Estrogen modulates the expression of myosin heavy chain in detrusor smooth muscle

The effect of low serum estrogen levels on urinary bladder function remains poorly understood. Using a rabbit model, we analyzed the effects of estrogen on the expression of the isoforms of myosin, the molecular motor for muscle contraction, in detrusor smooth muscle. Expression of myosin heavy chain (MHC) isoforms, which differ in the COOH-terminal (SM1 and SM2) and the NH(2)-terminal (SM-A and SM-B) regions as a result of alternative splicing of the mRNA at either the 3'- or 5'-ends, was analyzed in age-matched female rabbits that were sham operated, ovariectomized (Ovx), and given estrogen after ovariectomy (4 rabbits/group). Ovx rabbits showed a significant decrease in the overall MHC content per gram of wet detrusor smooth muscle compared with controls (P < 0.04), which was reversed by estrogen replacement (P < 0.02). MHC content, as a proportion of total milligram of protein in the bladder tissue extracted, was also increased in estrogen-treated Ovx rabbits. Quantitative competitive RT-PCR revealed 1.72-, 2.63-, and 5.82 x 10(6) copies of MHC mRNA/100 ng total mRNA in Ovx, control, and estrogen-treated rabbits, respectively (P < 0.01). RT-PCR analysis using oligonucleotides specific for the region containing the SM1/SM2 MHC alternative splice sites indicated a lower SM2-to-SM1 ratio in estrogen-treated compared with control and Ovx rabbits (P < 0.05). Similarly, SDS-PAGE analysis of extracted myosin from estrogen-treated rabbits revealed a significantly lower SM2-to-SM1 isoform ratio compared with control and Ovx rabbits (P < 0.05). Expression of the SM-A and SM-B isoforms was not affected. These results indicate that myosin content is increased upon estrogen replacement in Ovx rabbits and that the abundance of SM1 relative to SM2 is greater in estrogen-treated rabbits compared with normal and Ovx rabbits. These data suggest that estrogen affects alternative splicing at the 3'-end of the MHC pre-mRNA to increase the proportion of SM1 vs. SM2.     

Expression and function of COOH-terminal myosin heavy chain isoforms in mouse smooth muscle

Isoforms of the smooth muscle myosin motor, SM1 and SM2, differ in length at the carboxy terminal tail region. Their proportion changes with development, hormonal status and disease, but their function is unknown. We developed mice carrying the myosin heavy chain (MyHC) transgenes SM1, cMyc-tagged SM1, SM2, and V5-tagged SM2, and all transgenes corresponded to the SMa NH₂-terminal isoform. Transgene expression was targeted to smooth muscle by the smooth muscle -actin promoter. Immunoblot analysis showed substantial expression of the cMyc-tagged SM1 and V5-tagged SM2 MyHC protein in aorta and bladder and transgene mRNA was expressed in mice carrying unlabeled SM1 or SM2 transgenes. Despite significant protein expression of tagged MyHCs we found only small changes in the SM1:SM2 protein ratio. Significant changes in functional phenotype were observed in mice carrying unlabeled SM1 or SM2 transgenes. Force in aorta and bladder was increased (72 ± 14%, 92 ± 11%) in SM1 and decreased to 57 ± 1% and 80 ± 3% in SM2 transgenic mice. SM1 transgenic bladders had faster (1.8 ± 0.3 s) and SM2 slower (7.1 ± 0.5 s) rates of force redevelopment following a rapid step shortening. We hypothesize that small changes in the SM1:SM2 ratio could be amplified if they are associated with changes in thick filament assembly and underlie the altered contractility. These data provide evidence indicating an in vivo function for the COOH-terminal isoforms of smooth muscle myosin and suggest that the SM1:SM2 ratio is tightly regulated in smooth muscle tissues. myosin heavy chain; transgenic mice     

