Free E2 Research Articles

Dissipation and transformation of 17β-estradiol-17-sulfate in soil–water systems

In the environment, estrogen conjugates can be precursors to the endocrine-disrupting free estrogens, 17β-estradiol (E2) and estrone (E1). Compared to other estrogen conjugates, 17β-estradiol-17-sulfate (E2-17S) is detected at relatively high concentrations and frequencies in animal manure and surface runoff from fields receiving manure. To elucidate the lifecycle of manure-borne estrogens and their conjugates in the environment, the fate of radiolabelled E2-17S in agricultural soils was investigated using laboratory batch studies with soils of different organic carbon (OC) content (1.29% for topsoil versus 0.26% for subsoil). E2-17S was found relatively persistent in the aqueous phase throughout the duration of the 14 d experiment. The aqueous E2-17S persisted longer in the subsoil (half-lives (DT50)=64–173h) than the topsoil (DT50=4.9–26h), and the aqueous persistence of E2-17S depended on its initial concentration. The major transformation pathway was hydroxylation, yielding mono- and di-hydroxy-E2-17S (OH-E2-17S and diOH-E2-17S). Free estrogens, E2 and E1, were only observed in the sorbed phase of the soil at low concentrations (∼1% of applied dose), which demonstrated that deconjugation and subsequent oxidation had occurred. Although deconjugation was not a major pathway, E2-17S could be a precursor of free estrogens in the environment.

Sex Steroid Hormone Levels and Body Composition in Men

Previous studies indicate that testosterone (T) is positively correlated with lean mass and inversely correlated with fat mass in men; however, the directionality of these associations, as well as the association with other hormones including estradiol (E2) and SHBG, is unclear. We examined cross-sectional and longitudinal associations of E2, T, SHBG, and E2/T ratio with body composition among men ages 30 to 79 in the Boston Area Community Health/Bone Survey. Total, trunk, and appendicular lean and fat mass were measured by dual-energy x-ray absorptiometry at baseline, and weight and waist/hip circumference were measured at baseline and follow-up. Partial Pearson correlation coefficients were used to estimate the linear relationship between each body composition measure and log-transformed hormone variable. In cross-sectional analyses of 821 men, T, calculated free T, and SHBG were inversely correlated with fat mass, weight, body mass index, waist/hip circumference, and waist-to-hip ratio, with multivariable-adjusted correlations ranging from -0.13 to -0.37. Calculated free E2 was positively correlated with percentage total (r = .13) and trunk (r = .15) fat mass, and E2/T was positively correlated with all measures examined (r = .13-.40). There were no significant multivariable-adjusted longitudinal associations between baseline hormone levels and change in weight, body mass index, waist/hip circumference, or waist-to-hip ratio after an average follow-up of 4.8 years. We observed significant cross-sectional associations between hormone levels, including E2, T, and E2/T, and body composition measures in men. Longitudinal analyses showing no influence of baseline hormone levels on change in anthropometric measures imply that body composition affects hormone levels and not the reverse.

Short-term changes in serum sex steroid levels and cardiac function in healthy young men

Introduction: Male obesity is associated with an increase in estradiol (E2) and a decrease in testosterone (T). And, although sex steroids are associated with cardiovascular disease, direct effects on cardiac structure and function are hardly investigated in humans. Methodology: Twenty healthy men aged 20–40 years were randomized into two groups. One group was given an aromatase inhibitor (letrozole) only, thus obtaining a high testosterone and low E2 (group T). The other group received an aromatase inhibitor plus an E2 patch (dermestril), reaching a low testosterone and high E2 (group E). Serum levels of both testosterone and E2 remained within the normal reference range. The men underwent an echocardiography by a single cardiologist before the start of the intervention and after 7 days. Results: Total and free E2 serum levels were positively associated with ejection fraction (r=0.7, P=0.002 and r=0.6, P=0.007 respectively) at baseline in the whole group. In group E global circumferential strain decreased significantly from −25.3%±3.9 to −19.6%±2.5 after 1 week as compared to baseline (P=0.01). No significant changes in systolic function were observed in group T. Cardiac structure remained unaltered. Conclusion: In young healthy men, an increase in E2 and decrease in testosterone levels significantly decreased circumferential strain. The finding justifies larger studies of longer duration to discover the exact nature of the impact of changed sex steroids on cardiac function and remodelling in obesity.

