Both estrogen receptor subtypes, alpha and beta, attenuate cardiovascular remodeling in aldosterone salt-treated rats

Experimental and population-based studies indicate that female gender and estrogens protect the cardiovascular system against aldosterone-induced injury. Understanding the function of estrogens in heart disease requires more precise information on the role of both estrogen receptor (ER) subtypes, ERalpha and ERbeta. Therefore, we determined whether selective activation of ERalpha or of ERbeta would confer redundant, specific, or opposing effects on cardiovascular remodeling in aldosterone salt-treated rats. The ERalpha agonist 16alpha-LE2, the ERbeta agonist 8beta-VE2, and the nonselective estrogen receptor agonist 17beta-estradiol lowered elevated blood pressure, cardiac mass, and cardiac myocyte cross-sectional areas, as well as increased perivascular collagen accumulation and vascular osteopontin expression in ovariectomized rats receiving chronic aldosterone infusion plus a high-salt diet for 8 weeks. Uterus atrophy was prevented by 16alpha-LE2 and 17beta-estradiol but not by 8beta-VE2. Cardiac proteome analyses by 2D gel electrophoresis, mass spectrometry, and peptide sequencing identified specific subsets of proteins involved in cardiac contractility, energy metabolism, cellular stress response and extracellular matrix formation that were regulated in opposite directions by aldosterone salt treatment and by different estrogen receptor agonists. We conclude that activation of either ERalpha or ERbeta protects the cardiovascular system against the detrimental effects of aldosterone salt treatment and confers redundant, as well as specific, effects on cardiac protein expression. Nonfeminizing ERbeta agonists such as 8beta-VE2 have a therapeutic potential in the treatment of hypertensive heart disease.     

Ligand-dependent activation of ER{beta} lowers blood pressure and attenuates cardiac hypertrophy in ovariectomized spontaneously hypertensive rats

AIMS: The biological effects of oestrogens are mediated by two different oestrogen receptor (ER) subtypes, ERalpha and ERbeta, which might play different, redundant, or opposing roles in cardiovascular disease. Previously, we have shown that the selective ERalpha agonist 16alpha-LE2 improves vascular relaxation, attenuates cardiac hypertrophy, and increases cardiac output without lowering elevated blood pressure in spontaneously hypertensive rats (SHR). Because ERbeta-deficient mice exhibit elevated blood pressure and since the ERbeta agonist 8beta-VE2 attenuated hypertension in aldosterone-salt-treated rats, we have now tested the hypothesis that the isotype-selective ERbeta agonist 8beta-VE2 might be capable of lowering elevated blood pressure in ovariectomized SHR. METHODS AND RESULTS: Treatment of ovariectomized SHR with 8beta-VE2 for 12 weeks conferred no uterotrophic effects but lowered elevated systolic blood pressure (-38 +/- 5 mmHg, n = 31, P < 0.001 vs. placebo) as well as peripheral vascular resistance (-31.3 +/- 4.6%, P < 0.001 vs. placebo). 8beta-VE2 enhanced aortic ERbeta expression (+75.7 +/- 7.1%, P < 0.01 vs. placebo), improved NO-dependent vasorelaxation, augmented phosphorylation of the vasodilator-stimulated phosphoprotein in isolated aortic rings (P < 0.05 vs. placebo), increased cardiac output (+20.4 +/- 2.5%, P < 0.01 vs. placebo), and attenuated cardiac hypertrophy (-22.2 +/- 3.2%, p < 0.01 vs. placebo). 8beta-VE2, in contrast to oestradiol, did not enhance cardiac alpha-myosin heavy chain expression. CONCLUSION: Ligand-dependent activation of ERbeta confers blood pressure lowering effects in SHR that are superior to those of 17beta-estradiol or the ERalpha agonist 16alpha-LE2 and attenuates cardiac hypertrophy primarily by a reduction of cardiac afterload without promoting uterine growth.     