Mechanisms of 17beta-estradiol on the production of ET-1 in ovariectomized rats

In order to clarify the mechanism underlying the possible preventive effect of estrogen on atherogenesis, we investigated the role of 17beta-estradiol (E2) in the regulation of endothelin-1 (ET-1) production in ovariectomized rats, which may contribute to atherogenesis. Female Spragure-Dawly rats were randomly divided into three groups: sham-operated group (sham), ovariectomized group (OVX) and 17beta-estradiol replacement group (OVX + E2, 20 microg(-1).kg.d(-1),s.c.). 4 weeks after operation, the plasma concentration of ET-1, clearance of ET-1, functional ECE activity and preproET-1 mRNA expression in aorta were measured. Concentration of plasma ET-1 change from 107.8 +/- 18.3 pg/ml (sham) and 135.5 +/- 27.6 pg/ml (OVX + E2) to 190.7 +/- 25.5 pg/ml (OVX ) (n = 8, p < 0.05). There was no significant difference in the clearance of 125IET-1 among three groups (p > 0.05). Functional ECE activity was increased in OVX group in comparison to that in sham group (p < 0.05). The OVX increased the preproET-1 mRNA expression in sham, whereas treatment with estrogen reversed these changes (p < 0.05). The present study have shown that estrogen down-regulates plasma ET-1 levels by inhibiting the preproET-1 mRNA expression and functional ECE activity. Clearance of ET-1 was not affected. Inhibition of ET-1 production mediated by modulating ECE activity may be one of the novel mechanisms of the protective of estrogens on the cardiovascular system.     

The effects of smooth muscle caldesmon on actin filament motility.

The movement of reconstituted thin filaments over an immobilized surface of thiophosphorylated smooth muscle myosin was examined using an in vitro motility assay. Reconstituted thin filaments contained actin, tropomyosin, and either purified chicken gizzard caldesmon or the purified COOH-terminal actin-binding fragment of caldesmon. Control actin-tropomyosin filaments moved at a velocity of 2.3 +/- 0.5 microns/s. Neither intact caldesmon nor the COOH-terminal fragment, when maintained in the monomeric form by treatment with 10 mM dithiothreitol, had any effect on filament velocity; and yet both were potent inhibitors of actin-activated myosin ATPase activity, indicating that caldesmon primarily inhibits myosin binding as reported by Chalovich et al. (Chalovich, J. M., Hemric, M. E., and Velaz, L. (1990) Ann. N. Y. Acad. Sci. 599, 85-99). Inhibition of filament motion was, however, observed under conditions where cross-linking of caldesmon via disulfide bridges was present. To determine if monomeric caldesmon could "tether" actin filaments to the myosin surface by forming an actin-caldesmon-myosin complex as suggested by Chalovich et al., we looked for caldesmon-dependent filament binding and motility under conditions (80 mM KCl) where filament binding to myosin is weak and motility is not normally seen. At caldesmon concentrations > or = 0.26 microM, actin filament binding was increased and filament motion (2.6 +/- 0.6 microns/s) was observed. The enhanced motility seen with intact caldesmon was not observed with the addition of up to 26 microM COOH-terminal fragment. Moreover, a molar excess of the COOH-terminal fragment competitively reversed the enhanced binding seen with intact caldesmon. These results show that tethering of actin filaments to myosin by the formation of an actin-caldesmon-myosin complex enhanced productive acto-myosin interaction without placing a significant mechanical load on the moving filaments.     

Steroid-hormone regulation of myosin subunit expression in smooth and cardiac muscle

We investigated the effects of ovarectomy and the steroid hormones estrogen and testosterone on the in vivo expression of heavy (MHC) and light (MLC) chains of myosin in the heart, uterus, and aorta of rats. In the heart, ovarectomy decreased alpha-MHC expression, while both steroid hormones normalized it. Differential steroid hormone effects could be observed on myosin subunit expression of smooth muscle. Testosterone but not estrogen normalized the ovarectomy-induced decreased expression of SM1 and strongly increased the expression of 5′-inserted MHC in the uterus. Estrogen but not testosterone normalized the ovarectomy-induced diminished MLC17a expression. In contrast to the uterus, no steroid hormone effects on myosin subunit expression could be observed in the aorta. © 1995 Wiley-Liss, Inc.     

