Cryoelectron microscopy of Na+,K+-ATPase in the two E2P states with and without cardiotonic steroids.

Cryoelectron microscopy (cryo-EM) was applied to Na+,K+-ATPase (NKA) to determine the structures of two E2P states, one (E2PATP) formed by ATP and Mg2+ in the forward reaction, and the other (E2PPi) formed by inorganic phosphate (Pi) and Mg2+ in the backward reaction, with and without ouabain or istaroxime, representatives of classical and new-generation cardiotonic steroids (CTSs). These two E2P states exhibit different biochemical properties. In particular, K+-sensitive acceleration of the dephosphorylation reaction is not observed with E2PPi, attributed to the presence of a Mg2+ ion in the transmembrane cation binding sites. The cryo-EM structures of NKA demonstrate that the two E2P structures are nearly identical but Mg2+ in the transmembrane binding cavity is identified only in E2PPi, corroborating the idea that it should be denoted as E2PPi·Mg2+. We can now explain why the absence of transmembrane Mg2+ in E2PATP confers the K+ sensitivity in dephosphorylation. In addition, we show that ATP bridges the actuator (A) and nucleotide binding (N) domains, stabilizing the E2PATP state; CTS binding causes hardly any changes in the structure of NKA, both in E2PATP and E2PPi·Mg2+, indicating that the binding mechanism is conformational selection; and istaroxime binds to NKA, extending its aminoalkyloxime group deep into the cation binding site. This orientation is upside down compared to that of classical CTSs with respect to the steroid ring. Notably, mobile parts of NKA are resolved substantially better in the electron microscopy (EM) maps than in previous X-ray structures, including sugars sticking out from the ß-subunit and many phospholipid molecules.

Selective SERCA2a activator as a candidate for chronic heart failure therapy.

BACKGROUND: The sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2a) depression substantially contributes to diastolic dysfunction in heart failure (HF), suggesting that SERCA2a stimulation may be a mechanism-based HF therapy. Istaroxime is a drug endowed with both a SERCA2a stimulatory activity and a Na+/K+ pump inhibitory activity for acute HF treatment. Its main metabolite PST3093 shows a more favorable therapeutic profile as compared to the parent drug, but it is still unsuitable for chronic usage. Novel PST3093 derivatives have been recently developed for oral (chronic) HF treatment; compound 8 was selected among them and here characterized. METHODS: Effects of compound 8 were evaluated in a context of SERCA2a depression, by using streptozotocin-treated rats, a well-known model of diastolic dysfunction. The impact of SERCA2a stimulation by compound 8 was assessed at the cellular level ad in vivo, following i.v. infusion (acute effects) or oral administration (chronic effects). RESULTS: As expected from SERCA2a stimulation, compound 8 induced SR Ca2+ compartmentalization in STZ myocytes. In-vivo echocardiographic analysis during i.v. infusion and after repeated oral administration of compound 8, detected a significant improvement of diastolic function. Moreover, compound 8 did not affect electrical activity of healthy guinea-pig myocytes, in line with the absence of off-target effects. Finally, compound 8 was well tolerated in mice with no evidence of acute toxicity. CONCLUSIONS: The pharmacological evaluation of compound 8 indicates that it may be a safe and selective drug for a mechanism-based treatment of chronic HF by restoring SERCA2a activity.

Effects of maternal androgens and their metabolite etiocholanolone on prenatal development in birds.