The catechol estrogen, 4-hydroxyestrone, has tissue-specific estrogen actions

Recent data indicate that the catechol estrogen, 2-hydroxyestrone (2-OHE(1)), has no effect on any target tissue including bone, whereas 16 alpha-hydroxyestrone (16 alpha-OHE(1)) exerts tissue-selective estrogen agonist activity. The effect of the catechol estrogen, 4-hydroxyestrone (4-OHE(1)), putatively associated with tumorigenesis, has not been studied in the skeleton. The purpose of this study was to assess the effect of 4-OHE(1) on tibia, uterine and mammary gland histology and blood cholesterol in ovariectomized (OVX'd) growing rats. Ten-week-old female Sprague-Dawley rats were injected subcutaneously with 200 microg/kg BW per day with 4-OHE(1), 17 beta-estradiol (E(2)) or vehicle for three weeks. OVX resulted in uterine atrophy, increased body weight, radial bone growth and cancellous bone turnover, and hypercholesterolemia. E(2) prevented these changes with the expected exception that the subcutaneous infusion of this high dose of estrogen did not prevent the hypercholesterolemia. 4-OHE(1) prevented the increase in blood cholesterol and the increase in body weight. 4-OHE(1) appeared to have partial estrogen activity in the uterus; uterine weight and epithelial cell height were significantly greater than the OVX rats but significantly less (twofold) than the E(2) animals. Analysis of variance indicated that 4-OHE(1) slightly decreased the periosteal mineral apposition rate (P<0.05) compared with vehicle-treated rats but had no effect on double-labeled perimeter or bone formation rate. Similarly, 4-OHE(1) was a partial estrogen agonist on cancellous bone turnover. The data suggest that the catechol estrogen, 4-OHE(1), unlike 2-OHE(1), has estrogen activity. Furthermore, the profile of activity differs from that of 16 alpha-OHE(1). Our results suggest that estrogen metabolites may selectively influence estrogen-target tissues and, concomitantly, modulate estrogen-associated disease risk.     

Dose-dependent effects of tamoxifen on long bones in growing rats: influence of ovarian status

Tamoxifen is a nonsteroidal antiestrogen which has been reported by various investigators to have estrogen agonist and antagonist effects on rat bone. These different interpretations may be due to differences in the ovarian status, estrogen levels, and/or tamoxifen levels of the rats. To address this issue, a dose response was determined for the effects of tamoxifen on bone histomorphometry in intact female and ovariectomized (OVX) rats. The results were compared with those obtained after treatment of OVX rats with estrogen alone or a combination of estrogen and tamoxifen. OVX resulted in increases in growth rate (weight gain) and periosteal bone formation rate and decreases in uterine weight and cancellous bone fractional volume (BV/TV). Treatment of OVX rats with estrogen resulted in dose-dependent decreases in growth rate and periosteal bone formation rate as well as dose-dependent increases in uterine weight and BV/TV. Similarly, tamoxifen treatment resulted in dose-dependent decreases in overall growth rate and periosteal bone formation rate in both OVX and intact rats. Tamoxifen treatment prevented the decrease in BV/TV after OVX, although the highest dose of tamoxifen resulted in a small decrease in BV/TV in intact female rats. In contrast to estrogen, tamoxifen treatment prevented the increase in uterine weight in intact female rats as well as the decrease in uterine weight in OVX rats. Tamoxifen treatment did not alter the effects of 17 beta-estradiol on the periosteal bone formation rate in OVX rats, but reduced the increase in BV/TV to values similar to those in intact rats. These results are consistent with tamoxifen behaving as a partial estrogen agonist on rat bone.     

Tissue-selective effects of continuous release of 2-hydroxyestrone and 16alpha-hydroxyestrone on bone, uterus and mammary gland in ovariectomized growing rats.