Chronic estrogen treatment reduces vaso-constrictor responses in insulin resistant rats

Previous experiments have shown that chronic estrogen treatment via subcutaneous implants prevented insulin-induced blood pressure elevation and increased insulin sensitivity in ovariectomized female rats. In vitro vascular studies were performed using isolated mesenteric arteries to determine the effect of chronic estrogen and insulin treatments on vascular responses to vasoconstrictor agents. Female Wistar rats were assigned to the following groups: sham-operated, sham-operated plus insulin, sham-operated plus insulin plus estrogen, ovariectomized, ovariectomized plus insulin, and ovariectomized plus insulin plus estrogen. Chronic insulin and estrogen treatments were initiated with subcutaneous placement of insulin implants (2 U/d) and 17beta-estradiol implants (0.5 mg/pellet, 60 day release) at the back of the neck. After 8 weeks of treatment, mesenteric arteries were isolated for assessment of constrictor responses to norepinephrine and the thromboxane A2 analogue U46619 in the presence or absence of the endothelium. The results show that chronic estrogen treatment attenuated the vascular constrictor responses to norepinephrine and U46619 only in endothelium intact vessels. Incubation with insulin did not significantly affect norepinephrine-induced vascular smooth muscle contraction. The study provides evidence that the mechanism by which estrogen prevents insulin-induced blood pressure elevation in insulin-treated ovariectomized rats is by influencing endothelium-derived vasoactive factors such as thromboxane A2.     

The expression and functional activities of smooth muscle myosin and non‐muscle myosin isoforms in rat prostate

Benign prostatic hyperplasia (BPH) is mainly caused by increased prostatic smooth muscle (SM) tone and volume. SM myosin (SMM) and non‐muscle myosin (NMM) play important roles in mediating SM tone and cell proliferation, but these molecules have been less studied in the prostate. Rat prostate and cultured primary human prostate SM and epithelial cells were utilized. In vitro organ bath studies were performed to explore contractility of rat prostate. SMM isoforms, including SM myosin heavy chain (MHC) isoforms (SM1/2 and SM‐A/B) and myosin light chain 17 isoforms (LC_17a/b), and isoform ratios were determined via competitive RT‐PCR. SM MHC and NM MHC isoforms (NMMHC‐A, NMMHC‐B and NMMHC‐C) were further analysed via Western blotting and immunofluorescence microscopy. Prostatic SM generated significant force induced by phenylephrine with an intermediate tonicity between phasic bladder and tonic aorta type contractility. Correlating with this kind of intermediate tonicity, rat prostate mainly expressed LC_17a and SM1 but with relatively equal expression of SM‐A/SM‐B at the mRNA level. Meanwhile, isoforms of NMMHC‐A, B, C were also abundantly present in rat prostate with SMM present only in the stroma, while NMMHC‐A, B, C were present both in the stroma and endothelial. Additionally, the SMM selective inhibitor blebbistatin could potently relax phenylephrine pre‐contracted prostate SM. In conclusion, our novel data demonstrated the expression and functional activities of SMM and NMM isoforms in the rat prostate. It is suggested that the isoforms of SMM and NMM could play important roles in BPH development and bladder outlet obstruction.     

Blebbistain, a myosin II inhibitor, as a novel strategy to regulate detrusor contractility in a rat model of partial bladder outlet obstruction

