a reduction in glutathione content and only quercitrin counteracted a reduction in glutathione peroxidase activity. Both compounds displayed no effects on superoxide dismutase activity. A specific estrogen receptor antagonist, ICI 182780, did not abolish neuroprotective effects of quercitrin and 17β-estradiol. These findings suggested that quercitrin and 17β-estradiol attenuated Aβ_25–35-induced neurotoxicity in a comparable manner. Underlying neuroprotective mechanisms of both compounds were probably not related to estrogen receptor-mediated genomic mechanisms but might involve with their antioxidant and free radical scavenging properties.     

Pregnenolone and progesterone metabolism by the testes of Bufo arenarum

Sliced testis tissue from Bufo arenarum was incubated in the presence of [³H]pregnenolone. Testis fragments were also used for double isotope experiments using [³H]pregnenolone and [¹⁴C]progesterone. Specific activities were equated with the addition of radioinert pregnenolone. When yields of radiometabolites were analysed, pregnenolone was found to be a good precursor for C₁₉ steroids such as dehydroepiandrosterone, 5-androsten-3β,17β diol, testosterone, 5α-dihydrotestosterone and a C₂₁ steroid, 5α-pregnan-3,20 dione. Progesterone mainly converts to 5α-pregnan-3,20 dione, a steroid with unknown function in amphibians. The 5-ene pathway, including 5-androsten-3β,17β diol as intermediate, could be predominant for androgen biosynthesis. Testes bypass not only progesterone but also androstenedione for testosterone biosynthesis. Accepted: 17 April 1998     

Effect of Chronic Estradiol and Progesterone Treatments of Ovariectomized Rats on Brain Dopamine Uptake Sites

Abstract: Dopamine released from brain nerve terminals is mainly removed from the synaptic cleft by an uptake mechanism. Despite their functional importance, modulation of the dopamine uptake sites is still not well known. Steroid hormones were shown to modulate brain dopamine transmission. The aim of this study was thus to investigate in ovariectomized rats the effects of 17β-estradiol and progesterone treatments on brain dopamine uptake sites. Treatments consisted of 17β-estradiol (10 μg/0.2 ml), progesterone (0.72 mg/0.2 ml). 17β-estradiol + progesterone, or the vehicle (0.3% gelatin in saline solution) twice daily for 2 weeks. The steroid treatments left the affinity of [³H]GBR 12935 binding to striatal homogenates unchanged (ovariectomized rats, 0.823 ± 0.028 nM), whereas the density was increased by these steroids alone or in combination to a similar extent of 16-23%. Chronic treatment of ovariectomized rats with 17β-estradiol progesterone, or their combination increased to the same extent and uniformly [³H]-GBR 12935 binding in the striatum as measured by autoradiography; the increase was similar in the substantia nigra pars compacta, whereas no steroid effect was observed in the nucleus accumbens and in the substantia nigra pars reticulata. In summary, chronic exposure to 17β-estradiol and/ or progesterone increased dopamine uptake site density in the nigrostriatal dopaminergic system, whereas the nucleus accumbens and the substantia nigra pars reticulata were unaffected.