2-Hydroxyestrone (2-OHE(1)) and 16alpha-hydroxyestrone (16alpha-OHE(1)) have been reported to be risk factors for negative bone balance and breast cancer, respectively. The roles of these two metabolites of estrone as estrogen agonists or antagonists with respect to estrogen target tissues, or both, are poorly defined. The purpose of this study was to characterize metabolite and tissue-specific differences between the actions of hydroxylated estrones on selected reproductive and non-reproductive estrogen target tissues in growing rats. First, the effects of ovariectomy were determined. Ovariectomy had the expected effects, including increases in all dynamic bone measurements at the proximal tibial epiphysis, without induction of bone loss. Second, ovariectomized growing rats were continuously treated for 3 weeks with 2-OHE(1), 16alpha-OHE(1), 17beta-estradiol (E(2)), a combination of E(2) and 2-OHE(1) (E(2)+2-OHE(1)), or a combination of E(2) and 16alpha-OHE(1) (E(2)+16alpha-OHE(1)), using controlled release subcutaneous implanted pellets containing 5 mg 2-OHE(1), 5 mg 16alpha-OHE(1), 0.05 mg E(2) or placebo. E(2) reduced body weight gain and radial and longitudinal bone growth as well as indices of cancellous bone turnover, and increased serum cholesterol, uterine wet weight and epithelial cell height, and proliferative cell nuclear antigen labeling in mammary gland. The hydroxylated estrones did not alter uterine wet weight and 16alpha-OHE(1) antagonized the E(2)-stimulated increase in epithelial cell height. 2-OHE(1) had no effect on cortical bone, whereas 16alpha-OHE(1) was an estrogen agonist with respect to all cortical bone measurements. 16alpha-OHE(1) also behaved as an estrogen agonist with respect to serum cholesterol and cancellous bone measurements. 2-OHE(1) had no effect on most E(2)-regulated indices of cancellous bone growth and turnover, but was a weak estrogen agonist with respect to mineral apposition rate and bone formation rate. Neither estrogen metabolite influenced body weight gain. Third, weanling rats were treated for 1 week with vehicle, E(2) (200 microg/kg per day) or 16alpha-OHE(1) (30, 100, 300, 1000 and 3000 microg/kg per day) to confirm uterotropic effects of daily subcutaneous (s.c.) administration of 16alpha-OHE(1). 16alpha-OHE(1) increased uterine weight in a dose-response manner to values that did not differ from rats treated with E(2). We conclude that the estrogen metabolites 2-OHE(1) and 16alpha-OHE(1) have target tissue-specific biological activities which differ from one another as well as from E(2). These findings add further support to the concept that there are several classes of estrogens with distinct biological activities. Furthermore, differences in the route of administration could influence the tissue specificity of estrogen metabolites.     

Multihormonal control of pre-pro-somatostatin mRNA levels in the periventricular nucleus of the male and female rat hypothalamus

The influence of sex steroids as well as the possible involvement of dopaminergic pathways in the modulation of pre-pro-somatostatin (SS) mRNA levels was investigated by quantitative in situ hybridization in the hypothalamic periventricular nucleus (PeN) in adult male and female rats. In situ hybridization was performed using a [35S]-labeled cDNA probe encoding pre-proSS mRNA. Gonadectomy performed 14 days earlier decreased the mean number of silver grains/neuron corresponding to the relative pre-proSS mRNA levels by 22% in male and by 18-28% in female rats. A 14-day treatment with the nonaromatizable androgen dihydrotestosterone (DHT) increased the mean number of silver grains/neuron by 34-40% in gonadectomized animals of both sexes. Moreover, administration of 17 beta-estradiol (E2, 0.25 microgram twice daily) increased pre-proSS mRNA levels by 40% in ovariectomized (OVX) animals. Such treatment with E2 or DHT changed the frequency distribution profile of the hybridization signal intensity, thus increasing the percentage of highly labeled neurons (greater than or equal to 61 grains/neuron) by 10 to 12-fold. A 14-day treatment with the D2 dopamine receptor agonist bromocriptine (BRO) increased pre-proSS mRNA levels by 15 and 28% in intact female and OVX animals, respectively, while the dopaminergic antagonist haloperidol (HAL) decreased the value of this parameter by 20 and 30%. Furthermore, BRO increased pre-proSS mRNA levels by 10 and 20% in intact and castrated male rats, respectively, whereas HAL decreased pre-proSS mRNA levels by 25 and 14% in the same groups of animals. Administration of E2 in combination with HAL in OVX animals increased pre-proSS mRNA levels by 70% compared to those measured in OVX animals treated with HAL alone. In HAL-treated castrated male rats, administration of DHT increased the relative pre-proSS mRNA levels by 35% compared to those measured in castrated animals treated with HAL alone. The present data clearly demonstrate that androgens and estrogens as well as dopamine-mediated mechanisms could play a regulatory role in pre-proSS mRNA levels in somatostatinergic neurons in the hypothalamic PeN in both male and female rats.     

