Up-regulation of calcitonin gene-related peptide receptors underlying elevation of skin temperature in ovariectomized rats

We investigated the mechanism for the augmentation of the calcitonin gene-related peptide (CGRP)-induced elevation of skin temperature in ovariectomized (OVX) rats. I.v. injection of alphaCGRP (10 micro g/kg) elevated skin temperature of the hind paws. The elevation was significantly greater in OVX rats than in sham-operated rats and was inhibited by pretreatment with human CGRP(8-37) (100-1000 micro g/kg i.v.), a CGRP receptor antagonist, in a dose-dependent manner. In addition, ovariectomy not only potentiated vasorelaxation due to alphaCGRP but increased the number of CGRP receptors in mesenteric arteries. Further, the plasma concentration of endogenous CGRP was significantly lower in OVX rats. These results suggest that the low concentration of plasma CGRP due to ovarian hormone deficiency may induce the increase in the number of CGRP receptors due to up-regulation. Therefore, the increased number of CGRP receptors may be responsible for potentiation of exogenous alphaCGRP-induced elevation of skin temperature in OVX rats. The mechanism underlying the hot flashes observed in menopausal women may also involve, in part, the up-regulation of CGRP receptors following ovarian hormone deficiency.     

Genistein supplementation and estrogen replacement therapy improve endothelial dysfunction induced by ovariectomy in rats

BACKGROUND: We investigated the effect of genistein, a phytoestrogen derived from a soy diet with a flavonoid chemical structure, on endothelial dysfunction induced by estrogen deficiency in rats. METHODS: Female mature Sprague-Dawley rats were subjected to a bilateral ovariectomy (OVX rats). Sham-operated animals (Sham OVX rats) were used as controls. Three weeks after surgery animals were randomized to the following treatments: genistein (0.2 mg/kg/day, s.c. for 4 weeks), 17 beta-estradiol (20 micrograms/kg/day, s.c. for 4 weeks) or their respective vehicles. Mean arterial blood pressure (MAP), heart rate (HR), total plasma cholesterol, plasma estradiol, plasma genistein levels and uterine weights were studied. Furthermore, we investigated acetylcholine (ACh 10 nM-10 microM) and sodium nitroprusside: (SN 15-30 nM) induced relaxation of aortic rings as well as NG-L-arginine (L-NMA: 10-100 microM) induced vasoconstriction in phenylephrine precontracted aortic segments and calcium-dependent nitric oxide synthase (cNOS) activity in homogenates of lungs taken from both sham OVX and OVX rats. RESULTS: Untreated OVX rats had, compared with sham OVX animals, unchanged body weight, MAP, HR and plasma cholesterol. In contrast ovariectomy impaired endothelial responses, blunted L-NMA induced contraction (L-NMA 100 microM: Sham OVX = 2.1 +/- 0.2 g/mg tissue; OVX = 1.7 +/- 0.4 g/mg tissue) and reduced cNOS activity. Treatment with 17 beta-estradiol increased the hormone plasma levels, reverted the endothelial dysfunction and increased cNOS activity in lung homogenates. Genistein supplementation enhanced the circulating levels of the phytoestrogen and affected NOS activity and endothelial dysfunction to the same extent. CONCLUSIONS: Our data suggest that genistein and 17 beta-estradiol show overlapping effects on experimental endothelial dysfunction.     

Estrogen-dependent hypotensive effects of ethanol in conscious female rats

This study investigated the role of estrogen in the acute hemodynamic responses to intragastric (i.g.) ethanol in conscious female rats. Changes evoked by ethanol or equal volume of water in mean arterial pressure, heart rate, cardiac index, stroke volume (SV), and total peripheral resistance were followed in sham-operated, ovariectomy (OVX) vehicle-treated (OVX-veh), and OVX 17beta-estradiol (E2)-treated (OVX-E2) Sprague-Dawley rats. Plasma norepinephrine (NE) was measured as an index of sympathetic activity. In sham-operated rats, ethanol caused significant decreases in mean arterial pressure that were associated with significant reductions in cardiac index and SV, whereas total peripheral resistance was not changed. Measured plasma NE levels were not affected by ethanol except for a significant reduction observed one time. OVX abolished the hypotensive effect of ethanol and the associated decreases in cardiac output, SV, and plasma NE. Treatment of OVX rats with E2 restored the hypotensive and sympathoinhibitory (decreases in plasma NE) responses to ethanol. Blood ethanol concentrations were not affected by OVX or subsequent E2 administration. These findings suggest that intragastric ethanol elicits estrogen-dependent decreases in blood pressure in female rats, which results mainly from a reduction in cardiac output. The mechanism by which ethanol elicits E2-dependent hypotension remains to be determined.     