Sarcolipin Protein Interaction with Sarco(endo)plasmic Reticulum Ca2+ATPase (SERCA) Is Distinct from Phospholamban Protein, and Only Sarcolipin Can Promote Uncoupling of the SERCA Pump

Sarco(endo)plasmic reticulum Ca(2+)ATPase (SERCA) pump activity is modulated by phospholamban (PLB) and sarcolipin (SLN) in cardiac and skeletal muscle. Recent data suggest that SLN could play a role in muscle thermogenesis by promoting uncoupling of the SERCA pump (Lee, A.G. (2002) Curr. Opin. Struct. Biol. 12, 547-554 and Bal, N. C., Maurya, S. K., Sopariwala, D. H., Sahoo, S. K., Gupta, S. C., Shaikh, S. A., Pant, M., Rowland, L. A., Bombardier, E., Goonasekera, S. A., Tupling, A. R., Molkentin, J. D., and Periasamy, M. (2012) Nat. Med. 18, 1575-1579), but the mechanistic details are unknown. To better define how binding of SLN to SERCA promotes uncoupling of SERCA, we compared SLN and SERCA1 interaction with that of PLB in detail. The homo-bifunctional cross-linker (1,6-bismaleimidohexane) was employed to detect dynamic protein interaction during the SERCA cycle. Our studies reveal that SLN differs significantly from PLB: 1) SLN primarily affects the Vmax of SERCA-mediated Ca(2+) uptake but not the pump affinity for Ca(2+); 2) SLN can bind to SERCA in the presence of high Ca(2+), but PLB can only interact to the ATP-bound Ca(2+)-free E2 state; and 3) unlike PLB, SLN interacts with SERCA throughout the kinetic cycle and promotes uncoupling of the SERCA pump. Using SERCA transmembrane mutants, we additionally show that PLB and SLN can bind to the same groove but interact with a different set of residues on SERCA. These data collectively suggest that SLN is functionally distinct from PLB; its ability to interact with SERCA in the presence of Ca(2+) causes uncoupling of the SERCA pump and increased heat production.

Abstract B89: Endogenous hormone levels and lobular involution of the breast among healthy, postmenopausal women in the Komen Tissue Bank Study

Abstract Background: Terminal ductal lobular units (TDLUs), the functional units of the breast for lactation, are the structures from which most breast cancers arise. TDLU involution, reflecting atrophy and loss of TDLUs, has been related to lower subsequent breast cancer risk. The predictors of such breast tissue aging, however, are not well defined. Given that estrogens and other hormones play a central role in the etiology of postmenopausal breast cancers, we conducted a study to assess the relationship between four hormones and sex hormone-binding globulin (SHBG) with markers of TDLU involution. Methods: Subjects included healthy women who volunteered between 2009 and 2011 to participate in the Komen Tissue Bank Study. Participants completed a risk factor questionnaire, provided a blood sample, and donated breast tissue for research. Our analysis was restricted to postmenopausal women who were not currently taking menopausal hormones (n=153). A pathologist, masked to patient data, assessed tissue sections for the presence of TDLUs and, among sections containing TDLUs (n=88), measured TDLU diameters for up to ten TDLUs per section. Both presence of TDLUs and TDLU diameter are inversely related to involution. Chemiluminescent immunometric assays were used to measure serum levels of estradiol (E2), SHBG, follicle-stimulating hormone (FSH), and prolactin. Percent free E2 was calculated from measured E2 and SHBG levels. Logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for the presence of TDLUs (none or any) associated with tertiles of serum hormone and SHBG levels. Multivariable ordinal logistic regression was used to calculate ORs, 95% CIs, and p-trend for TDLU diameter categorized as tertiles (≥218.4, 218.4-281, and ≥281 microns) with serum hormone and SHBG levels. All models were adjusted for potential confounding factors. Results: The study population was 87.6% Caucasian with a mean age of 59.5 years, and >2/3 of the women were overweight or obese. Increasing percent free E2 was significantly associated with larger TDLU diameter (OR>2.53 vs. ≥1.99: 5.58, 95% CI: 1.48-21.07; p-trend=0.01), whereas increasing SHBG was associated with smaller TDLU diameter (OR>73nmol/L vs. ≥38 nmol/L: 0.19, 95% CI: 0.05-0.74; p-trend=0.02). Total E2 (p-trend=0.49), FSH (p-trend=0.85), and prolactin (p-trend=0.58) were not associated with TDLU diameter. We did not find significant relationships between the presence of TDLUs and any of the serum levels. Our observed relationships were not modified by age or other breast cancer risk factors, including BMI, years since menopause, and history of bilateral oophorectomy. Conclusion: Based on limited data, these findings suggest that sex hormones may play a role in TDLU involution. Thus, TDLU involution may represent one mechanism by which hormone levels influence breast cancer risk. TDLU measurements may therefore be helpful in assessing breast cancer risk as a marker of breast tissue aging. Analysis of the relationships between hormones and TDLU involution among premenopausal women may provide additional insights into early events in breast carcinogenesis. Citation Format: Zeina G. Khodr, Mark E. Sherman, Ruth M. Pfeiffer, Gretchen L. Gierach, Louise A. Brinton, Roni T. Falk, Deesha Patel, Laura Linville, Susan E. Clare, Daniel Visscher, Carolyn Mies, Stephen Hewitt, Anna Maria Storniolo, Jonine D. Figueroa. Endogenous hormone levels and lobular involution of the breast among healthy, postmenopausal women in the Komen Tissue Bank Study. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B89.

