Propyl Pyrazole Triol Research Articles

Sexual responses of the male rat medial preoptic area and medial amygdala to estrogen II: Site specific effects of selective estrogenic drugs

In the medial preoptic area (MPO) and medial amygdala (MEA), estradiol (E2) aromatized from testosterone (T) may act via either estrogen receptor (ER) α or ERβ to mediate mating in male rats. We tested the hypothesis that, in the MPO, ERα exclusively mediates sexual responses to E2 by monitoring mating in four groups of castrated male rats administered dihydrotestosterone (DHT) subcutaneously and MPO implants delivering either: cholesterol, E2, propyl pyrazole triol (PPT, ERα-agonist) or diarylpropionitrile (DPN, ER β-agonist); a fifth group of intact males served as DPN toxicity control, receiving DPN MPO implants. In a follow-up study, either 1-methyl-4-phenyl pyridinium (MPP, ERα-antagonist) or blank MPO cannulae were implanted in castrated male rats receiving T subcutaneously, whereas intact MPP toxicity controls received MPP MEA implants. PPT or E2 MPO implants maintained mating, but cholesterol or DPN MPO implants did not. Moreover, MPP MPO implants interfered with T reinstatement of mating suggesting that, in the MPO, ERα is necessary and sufficient for mating in androgen-maintained male rats and ERβ is not sufficient. Because it is unknown which ER subtype(s) mediate sexual responses of the MEA to E2, we examined mating following MEA implants of cholesterol, E2, PPT or DPN in four groups of castrated male rats administered DHT subcutaneously. E2 MEA implants maintained mounting but mating was significantly decreased in groups receiving PPT, DPN or cholesterol MEA implants suggesting that, unlike the MPO where ERα alone is essential, sexual responses of the MEA to E2 require simultaneous interactions among multiple ER subtypes.

Age-Dependent Reductions in Mitochondrial Respiration are Exacerbated by Calcium in the Female Rat Heart

Cardiovascular disease mortality increases rapidly after menopause by poorly defined mechanisms. Because mitochondrial function and Ca(2+) sensitivity are important regulators of cell death after myocardial ischemia, we sought to determine whether aging and/or estrogen deficiency (ovariectomy) increased mitochondrial Ca(2+) sensitivity. Mitochondrial respiration was measured in ventricular mitochondria isolated from adult (6 months; n = 26) and aged (24 months; n = 25), intact or ovariectomized female rats using the substrates α-ketoglutarate/malate (complex I); succinate/rotenone (complex II); ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine/antimycin (complex IV). State 2 and 3 respiration was initiated by sequential addition of mitochondria and adenosine diphosphate. Ca(2+) sensitivity was assessed by Ca(2+)-induced swelling of de-energized mitochondria and reduction in state 3 respiration. Propylpyrazole triol (PPT) was administered intraperitoneally 45 minutes before euthanasia to assess mitochondrial protective effects through estrogen receptor (ER) α activation. Aging decreased the respiratory control index (RCI; state 3/state 2) for complexes I and II by 12% and 8%, respectively, independent of ovary status (P < 0.05). Of interest, Ca(2+) induced a greater decrease (18%-30%; P < 0.05) in complex I state 3 respiration in aged and ovariectomized animals, and mitochondrial swelling occurred twice as quickly in aged (vs adult) female rats (P < 0.05). Pretreatment with PPT increased RCI by 8% and 7% at complexes I and II, respectively (P < 0.05) but surprisingly increased Ca(2+) sensitivity. Age-dependent decreases in RCI and sensitization to Ca(2+) may explain in part the age-associated reductions in female ischemic tolerance; however, protection afforded by ER agonism involves more complex mechanisms.