Effects of ovariectomy and 17beta-estradiol treatment on the renin-angiotensin system, blood pressure, and endothelial ultrastructure

The purpose of this study was to determine whether the renin-angiotensin system (RAS), nitric oxide (NO), atrial natriuretic peptide (ANP), blood pressure (BP), ultrastructural characteristics, and endothelium-dependent relaxation of thoracic aorta were modulated by the estrogen level. Rats were divided into 3 groups: ovariectomized (OVX); not ovariectomized (sham); and ovariectomized and treated with subcutaneous 17beta-estradiol (15 microg/kg/day, OVX+E(2)) (n=15-17 per group). For 13 weeks after surgery, blood pressure, serum estrogen, NO, plasma angiotensin II (Ang II), ANP, and renin activity levels were monitored. Thirteen weeks after surgery, the vasodilator responses of the aortic rings to acetylcholine and the ultrastructural characteristics of the thoracic aorta were determined. In the 9th and 13th week, OVX rats had a significantly higher blood pressure than the other two groups (p<0.05). Ovariectomy led to a significant decrease in plasma Ang II level and a significant increase in renin activity in OVX rats compared to sham rats; this effect could be reversed by estrogen treatment. In the 5th, 9th, and 13th weeks, the serum NO level was significantly lower in the OVX group than in the sham group (p<0.05); this effect could be reversed by estrogen treatment. Plasma ANP levels in the 9th and 13th weeks were significantly lower in the OVX group (p<0.05), and plasma ANP levels could be completely restored by estrogen treatment. Ovariectomy markedly reduced endothelium-dependent relaxation in response to acetylcholine in isolated rat thoracic aortic rings; chronic estrogen treatment significantly restored endothelium-dependent relaxation in response to acetylcholine. Under electron microscopy, the endothelial cells in OVX rats were swollen, even necrosed; estrogen treatment inhibited these changes. These results strongly suggest that estradiol protects rats from the development of hypertension and has a protective effect on the endothelium by increasing NO and ANP levels while decreasing renin activity. However, there was a discordance between the effects that estradiol had on angiotensin II and on blood pressure. This might be the result of negative feedback that ultimately results in the overall suppression of the RAS.     

Differential effects of 17beta-estradiol on function and expression of estrogen receptor alpha, estrogen receptor beta, and GPR30 in arteries and veins of patients with atherosclerosis

Venous complications have been implicated in the adverse effects of hormone replacement therapy. This study investigated acute effects of the natural estrogen, 17beta-estradiol, on function, estrogen receptors/GPR30 expression, and kinase activation in vascular rings and cultured smooth muscle cells from arteries and veins of patients with coronary artery disease. Changes in vascular tone of internal mammary arteries and saphenous veins exposed to the steroid were recorded. 17Beta-estradiol caused concentration-dependent, endothelium-independent relaxation in arteries (P<0.05 versus solvent control) but not in veins (P not significant). 17Beta-estradiol enhanced contractions to endothelin-1 in veins but not in arteries. The novel membrane estrogen receptor GPR30 was detected in both vessels. Moreover, gene expression of estrogen receptor beta was 10-fold higher than that of estrogen receptor alpha or GPR30 (P<0.05). Expression of all 3 of the receptors was reduced after exposure to 17beta-estradiol in arteries but not in veins (P<0.05). Basal phosphorylation levels of extracellular signal-regulated kinase were higher in venous than in arterial smooth muscle cells and were increased by 17beta-estradiol in arterial cells only. In summary, this is the first study to report that, in human arteries but not in veins, 17beta-estradiol acutely affects vascular tone, estrogen receptor expression, including GPR30, and extracellular signal-regulated kinase phosphorylation. These data indicate that effects of natural estrogens in humans differ between arterial and venous vascular beds, which may contribute to the vascular risks associated with menopause or hormone therapy.     