Adiposity and sex hormones across the menstrual cycle: the BioCycle Study

ObjectiveTo investigate the influence of adiposity on patterns of sex hormones across the menstrual cycle among regularly menstruating women.SubjectsThe BioCycle Study followed 239 healthy women for 1–2 menstrual cycles, with up to 8 visits per cycle timed using fertility monitors.MethodsSerum estradiol (E2), progesterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and sex hormone binding globulin (SHBG) were measured at each visit. Adiposity was measured by anthropometry and by dual energy x-ray absorptiometry. Differences in hormonal patterns by adiposity measures were estimated using nonlinear mixed models, which allow for comparisons in overall mean levels, amplitude (i.e. lowest to highest level within each cycle), and shifts in timing of peaks while adjusting for age, race, energy intake, and physical activity.ResultsCompared to normal weight women (n=154), obese women (BMI≥30 kg/m2, n=25) averaged lower levels of progesterone (−15%, P=0.003), LH (−17%, P=0.01), FSH (−23%, P=0.001) and higher free E2 (+22%, p=0.001) across the cycle. To lesser magnitudes, overweight women (BMI: 25–30, n=60) also exhibited differences in the same directions for mean levels of free E2, FSH, and LH. Obese women experienced greater changes in amplitude of LH (9%, p=0.002), and FSH (8%, p=0.004), but no differences were observed among overweight women. Higher central adiposity by top compared to bottom tertile of trunk-to-leg fat ratio by DXA was associated with lower total E2 (−14%, p=0.005) and FSH (−15%, p=0.001). Peaks in FSH and LH occurred later (~0.5 day) in the cycle among women with greater central adiposity.ConclusionGreater total and central adiposity were associated with changes in mean hormone levels. The greater amplitudes observed among obese women suggest compensatory mechanisms at work to maintain hormonal homeostasis. Central adiposity may be more important in influencing timing of hormonal peaks than total adiposity.

Estrogen receptors alpha (rs2234693 and rs9340799), and beta (rs4986938 and rs1256049) genes polymorphism in prostate cancer: Evidence for association with risk and histopathological tumor characteristics in Iranian men