Evidence for a permanent decline in tuberoinfundibular dopaminergic neuronal function after chronic estrogen treatment is terminated in Fischer 344 rats

Long-term 17 beta-estradiol (E2) treatment in rats decreases tuberoinfundibular dopaminergic (TIDA) neuronal function. The objective of this study was to determine if the decline in TIDA function after E2 treatment in Fischer 344 (F344) rats is sustained long after removal of E2. Ovariectomized (OVX) F344 rats were each implanted with an E2-containing or empty Silastic capsule for 4 weeks; the capsule was then removed, and 26 weeks later acute experiments were performed. Release of 3H from median eminence tissue in vitro in response to electrical stimulation after 3H-DA accumulation was not different between E2-treated rats and OVX controls, even though serum prolactin (PRL) was 4-fold greater in E2-treated animals. Acute administration of apomorphine hydrochloride, a DA receptor agonist, at 2 doses, reduced serum PRL values as much in E2-treated animals as in OVX control rats. Injection of morphine sulfate or nomifensine maleate, which directly influence TIDA neurons, resulted in nonsignificant serum PRL responses in animals long after E2 withdrawal as compared to the greater response in OVX control rats. To further evaluate TIDA neuronal function, OVX non-E2-treated rats and animals 26 weeks after E2 withdrawal received a 3-day E2 challenge which increased the stimulation-evoked release of 3H from the median eminence tissue in vitro 2-fold in OVX control rats but had no effect in rats given E2 26 weeks previously. The difference in the stimulation-evoked release occurred in the presence of similar circulating serum PRL levels in the two groups as a result of the 3-day E2 treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of angiotensinogen gene expression by estrogen.

OBJECTIVE: Clarification of the role of estrogen in the regulation of angiotensinogen gene expression in multiple tissues. DESIGN: The effect of 17 beta-estradiol (E2; 10 micrograms/100 mg body weight) administration in ovariectomized (OVX) rats upon angiotensinogen messenger RNA (mRNA) levels in multiple tissues was assessed. Confounding ovarian factors were thus removed by studying the animals in the castrate state. Controls consisted of OVX and intact female rats. METHODS: Adult female Sprague-Dawley rats were ovariectomized and experiments begun 21 days postsurgery. Animals were injected with E2 and studied after 0, 1, 4, and 24 h of treatment. Levels of angiotensinogen mRNA were determined by Northern blot analysis using beta-actin mRNA as an internal standard. RESULTS: A single angiotensinogen mRNA species with molecular size of approximately 1800 bp was observed in rat liver, aorta, kidney, cardiac atria, hypothalamus and whole brain. Little or no angiotensinogen mRNA was identified in the pituitary gland. Angiotensinogen mRNA was most abundant in rat liver, hypothalamus, aorta and progressively less abundant in whole brain, cardiac atria and kidney. A twofold induction of hepatic angiotensinogen mRNA levels in E2-OVX rats was observed by 4h. The angiotensinogen mRNA levels in kidney were threefold higher by 4 h compared with OVX control animals. In aorta, the angiotensinogen mRNA level was also threefold higher by 1 h after E2 treatment. No significant effect of estradiol treatment was observed in cardiac atria although the level of angiotensinogen mRNA was higher in intact female rats compared with OVX controls. CONCLUSION: These results suggest that estrogen modulates angiotensinogen gene expression in a tissue-specific manner.     