Acute Effects of Conjugate Equine Estrogen and Selective Estrogen Receptor‐α Agonist PPT on Postischemic Pial Arteriole Response in Rats

Study ObjectiveWe explored the effects of acute administration of conjugated equine estrogens (CEE), on postischemic pial arteriole dilatory capacity. Additionally, we determined determined if the beneficial effects of estrogen are mediated through estrogen receptor‐α (ER‐α) using the specific a specific agonist, propyl pyrazole triol (PPT).MethodsOvariectomized (OVX) female or male rats were anesthetized, and after placement of a closed cranial window over the parietal cortex, rats were subjected to 15‐min reversible forebrain ischemia, followed by 2 hr. reperfusion. OVX rats received intravenous CEE (1 mg/kg) or vehicle at the onset of reperfusion. Separate cohorts of OVX female or male rats were treated with the ER‐α agonist PPT intraperitoneally (2 mg/kg) 3 hours prior to ischemia. Pial artery dilatory capacity was tested before and after ischemia.ResultsAdministration of CEE improved vasodilatory response from −1.7 ± 4.43% to 15.31± 6.63% (p=0.0002). PPT augmented postischemic vasodilatory response in females from −2.63 ± 1.42% to 23.57 ± 11.3% (p= 0.005), and in male rats from −0.38 ± 2.88% to 30.84 ± 11.66% (p=0.007).ConclusionAcute administration of CEE or PPT restores postischemic pial arteriole vasodilatory reactivity to Ach. These findings suggest that the effects acute estrogen treatment after ischemic cerebral injury may be modulated via ER‐α.

Estrogen receptor (ESR) 2 partially offsets the absence of ESR1 in gonadotropes of pituitary-specific Esr1 knockout female mice

Estrogen receptor 1 and 2 (ESR1 and 2) mediate estrogen (E) action on gonadotrope function. While much is known about the effects of ESR1 on the gonadotrope, there is still some controversy regarding the effects of ESR2. To investigate the role of ESR2 in the gonadotrope, 45-day-old female mice of two different genotypes were used: wild type (WT) and pituitary (gonadotropes and thyrotropes)-specific Esr1 knockout (KO). All mice were ovariectomized (OVX) and 15 days later injected over 3 days with 2.5 μg 17β-estradiol (E(2)), 0.2 mg of the selective ESR1 or 2 agonists, propylpyrazole triol and diarylpropionitrile, respectively, or 0.1 ml oil. The day after treatment, anterior pituitary glands were dissected out for evaluation of gonadotrope ultrastructural morphology and pituitary immunohistochemical expression of progesterone receptor (Pgr (Pr)). Blood was collected and serum LH levels were assessed. Activation of ESR1 in WT mice resulted in the following: i) uterine ballooning and vaginal cornification, ii) negative feedback on LH secretion, iii) increased number of homogeneous (functional) gonadotropes, and iv) pituitary Pgr expression (35.9±2.0% of pituitary cells). Activation of ESR1 in KO mice induced normal uterine, vaginal, and LH secretion responses, but failed to increase the number of functional gonadotropes, and induced significantly lower Pgr expression (21.0±3.0% of pituitary cells) than in WT mice. Whilst activation of ESR2 had no significant effects in WT mice, it doubled the number of functional gonadotropes exhibited by KO mice injected with oil. It is concluded that E(2) exerted its action in KO mouse gonadotropes via ESR2.

Defeminization of Brain Functions by a Single Injection of Estrogen Receptor α or β Agonist in Neonatal Female Rats

Sexual differentiation of brain function is regulated by estrogen in the perinatal period of rodents. However, the role of the estrogen receptor subtypes ERα and ERβ is still in question. Accordingly, the effects of neonatal treatment with the ERα agonist propyl pyrazole triol (PPT) or the ERβ agonist diarylpropionitrile (DPN) on female reproductive functions were investigated in rats. Female rats were injected subcutaneously with 100–500 µg/10 g body weight (b.w.) PPT or DPN, 100 µg/10 g b.w. estradiol (E₂), or saline at day 5 (birth day = day 1), and then vaginal opening and vaginal smears were examined. On day 60, their ovaries were removed and lordosis behavior was observed after subcutaneous implantation of a silicon tube containing E₂. As a result, in most PPT and all E₂ rats, vaginal opening was advanced and an irregular estrous cycle was observed. In contrast, in most rats of the DPN groups, vaginal opening was comparable to that of the control and there was a regular estrous cycle. Lordosis tests revealed that the mean lordosis quotients (LQs) in the 250- and 500-µg PPT groups was lower than in the saline group, but higher than in the E₂ group. Mean LQs in all DPN groups were comparable to those in the saline group. These results suggest that ERα plays a major role in masculinization of the system regulating the estrous cycle in the rat brain. In behavioral defeminization of the lordosis-regulation system, ERα was also found to be the main target of estrogen.