Offspring phenotypes can be affected by maternal testosterone and androstenedione (A4), which are considered a tool of mothers to adjust offspring to a fluctuating environment. Yet testosterone and A4 are very rapidly metabolized by developing avian embryos, suggesting that either the maternal testosterone and A4 have potent organizational effects on the embryos extremely early before being metabolized or it is the metabolites that evoke phenotypic variation in the offspring. One of the metabolites, etiocholanolone, increases substantially during early embryonic development and is a likely candidate for mediating maternal effects as it can promote erythropoiesis. To investigate and compare the effects of testosterone and A4 with the possible effects of etiocholanolone during prenatal embryonic development, we increased their levels in black-headed gull eggs (Larus ridibundus), and used sham-injected eggs as controls. This species usually has 3-egg clutches in which maternal androgen levels increase with the egg-laying sequence. We analysed embryonic heart rate, peri-hatching biometric traits, the ratio of white to red blood cells (W/R ratio) and bursa development. We found that testosterone and A4 treatment increased embryonic heart rate irrespective of egg-laying sequence and decreased bill length and W/R ratio, whereas etiocholanolone did not mimic these effects. Instead, etiocholanolone treatment decreased tarsus length and brain mass. Our finding that etiocholanolone does not mimic the effects induced by testosterone and A4 suggests that the embryonic metabolism of maternal testosterone and A4 can potentially diversify the function of these maternal androgens.

Istaroxime Metabolite PST3093 Selectively Stimulates SERCA2a and Reverses Disease-Induced Changes in Cardiac Function.

Heart failure (HF) therapeutic toolkit would strongly benefit from the availability of ino-lusitropic agents with a favorable pharmacodynamics and safety profile. Istaroxime is a promising agent, which combines Na+/K+ pump inhibition with sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) stimulation; however, it has a very short half-life and extensive metabolism to a molecule named PST3093. The present work aims to investigate whether PST3093 still retains the pharmacodynamic and pharmacokinetic properties of its parent compound. We studied PST3093 for its effects on SERCA2a and Na+/K+ ATPase activities, Ca2+ dynamics in isolated myocytes, and hemodynamic effects in an in vivo rat model of diabetic [streptozotocin (STZ)-induced] cardiomyopathy. Istaroxime infusion in HF patients led to accumulation of PST3093 in the plasma; clearance was substantially slower for PST3093 than for istaroxime. In cardiac rat preparations, PST3093 did not inhibit the Na+/K+ ATPase activity but retained SERCA2a stimulatory activity. In in vivo echocardiographic assessment, PST3093 improved overall cardiac performance and reversed most STZ-induced abnormalities. PST3093 intravenous toxicity was considerably lower than that of istaroxime, and it failed to significantly interact with 50 off-targets. Overall, PST3093 is a "selective" SERCA2a activator, the prototype of a novel pharmacodynamic category with a potential in the ino-lusitropic approach to HF with prevailing diastolic dysfunction. Its pharmacodynamics are peculiar, and its pharmacokinetics are suitable to prolong the cardiac beneficial effect of istaroxime infusion. SIGNIFICANCE STATEMENT: Heart failure (HF) treatment would benefit from the availability of ino-lusitropic agents with favourable profiles. PST3093 is the main metabolite of istaroxime, a promising agent combining Na+/K+ pump inhibition and sarcoplasmic reticulum Ca2+ ATPase2a (SERCA2a) stimulation. PST3093 shows a longer half-life in human circulation compared to istaroxime, selectively activates SERCA2a, and improves cardiac performance in a model of diabetic cardiomyopathy. Overall, PST3093 as a selective SERCA2a activator can be considered the prototype of a novel pharmacodynamic category for HF treatment.

Safety and Efficacy of Istaroxime 1.0 and 1.5 µg/kg/min for Patients With Pre-Cardiogenic Shock.