Effects of estradiol and raloxifene analog on brain, adrenal and serum allopregnanolone content in fertile and ovariectomized female rats

Allopregnanolone is a neuroactive steroid synthesized in rat gonads, adrenal cortex, and central nervous system. It has been suggested that sex steroid hormones might influence allopregnanolone concentrations but no clear data have ever been reported. The aim of the present study was to investigate the effects of administration of 17beta-estradiol (17beta-E2), the raloxifene analog LY-117018 or their combination on allopregnanolone levels in fertile and ovariectomized (OVX) rats. Thirteen groups of 12 Wistar female rats each received either 17beta-E2 (0.1 or 1 microg/day) or LY-117018 (25, 250, and 1,250 microg/day), or 17beta-E2 1 microg/day plus LY-117018: 25, 250, and 1,250 microg/day for 14 days. The rats were then sacrificed and allopregnanolone content was assessed in the hypothalamus, hippocampus, pituitary, adrenals, and serum. Ovariectomy determined a significant decrease in allopregnanolone content in the hypothalamus, hippocampus, pituitary, and serum, while increasing it in the adrenals (p<0.01). In OVX rats, the administration of either 17beta-E2 or LY- 117018 restored ovariectomy-induced allopregnanolone changes. The administration of LY-117018 in addition to 17beta-E2 to OVX animals suppressed the increase in allopregnanolone levels determined by 17beta-E2 in the hippocampus, hypothalamus, and pituitary, but not in the adrenals and serum. In fertile rats, the administration of LY-117018 reproduced the effects of ovariectomy. This study shows that the raloxifene analog LY-117018 has an estrogen-like action on the central nervous system of OVX rats when administered alone, while it acts as an antiestrogen in the presence of 17beta-E2, both in OVX animals treated with 17beta-E2 and in fertile rats. A different effect was observed in the adrenal glands. The mechanism of action of this compound has still to be clarified.     

Estrogens up-regulate the Fas/FasL apoptotic pathway in lactotropes

The Fas/FasL system provides the major apoptotic mechanism for many cell types, participating in cell turnover in hormone-dependent tissues. In the present study, we localized both Fas and FasL in anterior pituitary cells, mainly in lactotropes and somatotropes. The percentage of anterior pituitary cells showing immunoreactivity for Fas or FasL was higher in cells from rats killed in proestrus than in diestrus. Also, the proportion of pituitary cells from ovariectomized (OVX) rats expressing Fas or FasL increased in the presence of 17beta-estradiol (10(-9) M). This steroid increased the percentage of lactotropes with immunoreactivity for Fas or FasL and the percentage of somatotropes expressing Fas. Activation of Fas by an agonist anti-Fas antibody (Mab-Fas) decreased the vi-ability-3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT assay)-of anterior pituitary cells from OVX rats cultured in the presence of 17beta-estradiol. Also, membrane-bound FasL decreased cell viability-[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay (MTS assay)-only when anterior pituitary cells from OVX rats were incubated with 17beta-estradiol. Moreover, FasL increased the percentage of hypodiploid anterior pituitary cells (flow cytometry). Mab-Fas increased the percentage of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL)-positive pituitary cells and lactotropes from OVX rats only when cells were incubated in the presence of 17beta-estradiol. Also, Mab-Fas triggered apoptosis of anterior pituitary cells from rats killed at proestrus but not at diestrus. Our results show that 17beta-estradiol up-regulates the expression of the Fas/FasL system in anterior pituitary cells and increases Fas-induced apoptosis in lactotropes, suggesting that Fas-induced apoptosis could be involved in the pituitary cell renewal during the estrous cycle.     