Treatment with bisphenol A and methoxychlor results in the growth of human breast cancer cells and alteration of the expression of cell cycle-related genes, cyclin D1 and p21, via an estrogen receptor-dependent signaling pathway

Various endocrine disrupting chemicals (EDCs) are exogenous compounds found in the environment and have the potential to interfere with the endocrine system and hormonal regulation. Among EDCs, bisphenolA (BPA) and 1,1,1-trichloro-2,2-bis(4-methoxyphenol)-ethane [methoxychlor (MXC)] have estrogenic activity resulting in a variety of dysfunctions in the E2-mediated response by binding to estrogen receptors (ERs), causing human health problems such as abnormal reproduction and carcinogenesis. In this study, we investigated the effects of BPA and MXC on cell proliferation facilitated by ER signaling in human breast cancer cells. MCF-7 cells are known to be ERα-positive and to be a highly E2-responsive cancer cell line; these cells are, therefore, a useful invitro model for detecting estrogenic activity in response to EDCs. We evaluated cancer cell proliferation following BPA and MXC treatment using an MTT assay. We analyzed alterations in the expression of genes associated with the cell cycle in MCF-7 cells by semi-quantitative reverse-transcription PCR following treatment with BPA or MXC compared to EtOH. To determine whether BPA and MXC stimulate cancer cell growth though ER signaling, we co-treated the cells with agonists (propyl pyrazoletriol, PPT; and diarylpropionitrile, DPN) or an antagonist (ICI 182,780) of ER signaling and reduced ERα gene expression via siRNA in MCF-7 cells before treatment with EDCs. These studies confirmed the carcinogenicity of EDCs invitro. As a result, BPA and MXC induced the cancer cell proliferation by the upregulation of genes that promote the cell cycle and the downregulation of anti-proliferative genes, especially ones affecting the G1/S transition via ERα signaling. These collective results confirm the carcinogenicity of these EDCs invitro. Further studies are required to determine whether EDCs promote carcinogenesis invivo.

P21 and Notch Signalings in the Persistently Altered Vagina Induced by Neonatal Diethylstilbestrol Exposure in Mice

Female reproductive organs show organ-specific morphological changes during estrous cycles. Perinatal exposure to natural and synthetic estrogens including diethylstilbestrol (DES) or estrogenic chemicals induces estrogen-independent persistent proliferation of vaginal epithelium in mice. To understand the underlying mechanism of the estrogen-independent persistent vaginal changes induced by perinatal DES exposure, we examined global gene expressions in the vaginae of ovariectomized adult mice exposed neonatally to DES using a microarray. The cell cycle-related gene, p21, a cyclin-dependent kinase inhibitor, showed upregulation in the vagina, and p21 protein was localized in the basal layer of the vaginal epithelium in mice exposed neonatally to DES and an estrogen receptor α agonist, propyl pyrazole triol (PPT). The expressions of Notch receptors and Notch ligands were unchanged; however, the mRNAs of hairy-related basic helix-loop-helix (bHLH) transcription factor genes, Hes1, Hey1 and Heyl were persistently downregulated in the vagina, indicating estrogen-independent epithelial cell proliferation in mice exposed neonatally to DES and PPT.

Estrogen prevents increased hepatic aquaporin-9 expression and glycerol uptake during starvation

