ERα Agonist Propyl Pyrazole Triol Research Articles

G protein‐coupled estrogen receptor‐1 enhances excitatory synaptic responses in the entorhinal cortex

Activation of estrogen receptors is thought to modulate cognitive function in the hippocampus, prefrontal cortex, and striatum by affecting both excitatory and inhibitory synaptic transmission. The entorhinal cortex is a major source of cortical sensory and associational input to the hippocampus, but it is unclear whether either estrogens or progestogens may modulate cognitive function through effects on synaptic transmission in the entorhinal cortex. This study assessed the effects of the brief application of either 17-β estradiol (E2) or progesterone on excitatory glutamatergic synaptic transmission in the female rat entorhinal cortex in vitro. Rats were ovariectomized on postnatal day (PD) 63 and also received subdermal E2 implants to maintain constant low levels of circulating E2 on par with estrus. Electrophysiological recordings from brain slices were obtained between PD70 and PD86, and field excitatory postsynaptic potentials (fEPSPs) reflecting the activation of the superficial layers of the entorhinal cortex were evoked by the stimulation of layer I afferents. The application of E2 (10nM) for 20 min resulted in a small increase in the amplitude of fEPSPs that reversed during the 30-min washout period. The application of the ERα agonist propylpyrazoletriol (PPT) (100 nM) or the β agonist DPN (1μM) did not significantly affect synaptic responses. However, the application of the G protein-coupled estrogen receptor-1 (GPER1) agonist G1 (100 nM) induced a reversible increase in fEPSP amplitude similar to that induced by E2. Furthermore, the potentiation of responses induced by G1 was blocked by the GPER1 antagonist G15 (1μM). Application of progesterone (100 nM) or its metabolite allopregnanolone (1μM) did not significantly affect synaptic responses. The potentiation of synaptic transmission in the entorhinal cortex induced by the activation of GPER1 receptors may contribute to the modulation of cognitive function in female rats.

Estrogen activates endothelial exocytosis

Estrogen therapy is used to treat patients with post-menopausal symptoms, such as hot flashes and dyspareunia. Estrogen therapy also decreases the risk of fractures from osteoporosis in post-menopausal women. However, estrogen increases the risk of venous thromboembolic events, such as pulmonary embolism, but the pathways through which estrogen increase the risk of thromboembolism is unknown. Here, we show that estrogen elicits endothelial exocytosis, the key step in vascular thrombosis and inflammation. Exogenous 17β-estradiol (E2) stimulated endothelial exocytosis of Weibel-Palade bodies (WPBs), releasing von Willebrand factor (vWF) and interleukin-8 (IL-8). Conversely, the estrogen antagonist ICI-182,780 interfered with E2-induced endothelial exocytosis. The ERα agonist propyl pyrazole triol (PPT) but not the ERβ agonist diarylpropionitrile (DPN) induced vWF release, while ERα silencing counteracted vWF release by E2, suggesting that ERα mediates this effect. Exocytosis triggered by E2 occurred rapidly within 15 min and was not inhibited by either actinomycin D or cycloheximide. On the contrary, it was inhibited by the pre-treatment of U0126 or SB203580, an ERK or a p38 inhibitor, respectively, suggesting that E2-induced endothelial exocytosis is non-genomically mediated by the MAP kinase pathway. Finally, E2 treatment enhanced platelet adhesion to endothelial cells ex vivo, which was interfered with the pre-treatment of ICI-182,780 or U0126. Taken together, our data show that estrogen activates endothelial exocytosis non-genomically through the ERα-MAP kinase pathway. Our data suggest that adverse cardiovascular effects such as vascular inflammation and thrombosis should be considered in patients before menopausal hormone treatment.

Atheroprotective effects of 17β-oestradiol are mediated by peroxisome proliferator-activated receptor γ in human coronary artery smooth muscle cells.