Partial bladder outlet obstruction (PBOO), a common urologic pathology mostly caused by benign prostatic hyperplasia, can coexist in 40-45% of patients with overactive bladder (OAB) and is associated with detrusor overactivity (DO). PBOO that induces DO results in alteration in bladder myosin II type and isoform composition. Blebbistatin (BLEB) is a myosin II inhibitor we recently demonstrated potently relaxed normal detrusor smooth muscle (SM) and reports suggest varied BLEB efficacy for different SM myosin (SMM) isoforms and/or SMM vs nonmuscle myosin (NMM). We hypothesize BLEB inhibition of myosin II as a novel contraction protein targeted strategy to regulate DO. Using a surgically-induced male rat PBOO model, organ bath contractility, competitive and Real-Time-RT-PCR were performed. It was found that obstructed-bladder weight significantly increased 2.74-fold while in vitro contractility of detrusor to various stimuli was impaired ～50% along with decreased shortening velocity. Obstruction also altered detrusor spontaneous activities with significantly increased amplitude but depressed frequency. PBOO switched bladder from a phasic-type to a more tonic-type SM. Expression of 5' myosin heavy chain (MHC) alternatively spliced isoform SM-A (associated with tonic-type SM) increased 3-fold while 3' MHC SM1 and essential light chain isoform MLC(17b) also exhibited increased relative expression. Total SMMHC expression was decreased by 25% while the expression of NMM IIB (SMemb) was greatly increased by 4.5-fold. BLEB was found to completely relax detrusor strips from both sham-operated and PBOO rats pre-contracted with KCl, carbachol or electrical field stimulation although sensitivity was slightly decreased (20%) only at lower doses for PBOO. Thus we provide the first thorough characterization of the response of rat bladder myosin to PBOO and demonstrate complete BLEB-induced PBOO bladder SM relaxation. Furthermore, the present study provides valuable evidence that BLEB may be a novel type of potential therapeutic agent for regulation of myogenic and nerve-evoked DO in OAB.     

Estrogen potentiates vasopressin-induced contraction of female rat aorta by enhancing cyclooxygenase-2 and thromboxane function

To determine the roles of estrogen and constrictor prostanoids in vasopressin (VP)-induced contraction of female rat aorta, vascular reactivity to VP was determined in thoracic aortas of intact, ovariectomized, and ovariectomized + estrogen-replaced female rats in the presence of indomethacin (Indo), NS-398, SQ-29,548, or vehicle control. The effects of estrogen on vascular reactivity to the thromboxane A(2) analog U-46619 were also examined. Maximal contractile response to VP in intact female rats (5,567 +/- 276 mg/mg of aortic ring wt) was markedly attenuated by ovariectomy (2,485 +/- 394 mg; P < 0.001) and restored by estrogen replacement with 17beta-estradiol (5,059 +/- 194 mg; P > 0.1). Indo and NS-398 significantly attenuated maximal responses to VP in intact female rats to a similar extent [3,176 +/- 179 (P < 0.0001) and 3,258 +/- 152 mg (P < 0.0001), respectively]. Ovariectomy abolished and estrogen replacement restored the inhibitory effects of Indo, NS-398, and SQ-29,548. Contractile responses of rat aorta to U-46619 were significantly greater (P < 0.0001) in females (5,040 +/- 238 mg) than in males (3,679 +/- 96 mg). Ovariectomy markedly attenuated (3,923 +/- 84 mg; P < 0.01) and estrogen replacement restored (5,024 +/- 155 mg; P > 0.1) responses to U-46619 in female aortas. These data reveal that estrogen is an important regulator of the contractile responses of female rat aorta to VP, which appears to potentiate both cyclooxygenase-2 and constrictor prostanoid function in the vascular wall.     

SM22alpha is required for agonist-induced regulation of contractility: evidence from SM22alpha knockout mice

The present study was undertaken to determine whether SM22alpha participates in the regulation of vascular smooth muscle contractility using SM22alpha knockout mice and, if so, to investigate the mechanisms involved. Aortic ring preparations were mounted and equilibrated in organ baths for 60 min before observing contractile responses to 50 mM KCl, and then exposed to contractile agents such as phenylephrine and phorbol ester. Measurement of isometric contractions using a computerized data acquisition system was combined with molecular or cellular experiments. Interestingly, the aortas from SM22alpha-deficient mice (SM22(-/-LacZ)) displayed an almost three-fold increase in the level of SM22beta protein compared to wild-type mice, but no change in the levels of caldesmon, actin, desmin or calponin. Ca2+-independent contraction in response to phenylephrine or phorbol ester was significantly decreased in the SM22alpha-deficient mice, whereas in the presence of Ca2+ neither contraction nor subcellular translocation of myosin light chain kinase (MLCK) in response to phenylephrine or 50 mM KCl was significantly affected. A decrease in phosphorylation of extracellular signal regulated kinase (ERK) 1/2 was observed in the SM22alpha-deficient mice and this may be related to the decreased vascular contractility. Taken together, this study provides evidence for a pivotal role of SM22alpha in the regulation of Ca2+-independent vascular contractility.     