In starvation, glycerol is released from adipose tissue and serves as an important precursor for hepatic gluconeogenesis. By unknown sex-specific mechanisms, women suppress the endogenous glucose production better than men and respond to metabolic stress with higher plasma glycerol levels. Hepatic glycerol uptake is facilitated by aquaporin-9 (AQP9), a broad-selectivity neutral solute channel, and represents an insulin-regulated step in supplying gluconeogenesis with glycerol. In the present study, hepatic AQP9 abundance was increased 2.6-fold in starved male rats as assessed by immunoblotting and immunohistochemistry. By contrast, starvation had no significant effect on hepatic AQP9 expression in female rats. Coordinately, plasma glycerol levels remained unchanged with starvation in male rats, whereas it was increased in female rats. The different responses to starvation were paralleled by higher glycerol permeability in basolateral hepatocyte membranes from starved male rats compared with starved females. Ovariectomy led to a starvation-response pattern identical to that observed in male rats with increased hepatic AQP9 expression and unchanged plasma glycerol levels. In cultured hepatocytes, 17β-estradiol and the selective estrogen receptor α-agonist, propyl pyrazole triol, caused a decrease in AQP9 expression. Our results support that a sex-specific regulation of the hepatic glycerol channel AQP9 during starvation contributes to the higher plasma glycerol levels observed in women during fasting and possibly results in a lower cytosolic availability of glycerol. Furthermore, the sexual dimorphism in the hepatic handling of glycerol during starvation might be explained by 17β-estradiol preventing the starvation-induced increase in hepatic AQP9 abundance.

Estradiol inhibits hyaluronic acid synthase 1 expression in human vascular smooth muscle cells

Epidemiological and clinical data suggest that estrogen retards the progression of atherosclerosis. This study aims to elucidate whether the phenotypic regulation of human vascular smooth muscle cells (VSMC) by estrogen may involve effects on the hyaluronan matrix. VSMC were synchronized by serum withdrawal and subsequently stimulated with 0.001, 0.01, 0.1 and 1μM estradiol (E(2)) in the presence or absence of platelet-derived growth factor BB (PDGF-BB) for 24h. E(2) reduced mRNA-expression of hyaluronic acid synthase (HAS) 1 in the presence and absence of PDGF-BB. In contrast, HAS2- and HAS3-mRNA-expression were not affected. This E(2)-mediated effect on HAS1 mRNA-expression was accompanied by reduced hyaluronan secretion and a shift of HA toward lower molecular weight as evidenced by molecular sieve chromatography. The downregulation of HAS1 was abrogated by the estrogen receptor (ER) α and β antagonist ICI182780 and could be mimicked by the ERα-agonist propyl-pyrazole triol (PPT). On the contrary, the ERβ-agonist diarylpropionitrile (DPN) had no effect on HAS1 mRNA-expression. To investigate whether the downregulation of HAS1 was causally involved in the phenotypic regulation of human VSMC by E(2), lentiviral overexpression of HAS1 was conducted. Overexpression of HAS1 abrogated the inhibition of sustained ERK1/2 phosphorylation and in turn inhibition of DNA-synthesis by E(2). For the first time this study provides strong evidence that HAS1-driven HA-synthesis is a target of E(2) in human VSMC and that E(2) mediates part of its anti-proliferative effects through an ERα-dependent inhibition of HA-synthesis.

Estradiol Modulates Effort-Based Decision Making in Female Rats

Disorders of the dopamine system, such as schizophrenia or stimulant addiction, are associated with impairments in different forms of cost/benefit decision making. The neural circuitry (ie amygdala, prefrontal cortex, nucleus accumbens) underlying these functions receives dopamine input, which is thought to have a central role in mediating cost/benefit decisions. Estradiol modulates dopamine activity, and estrogen receptors (ERs) are found within this neurocircuitry, suggesting that decision making may be influenced by estradiol. The present study examined the contribution of estradiol and selective ERα and β agonists on cost/benefit decision making in adult female Long-Evans rats. An effort-discounting task was utilized, where rats could either emit a single response on a low-reward lever to receive two pellets, or make 2, 5, 10, or 20 responses on a high-reward lever to obtain four pellets. Ovariectomy increased the choice on the high-reward lever, whereas replacement with high (10 μg), but not low (0.3 μg), levels of estradiol benzoate reduced the choice on the high-reward lever. Interestingly, both an ERα agonist (propyl-pyrazole triol (PPT)) and an ERβ agonist (diarylpropionitrile (DPN)) increased choice on the high-reward lever when administered independently, but when these two agonists were combined, a decrease in choice for the high-reward lever was observed. The effects of estradiol, PPT, and DPN were more pronounced 24 h post-administration, suggesting that these effects may be genomic in nature. Together, these results demonstrate that estradiol modulates cost/benefit decision making in females, whereby concomitant activation of ERα and β receptors shifts the decision criteria and reduces preference for larger, yet more costly rewards.