Introduction17β-oestradiol (E2) mediates vasculoprotection in various preclinical and clinical models of atherosclerosis and neointimal hyperplasia. However, the molecular mechanisms underlying these effects are still not fully elucidated. Previous studies have demonstrated the essential role of the peroxisome-proliferator-activated-receptor-γ (PPARγ) in mediating vasculoprotective effects of E2 in vivo. The aim of the current study was to investigate whether PPARγ mediates vasculoprotective mechanisms of E2 in human coronary artery smooth muscle cells (HCASMC).Material and methodsPrimary HCASMC were stimulated with E2 (10 nM), the selective oestrogen receptor α (ERα) agonist propylpyrazole triol (PPT) (50 nM) and the selective ERα antagonist methyl-piperidino-pyrazole (MPP) (1 µM), respectively. Changes in PPARγ mRNA, protein expression, and DNA binding affinity were assessed.ResultsE2 significantly increased PPARγ expression in HCASMC (1.95 ±0.41-fold; n = 5; p = 0.0335). This effect was mimicked by ERα agonist PPT (1.63 ±0.27-fold; n = 7; p = 0.0489) and was abrogated by co-incubation with ERα antagonist MPP (1.17 ±0.18-fold; n = 3; pvs. control > 0.05). PPARγ-DNA binding activity to PPRE remained unchanged upon stimulation with E2 (0.94 ±0.11-fold; n = 4; pvs. control > 0.05). Pharmacological inhibition of PI3K/Akt by LY294002 abrogated E2-induced expression of PPARγ (0.24 ±0.09-fold; n = 3; pvs. E2 = 0.0017).ConclusionsThe present study identifies PPARγ as an important downstream mediator of E2-related atheroprotective effects in HCASMC. PPARγ agonism might be a promising therapeutic strategy to prevent neointimal hyperplasia and consecutive cardiovascular events in postmenopausal women with depleted E2 plasma levels.

Selective activation of estrogen receptors α and β: Implications for depressive-like phenotypes in female mice exposed to chronic unpredictable stress

The estrogen receptor (ER) mechanisms by which 17β-estradiol influences depressive-like behaviour have primarily been investigated acutely and not within an animal model of depression. Therefore, the current study aimed to dissect the contribution of ERα and ERβ to the effects of 17β-estradiol under non-stress and chronic stress conditions. Ovariectomized (OVX) or sham-operated mice were treated chronically (47 days) with 17β-estradiol (E2), the ERβ agonist diarylpropionitrile (DPN), the ERα agonist propylpyrazole-triol (PPT), or vehicle. On day 15 of treatment, mice from each group were assigned to chronic unpredictable stress (CUS; 28 days) or non-CUS conditions. Mice were assessed for anxiety- and depressive-like behaviour and hypothalamic-pituitary-adrenal (HPA) axis function. Cytokine and chemokine levels, and postsynaptic density protein 95 were measured in the hippocampus and frontal cortex, and adult hippocampal neurogenesis was assessed. Overall, the effects of CUS were more robust that those of estrogenic treatments, as seen by increased immobility in the tail suspension test (TST), reduced PSD-95 expression, reduced neurogenesis in the ventral hippocampus, and HPA axis negative feedback dysregulation. However, we also observe CUS-dependent and -independent effects of ovarian status and estrogenic treatments. The effects of CUS on PSD-95 expression, the cytokine milieu, and in TST were largely driven by PPT and DPN, indicating that these treatments were not protective. Independent of CUS, estradiol increased neurogenesis in the dorsal hippocampus, blunted the corticosterone response to an acute stressor, and increased anxiety-like behaviour. These findings provide insights into the complexities of estrogen signaling in modulating depressive-like phenotypes under non-stress and chronic stress conditions.

Medial preoptic estrogen receptor-beta blunts the estrogen receptor-alpha mediated increases in wheel-running behavior of female rats