Estrogen, nitric oxide, and hypertension differentially modulate agonist-induced contractile responses in female transgenic (mRen2)27 hypertensive rats

Clinical trials revealed that estrogen may result in cardiovascular risk in patients with coronary heart disease, despite earlier studies demonstrating that estrogen provided cardiovascular protection. It is possible that the preexisting condition of hypertension and the ability of estrogen to activate the renin-angiotensin system could confound its beneficial effects. Our hypothesis is that the attenuation of estrogen to agonist-induced vasoconstrictor responses through the activation of nitric oxide (NO) synthase (NOS) is impaired by hypertension. We investigated the effects of 17beta-estradiol (E(2)) replacement in normotensive Sprague-Dawley (SD) and (mRen2)27 hypertensive transgenic (TG) rats on contractile responses to three vasoconstrictors, angiotensin II (ANG II), serotonin (5-HT), and phenylephrine (PE), and on the modulatory role of vascular NO to these responses. The aorta was isolated from ovariectomized SD and TG rats treated chronically with 5 mg E(2) or placebo (P). The isometric tension of the aortic rings was measured in organ chambers, and endothelial NOS (eNOS) in the rat aorta was detected using Western blot analysis. E(2) treatment increased eNOS expression in the SD and TG aorta and reduced ANG II- and 5-HT- but not PE-induced contractions in SD and TG rats. The inhibition of NOS with N(omega)-nitro-L-arginine methyl ester enhanced ANG II-, 5-HT-, and PE-induced contractions in P-treated and ANG II and PE responses in E(2)-treated SD and TG rats. Only the responses to 5-HT were augmented in hypertensive rats. In conclusion, this study shows that the preexisting condition of hypertension augmented the vascular responsiveness of 5-HT, whereas the attenuation of estrogen by ANG II and 5-HT vascular responses was not impaired by hypertension. The adrenergic agonist was unresponsive to estrogen treatment. The contribution of NO as a factor contributing to the relative refractoriness of the vascular responses is dependent on the nature of the vasoconstrictor and/or the presence of estrogen.     

Smooth muscle myosin expression, isoform composition, and functional activities in rat corpus cavernosum altered by the streptozotocin-induced type 1 diabetes

Diabetes mellitus (DM) is a quite common chronic disease, and the prevalence of erectile dysfunction (ED) is three times higher in this large population. Although diabetes-related ED has been studied extensively, the actin-myosin contractile apparatus was not examined. The mRNAs encoding smooth muscle myosin (SMM) heavy chains (MHC) and essential light chains (LC(17)) exist as several different alternatively spliced isoforms with distinct contractile properties. Recently, we provided novel data that blebbistatin (BLEB), a specific myosin II inhibitor, potently relaxed corpus cavernosum smooth muscle (CCSM). In this study, we examine whether diabetes alters SMM expression, alternative splicing, and/or functional activities, including sensitivity to BLEB. By using streptozotocin (STZ)-induced 2-mo diabetic rats, functional activities were tested in vivo by intracavernous pressure (ICP) recording during cavernous nerve stimulation and in vitro via organ bath contractility studies. SMM isoform composition was analyzed by competitive RT-PCR and total SMM, myocardin, and embryonic SMM (SMemb) expression by real-time RT-PCR. Results revealed that the blood glucose level of STZ rats was 407.0 vs. 129.5 mg/dl (control). STZ rats exhibited ED confirmed by significantly increased CCSM contractile response to phenylephrine and decreased ICP response. For STZ rats, SM-B, LC(17a) and SM2 isoforms, total SMM, and myocardin expression increased, whereas SM-A, LC(17b), and SM1 isoforms were decreased, with SMemb unchanged. BLEB was significantly more effective in relaxing STZ CCSM both in vitro and in vivo. Thus we demonstrated a novel diabetes-specific effect on alternative splicing of the SMM heavy chain and essential light chain genes to a SMM isoform composition favoring a heightened contractility and ED. A switch to a more contractile phenotype was supported further by total SMM expression increase. Moreover, the change in CCSM phenotype was associated with an increased sensitivity to BLEB, which may serve as a novel pharmacotherapy for ED.     