Does a Nonclassical Signaling Mechanism Underlie an Increase of Estradiol-Mediated Gonadotropin-Releasing Hormone Receptor Binding in Ovine Pituitary Cells?1

Estradiol-17beta (E2) is the major regulator of GnRH receptor (GnRHR) gene expression and number during the periovulatory period; however, the mechanisms underlying E2 regulation of the GNRHR gene remain undefined. Herein, we find that E2 conjugated to BSA (E2-BSA) mimics the stimulatory effect of E2 on GnRH binding in primary cultures of ovine pituitary cells. The time course for maximal GnRH analog binding was similar for both E2 and E2-BSA. The ability of E2 and E2-BSA to increase GnRH analog binding was blocked by the estrogen receptor (ER) antagonist ICI 182,780. Also, increased GnRH analog binding in response to E2 and the selective ESR1 agonist propylpyrazole triol was blocked by expression of a dominant-negative form of ESR1 (L540Q). Thus, membrane-associated ESR1 is the likely candidate for mediating E2 activation of the GNRHR gene. As cAMP response element binding protein (CREB) is an established target for E2 activation in gonadotrophs, we next explored a potential role for this protein as an intracellular mediator of the E2 signal. Consistent with this possibility, adenoviral-mediated expression of a dominant-negative form of CREB (A-CREB) completely abolished the ability of E2 to increase GnRH analog binding in primary cultures of ovine pituitary cells. Finally, the presence of membrane-associated E2 binding sites on ovine pituitary cells was demonstrated using a fluorescein isothiocyanate conjugate of E2-BSA. We suggest that E2 regulation of GnRHR number during the preovulatory period reflects a membrane site of action and may proceed through a nonclassical signaling mechanism, specifically a CREB-dependent pathway.

Phospholipid hydroperoxide glutathione peroxidase gene is regulated via an estrogen and estrogen receptor signaling in cultured mouse fetuses

Although it has been suggested that the transcription of phospholipid hydroperoxide glutathione peroxidase (PHGPx), an essential antioxidant selenoenzyme, may be affected by the estrogen state in mammals, the direct mechanism underlying the regulation of the PHGPx gene by estrogens in mammalian tissues remains to be clearly elucidated. In this study, we evaluated the expression of the PHGPx mRNA in cultured mouse fetuses (embryonic days 8.5-10.5) exposed to 17β-estradiol (E(2); 0.1, 1, 10, 100, and 1,000 ng/ml); estrogen receptor (ER) agonists [propyl pyrazole triol (PPT, an ERα-selective ligand, 1 μl/ml) and diarylpropionitrile (DPN, an ERβ-selective ligand, 1 μl/ml)]; and/or ER antagonist [ICI 182,780 (ICI, 1 μl/ml)] using a whole embryo culture system. E(2)-alone treatment significantly stimulated the expressions of both ERα and ERβ mRNAs in all the cultured fetuses (p < 0.05), although the ERβ mRNA levels were higher than ERα mRNA. PHGPx mRNA expression was significantly increased in all the fetuses treated with E(2) (1-1,000 ng/ml), PPT, and DPN (p < 0.05). Furthermore, pretreatment with ICI completely blocked the E(2)-induced PHGPx mRNA expression in the fetuses. In addition, the mRNA levels of cytosolic GPx, the other intracellular antioxidant selenoenzyme, did not differ significantly from the controls by an exposure to those agents. These results suggest that the PHGPx gene is regulated via an estrogen and ER signal pathway in the cultured mouse fetus.