Estrogens are believed to enhance rodent voluntary wheel-running through medial preoptic (mPOA) estrogen receptor α (ERα) signaling, with little role attributed to estrogen receptor β (ERβ). Systemic ERβ activation has been shown to mitigate ERα driven increases in wheel-running. Therefore, the present goal was to determine whether ERβ signaling in the mPOA plays a similar modulatory role over ERα. We utilized outbred wild-type (WT) and rats selectively bred for low voluntary running (LVR) behavior to address whether mPOA ERβ signaling blunts ERα driven wheel-running behavior and immediate-early gene (Fos, Zif268, and Homer1) mRNA induction. Further, we addressed baseline mPOA mRNA expressions and circulating 17β-estradiol levels between female WT and LVR rats. Following ovariectomy, WT rats reduced running behavior ∼40 %, with no effect in LVR rats. Intra-medial preoptic injection of the ERα-agonist propylpyrazoletriol (PPT) increased wheel-running ∼3.5-fold in WT rats, while injections of the ERβ-agonist diarylpropionitrile (DPN) or a combination of the two agonists had no effect. Similarly, ERα-agonism (PPT) increased Fos and Homer1 induction ∼3-fold in WT and LVR isolated mPOA neurons, with no effect of the ERβ-agonist DPN alone or in combination with PPT, suggesting medial-preoptic ERβ activity may blunt ERα signaling. LVR rats exhibited higher mPOA mRNA expressions of Esr1, Esr2 and Cyp19a1, lower normalized uterine wet weights and lower 17β-estradiol plasma levels compared to WT, suggesting their low running may be due to low circulating estrogen levels. Collectively, these findings highlight mPOA ERβ as a potential neuro-molecular modulator of the estrogenic control of wheel-running behavior.

17β-Estradiol Activates HSF1 via MAPK Signaling in ERα-Positive Breast Cancer Cells.

Heat Shock Factor 1 (HSF1) is a key regulator of gene expression during acute environmental stress that enables the cell survival, which is also involved in different cancer-related processes. A high level of HSF1 in estrogen receptor (ER)-positive breast cancer patients correlated with a worse prognosis. Here we demonstrated that 17β-estradiol (E2), as well as xenoestrogen bisphenol A and ERα agonist propyl pyrazole triol, led to HSF1 phosphorylation on S326 in ERα positive but not in ERα-negative mammary breast cancer cells. Furthermore, we showed that MAPK signaling (via MEK1/2) but not mTOR signaling was involved in E2/ERα-dependent activation of HSF1. E2­activated HSF1 was transcriptionally potent and several genes essential for breast cancer cells growth and/or ERα action, including HSPB8, LHX4, PRKCE, WWC1, and GREB1, were activated by E2 in a HSF1-dependent manner. Our findings suggest a hypothetical positive feedback loop between E2/ERα and HSF1 signaling, which may support the growth of estrogen-dependent tumors.

P720Atheroprotective effects of 17beta-estradiol are mediated by PPARgamma in human coronary artery smooth muscle cells

Abstract Background 17β-estradiol (E2) mediates vasculoprotection in various preclinical and clinical models of atherosclerosis and neointimal hyperplasia. However, the molecular mechanisms underlying these effects are still not fully elucidated. Previous studies have demonstrated the essential role of the peroxisome-proliferator-activated-receptor-γ (PPARγ) in mediating vasculoprotective effects of E2 in vivo. The aim of the current study was to investigate whether PPARγ is implicated in mediating vasculoprotective mechanisms of E2 in human coronary artery smooth muscle cells (HCASMC). Methods Primary HCASMC were purchased and stimulated with E2 [10 nM], the selective estrogen receptor α (ERα) agonist propylpyrazole triol (PPT) [50 nM] and the selective ERα antagonist methyl-piperidino-pyrazole (MPP) [1 μM], respectively. Changes in PPARγ mRNA and protein expression upon stimulation of ERα were assessed by qPCR and Western blot analyses. Nuclear PPARγ protein expression and DNA binding affinity was assessed after the isolation of the nuclear protein fraction. Hereafter, HCASMC were incubated with E2, PPARγ-antagonist GW9662 [1 μM – 30 μM], or both. HCASMC proliferation was assessed by nuclear BrdU staining and reactive oxygen species (ROS) formation was assessed by L-012- and DCF-DA assays. Results E2 significantly increased PPARγ expression in HCASMC (1.95±0.41 –fold; n=5; p=0.0335). This effect was mimicked by ERα agonist PPT (1.63±0.27 –fold; n=7; p=0.0489) and was abrogated by co-incubation with ERα antagonist MPP (1.17±0.18 –fold; n=3; pvs. control >0.05). Nuclear PPARγ expression was enhanced by E2 (1.53±0.16 –fold; n=4; pvs. control = 0.0074; Fig. 2A) whereas PPARγ's DNA binding activity to PPRE remained unchanged upon stimulation with E2 (0.94±0.11 –fold; n=4; pvs. control >0.05). Pharmacological inhibition of PI3K/Akt by LY294002 abrogated E2-induced expression of PPARγ (0.24±0.09 –fold; n=3; pvs. E2 = 0.0017), arguing for a PI3K/Akt-dependent activation by E2. The role of PPARγ in mediating vasculoprotective effects of E2 was assessed in functional assays using PPARγ-antagonist GW9662. E2 diminished HCASMC proliferation which was restored by GW9662. While E2 only slightly decreased ROS production by HCASMC, GW9662 significantly increased ROS levels (1,036±169 RLU x s–1 x cell–1 versus 561±99 RLU x s–1 x cell–1; n=5–6; p=0.0287). Conclusion In summary, the present study identifies PPARγ as a downstream mediator of E2-related atheroprotective effects in HCASMC. 17β-estradiol regulates vascular PPARγ-expression in HCASMC via the ERα receptor and the PI3K/Akt pathway. PPARγ agonism might be a promising therapeutic strategy to prevent cardiovascular events in postmenopausal women with depleted E2 plasma levels. Acknowledgement/Funding This work was supported by the Bonfor program of the University of Bonn [grant number O-109.0057 to JJ].