We evaluated the effect of estrogen receptor (ER)-α and ER-β genes polymorphisms on development of prostate cancer (PCa) and its correlation with serum reproductive hormones and with clinicopathological characteristics in a sample of Iranian men. One hundred sixty-two men with PCa (mean age 63.7 ± 3.4 years) and 324 age-matched healthy controls (mean age 63.1 ± 3.2 years) were recruited in this study. Genotypes for ER-α and ER-β genes polymorphisms were identified by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Serum levels of reproductive hormones were also measured. Of PCa patients, 38.3%, and 61.7% had localized and advanced tumor, and 45.7%, and 54.3%, had low grade and high-grade cancer, respectively. There was a significant difference in genotype frequency distribution of ER-α gene polymorphism (P = 0.002), and ER-β gene polymorphism (P = 0.003) between cancer patients and controls. The ER-α Pvull C allele carriers (TC or CC) had a significantly increased risk of PCa compared with the TT homozygotes [odds ratio (OR) 3.12; 95% confidence interval (CI) 1.87-5.84, and OR = 4.73, 95% CI:2.44-7.33, respectively]. It was also found that the ER-α XbaI AG (OR = 4.36; 95% CI:2.47-6.68; P = 0.001) and ER-β AluI AG (OR = 2.66, 95% CI:1.61-4.16; P = 0.004) genotypes were significantly associated with increased risk of PCa. The ER-β RsaI genotype was not associated with PCa. Baseline serum free E2 levels tended to be lower in men with PCa (0.35 ± 0.04 pg/ml) compared to healthy men (0.48 ± 0.05 pg/ml). Genotypes which confer susceptibility for developing PCa, accompanied with lowest serum levels of free E2. In the Iranian population, genetic polymorphisms of the ER-α and ER-β genes may be involved in the etiology of PCa.

Inhibition of mitochondrial Na+-dependent Ca2+ efflux by 17β-estradiol in the rat hippocampus

Our results, as well as those of others, have indicated that 17β-estradiol (E2) exerts its nongenomic effects in neuronal cells by affecting plasma membrane Ca(2+) flux. In neuronal cells mitochondria possess Ca(2+) buffering properties as they both sequester and release Ca(2+). The goal of this study was to examine the rapid non-genomic effect of E2 on mitochondrial Ca(2+) transport in hippocampal synaptosomes from ovariectomised rats. In addition, we aimed to determine if, and to what extent, E2 receptors participated in mitochondrial Ca(2+) transport modulation by E2 in vitro. E2-specific binding and Ca(2+) transport was monitored. At physiological E2 concentrations (0.1-1.5 nmol/L), specific E2 binding to mitochondria isolated from hippocampal synaptosomes was detected with a B(max.) and K(m) of 37.6±2.6 fmol/mg protein and 0.69±0.14 nmol/L of free E2, respectively. The main mitochondrial Ca(2+) influx mechanism is the Ruthenium Red-sensitive uniporter driven by mitochondrial membrane potential. Despite no effect of E2 on Ca(2+) influx, a physiological E2 concentration (0.5 nmol/L) protected mitochondrial membrane potential and consequently Ca(2+) influx from the uncoupling agent carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (1 μmol/L). In neuronal cells the predominant mitochondrial Ca(2+) efflux mechanism is the Na(+)/Ca(2+) exchanger. E2 caused Ca(2+) efflux inhibition (by 46%) coupled with increased affinity of the Na(+)/Ca(2+) exchanger for Na(+). Using E2 receptor (ERα and ERβ) antagonists and agonists, we confirmed ERβ's involvement in E2-induced mitochondrial membrane potential protection as well as Ca(2+) efflux inhibition. In summary, our results indicate that the non-genomic neuromodulatory role of E2 in rat hippocampus is achieved by affecting mitochondrial Ca(2+) transport via, in part, mitochondrial ERβ.

Non-nuclear Estrogen Receptor Signaling in the Endothelium

In addition to the classical function of estrogen receptors (ER) as transcription factors, evidence continues to accumulate that they mediate non-nuclear processes in numerous cell types, including the endothelium, in which they activate endothelial NO synthase. Non-nuclear ER signaling entails unique post-translational modifications and protein-protein interactions of the receptor with adaptor molecules, kinases, and G proteins. Recent in vitro and in vivo studies in mice using an estrogen-dendrimer conjugate that is excluded from the nucleus indicate that non-nuclear ER activation underlies the migration and growth responses of endothelial cells to estrogen but not the growth responses of endometrial or breast cancer cells to the hormone. In this minireview, the features of ERα and protein-protein interactions that enable it to invoke extranuclear signaling in the endothelium and the consequences of that signaling are discussed.