Estrogen receptor‐α partially mediates estrogen effect on muscle Hsp70 levels

We examined the influence of estrogen receptor –α (ERα) activation on estrogen mediated augmentation of heat shock protein 70 (HSP70) content in skeletal muscle. Ovariectomized female rats were administered either estrogen (EST), an ERα agonist (propyl pyrazole triol, PPT), both (EST+PPT), or a sham and served either as unexercised controls or were run downhill (17 m·min-1, −13.5° grade) for 90 min. At 72 hrs post-exercise, soleus muscles were surgically removed, frozen and sectioned and Hsp70 immunohistochemistry and muscle fibre typing performed. In type I muscle fibres, unexercised EST, PPT and EST+PPT groups exhibited elevated (P<0.05) Hsp70 levels relative to unexercised sham animals with EST and EST+PPT also higher (P<0.05) than PPT. Exercise induced elevations (P<0.05) in Hsp70 concentration relative to unexercised animals in sham and PPT groups to levels equal to resting values in the EST and EST+PPT groups in which exercise induced no further changes in Hsp70. The effects of exercise, EST or PPT were not as clear in other muscle fibre types. Since PPT administration alone succeeded in partially augmenting resting muscle Hsp70 levels relative to the greater effect of EST or EST+PPT, we conclude that the ability of EST to augment resting type I muscle Hsp70 content to levels otherwise induced by exercise is partially mediated by the muscle estrogen α-receptor. Supported by NSERC Discovery grants to PMT & ART

Estrogen receptor ligands counteract cognitive deficits caused by androgen deprivation in male rats

Androgen deprivation causes impairment of cognitive tasks in rodents and humans, and this deficit can be reverted by androgen replacement therapy. Part of the effects of androgens in the male may be mediated by their local metabolism to estradiol or 3-alpha androstanediol within the brain and the consequent activation of estrogen receptors. In this study we have assessed whether the administration of estradiol benzoate, the estrogen receptor β selective agonist diarylpropionitrile or the estrogen receptor α selective agonist propyl pyrazole triol affect performance of androgen-deprived male Wistar rats in the cross-maze test. In addition, we tested the effect of raloxifene and tamoxifen, two selective estrogen receptor modulators used in clinical practice. The behavior of the rats was assessed 2 weeks after orchidectomy or sham surgery. Orchidectomy impaired acquisition in the cross-maze test. Estradiol benzoate and the selective estrogen receptor β agonist significantly improved acquisition in the cross-maze test compared to orchidectomized animals injected with vehicle. Raloxifene and tamoxifen at a dose of 1 mg/kg, but not at doses of 0.5 or 2 mg/kg, also improved acquisition of orchidectomized animals. Our findings suggest that estrogenic compounds with affinity for estrogen receptor β and selective estrogen receptor modulators, such as raloxifene and tamoxifen, may represent good candidates to promote cognitive performance in androgen-deprived males.

Rapid Effects of Estrogen Receptor α and β Selective Agonists on Learning and Dendritic Spines in Female Mice

Estrogen receptor (ER) agonists rapidly affect neural plasticity within 1 h, suggesting they play a functional role in learning and memory. However, behavioral learning experiments on such a rapid time scale are lacking. Therefore we investigated whether the ERα agonist propyl pyrazole triol (PPT) and ERβ agonist diarylpropionitrile (DPN) could affect social recognition, object recognition, or object placement learning within 40 min of drug administration. At the same time, we examined their effects on CA1 hippocampal dendritic spines. Ovariectomized female CD1 mice were administered a range of PPT or DPN doses (0, 30, 50, 75, or 150 μg/mouse). PPT at the middle doses improved social recognition, facilitated object recognition and placement at a dose of 75 μg, and increased dendritic spine density in the stratum radiatum and lacunosum-moleculare. In contrast, DPN impaired social recognition at higher doses, did not affect object recognition, but slightly facilitated object placement learning at the 75-μg dose. DPN did not affect spines in the stratum radiatum but decreased spine density and increased spine length in the lacunosum-moleculare. This suggests that rapid estrogen-mediated learning enhancements may predominantly be mediated through ERα, while the effects of DPN are weaker and may depend on the learning paradigm. The role of ERα and ERβ in learning and memory may vary depending on the timing of drug administration, as genomic studies often implicate ERβ in enhancing effects on learning and memory. To our knowledge, this is the first report of estrogens' effects on learning within such a short time frame.