Behavioral responses to emotional challenges in female rats living in a seminatural environment: The role of estrogen receptors

Estrogen receptors (ERs) are involved in sexual as well as non-sexual behaviors. In the present study we assessed the effects of stimuli inducing positive or negative affect on sociosexual, exploratory and fear-related behaviors of female rats housed in groups (4 females, 3 males) in a seminatural environment. Ovariectomized females were treated with oil, 17β‑estradiol benzoate (EB, 18 μg/kg), the ERα agonist propylpyrazoletriol (PPT), or the ERβ agonist diarylpropionitrile (DPN) (both 2 × 10 mg/rat). On the test day, the females were exposed to a sequence of events consisting of lavender odor, Mozart's Sonata for Two Pianos K448, chocolate pellets, white noise and fox odor (2,3,5‑Trimethyl‑3‑thiazoline, TMT). All these events are known to induce positive or negative affect. Behavior was carefully observed from the video record. White noise suppressed sexual behaviors and reduced the time spent in the open area of the environment. TMT had no consistent effect whereas exposure to music caused avoidance of the open area. Exposure to chocolate increased exploratory and social behavior. Lavender odor enhanced exploratory behavior. PPT and EB stimulated sexual behaviors, whereas DPN was ineffective. Co-occurrence analyses of the sequence of behavioral patterns revealed that PPT and EB consistently belonged to clusters different from oil and DPN, whereas DPN was separate from oil only under fear-inducing experimental conditions. These data, from a procedure with external validity, confirm that the ERα is crucial for sexual behaviors, that these behaviors are reduced under stressful conditions, and that the ERβ may have some role in fear-related behaviors.

Estrogen receptor agonists alleviate cardiac and renal oxidative injury in rats with renovascular hypertension

ABSTRACTAlthough endogenous estrogen is known to offer cardiac and vascular protection, the involvement of estrogen receptors in mediating the protective effect of estrogen on hypertension-induced cardiovascular and renal injury is not fully explained. We aimed to investigate the effects of estrogen receptor (ER) agonists on oxidative injury, cardiovascular and renal functions of rats with renovascular hypertension (RVH). Female Sprague-Dawley rats were randomly divided as control and RVH groups, and RVH groups had either ovariectomy (OVX) or sham-OVX. Sham-OVX-RVH and OVX-RVH groups received either ERβ agonist diarylpropiolnitrile (1 mg/kg/day) or ERα agonist propyl pyrazole triol (1 mg/kg/day) for 6 weeks starting at the third week following the surgery. At the end of the 9th week, systolic blood pressures were recorded, cardiac functions were determined, and the contraction/relaxation responses of aortic rings were obtained. Serum creatinine levels, tissue malondialdehyde, glutathione, superoxide dismutase, catalase levels, and myeloperoxidase activity in heart and kidney samples were analyzed, and Na+, K+-ATPase activity was measured in kidney samples. In both sham-OVX and OVX rats, both agonists reduced blood pressure and reversed the impaired contractile performance of the heart, while ERβ agonist improved renal functions in both the OVX and non-OVX rats. Both agonists reduced neutrophil infiltration, lipid peroxidation, and elevated antioxidant levels in the heart, but a more ERβ-mediated protective effect was observed in the kidney. Our data suggest that activation of ERβ might play a role in preserving the function of the stenotic kidney and delaying the progression of renal injury, while both receptors mediate similar cardioprotective effects.