Calcitonin versus Clodronate in the Prevention of Ovariectomy-Induced Osteopenia in Rats

The effects of salmon calcitonin and clodronate were compared in ovariectomised rats. Sixty female Wistar rats (∼260 g in weight) were fed the same diet and had the same living conditions. The rats were divided into the following groups: 15 rats with sham ovariectomy and no drug treatment (Sham-OVX); 45 rats with bilateral ovariectomy subdivided into 15 rats not receiving drug treatment (OVX group), 15 rats treated with subcutaneous salmon calcitonin, 2 U/kg/day every 2 days (OVX + CT group) and 15 rats treated with subcutaneous clodronate, 5 mg/kg/day every 2 days (OVX + Cl group). Sixty days after surgery, the rats were sacrificed and their femurs and fifth lumbar vertebrae were dissected and cleaned of soft tissue. Femur length, vertebral height, and bone mineral content and bone mineral density of the femur and fifth lumbar vertebra by dual-energy X-ray absorptiometry were measured. Calcitonin had a significant and stronger effect in preventing ovariectomy-induced osteopenia in the femur (OVX + CT vs OVX groups, p<0.0001); both calcitonin and clodronate had a significant effect on the fifth lumbar vertebra, which was greater in the calcitonin group (OVX + CT vs OVX + Cl groups, p<0.005). These findings indicate that calcitonin has a protective effect on both the axial (trabecular bone) and peripheral (cortical bone) skeletons, but clodronate only has a protective effect on the axial skeleton. Received: 28 May 1999 / Accepted: 29 July 1999     

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.     

Raloxifene, a selective estrogen receptor modulator, reduces carrageenan-induced acute inflammation in normal and ovariectomized rats

Raloxifene (RAL) is a selective estrogen receptor modulator presenting tissue-specific agonist activity. The aim of this study was to examine whether RAL has an estrogenic effect on carrageenan-induced acute inflammation. Adult female rats were ovariectomized (OVX) 7 wk before edema or pleurisy to deplete circulating estrogens. Edema formation and selected inflammatory markers in inflamed paw tissue were measured in intact (sham-operated) and OVX rats. Groups of OVX rats were treated with RAL (1, 3, or 10 mg/kg) or 17beta-estradiol (E2, 25 microg/kg), and these treatments began 2 d after surgery and continued until carrageenan paw edema or pleurisy. Ovariectomy amplifies the inflammation, and we found that RAL, as well as E2, attenuates inflammation and tissue damage associated with paw edema and pleurisy. In treated rats, there is a decrease in edema development and formation, and in polymorphonuclear cell infiltration and migration, as shown by myeloperoxidase measurement and cell counting. RAL and E2 treatments decrease cyclooxygenase-2 and inducible nitric oxide synthase expression in inflamed areas and counteract the inhibition of peroxisome proliferators-activated receptor-gamma expression caused by ovariectomy, restoring this receptor protein expression to sham-operated levels and identifying a possible peroxisome proliferators-activated receptor-dependent antiinflammatory effect of these drugs. Moreover, RAL and E2 increase cytoprotective heat shock protein 72 expression, which seems to be closely associated with the remission of the inflammatory reaction. In addition, we confirm the antiinflammatory effect of RAL in male rats, using a single administration of RAL or E2.     