The luteinizing hormone surge is preceded by an estrogen-induced increase of hypothalamic progesterone in ovariectomized and adrenalectomized rats

As circulating estrogen levels rise on the afternoon of proestrus, they stimulate the hypothalamo-pituitary axis. This estrogen positive feedback is pivotal to stimulate the luteinizing hormone (LH) surge required for ovulation and luteinization of ovarian follicles. In addition to estrogen, pre-LH surge progesterone is critical for an LH surge as was demonstrated by blocking progesterone synthesis. In ovariectomized (OVX) rats treated with trilostane, a blocker of the enzyme 3beta-hydroxysteroid dehydrogenase (3beta-HSD) that catalyzes the conversion of pregnenolone to progesterone, estrogen did not induce an LH surge. Further, estrogen induced an LH surge in OVX and adrenalectomized (ADX) rats, indicating that the source of progesterone was neither the ovary nor adrenal gland. This estrogen-only LH surge was inhibited by pretreatment with trilostane, indicating that although the adrenal gland and ovary were not necessary for positive feedback, progesterone synthesis was critical for estrogen-induced positive feedback in an OVX/ADX rat. This suggested that the LH surge is dependent on the pre-LH surge synthesis of progesterone. Estrogen-induced progesterone receptors in the hypothalamus are vital for the LH surge, so a potential location for progesterone synthesis is the hypothalamus. OVX/ADX female rats were treated with 17beta-estradiol (50 microg) and progesterone levels were assayed by RIA. Progesterone levels were elevated in hypothalamic tissue following estrogen treatment. No increases in tissue progesterone levels were found in parietal cortex, cerebellum, medulla, pituitary or plasma. Additionally, male rats that do not have an estrogen positive feedback-induced LH surge were examined. Castrated/ADX male rats had no increase in hypothalamic progesterone levels after estrogen treatment. Together, these data strongly suggest that estrogen enhances neuroprogesterone synthesis in the hypothalamus that is involved in the positive feedback regulating the LH surge.     

Effects of long-term treatment with resveratrol and subcutaneous and oral estradiol administration on the pituitary-thyroid-axis.

The lack of estrogen during menopause is associated with various symptoms including osteoporosis, cardiovascular diseases, and menopausal symptoms. For many years, conventional hormone replacement therapy has been successfully used to treat these conditions. However, in light of recent studies that draw attention to potential hazards of conventional HRT, various attempts were undertaken to search for alternatives of classical HRT. Phytoestrogens are supposed to ameliorate various discomforts associated with menopause. Resveratrol (RES) is present in red wine, grapes and peanuts and has been implicated in cardioprotection and prevention of adverse side effects observed after regular HRT. As the pituitary-thyroid axis is a target of estrogen action, we first assessed the effects of E2 administration on thyroid hormone stimulating hormone releasing hormone (TRH)-induced thyroid stimulating hormone (TSH) secretion from pituitary cell cultures in vitro. Our data reveal that E2 treatment augments the TRH-induced TSH secretion. We furthermore designed a long-term study of three months to assess the effects of subcutaneous and oral administration of 17beta-estradiol (E2), as well as the actions of RES on the pituitary-thyroid axis in ovariectomized (OVX) female rats. Our results demonstrate that serum levels of 1.0 and 8.1 microM RES lead to a significant increase in total serum triiodthyronine (T3) levels. OVX induces TSHbeta mRNA in the adenohypohysis and E2 treatment attenuates this effect. Treatment of rats with subcutaneous implants of E2 does not affect the pituitary-thyroid axis, whereas orally applied E2 benzoate (E2B) increases plasma TSH and total thyroxine (T4) in OVX rats. In all animals, we could not detect changes in thyroid morphology as assessed by hematoxylin-eosin (HE) and Perjod-Acid Schiff's (PAS) staining.     