Testosterone but not estradiol level is positively related to muscle strength and physical performance independent of muscle mass: a cross-sectional study in 1489 older men

To examine the relationship between different measures of testosterone and estradiol (E(2)), muscle mass, muscle strength, and physical performance; and to test whether the association of sex hormone level with muscle strength and physical performance was independent of muscle mass. A cross-sectional survey on 1489 community-dwelling men older than 64 years of age. Serum levels of testosterone and E(2) were measured by mass spectrometry, and sex hormone-binding globulin (SHBG) levels were measured by immunoradioassay. Muscle mass was examined by dual-energy X-ray absorptiometry and physical performance was assessed by hand-grip strength, gait speed, step length and chair-stand test. Appendicular skeletal mass (ASM) was positively associated with total testosterone (TT; P<0.001), free testosterone (FT; P<0.001), and total E(2) (P<0.001) but not with free E(2) (P=0.102). After adjustment for age, serum SHBG and relative ASM, both TT and FT were significantly associated with grip strength, narrow-walk speed and the composite neuromuscular score. Higher total E(2), but not free E(2) was associated with lower grip strength (P<0.05) after adjustment for age, FT, SHBG and relative ASM. Testosterone level was related to both muscle mass, strength and physical performance. Total E(2) level, though related to muscle mass positively, affected muscle strength adversely in older men.

Impact of Serum Thyroid Hormones and Estrogen Status on the Risk of Breast Cancer in Kashmiri Women

Estrogen involvement in breast cancer has been established; however, the association between breast cancer and thyroid diseases is controversial. Estrogen-like effects of thyroid hormone on breast cancer cell growth in culture have been reported. The aim of our study was to investigate a possible correlation between the genesis of breast cancer and the levels of serum thyroid hormones. we measured the percentage of free estradiol (E2) and the amounts of sex hormone-binding globulin (SHBG) and thyroid hormones in serum samples from Kashmiri patients with breast cancer (n=50) and normal controls (n=25). The patients were found to have significantly higher free E2 and significantly lower SHBG than controls. Moreover, the serum levels of free triiodothyronine (FT3) and free thyroxine (FT4) were lower in the patients than in controls, while the serum levels of TSH and TBG in the patients were not significantly different from those in controls. These results suggest that the reduction in the serum FT3, and FT4 levels, which is independent of changes in the serum level of free E2, may be one of the risk factors for breast cancer in Kashmir.

Mechanism of E1-E2 Interaction for the Inhibition of Ubl Adenylation

Conjugates of ubiquitin or its homologues to other proteins occur by strictly ordered steps with ordered addition of substrates for each step. High concentrations of E2 were shown to inhibit the formation of E2∼Ubl thioester and Ubl∼target conjugates. We investigated the mechanism of such inhibitory effect of the SUMO E2 and whether the E2 has two binding sites on its E1, one for the inhibitory effect and one for productive SUMOylation. NMR methods in combination with mutagenesis and biochemical assays revealed that Ubc9 binds to two flexible domains of its free E1 simultaneously, suggesting extensive domain movements in the free E1. Further, interaction of free E1 and E2 inhibits SUMO adenylation, and the interfaces responsible for the inhibition were the same as those required for productive transfer of SUMO from E1 to E2. This study indicates a conformational flexibility-dependent mechanism to control the strictly ordered steps in Ubl modifications.

The Effects of Injected Testosterone Dose and Age on the Conversion of Testosterone to Estradiol and Dihydrotestosterone in Young and Older Men

During testosterone (T) therapy, T is partly converted to 17beta-estradiol (E2) and 5alpha-dihydrotestosterone (DHT). Effects of age, testosterone dose, and body composition on total and free E2 and DHT levels are unknown. We evaluated age and dose-related differences in E2 and DHT levels in response to graded doses of testosterone enanthate in young and older men. Fifty-one young (aged 19-35 yr) and 52 older (aged 59-75 yr) men completed treatment with monthly injections of a GnRH agonist plus randomly assigned weekly doses of testosterone enanthate (25, 50, 125, 300, or 600 mg) for 5 months. During testosterone administration, total and free E2 levels increased dose-dependently (dose effect, P<0.001) in both young and older men. Total and free E2 levels and E2:T ratios during T administration were higher in older than young men, but age-related differences in free E2 and free E2:T ratios were not significant after adjusting for testosterone levels, percentage fat mass, and SHBG. DHT levels and DHT:T ratios were dose-related but did not differ between young and older men. Mechanistic modeling of free hormone data revealed that the conversions of T to E2 and DHT were both consistent with saturable Michaelis-Menten kinetics. The in vivo Km values were estimated to be 1.83 nm for aromatase and 3.35 nm for 5alpha-reductase, independent of age. The Vmax parameter for E2 was 40% higher in older men than younger men, but Vmax for DHT was not significantly different between age groups. During im testosterone administration, E2 and DHT levels exhibit saturable increases with dose. The rate of whole body aromatization is higher in older men, partly related to their higher percentage fat mass, SHBG, and testosterone levels.