Resveratrol interacts with estrogen receptor-β to inhibit cell replicative growth and enhance stress resistance by upregulating mitochondrial superoxide dismutase

trans-Resveratrol (RES) is one of a number of dietary polyphenols that have been reported to beneficially affect human physiology. Although numerous studies have attributed this to direct interactions between RES and histone deacetylases, recently the reliability of these results has been questioned. We have shown that the mitochondrial superoxide dismutase (MnSOD) is substantially upregulated in RES-treated cells. Here we explore the mechanisms underlying this, showing that two of RES's more interesting effects, inhibition of replication and enhancement of stress resistance, are mediated by MnSOD upregulation in three cell lines: MRC5 human lung fibroblasts, C2C12 mouse myoblasts, and SHSY5Y human neuroblastoma cells. When small interfering RNA was used to prevent induction of MnSOD expression, the effects of RES on population doubling time of cells in culture, and resistance to cell death after exposure to hydrogen peroxide or paraquat, were abolished. Interestingly, the RES-induced upregulation of MnSOD levels could be prevented by the estrogen receptor antagonist ICI 182780. RES's effects also could be reproduced using estradiol or the estrogen receptor-β agonist diarylpropionitrile, but not using the estrogen receptor-α agonist propylpyrazole triol. Thus, we suggest that RES interacts with estrogen receptor-β to induce the upregulation of MnSOD, which affects cell cycle progression and stress resistance. These results have important implications for our understanding of RES's biological activities and potential applications to human health.

Role of Estrogen Receptor-α in the Regulation of Claudin-6 Expression in Breast Cancer Cells

PurposeIn our previous studies we showed that upregulating claudin-6 (CLDN6) expression may contribute to preventing breast cancer, and that 17β-estradiol induces a concentration- and time-related effect on CLDN6 mRNA and protein expression in MCF-7 cells. However, the mechanisms of 17β-estradiol regulation of CLDN6 are still unclear. We determined the role of estrogen receptors in the regulation of CLDN6 expression in human breast cancer tissues and a cell line.MethodsCLDN6, estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) expression in breast cancer tissues were examined using immunohistochemistry. The human breast cancer cell line, MCF-7, which expresses ERα but not ERβ was used. CLDN6 and ERα expression were measured by reverse transcriptase-PCR, Western blotting and immunofluorescent staining. Treatments with propyl pyrazole triol (PPT) and ICI 182, 780 (ICI) were performed.ResultsThe results revealed that CLDN6 expression was related to ERα in breast cancer tissues (p=0.033). PPT, an ERα-selective ligand, upregulated CLDN6 expression at 10-5 mol/L after 24 hours. The effect of PPT on regulating CLDN6 expression in MCF-7 cells was blocked by ICI.ConclusionThese findings suggest that Erα reulates CLDN6 expression in breast cancer tissues and that 17β-estradiol induces CLDN6 expression through an ERα pathway in MCF-7 cells.

Estradiol and ERβ agonists enhance recognition memory, and DPN, an ERβ agonist, alters brain monoamines

Effects of estradiol benzoate (EB), ERα-selective agonist, propyl pyrazole triol (PPT) and ERβ-selective agonists, diarylpropionitrile (DPN) and Compound 19 (C-19) on memory were investigated in OVX rats using object recognition (OR) and placement (OP) memory tasks. Treatments were acute (behavior 4 h later) or sub chronic (daily injections for 2 days with behavior 48 h later). Objects were explored in sample trials (T1), and discrimination between sample (old) and new object/location in recognition trials (T2) was examined after 2–4 h inter-trial delays. Subjects treated sub chronically with EB, DPN, and C-19, but not PPT, discriminated between old and new objects and objects in old and new locations, suggesting that, at these doses and duration of treatments, estrogenic interactions with ERβ contribute to enhancements in recognition memory. Acute injections of DPN, but not PPT, immediately after T1, also enhanced discrimination for both tasks (C19 was not investigated). Effects of EB, DPN and PPT on anxiety and locomotion, measured on elevated plus maze and open field, did not appear to account for the mnemonic enhancements. Monoamines and metabolites were measured following DPN treatment in subjects that did not receive behavioral testing. DPN was associated with alterations in monoamines in several brain areas: indexed by the metabolite, 3-methoxy-4-hydroxyphenylglycol (MHPG), or the MHPG/norepinephrine (NE) ratio, NE activity was increased by 60–130% in the prefrontal cortex (PFC) and ventral hippocampus, and NE activity was decreased by 40–80% in the v. diagonal bands and CA1. Levels of the dopamine (DA) metabolite, homovanillic acid (HVA), increased 100% in the PFC and decreased by 50% in the dentate gyrus following DPN treatment. The metabolite of serotonin, 5-hydroxyindole acetic acid (5-HIAA), was increased in the PFC and CA3, by approximately 20%. No monoaminergic changes were noted in striatum or medial septum. Results suggest that ERβ mediates sub chronic and acute effects of estrogens on recognition memory and that memory enhancements by DPN may occur, in part, through alterations in monoaminergic containing systems primarily in PFC and hippocampus.