Estrogenic action on arterial smooth muscle: permissive for maintenance of CRHR2 expression

Urocortin (Ucn), a member of CRH family, has been implicated to be one of the endogenous regulators in the cardiovascular system and exerts its effects locally via an autocrine/paracrine fashion. Previous studies have shown the gender difference in CRH-induced vasodilation in human skin, which is related to the concentration of estrogens during the menstrual cycle. The aim of this study was to investigate whether estrogens modulate Ucn/CRH receptor type 2 (CRHR2) expression in vascular smooth muscle, thereby leading to vasodilation. We performed sham operation or bilateral ovariectomy (OVX) on female Sprague Dawley rats. OVX rats were sc administered 17β-estradiol (E?) at a dose of 30 μg/kg·d or with placebo for 12 wk. Primary smooth muscle cells of aorta were used for the in vitro study. It was found that the Ucn-induced vasodilation and CRHR2 expression were decreased in OVX rats and restored by E? replacement treatment for 12 wk. E? increased the expression of CRHR2 in cultured smooth muscle cells, which was blocked by estrogen receptor-β antagonist. Ucn significantly suppressed the phenylephrine-induced phospholipase Cβ3 activation, inositol 1,4,5-trisphosphate (IP?) production, and intracellular Ca2? elevation. Ucn stimulated the expression of active GTP-bound Gαs protein and cAMP production. The suppressive effects of Ucn on phenylephrine-induced IP? production and intracellular Ca2? elevation were blocked by the inhibitors of adenylate cyclase and protein kinase A. Our results demonstrate that estrogen maintains the expression of CRHR2 in aorta smooth muscle, thereby enhancing vasodilator actions of Ucn. Ucn exerts its vasorelaxant effects via Gαs-cAMP-protein kinase A signaling, leading to down-regulation of the phospholipase Cβ-IP?-Ca2? signaling pathway.     

Effects of steroid hormones on synaptosomal ectonucleotidase activities from hippocampus and cortex of adult female rats

Over the last few years, the effects of steroid hormones on the brain have been intensively discussed. It has been demonstrated that ATP (acting as a neurotransmitter) is hydrolyzed to adenosine in the synaptic cleft by the conjugated action of ectonucleotidases, which include an enzyme of the E-NTPDase family (NTPDase3, apyrase, EC 3.6.1.5) and a 5'-nucleotidase (EC 3.1.3.5). The 5'-nucleotidase enzyme is able to hydrolyze AMP as well as other monophosphate nucleotides. The importance of this enzyme in the central nervous system is to participate in the adenosine formation, a nucleoside with neuroprotective properties and modulatory effects. However, several questions have been raised about the mechanisms of steroid hormones and the possible neuroprotective effects of estrogen. Thus, we examined the effects of gonadal steroid hormone deprivation, induced by ovary removal (OVX) and estradiol replacement therapy, on the ectonucleotidase activities in synaptosomes from hippocampus and cerebral cortex of adult rats. ATP and ADP hydrolysis in synaptosomes from cerebral cortex and hippocampus did not change as a function of OVX and results demonstrated an increase in AMP hydrolysis (82%) in the animals submitted to OVX in cerebral cortex, but not in hippocampus, when compared to control and sham-operated groups. Estradiol replacement therapy reversed this effect. RT-PCR analysis showed that the enhancement of enzyme activity in cerebral cortex could be explained by the higher expression of 5'-nucleotidase, following OVX. The hormones 17beta-estradiol (cyclodextrin-encapsulated 17beta-estradiol), DHEAS, and pregnenolone (1.0, 2.5, and 5.0 microM) did not alter the nucleotide hydrolysis, in vitro, in synaptosomes from cortex and hippocampus of female adult rats. Results presented, herein, should be considered relevant for hormone replacement therapy, since much controversy exists surrounding this area and the relationship between adenosine and sex steroids is still poorly understood.     

Estrogen and exercise may enhance beta-cell function and mass via insulin receptor substrate 2 induction in ovariectomized diabetic rats