Estradiol and Metabolic Syndrome in Older Italian Men: The InCHIANTI Study

The increasing prevalence of metabolic syndrome (MS) with age in older men has been linked with decreasing testosterone levels. Interestingly, while testosterone levels decline with age, estradiol (E2) levels remain relatively stable, resulting in a decreased testosterone:E2 ratio. Because E2 levels tend to be elevated in morbid obesity, insulin resistance, and diabetes, it is reasonable to hypothesize that high E2 levels are associated with MS in older men. We studied the relationship of total and free E2 with MS after adjustment for multiple confounders, including age, BMI, smoking, alcohol consumption, physical activity, interleukin-6 (IL-6), fasting insulin, and testosterone. Men 65 years or older (age range, 65-96; n = 452) had complete data on E2, testosterone, fasting insulin, sex hormone-binding globulin, IL-6, and albumin. Concentrations of free E2 and free testosterone were calculated using the mass action equations. MS was defined according to Adult Treatment Panel III (ATP-III). Participants with MS had significantly higher serum free and total E2 (P < .001) (P = .003). After adjusting for confounders, including age, smoking, alcohol consumption, physical activity, log(IL-6), and log(insulin), participants with higher log(total E2) (odds ratio [OR], 2.31; 95% confidence interval [95% CI], 1.39-4.70; P = .02) and higher log(free E2) (OR, 2.69; 1.38-5.24; P < .001) had an increased risk of having MS. Log(free E2) (P = .04) maintained significant correlation with MS, even after further adjustment for BMI. In older men, high E2 is independently associated with MS. Whether confirmed in other studies, assessment of E2 should be also considered in older men. Whether changes in this hormonal pattern play a role in the development of MS should be further tested in longitudinal studies.

Seizure frequency and sex steroids in women with partial epilepsy on antiepileptic therapy

Neuroactive sex steroids influence neuron excitability, which is enhanced by estradiol (E2) and decreased by progesterone (Pg). In epilepsy, the production, metabolism, biologic availability, and activity of sex hormones may be affected by seizures themselves or by antiepileptic drugs (AEDs). This cross-sectional observational study was aimed at evaluating the relationships between sex steroids, seizure frequency, and other clinical parameters in women with partial epilepsy (PE) on AED treatments. Serum E2, Pg, sex hormone binding globulin (SHBG) levels, free E2 (fE2), and E2/Pg ratios were determined during the follicular and luteal phases in 72 adult women with PE, and in 30 healthy controls. Hormonal data were correlated with seizure frequency, age, body weight, body mass index (BMI), disease onset and duration, and AED therapies. In patients, E2, fE2, and Pg levels were lower in both ovarian phases, whereas those of SHBG were higher than in controls. No significant changes in hormone levels and in prevalence of anovulatory cycles were observed between patients grouped according to their seizure frequency. However, when compared with those in healthy controls, luteal fE2 and Pg levels were chiefly impaired in women with more frequent seizures, mostly undergoing AED polytherapies, but not in those with absent or rarer seizures. The actual changes in sex steroid levels and E2/Pg ratios did not explain an increased seizure frequency in adult women with AED-treated PE, but patients with more severe disease showed more relevant changes in their sex hormone profile and impaired Pg levels during the luteal phase.