Estradiol replacement reverses ovariectomy-induced muscle contractile and myosin dysfunction in mature female mice.

Skeletal muscle contractility and myosin function decline following ovariectomy in mature female mice. In the present study we tested the hypothesis that estradiol replacement can reverse those declines. Four-month-old female C57BL/6 mice (n = 69) were ovariectomized (OVX) or sham operated. Some mice were treated immediately with placebo or 17beta-estradiol (OVX + E(2)) while other mice were treated 30 days postsurgery. Thirty or sixty days postsurgery, soleus muscles were assessed in vitro for contractile function and susceptibility to eccentric contraction-induced injury. Myosin structural dynamics was analyzed in extensor digitorum longus (EDL) muscles by electron paramagnetic resonance spectroscopy. Maximal isometric tetanic force was affected by estradiol status (P < 0.001) being approximately 10% less in soleus muscles from OVX compared with sham-operated mice [168 mN (SD 16.7) vs. 180 mN (SD 14.4)] and was restored in OVX + E(2) mice [187 mN (SD 17.6)]. The fraction of strong-binding myosin during contraction was also affected (P = 0.045) and was approximately 15% lower in EDL muscles from OVX compared with OVX + E(2) mice [0.263 (SD 0.034) vs. 0.311 (SD 0.022)]. Plasma estradiol levels were correlated with maximal isometric tetanic force (r = 0.458; P < 0.001) and active stiffness (r = 0.329; P = 0.044), indicating that circulating estradiol influenced muscle and myosin function. Estradiol was not effective in protecting muscle against an acute eccentric contraction-induced injury (P >or= 0.401) but did restore ovariectomy-induced increases in muscle wet mass caused by fluid accumulation. Collectively, estradiol had a beneficial effect on female mouse skeletal muscle.     

17β—雌二醇下调血管平滑肌内皮素A型受体的表达

为进一步探讨雌激素对心血管的保护作用,实验在双侧卵巢去势大鼠模型和培养的血管平滑肌细胞（VSMCs)上,观察17β－雌二醇（E2）对血管反应性及VSMCs增殖的影响,以RT－PCR和Western blot检测内皮素受体（ETAR）的表达,结果显示：去势雌性大鼠血管对内皮素（ET－1）的反应性明显增高,ETAR特异性受体阻断剂BQ123能完全阻断ET－1对VSMCs增殖的影响,E2能明显抑制ET－1对VSMCs增殖的作用,RT－PCR结果显示E2能抑制ETAR mRNA的表达,Western blot进一步证实E2能抑制ETAR蛋白表达,E2受体阻断剂Tamoxifen能部分抑制ET－1对VSMCs的增殖及ETAR的mRNA和蛋白 的表达。以上结果提示;ET－1促VSMCs增殖的效应主要是由ETAR介导的,雌激素能通过下调ETAR来抑制ET－1对VSMCs 促增殖的作用和血管对ET－1的反应,且此作用与雌激素受体有关。     

Effect of steroids and the estrous cycle on uterine neuromedin U receptor expression

We investigated the role of estrogen and progesterone on rat uterine NmU receptor expression in both intact and ovariectomized animals and examined receptor expression through the estrous cycle. Chronic administration of beta-estradiol 3-benzoate (E2) or progesterone in intact animals was devoid of any effect. RU486 caused a 2-fold up-regulation in NmU receptor density. Ovariectomy caused a 60% decrease in receptor density, but chronic E2 administration to ovariectomized rats significantly increased NmU receptor density. The estrous cycle had no significant effect on NmU receptor density. These results suggest that NmU receptor expression is estrogen-dependent, whereas progesterone or a progestin-induced factor is involved in the modulation of this expression.     