The prevalence and progression of type 2 diabetes have increased remarkably in postmenopausal women. Although estrogen replacement and exercise have been studied for their effect in modulating insulin sensitivity in the case of insufficient estrogen states, their effects on beta-cell function and mass have not been studied. Ovariectomized (OVX) female rats with 90% pancreatectomy were given a 30% fat diet for 8 wk with a corresponding administration of 17beta-estradiol (30 microg/kg body weight) and/or regular exercise. Amelioration of insulin resistance by estrogen replacement or exercise was closely related to body weight reduction. Insulin secretion in first and second phases was lower in OVX during hyperglycemic clamp, which was improved by estrogen replacement and exercise but not by weight reduction induced by restricted diets. Both estrogen replacement and exercise overcame reduced pancreatic beta-cell mass in OVX rats via increased proliferation and decreased apoptosis of beta-cells, but they did not exhibit an additive effect. However, restricted diets did not stimulate beta-cell proliferation. Increased beta-cell proliferation was associated with the induction of insulin receptor substrate-2 and pancreatic homeodomain protein-1 via the activation of the cAMP response element binding protein. Estrogen replacement and exercise shared a common pathway, which led to the improvement of beta-cell function and mass, via cAMP response element binding protein activation, explaining the lack of an additive effect with combined treatments. In conclusion, decreased beta-cell mass leading to impaired insulin secretion triggers glucose dysregulation in estrogen insufficiency, regardless of body fat. Regular moderate exercise eliminates the risk factors of contracting diabetes in the postmenopausal state.     

Comparative effects of 17beta-estradiol and phytoestrogens in the regulation of endometrial functions in the rodent uterus

The present study aimed at improving our understanding of the effects of 17beta-estradiol and phytoestrogens on the uterine tissue, by evaluating tissue-specific modulation of molecules related to cell-cycle control and angiogenesis. Specifically, the uterine expression of Ki67, peroxisome proliferator-activated receptor gamma (PPARgamma), and vascular endothelial growth factor receptor-2 (VEGFR-2), was examined by immunohistochemical analysis. Ovariectomized (OVX) rats were treated with either the vehicle, a phytoestrogen- containing soy extract (SSE) (100 mg/kg/day pos), or 17beta-estradiol (0.5 mg/kg/day pos); a sham control group (SHAM) was also included in the study. At necropsy, uteri were weighed, collected, and subsequently processed for histopathology or immunohistochemistry. SSE-treated rats did not show any significant change either in the weight or in histological features of the uterus when compared to OVX controls; the epithelial expression of proliferation marker Ki67 was seen to be significantly reduced, in comparison to both SHAM and OVX rats. Conversely, 17beta-estradiol significantly increased uterine weight, induced hyperplasia in the majority of rats, and enhanced Ki67 epithelial expression. The regulation of PPARgamma expression, reduced after ovariectomy, was similar in SSE- and 17beta-estradiol-treated rats, showing a further significant decrease in stromal immunostaining, in comparison to OVX controls. VEGFR-2 epithelial immunostaining, slightly reduced following ovariectomy, was highly increased on 17beta-estradiol treatment, while following SSE, the pattern of staining observed was similar to that of OVX controls. Data from this study show that PPARgamma and VEGFR-2 represent additional targets by which sex steroid estrogen and plant-derived phytoestrogens may, at certain doses, differentially regulate endometrial functions.     

Ovarian hormone modulates 5-hydroxytryptamine 3 receptors mRNA expression in rat colon with restraint stress-induced bowel dysfunction

AIM: To examine the effects of ovarian hormone on the expression of 5-hydroxytryptamine 3 receptors (5-HT3R) in rat colon of restraint stress-induced bowel dysfunction. METHODS: Twenty-four female Sprague-Dawley rats were randomly divided into three groups of 8 each: sham operation, ovariectomy (OVX) and ovariectomy with estrogen (E2) and progesterone (P) replacement therapy (OVX+E2+P). The rats were subjected to 1-h restraint stress 4 wk after operation. The changes of defecation were monitored by collection of fecal pellets. The gonadal steroids were measured in duplicate by radioimmunoassay (RIA). The expression of 5-HT3R mRNA in the colon was studied by RT-PCR. RESULTS: Compared with sham group and OVX+E2+P group, OVX group showed increase in fecal pellets and decrease in the time of vitreous pellets excretion (P<0.01). Serum levels of E2 and P were suppressed in OVX group and restored following treatment with ovarian steroids (P<0.01), and the levels of 5-HT3R mRNA in the colon of ovariectomized rats were significantly increased, the expression of 5-HT3R mRNA was significantly decreased in hormone replacement therapy group (P<0.01). CONCLUSION: Ovarian hormone plays a role in the regulation of 5-HT3R expressions in restraint stress-induced bowel dysfunction of rats. The interactions between ovarian steroids and gastrointestinal tract may have major pathophysiological implications in 5-HT-related disorders, such as irritable bowel syndrome (IBS).     