Activation of the G protein-coupled estrogen receptor, but not estrogen receptor α or β, rapidly enhances social learning

Social learning is a highly adaptive process by which an animal acquires information from a conspecific. While estrogens are known to modulate learning and memory, much of this research focuses on individual learning. Estrogens have been shown to enhance social learning on a long-term time scale, likely via genomic mechanisms. Estrogens have also been shown to affect individual learning on a rapid time scale through cell-signaling cascades, rather than via genomic effects, suggesting they may also rapidly influence social learning. We therefore investigated the effects of 17β-estradiol and involvement of the estrogen receptors (ERs) using the ERα agonist propyl pyrazole triol, the ERβ agonist diarylpropionitrile, and the G protein-coupled ER 1 (GPER1) agonist G1 on the social transmission of food preferences (STFP) task, within a time scale that focused on the rapid effects of estrogens. General ER activation with 17β-estradiol resulted in a modest facilitation of social learning, with mice showing a preference up to 30min of testing. Specific activation of the GPER1 also rapidly enhanced social learning, with mice showing a socially learned preference up to 2h of testing. ERα activation instead shortened the expression of a socially learned food preference, while ERβ activation had little to no effects. Thus, rapid estrogenic modulation of social learning in the STFP may be the outcome of competing action at the three main receptors. Hence, estrogens' rapid effects on social learning likely depend on the specific ERs present in brain regions recruited during social learning.

Estrogen Alleviates Acetic Acid-Induced Gastric or Colonic Damage via Both ERα- and ERβ-Mediated and Direct Antioxidant Mechanisms in Rats

In order to demonstrate the possible protective effects of estrogen receptor (ER)-α and ERβ receptor subtypes in the pathogenesis of colonic and gastric oxidant damage, experimental ulcer and colitis were induced by acetic acid, and the animals were randomly divided as colitis, ulcer, and their corresponding non-ulcer and non-colitis control groups. Each group of rats was treated intramuscularly with the vehicle, selective ERα agonist propylpyrazole-triol (1mg/kg), ERβ agonist diarylpropionitrile (1mg/kg), non-selective ER agonist 17β estradiol (E2; 1mg/kg), or E2 plus non-selective ER antagonist ICI-182780 (1mg/kg). The results revealed that induction of ulcer or colitis resulted in systemic inflammation as assessed by increased levels of plasma TNF-α and IL-6 levels. In both tissues, the presence of oxidant damage was verified by histological analysis and elevated myleoperoxidase activity. In the colitis and ulcer groups, both ER agonists and the non-selective E2 reversed the oxidative damage in a similar manner. These findings indicate that estrogen acts via both ERα- and ERβ-mediated and direct antioxidant mechanisms, where both ER subtypes play equal and efficient roles in the anti-inflammatory action of estrogen, in limiting the migration of neutrophils to the inflamed tissue, reducing the release and activation of cytokines and thereby alleviating tissue damage.

Intrahypothalamic Estradiol Modulates Hypothalamus-Pituitary-Adrenal-Axis Activity in Female Rats