Supraphysiological level of estrogen exposure in vivo increases lymphoid cell death in mice

Estrogen can enhance or reduce lymphocyte functions in vitro depending on dose and exposure duration. The purpose of this study was to determine the effect of in vivo 17 beta-estradiol (E2) on apoptosis and necrosis in lymphoid tissue of female C567BL/6 mice. Animals were ovariectomized (OVX), ovariectomized and 17 beta-estradiol supplemented (OVX + E2; 71 micrograms E2 per day for 14 days), sham ovariectomized (SHAM), or unhandled controls (CONTROL). Thymus and spleen were removed aseptically, cells dispersed into single cell suspensions in RPMI-1640, and measures of cell damage performed: an annexin V flow cytometric assay for markers of apoptosis and an enzyme-linked immunoassay for measures of DNA fragmentation and necrosis. OVX + E2 mice had 620 +/- 72 pg/ml 17 beta-estradiol in serum in contrast to OVX mice which had 7.6 +/- 5 pg/ml, the SHAM mice which had 2.8 +/- 1 pg/ml of serum E2, and the CONTROL mice which had 3.9 +/- 0.8 pg/ml of serum E2 (p < 0.001). There was a significantly lower percentage of viable thymocytes in OVX + E2 mice compared to the other treatment conditions (p < 0.001, respectively). There was also a significantly higher percentage of annexin V positive thymocytes in OVX + E2 mice (p < 0.005). Measures of DNA fragmentation by ELISA were higher in splenocytes from OVX + E2 mice than in the OVX, SHAM or CONTROL mice (p < 0.005). These results suggest that supraphysiological levels of estrogen in vivo induce damage in lymphoid cells; however, the impact of estrogen associated lymphoid tissue damage on specific immune functions remains to be determined.     

Regulation of leptin by steroid hormones in rat adipose tissue.

We investigated if steroid hormones regulate the secretion and the expression of leptin in female and male rat adipose tissue fragments in vitro. Dexamethasone time and dose-dependently increased the secretion and mRNA expression of leptin with a half-maximal stimulation of approximately 1 nM. A time-course revealed a maximal stimulatory effect of 17 beta-estradiol after 24 hours. In male adipose tissue 17 beta-estradiol increased leptin secretion (32% by 50 nM 17 beta-estradiol, P = 0.07 and 34% by 500 nM 17 beta-estradiol, P < 1780.05) after 24 hours. An additional effect of estrogen was seen in the dexamethasone (50 nM) stimulated cells (38% with 50 nM 17 beta-estradiol, P < 0.05 and 48% by 500 nM 17 beta-estradiol, P < 0.05). Basal secretion of leptin was equal in female and male adipose tissue, whereas the effects of 17 beta-estradiol (50 nM) and dexamethasone were significantly increased in female as compared with male adipose tissue. Progesterone, testosterone, dihydrotestosterone and dehydroepiandrostendione-sulfate neither affected leptin secretion in male nor female adipose tissue in vitro. Furthermore, to investigate the effect of estrogen female rats were ovariectomized (OVX) and the adipose tissue was incubated in vitro and compared with adipose tissue leptin secretion from sham operated rats (SHAM), and with ovariectomized rats treated with 17 beta-estradiol (EST). A decreased basal and dexamethasone-stimulated leptin secretion from OVX rats compared with SHAM rats was found (P < 0.005) whereas 17 beta-estradiol treatment of ovariectomized rats maintained a normal leptin secretion. However, the dexamethasone stimulation was equally increased above basal levels in SHAM, OVX and EST rats (3.7 +/- 1.2, 2.9 +/- 0.8, 4.2 +/- 1.4, NS, ANOVA) respectively.     

Dinitrophenylation of rabbit skeletal actomyosin.