降低雌激素水平对大鼠降钙素基因相关肽合成与释放的影响

目的　探索女性绝经后雌激素水平降低对降钙素基因相关肽 (CGRP)的影响。方法　采用大鼠去卵巢模型及苯甲酸雌二醇皮下注射进行替代治疗 ,分别于治疗 1个月、2个月时 ,用CGRP特异性放免分析的方法测定血清、组织 (腹主动脉、十二指肠、背根神经节 )及外周神经刺激 (PNS)后离体肠系膜血管灌流液中的CGRP样免疫活性 (CGRP LI)物质。结果　 1个月时 ,非替代治疗组血清、腹主动脉、十二指肠组织中CGRP LI[(2 7 64± 4 63 )pg/μl,(4 12± 1 5 1)pg/μg,(4 67± 0 71)pg/μg]明显高于假手术组 [(15 79± 3 0 2 )pg/μg ,(0 68± 0 2 9)pg/μg,(2 13± 0 3 5 )pg/μg) ,P <0 0 5 ;非替代治疗组肠系膜灌流液基础以及PNS(4Hz、16Hz)引起的CGRP释放 [(7 880± 0 4 49)pg/ml,(16 2 5 0±1 73 4)pg/ml,(19 80 7± 1 62 1)pg/ml)明显高于假手术组 [(5 2 14± 0 914 )pg/ml,(11 2 89± 1 0 3 5 )pg/ml,(13 3 70± 0 60 6)pg/ml],P <0 0 5 ,雌激素替代后恢复至对照水平。 2个月时去卵巢引起的CGRP组织含量和释放的变化均消失。结论　CGRP在绝经后短期内通过合成与释放的增加 ,加强了对心血管系统的保护 ,但这种作用是短暂的。     

Suppressive action of orexin A on pulsatile luteinizing hormone secretion is potentiated by a low dose of estrogen in ovariectomized rats

Orexins are hypothalamic neuropeptides which stimulate luteinizing hormone (LH) secretion in estrogen- and progesterone-treated ovariectomized (OVX) rats and suppress it in OVX rats not treated with estrogen, suggesting a modulation by estrogen of the response to orexins. We examined the effects of orexin A on pulsatile LH secretion in OVX rats treated with a very small dose of estrogen so as to maintain the pulsatile secretion of LH. The estrogen treatment was done 24 h before the blood sampling by subcutaneously implanting a silicone tube (id = 1.5 mm, od = 2.5 mm, length = 25 mm) containing 17beta-estradiol (E(2)) dissolved in sesame oil at 20 microg/ml. In OVX rats treated with sesame oil as a control, the intracerebroventricular (icv) injection of orexin A (0.3 nmol, dissolved in 3 microl artificial cerebrospinal fluid) had no significant effect on the parameters of pulsatile LH secretion, i.e., pulse frequency and pulse amplitude, although it caused a small but statistically significant decrease in overall mean LH concentrations within 1 h. In OVX rats treated with E(2), the icv injection of orexin A significantly suppressed the pulsatile LH secretion; the frequency decreased for more than 2 h, inducing a rapid decline in overall mean LH concentrations. In view of the finding that a much higher dose of orexin A suppresses pulsatile LH secretion in OVX rats not treated with E(2), we suggest that the suppressive action of orexin A on pulsatile LH secretion is potentiated by estrogen.     

Clinically used estrogens differentially inhibit human aortic smooth muscle cell growth and mitogen-activated protein kinase activity

Some estrogenic compounds modify vascular smooth muscle cell (SMC) biology; however, whether such effects are mediated in part by estrogen receptors is unknown. The purpose of this study was to evaluate whether the actions of clinically used estrogens on human aortic SMC biology are mediated by estrogen receptors. We examined the effects of various clinically used estrogens in the presence and absence of ICI 182,780, an estrogen receptor antagonist, on cultured human aortic SMC DNA synthesis ([(3)H]thymidine incorporation), cellular proliferation (cell counting), cell migration (modified Boyden chamber), collagen synthesis ([(3)H]proline incorporation), and mitogen-activated protein kinase activity. FCS-induced DNA synthesis, cell proliferation, collagen synthesis, platelet-derived growth factor-induced SMC migration, and mitogen-activated protein kinase activity were significantly inhibited by physiological (10(-9) mol/L) concentrations of 17beta-estradiol and low concentrations (10(-8) to 10(-7) mol/L) of 17beta-estradiol, estradiol valerate, estradiol cypionate, and estradiol benzoate but not by estrone, estriol, 17alpha-estradiol, or estrone sulfate. The inhibitory effects of 17beta-estradiol and other inhibitory estrogens were completely reversed by 100 micromol/L ICI 182,780, and the rank-order potency of various estrogens to inhibit SMC biology matched their rank-order affinity for estrogen receptors. The inhibitory effects of estrogens on SMC biology are in part receptor-mediated. Because the cardioprotective effects of hormone replacement therapy are most likely mediated by modification of SMC biology, whether hormone replacement therapy protects a given postmenopausal woman against cardiovascular disease will depend partially on the affinity of the estrogen for estrogen receptors in vascular SMCs.     