Impact of estrogen receptor alpha and beta agonists on delayed alternation in middle-aged rats

Estrogens act in the adult brain to modulate cognition, enhancing performance on some learning tests and impairing performance on others. Our previous research has revealed an impairing effect of chronic 17β-estradiol treatment in young and aged rats on a prefrontally-mediated working memory task, delayed spatial alternation (DSA). Little is known about the mechanisms of these impairing effects. The current study examined the effects of selective estrogen receptor (ER) α or ERβ activation on DSA performance in middle-aged female rats. Ovariectomized 12 month old Long–Evans (LE) rats were treated by subcutaneous injection with the ERα agonist propyl pyrazole triol (PPT) or the ERβ agonist diarylpropionitrile (DPN) at 0.02, 0.08, or 0.20 mg/kg/day, or with oil vehicle and tested on an operant variable delay DSA task. A 17β-estradiol group (10% in cholesterol) was included as a positive control group. We replicated our previous finding of a 17β-estradiol induced deficit on DSA performance and this effect was paralleled by low dose (0.02 mg/kg/day) DPN treatment. Higher doses of DPN failed to produce a significant change in performance. The highest dose of PPT (0.20 mg/kg/day) also impaired performance, but this effect was subtle and limited to the longest delay during the final block of testing. These data confirm our earlier findings that chronic 17β-estradiol treatment has an impairing effect on the DSA task, and suggest that ERβ activation may underlie the deficit.

17β-ESTRADIOL MEDIATED PROTECTION AGAINST VASCULAR LEAK AFTER HEMORRHAGIC SHOCK

Vascular hyperpermeability is a clinical complication associated with hemorrhagic shock (HS) and occurs mainly because of the disruption of the adherens junctional complex. The objective of this study was to understand the role of 17beta-estradiol in HS-induced hyperpermeability particularly focusing on estrogen receptors. In male Sprague-Dawley rats, HS was induced by withdrawing blood to reduce the mean arterial pressure to 40 mmHg for 1 hour followed by 1 hour of resuscitation to 90 mmHg. The study groups were 17beta-estradiol, tamoxifen, fulvestrant plus 17beta-estradiol, propyl pyrazole triol plus 17beta-estradiol, and diarylpropionitrile plus 17beta-estradiol. Intravital microscopy was used to study changes in mesenteric postcapillary venules. Mitochondrial reactive oxygen species formation was studied in vivo using dihydrorhodamine 123. The mitochondrial transmembrane potential was studied using the fluorescent cationic probe 5,5',6,6'tetrachloro-1,1',3,3'tetraethylbenzimidazolyl carbocyanine iodide (JC-1). The mesenteric microvasculature was analyzed for cytochrome c levels by enzyme-linked immunosorbent assay and caspase-3 activity by a fluorometric assay. Our results demonstrated that 17beta-estradiol attenuated HS-induced hyperpermeability. Fulvestrant reversed this protective effect (P < 0.05). Tamoxifen 5 mg/kg attenuated HS-induced hyperpermeability, whereas 10 mg/kg induced permeability (P < 0.05). Both alpha and beta estrogen receptor agonists inhibited HS-induced hyperpermeability (P < 0.05). 17beta-Estradiol decreased HS-induced reactive oxygen species formation and restored mitochondrial transmembrane potential. 17beta-Estradiol decreased both cytosolic cytochrome c level and activation of caspase-3 (P < 0.05). These findings suggest that 17beta-estradiol protects the microvasculature after HS, and that this protection may be mediated through the alpha and beta estrogen receptors.