Estrogen plays an important role in the regulation of the hypothalamus-pituitary-adrenal (HPA)-axis, but the neuroendocrine pathways and the role of estrogen receptor (ER) subtypes involved in specific aspects of this interaction remain unknown. In a first set of experiments, we administered estradiol (E2) intravenously, intracerebroventricularly, and by intrahypothalamic microdialysis to ovariectomized rats to measure plasma corticosterone (CORT) concentrations from carotid artery blood. Systemic infusion of E2 did not increase plasma CORT, but intracerebroventricular E2 induced a 3-fold CORT increase (P = 0.012). Local E2 infusions in the hypothalamic paraventricular nucleus (PVN) significantly increased plasma CORT (P < 0.001). A similar CORT increase was seen after PVN infusion of the ERα agonist propylpyrazoletriol, whereas the ERβ agonist diarylpropiolnitrile had no effect. In a second set of experiments, we investigated whether E2 modulates the HPA-axis response to acute stress by administering E2 agonists or its antagonist ICI 182,780 into the PVN during restraint stress exposure. After 30 min of stress exposure, plasma CORT had increased 5.0-fold (P < 0.001). E2 and propylpyrazoletriol administration in the PVN enhanced the stress-induced plasma CORT increase (8-fold vs. baseline), whereas ICI 182,780 and diarylpropiolnitrile reduced it, as compared with both E2 and vehicle administration in the PVN. In conclusion, central E2 modulates HPA-axis activity both in the basal state and during restraint stress. In the basal condition, the stimulation is mediated by ERα-sensitive neurons, whereas during stress, it is mediated by both ERα and ERβ.

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.

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.

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.

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.

Endothelium-independent vasorelaxation by the selective alpha estrogen receptor agonist propyl pyrazole triol in rat aortic smooth muscle

Abstract Objectives This study investigated the signalling mechanism of the relaxant responses to the estrogen receptor alpha (ERα) agonist PPT (propyl pyrazole triol) in endothelium-denuded rat aortic rings. Methods Several compounds, including protein kinase G (PKG) inhibitors and potassium channel inhibitors, were tested against PPT-dependent rat aortic relaxation. Cyclic GMP and cytosolic calcium responses to PPT in isolated aortic smooth muscle were investigated in parallel. Key findings PPT vasorelaxation was largely reduced by the selective ERα antagonist methyl-piperidinopyrazole (MPP; −91.6 ± 2.5%), by the selective PKG inhibitor Rp-8-Br-cGMP (−78.6 ± 4.9%), by the specific soluble guanylyl cyclase inhibitor ODQ (1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one; −85.3 ± 5.2%) and to a lesser extent by the selective BKCa (large-conductance calcium- and voltage-activated potassium channel) inhibitor iberiotoxin (−59.3%), the selective IKCa (intermediate-conductance calcium-activated potassium channel) inhibitor TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole; −50.7%) and the voltage-gated potassium channel inhibitor 4-aminopyridine (−40.8%). In isolated aortic smooth muscle, PPT strongly enhanced the cyclic GMP content (+144%) and Rp-8-Br-cGMP largely reduced the PPT-dependent calcium signal (−80.8%). Conclusions ERα receptor stimulation in rat aortic smooth muscle evokes a PKG-signalling pathway, likely triggering relaxation by BKCa and IKCa channel opening.

Estradiol after Cardiac Arrest and Cardiopulmonary Resuscitation is Neuroprotective and Mediated through Estrogen Receptor-β

We evaluated long-term administration of estrogen after cardiac arrest and cardiopulmonary resuscitation (CA/CPR) on neurohistopathological and behavioral outcome. We also examined the effect of estrogen receptor (ER) stimulation using ER-alpha agonist propyl pyrazole triol (PPT) and ER-beta agonist diarylpropionitrile (DPN) on neuronal survival after CA/CPR to determine whether possible neuroprotective effects of estrogen are ER-mediated. Male C57Bl/6 mice underwent 10 mins of CA/CPR and 3-day survival. In protocol 1, intravenous injection of vehicle (NaCl 0.9%) and 0.5 or 2.5 microg 17beta-estradiol (E2 loading dose) was performed followed by subcutaneous implants containing vehicle (oil) or E2 (12.6 microg), according to a treatment group. In experimental protocol 2, mice were injected (intravenously) with the ER-alpha agonist PPT or ER-beta agonist DPN followed by Alzet pump implants (subcutaneously) containing PPT (200 microg) or DPN (800 microg). Long-term E2 administration reduced neuronal injury in the striatum after administration of either loading dose (41%+/-19%, 35%+/-26% of injured neurons), as compared with vehicle (68%+/-7%, P<0.01), with no effect in the hippocampal CA1 field. In protocol 2, treatment with ER-beta agonist DPN reduced neuronal injury in the striatum (51%+/-13% injured neurons) as compared with ER-alpha agonist PPT (68%+/-10%) and vehicle (69%+/-11%; P<0.01). Estrogen receptor-beta agonist DPN reduced neuronal injury in the hippocampal CA1 field (29%+/-22% injured neurons) as compared with ER-alpha agonist PPT treatment (62%+/-33%; P<0.05). Injury was not different in hippocampal CA1 between vehicle and ER-alpha agonist-treated animals. We conclude that long-term E2 administration after CA/CPR is neuroprotective and that this effect is most likely mediated via ER-beta.