Estrogen and tamoxifen modulate cerebrovascular tone in ovariectomized female rats

Postmenopausal estrogen deficiency increases the incidence of cerebrovascular disease. However, hormone replacement therapy is associated with an increased cardiovascular risk. Tamoxifen is a selective estrogen receptor modulator with estrogenic effects on cardiovascular risk factors, but its long-term impacts on cerebral vasculature are unknown. We hypothesized that chronic 17beta-estradiol or tamoxifen treatment exerted similar effects in reducing cerebrovascular tension in ovariectomized rats. We therefore determine whether (1) chronic 17beta-estradiol treatment could influence vasomotor activities, (2) chronic tamoxifen therapy could exert an estrogen-like or estrogen-antagonistic effect, and (3) acute exposure to estrogen could mimic the effect of 17beta-estradiol. Isometric tension was measured in cerebral arteries from female rat groups: control, ovariectomy, ovariectomy plus 17beta-estradiol treatment, ovariectomy plus tamoxifen treatment, and ovariectomized rats treated with tamoxifen and 17beta-estradiol. Ovariectomy enhanced cerebrovascular contractions to endothelin-1 or CaCl2, but not to U46619 or phenylephrine. 17beta-Estradiol therapy reversed these effects. Chronic tamoxifen treatment exerted estrogen-like actions by reversing ovariectomy-induced enhancement of vessel tone without antagonizing the effect of chronic 17beta-estradiol treatment. Ovariectomy enhanced the relaxing potency of nicardipine, and 17beta-estradiol treatment prevented this effect. Acute exposure to 10(-9) mol/L 17beta-estradiol or 10(-8) mol/L tamoxifen did not modulate contractions in rings from nonoperated female rats. In conclusion, ovariectomy differentially enhances agonist-induced cerebrovascular tone, an effect that was reversed by estrogen therapy. Tamoxifen does not act as an estrogen antagonist; instead, it functions as an estrogen agonist during estrogen deficiency. Thus, tamoxifen may confer beneficial effects similar to estrogen in cerebrovascular vessels.     

Estrogens protect myocardium against ischemia/reperfusion insult by up-regulation of CRH receptor type 2 in female rats

Background

The mechanism underlying estrogen cardioprotection remains largely unknown. Urocortin (UCN), a member of corticotropin-releasing hormone (CRH) family, is one of endogenous cardioprotective factors. The goal of present study is to investigate whether estrogens regulate UCN and its receptor CRH receptor type 2 (CRHR2) in female rat heart.

Methods

17β-estradiol (E2) was subcutaneously administrated to ovariectomized (OVX) rats for eight weeks. UCN was administrated before simulated myocardial ischemia/reperfusion (I/R). Cell damage was assessed by measurement of infarct size, activity of serum creatine kinase (CK) and lactate dehydrogenase (LDH) and percentage of TUNEL staining in myocardium. The mRNA and protein levels of UCN and CRHR2 were determined in sham operated and OVX rats with or without E2 replacement. DNA methylation frequency of CRHR2 gene promoter was determined by bisulfite-sequencing.

Results

UCN administration reduced infarct size, LDH and CK level and percentage of TUNEL staining upon I/R injury. The cardioprotective effects of UCN were abrogated in OVX rats and E2 replacement restored UCN-induced cardioprotection.CRHR2 mRNA and protein expression were down-regulated more than 40% in OVX rats, both of which were restored by E2 replacement. UCN mRNA and protein levels were not affected by ovariectomy and E2 replacement. Hypermethylation in CRHR2 promoter was found in OVX rats, and two of the methylated CpG sites were seated at cis-acting elements. Hypermethylation induced by OVX could also be ameliorated by E2 replacement.