Estrogen treatment prevents osteopenia and depresses bone turnover in ovariectomized rats

Female Sprague-Dawley rats were subjected to bilateral ovariectomy (OVX) or sham surgery (control). Groups of ovariectomized (OVX) and control rats were injected daily with low, medium, or high doses of 17 beta-estradiol (10, 25, or 50 micrograms/kg BW, respectively). An additional group of OVX and control rats was injected daily with vehicle alone. All rats were killed 35 days after OVX, and their proximal tibiae were processed undecalcified for quantitative bone histomorphometry. Trabecular bone volume was markedly reduced in vehicle-treated OVX rats relative to that in control rats (12.1% vs. 26.7%). This bone loss was associated with a 2-fold increase in osteoclast surface and a 4-fold increase in osteoblast surface. The bone formation rate, studied with fluorochrome labeling, was also significantly elevated in vehicle-treated OVX rats (0.111 vs. 0.026 micron3/micron2.day). In contrast, treatment of OVX rats with the three doses of estradiol resulted in normalization of tibial trabecular bone volume and a decline in histomorphometric indices of bone resorption and formation. Our results indicate that estrogen treatment provides complete protection against osteopenia in OVX rats. The protective mechanism involves estrogenic suppression of bone turnover. These findings are consistent with the skeletal effects of estrogen therapy in postmenopausal women.     

Testosterone prevents the heat shock-induced overactivation of glycogen synthase kinase-3β but not of cyclin-dependent kinase 5 and c-Jun NH2-terminal kinase and concomitantly abolishes hyperphosphorylation of τ: Implications for Alzheimer's disease

We have shown previously that glycogen synthase kinase-3 beta (GSK-3 beta), cyclin-dependent kinase 5, and c-Jun NH(2)-terminal kinase become overactivated and hyperphosphorylate tau in heat-shocked female rats. This hyperphosphorylation of tau is estrogen-independent, prevented by androgens, and similar to Alzheimer's disease. In this study, ovariectomized (OVX) Sprague-Dawley rats (n = 75) received daily injections of 10 microg of 17 beta-estradiol benzoate (EB), or 250 microg of testosterone propionate (TP), or both EB and TP, or sesame oil (SO) vehicle for 4-6 weeks. In kinase assays of forebrain homogenates, overactivation of GSK-3 beta at 0-6 h after heat shock toward human recombinant tau, bovine tau, and phosphoglycogen synthase peptide 2 was prevented in OVX + TP and OVX + (EB + TP) but not in sham-OVX + SO, OVX + SO, and OVX + EB. Abs against inactive (pSer(9)) and activity-enhanced (pTyr(216)) GSK-3 beta showed marked increase of pSer(9)- and decrease of pTyr(216)-GSK-3 beta in both OVX + TP and OVX + (EB + TP) but not in sham-OVX + SO, OVX + SO, and OVX + EB. EB enhanced the overactivation of cyclin-dependent kinase 5. The activity of c-Jun NH(2)-terminal kinase was gonadal hormone-independent. The serum concentrations of testosterone and 17 beta-estradiol were 2.53 ng/ml and 201 pg/ml in OVX + TP and OVX + EB, respectively. These findings demonstrate that testosterone prevents the hyperphosphorylation of tau by inhibiting the heat shock-induced overactivation of GSK-3 beta and suggest that androgens given to aging men or, in combination with estrogens, to postmenopausal women could prevent or delay Alzheimer's disease.     

氟化钠对去卵巢大鼠胫骨作用的骨形态计量学研究

目的 了解卵巢切作大鼠胫骨对氟化钠的治疗反应。方法 将２４只雌性大鼠随机分成３组,单纯切卵巢组、对照组和氟化钠治疗组。对单纯切卵巢组和氟化钠治疗组大鼠行卵巢切除手术,建立骨质疏松动物模型,氟化钠治疗组于卵巢切除术后１个月予氟化钠,给药３个月后处死。用骨形态计量学方法检测卵巢切除大鼠无后肢胫骨对氟化钠的治疗反应。结果 单纯卵巢切除组大鼠骨小梁体积,平均骨小梁密度较对照组显著较减少（Ｐ〈０．０５）,四     

Chronic estrogen-induced alterations in adrenocorticotropin and corticosterone secretion, and glucocorticoid receptor-mediated functions in female rats.