Rapid Estrogen Receptor-α Activation Improves Ischemic Tolerance in Aged Female Rats through a Novel Protein Kinase Cε-Dependent Mechanism

The effects of estrogen deficiency on the loss of cardioprotection with advancing age are complex and poorly understood. A major focus of the current study was to uncover a cardioprotective role for rapid, nongenomic estrogen receptor (ER) signaling in the aged female myocardium. We hypothesized that selective ERalpha activation in aged females would reduce infarct size in part, through reversal of age-associated reductions in mitochondrial protein kinase Cepsilon (PKCepsilon). Hearts isolated from adult (6 month old) and aged (23-24 months old) female F344 rats with ovaries removed (n = 20 per group) were subjected to ischemia/reperfusion (47 min global ischemia). Rats were injected sc with the ERalpha agonist propylpyrazole triol (PPT) or vehicle 45 min before heart isolation (5 microg/kg). Infarct size was greatest in aged vs. adult ovariectomized rats, significantly reduced by PPT, and the protection reversed by prior administration of the ER inhibitor ICI 182,780 (3 mg/kg). Increased ERalpha particulate targeting occurred after PPT in conjunction with reversal of age-related reductions in nuclear PKCepsilon, mitochondrial PKCepsilon and pAkt (P < 0.05). PPT also increased mRNA levels for the PKCepsilon anchoring protein, receptor for activated C kinase2 (RACK2; P < 0.05). Our data suggest, for the first time, that selective ERalpha activation reduces ischemic injury in the aged, estrogen-deficient heart through a mechanism involving nongenomic redistribution of ERalpha and PKCepsilon activation. A novel feed-forward transcriptional mechanism to potentially enhance PKCepsilon-RACK2 interactions was also observed. Collectively, our findings may provide key insight into developing targeted therapeutic interventions in postmenopausal women to reduce ischemia/reperfusion injury, including selective ERalpha mimetics.

Ethanol impairs estrogen receptor signaling resulting in accelerated activation of senescence pathways, whereas estradiol attenuates the effects of ethanol in osteoblasts.

Epidemiological and animal studies have suggested that chronic alcohol consumption is a major risk factor for osteoporosis. Using bone from cycling female rats infused chronically with ethanol (EtOH) in vivo and osteoblastic cells in vitro, we found that EtOH significantly increased estrogen receptor α (ERα) and β (ERβ) mRNA and ERα protein levels. Treatment with 17β-estradiol (E2) in vivo and in vitro interfered with these effects of EtOH on bone and osteoblastic cells. ERα agonist propylpyrazoletriol (PPT) and ERβ agonist diarylpropionitrile (DPN) attenuated EtOH-induced ERα and ERβ gene overexpression, respectively. Similar to the ER antagonist ICI 182780, EtOH blocked nuclear translocation of ERα-ECFP in the presence of E2 in UMR-106 osteoblastic cells. EtOH also downregulated ERE-luc reporter activity. On the other hand, EtOH by itself upregulated some common ERα- and ERβ-mediated genes apparently by an ER-independent pathway. EtOH also transactivated the luciferase activity of the p21 promoter region independent of additional exogenous ERα, activated p21 and p53, and stimulated senescence-associated β-galactosidase activity in rat stromal osteoblasts. E2 treatment attenuated these EtOH actions. We conclude that inhibitory cross-talk between EtOH and E2 in osteoblasts on ERs, p53/p21, and cell senescence provides a pathophysiologic mechanism underlying bone loss and the protective effects of estrogens in alcohol-exposed females.