Conclusion

Estrogens maintain CRHR2 expression in myocardium, which may through an epigenetic mechanism, and enhance UCN-induced cardioprotective effects against I/R injury.     

Effects of physical training on body composition and organ weights in ovariectomized and hyperestrogenic rats

OBJECTIVE: To investigate whether regular endurance-type exercise can benefit rats submitted to a model of ovariectomy (OVX)-induced obesity with or without estrogen replacement. SUBJECTS: OVX Sprague-Dawley rats were compared to an ovariectomized-estradiol-treated group (OVXE2) and a Sham-operated (Sham) group. Each of these groups were subdivided into a sedentary and a treadmill-trained (8 wk) group. DESIGN AND MEASUREMENTS: An experimental study in which various parameters, including fat depots, blood lipids and several organ weights were measured. RESULTS: Plasma levels of 17beta-estradiol and uterus weights were significantly (P<0.05) lower in OVX compared to Sham and significantly (P<0.01) higher in OVXE2 (hyperestrogenic) compared to Sham rats. Body weights were significantly (P<0.01) different among groups, in the following decreasing order: OVX, Sham and OVXE2. The average daily food intake and food efficiency were significantly (P<0.01) increased in OVX compared to Sham, whereas estradiol treatment diminished this effect (P<0.01). Exercise training did not alter any of the above-mentioned variables in any of the three estrogen groups. Mesenteric and subcutaneous fat weights were significantly (P<0.01) increased by OVX. This increase was abolished by estrogen replacement or by exercise training. Exercise training also decreased fat weights in OVXE2 and Sham rats. OVX resulted in a decrease in the weights of several other tissues (femur, heart, lungs, liver and adrenal glands) while hyperestrogenic replacement resulted in an increase in weight of all measured tissues. Aside from fat depots, exercise training did not affect any of the tissue weights with the exception for an increase in the weight of the plantaris muscle and adrenal glands and a decrease in lung weight in all three estrogen groups. CONCLUSION: In OVX animals, exercise training may bring about positive changes in body composition (ie reduction in fat weights) despite an ovariectomy-induced increase in body weight.     

17alpha-estradiol: a brain-active estrogen?

The estrogen 17beta-estradiol has profound effects on the brain throughout life, whereas 17alpha-estradiol, the natural optical isomer, is generally considered less active because it binds less avidly to estrogen receptors. On the contrary, recent studies in the brain document that 17alpha-estradiol elicits rapid and sustained activation of the MAPK/ERK and phosphatidylinositol 3-kinase-Akt signaling pathways; is neuroprotective, after an ischemic stroke and oxidative stress, and in transgenic mice with Alzheimer's disease; and influences spatial memory and hippocampal-dependent synaptic plasticity. The present study measured the endogenous content of 17alpha-estradiol in the brain and further clarified its actions and kinetics. Here we report that: 1) endogenous levels of 17alpha-estradiol and its precursor estrone are significantly elevated in the postnatal and adult mouse brain and adrenal gland of both sexes, as determined by liquid chromatography/tandem mass spectrometry; 2) 17alpha-estradiol and 17beta-estradiol bind estrogen receptors with similar binding affinities; 3) 17alpha-estradiol transactivates an estrogen-responsive reporter gene; and 4) unlike 17beta-estradiol, 17alpha-estradiol does not bind alpha-fetoprotein or SHBG, the estrogen-binding plasma proteins of the developing rodent and primate, respectively. 17alpha-Estradiol was also found in the brains of gonadectomized or gonadectomized/adrenalectomized mice, supporting the hypothesis that 17alpha-estradiol is locally synthesized in the brain. These findings challenge the view that 17alpha-estradiol is without biological significance and suggest that 17alpha-estradiol and its selective receptor, ER-X, are not part of a classical hormone/receptor endocrine system but of a system with important autocrine/paracrine functions in the developing and adult brain. 17alpha-Estradiol may have enormous implications for hormone replacement strategies at the menopause and in the treatment of such neurodegenerative disorders as Alzheimer's disease and ischemic stroke.