The effect of estrogen (E) on the hypothalamic-pituitary-adrenal axis was investigated in female Sprague-Dawley rats. Animals were bilaterally ovariectomized (OVX), and a Silastic capsule (0.5 cm) containing 17 beta-estradiol was sc implanted. Control animals received a blank capsule. Animals were killed 21 days later. In E-treated rats, we found significantly higher corticosterone (CORT) peak levels 20 min after a 5-sec footshock (1.0 mamp) or exposure to ether vapors (P less than 0.05) compared to those in OVX controls. In addition, the recovery of the ACTH and CORT responses to footshock stress was significantly prolonged (P less than 0.05) in the presence of E. Furthermore, the ACTH and CORT secretory responses to ether stress could be suppressed by exogenous RU 28362 (a specific glucocorticoid receptor agonist; 40 micrograms/100 g BW for 4 days) in OVX controls (P less than 0.05), but not in E-treated animals. These data suggest that E can impair glucocorticoid receptor-mediated delayed or slow negative feedback. Consequently, we examined the influence of E on mineralocorticoid and glucocorticoid receptor concentrations using in vitro binding assays. E did not alter mineralocorticoid or glucocorticoid receptor concentrations in any of the brain regions examined. The administration of RU 28362 (40 micrograms/100 g BW for 4 days) to OVX control or E-treated rats significantly down-regulated hippocampal glucocorticoid receptor (P less than 0.02) in control rats only. In contrast, aldosterone administration (40 micrograms/100 g BW for 4 days) significantly down-regulated hippocampal glucocorticoid receptor (P less than 0.0008) in both control and E-treated animals. Thus, E treatment results in a loss of the glucocorticoid receptor's ability to autoregulate; this suggests that E may cause a functional impairment of the glucocorticoid receptor even though receptor binding appears normal. These findings suggest that hyperactivation of the hypothalamic-pituitary-adrenal axis after stress in E-treated rats is due in part to impaired glucocorticoid receptor-mediated slow negative feedback.     

Opposite effects of insulin-like growth factor-I on the formation of trabecular and cortical bone in adult female rats.

We have previously found that in intact adult female rats, 17 beta-estradiol (E2) stimulates trabecular bone formation, which can be detected by triple fluorochrome labeling within a few days of commencing E2 treatment. We have also observed that E2 stimulates trabecular bone formation in ovariectomised (Ovx) rats given 3-amino-1-hydroxypropylidene bisphosphonate (AHPrBP). This anabolic action of E2 is presumably mediated through the local release of growth factors, of which insulin-like growth factor-I (IGF-I) is a strong contender. To assess the role of IGF-I as mediator of the anabolic action of E2 on rat trabecular bone, we examined the effect of daily sc administration of IGF-I (200 micrograms/kg) for 17 days on trabecular bone formation at the proximal tibial metaphysis in experiments using the above two models. Intact animals received fluorochrome labels on days 1, 8, and 15 of IGF-I treatment, and Ovx animals received AHPrBP (0.3 mg/kg) on days 1, 8, and 15 and fluorochrome labels on days 8 and 15. Trabecular surfaces covered by first and second labels only (termed arrested surfaces), second and third labels only (induction surfaces), and all three labels (persisting surfaces) were assessed in intact animals; double labeled surfaces were assessed in Ovx animals. We found that IGF-I increased the longitudinal growth rate in both intact rats and Ovx animals given AHPrBP. However, to our surprise, we found that IGF-I inhibited trabecular bone formation in intact animals, reducing both arrested and persisting trabecular surfaces by approximately 50%. This was associated with a doubling of periosteal bone formation, as measured at the tibial diaphysis. In Ovx animals given AHPrBP, the AHPrBP markedly inhibited trabecular bone formation compared with that in Ovx controls, and IGF-I was without effect. We conclude that, in contrast to E2, IGF-I acts to increase the length and circumference of long bones and, perhaps through reactive mechanisms such as mechanical compensation, reduces rather than stimulates trabecular bone formation.     

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.