Dynamic Partnership between KCNQ1 and KCNE Subunits

Background: (KCN)Q1 and (KCN)E1 associate to form IKs channels important for cardiac electrical stability. (KCN)E2 is also expressed in the heart and can associate with Q1 or Q1/E1 to suppress the current amplitude. What regulate the partnerships of Q1/Ex (x = 1 or 2) and their stability are not clear. Methods: We use COS-7 & oocyte expression systems to test (a) Q1/Ex stability by pulse-chase experiments, (b) ability of E1 or E2 to traffic to cell surface without/with Q1, and (c) ability of free E1 or E2 peptides to associate with lone Q1 channels in cell membrane. Results: Pulse chase-experiments in COS-7 cells show that Q1 has a mean turnover τ (τturnover) of 13 hr, while Q1-associated E1 (co-immunoprecipitated with Q1) has a τturnover of 6 - 7 hr, suggesting E1 turnover in Q1/E1 complexes. Biotinylation experiments show that E1 expressed alone in COS-7 traffics to the cell surface inefficiently. Q1 coexpression leads to a 5-fold increase in cell surface E1. In contrast, E2 expressed alone in COS-7 traffics more efficiently to cell surface, and Q1 coexpression does not further increase cell surface E2. Injecting vesicles made from E1-expressing COS-7 cells into control oocytes (expressing native Q1) or oocytes pre-injected with Q1 cRNA produces an IKs-like current after a few hours, while injecting ‘empty’ vesicles from untransfected COS-7 cells has not effects. Likewise, injecting vesicles from COS-7 cells expressing E2 (with an extracellular epitope tag) into Q1-expressing oocytes leads to a decrease in current amplitude and appearance of cell surface epitope. Thus, vesicle-delivered E1 or E2 peptides can associate with lone Q1 channels and modulate their function. Proposal: In cardiac mycoytes coexpressing Q1, E1 & E2, both KCNE subunits can associate with Q1 and modulate cardiac IKs channel function in a dynamic fashion.

Gender-Related Differences in Muscle Injury, Oxidative Stress, and Apoptosis

Due to its alleged antioxidant properties, 17beta-estradiol (E2) may protect against muscle injury, oxidative stress, and apoptosis. This study sought to determine whether such mechanisms existed between genders for muscle injury, oxidative stress, and apoptosis after eccentric exercise. Eight men and eight women (no oral contraceptive use; midluteal phase of menstrual cycle) performed 7 x 10 eccentric repetitions of the knee extensors at 150% 1RM. Strength, soreness, and blood samples were taken before exercise and 6, 24, 48, and 72 h after exercise while muscle samples were collected before and 6 and 24 h after exercise. Blood samples were assayed for free E2, lactate dehydrogenase (LDH), superoxide dismutase (SOD), and 8-isoprostane (8-iso). Muscle samples were assayed for mitochondrial apoptosis (e.g., bax, bcl-2, cytochrome c, and cell death), total DNA content, and myofibrillar protein content. Men reported greater soreness levels at 24, 48, and 72 h after exercise, whereas strength changes were similar among genders. At baseline and independent of exercise, females had higher E2 (P < 0.001) and SOD in conjunction with lower 8-iso levels when compared with men. Bax increased in both genders, whereas bcl-2 increased only in women with no cytochrome c changes for either gender after exercise. The bax/bcl-2 ratio in women significantly decreased after 6 h (P = 0.03) and returned to baseline levels after 24 h. Men exhibited greater cell death at all time points (P < 0.05), whereas myofibrillar protein content and total DNA content decreased in both genders at 24 h after exercise. No changes in LDH were found (P > 0.05). Although more research is needed, differences between gender may provide greater endogenous protection against oxidative stress and apoptosis.

Relations between endogenous androgens and estrogens in postmenopausal women with suspected ischemic heart disease.

Because androgens are obligatory precursors of estrogens, it is reasonable to assume that their serum concentrations would exhibit positive correlations. If so, then epidemiologic studies that examine the association between androgens and pathological processes should adjust the results for the independent effect of estrogens. The objective of the study was to examine the interrelationships among testosterone (T), androstenedione, estradiol (E2), estrone, and SHBG in postmenopausal women. This was a cross-sectional study of women participating in the National Heart, Blood, and Lung Institute-sponsored Women's Ischemia Syndrome Evaluation study. The study was conducted at four academic medical centers. A total of 284 postmenopausal women with chest pain symptoms or suspected myocardial ischemia. Post hoc analysis of the relationships among sex steroid hormones with insulin resistance, body mass index (BMI), and presence or absence of coronary artery disease as determined by coronary angiography. BMI was significantly associated with insulin resistance, total E2, free E2, bioavailable E2, and free T. Highly significant correlations were found for total T, free T, and androstenedione with total E2, free E2, bioavailable E2, and estrone and persisted after adjustment for BMI and insulin resistance. A significant relationship was present between total and free T and the presence of coronary artery disease after adjustment for the effect of E2. Serum levels of androgens and estrogens track closely in postmenopausal women referred for coronary angiography for suspected myocardial ischemia. Epidemiological studies that relate sex steroid hormones to physiological or pathological processes need to control for the independent effect of both estrogens and androgens.