Dinitrophenylated reconstituted or natural actomyosin effected changes in the Ca2+ sensitivity which were dependent upon the ionic strength of the reaction medium. Dinitrophenylation of reconstituted actomyosin in 0.6 M KCl led to the incorporation of 2-6 mol of the reagent per 5-10(5) g of protein and it possessed considerable Ca2+ sensitivity. Dinitrophenylated natural actomyosin under the same conditions lost most of its Ca2+ sensitivity when 1.3-5.4 mol of the dinitrophenyl group were bound. The myosin from these modified actomyosins did not lose Ca2+ sensitivity and the myosin was labeled only with 0.4-1.7 mol of the dinitrophenyl group. Dinitrophenylation of both kinds of actomyosin in 0.06 M KCl abolished the Ca2+ sensitivity; the myosin from the modified actomyosins also lost Ca2+ sensitivity. Myosin alone was more susceptible to a loss of Ca2+ sensitivity than myosin in actomyosin. Actin protected the ability of myosin to sense Ca2+ regulated actin in modified actomyosin at 0.6 M KCl but not at 0.06 M KCl. Actomyosin dinitrophenylated in the presence of ATP lost Ca2+ sensitivity. However, the myosin from this actomyosin possessed Ca2+ sensitivity. Thiolysis of the dinitrophenylated actomyosin by 2-mercaptoethanol at low ionic strength did not restore the Ca2+ sensitivity of this actomyosin or its myosin although there was a significant loss of the dinitrophenyl group.     

The effect of ovariectomy on the contractile response of the rat isolated detrusor muscle and urethra

Contractile responses induced by carbachol on the detrusor muscle and by noradrenaline on the isolated urethra were compared between ovariectomized rats pretreated with estradiol (50 μg/animal s.c. twice daily for five days), untreated ovariectomized rats and intact animals. In the detrusor muscle, contractions induced by 30μM carbachol, when normalized with respect to KCl 100 mM-induced contraction, were similar for the three groups. Furthermore, contractions induced by 100 μM noradrenaline in the isolated urethra were not significatively different between groups. However, the pD₂ value for noradrenaline was greater in urethral tissue from ovariectomized rats compared with ovariectomized -estrogen treated and control rats. A similar result was found for pD₂ values for carbachol-induced contractions on the detrusor muscle. These results suggest that ovariectomy increases the sensitivity of the urinary bladder and urethra to the contractile effects of carbachol and noradrenaline, respectively and that this effect is reversed by in vivo estrogen pretreatment.     

Immunological analysis of the biochemical properties of the uterine estrogen receptor

Immunohistochemical and immunochemical analysis using Western blot techniques were carried out with estrogen receptor (ER) monoclonal antibody H-222 to 1) clarify the "nuclear translocation" phenomenon of ER, 2) elucidate the primary nuclear binding site of ER, and 3) to evaluate the binding force between ER and its nuclear binding site in the uterus of ovariectomized adult mice. Exclusive nuclear localization of ER was recognized in the epithelial cells, stroma cells, and smooth muscle cells. Uterine tissues prepared from animals injected with saline, 17 beta-estradiol (E2), estriol (E3), and diethylstilbestrol (DES) exhibited almost the same ER immunostaining when they were fixed prior to sectioning (prefixation method) and frozen sections were used. On the other hand, when fresh-frozen sections were fixed before or after incubation with various solutions (postfixation method) and then treated with various salt solutions, greater differences were seen in immunostaining of ER between saline-injected and hormone-treated animals. Immunostaining of ER in control animals was low after incubation with PBS (0.01 M phosphate buffer containing 0.16 M NaCl, pH 7.2), whereas uterine tissue from hormone-injected mice showed strong nuclear immunostaining after this treatment. After treatment with 0.4 M KCl or 0.5 M NaCl, immunostaining in the uterus of both hormone-injected and control animals was completely abolished. DNase treatment caused an almost complete loss of immunostaining of ER; however, RNase digestion slightly increased immunoreactivity in both E2-injected and control animals. Quantitative analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot techniques showed that after incubation of tissue sections for 30 min with PBS, 0.4 M KCl, or DNase, 60%, 10%, and 30% of ER were present, respectively, compared to amount of ER present in unincubated sections. These findings suggest the following for the ER in uterine tissue; nuclear occupancy is a phenomenon that occurs due to a differential affinity between occupied and unoccupied receptors in the nucleus; after hormone treatment, the receptor levels do not fluctuate in the nucleus to the extent demonstrated by binding assays; and the properties of the ER detected in the immunohistochemical analysis are identical to those observed in biochemical studies.