Side-Chain Protonation and Mobility in the Sarcoplasmic Reticulum Ca 2+-ATPase: Implications for Proton Countertransport and Ca 2+ Release

Protonation of acidic residues in the sarcoplasmic reticulum Ca 2+-ATPase (SERCA 1a) was studied by multiconformation continuum electrostatic calculations in the Ca 2+-bound state Ca 2E1, in the Ca 2+-free state E2(TG) with bound thapsigargin, and in the E2P (ADP-insensitive phosphoenzyme) analog state with Mg F 4 2 − E 2 ( TG + Mg F 4 2 − ) . Around physiological pH, all acidic Ca 2+ ligands (Glu 309, Glu 771, Asp 800, and Glu 908) were unprotonated in Ca 2E1; in E2(TG) and E 2 ( TG + Mg F 4 2 − ) Glu 771, Asp 800, and Glu 908 were protonated. Glu 771 and Glu 908 had calculated pK a values larger than 14 in E2(TG) and E 2 ( TG + Mg F 4 2 − ) , whereas Asp 800 titrated with calculated pK a values near 7.5. Glu 309 had very different pK a values in the Ca 2+-free states: 8.4 in E 2 ( TG + Mg F 4 2 − ) and 4.7 in E2(TG) because of a different local backbone conformation. This indicates that Glu 309 can switch between a high and a low pK a mode, depending on the local backbone conformation. Protonated Glu 309 occupied predominantly two main, very differently orientated side-chain conformations in E 2 ( TG + Mg F 4 2 − ) : one oriented inward toward the other Ca 2+ ligands and one oriented outward toward a protein channel that seems to be in contact with the cytoplasm. Upon deprotonation, Glu 309 adopted completely the outwardly orientated side-chain conformation. The contact of Glu 309 with the cytoplasm in E 2 ( TG + Mg F 4 2 − ) makes this residue unlikely to bind lumenal protons. Instead it might serve as a proton shuttle between Ca 2+-binding site I and the cytoplasm. Glu 771, Asp 800, and Glu 908 are proposed to take part in proton countertransport.

Differences in Free Estradiol and Sex Hormone-Binding Globulin in Women with and without Premenstrual Dysphoric Disorder

Over the years, different hypotheses involving the ovarian steroid hormones have been proposed to explain the luteal phase occurrence of severe premenstrual syndrome symptoms. Although it had been strongly suspected that differences in the concentrations of the ovarian steroids may underlie the mood and psychological imbalance of this disorder, the evidence for this hypothesis has been inconsistent and remains controversial. Our objective was to measure the ovarian steroid hormones across the menstrual cycle in women with and without luteal phase symptoms consistent with premenstrual dysphoric disorder (PMDD). We measured estradiol (E2), progesterone, and SHBG in women with and without PMDD using a cross-sectional and prospective experimental design. Participating women underwent 2-month self-assessment symptom screening and 1-month hormonal evaluation. Overall means for LH, progesterone, E2, peak E2, and free E2 were not different between groups. Across the menstrual cycle, overall percent free E2 was significantly lower and SHBG significantly greater in the PMDD group compared with controls (1.39 +/- 0.26 vs. 1.50 +/- 0.28, P = 0.03; 61.4 +/- 25.1 vs. 52.4 +/- 21.3 nmol/liter, P = 0.046, respectively). During the luteal phase, free E2 was significantly lower in the PMDD group compared with controls (PMDD 7.6 +/- 7.0 vs. controls 8.9 +/- 8.4 pmol/liter; P = 0.032). For both follicular and luteal phases, SHBG was significantly higher in the PMDD group (follicular phase 60.5 +/- 31.7 vs. 51.4 +/- 38.2 nmol/liter, P = 0.047; luteal phase 65.1 +/- 32.3 vs. 55.1 +/- 38.9 nmol/liter, P =0.03). In both groups, SHBG significantly increased from the follicular to luteal phase. Luteal phase concentrations of free E2, percent free E2, and SHBG differ significantly between women with and without PMDD.