INTRODUCTION: Istaroxime was shown, in a small study, to increase systolic blood pressure (SBP) in patients with pre-cardiogenic shock (CS) due to acute heart failure (AHF). OBJECTIVES: In the current analysis, we describe the effects of 2 doses of istaroxime 1.0 (Ista-1) and 1.5 µg/kg/min (Ista-1.5). METHODS: The target dose of istaroxime, administered in a double-blind, placebo-controlled fashion, was 1.5 µg/kg/min in the first cohort (n = 24), and it was reduced to 1.0 µg/kg/min in subsequent patients (n = 36). RESULTS: Ista-1 was associated with numerically larger effects on SBP area under the curve, with a 93.6% relative increase from baseline during the first 6 hours with Ista-1 vs 39.5% for Ista-1.5, and with a 49.4% and 24.3% relative increase, respectively, at 24 hours. Compared to placebo, Ista-1.5 had more worsening HF events until day 5 and fewer days alive out of hospital (DAOH) through day 30. Ista-1 had no worsening HF events, and DAOH to day 30 were significantly increased. Effects on echocardiographic measures were similar, although decreases in left ventricular end systolic and diastolic volumes were numerically larger in the Ista-1 group. Ista-1, but not Ista-1.5, showed numerically smaller creatinine increases and larger decreases in natriuretic peptides as compared to placebo. There were 5 serious adverse events in Ista-1.5 (4 of which were cardiac) but only 1 in Ista-1. CONCLUSIONS: In patients with pre-CS due to AHF, istaroxime 1.0 µg/kg/min induced beneficial effects on SBP and DAOH. Clinical benefits appear to be reached at dosages less than 1.5 ug/kg/min.

Early conversion of maternal androgens affects the embryo already in the first week of development.

Maternal androgen exposure has potent effects on offspring development. As substantial levels of maternal androgens are deposited in avian egg yolks, avian eggs are frequently used to study maternal effects, with a strong focus on post-natal development. However, the underlying pathways are largely unknown. Since the hormones are taken up during the embryonic phase, and these are rapidly metabolized by avian embryos into metabolites such as etiocholanolone, we studied the effects of yolk androgens (testosterone and androstenedione) and their metabolite etiocholanolone during the first few days of embryonic development. As embryonic heart rate is often used as an indicator of embryonic development, we measured the heart rate from day 3 to day 6 of incubation by using a shell-less culture technique in rock pigeon eggs (Columba livia). Increased androgen exposure increased heart rate, and increased etiocholanolone mimicked this effect, albeit in a small sample size. This indicates that exposure to maternal androgens increases embryonic overall metabolism which may account for the developmental outcomes found in previous studies such as increased growth. Moreover, etiocholanolone is likely to be an important metabolite in a non-genomic pathway underlying the androgen-mediated maternal effect.

Evaluation of the Anticancer and Biological Activities of Istaroxime via Ex Vivo Analyses, Molecular Docking and Conceptual Density Functional Theory Computations.

Cancer is a disease that occurs as a result of abnormal or uncontrolled growth of cells due to DNA damage, among many other causes. Certain cancer treatments aim to increase the excess of DNA breaks to such an extent that they cannot escape from the general mechanism of cell checkpoints, leading to the apoptosis of mutant cells. In this study, one of the Sarco-endoplasmic reticulum Ca2+ATPase (SERCA2a) inhibitors, Istaroxime, was investigated. There has been very limited number of articles so far reporting Istaroxime's anticancer activity; thus, we aimed to evaluate the anticancer effects of Istaroxime by cell proliferation assay and revealed the cytotoxic activity of the compound. We further determined the interaction of Istaroxime with topoisomerase enzymes through enzyme activity tests and detailed molecular modeling analysis. Istaroxime exhibited an antiproliferative effect on A549, MCF7, and PC3 cell lines and inhibited Topoisomerase I, suggesting that Istaroxime can act as a Topoisomerase I inhibitor under in vitro conditions. Molecular docking analysis supported the experimental observations. A chemical reactivity analysis of the Istaroxime molecule was made in the light of Density Functional Theory computations. For this aim, important chemical reactivity descriptors such as hardness, electronegativity, and electrophilicity were computed and discussed as detailed.

Testing the role of testosterone versus estrogens in mediating reproductive transitions in female rhesus macaques.

In male vertebrates, testosterone is generally known to coordinate reproductive trade-offs, in part by promoting the transition to the next reproduction at the expense of current parental care. The role of testosterone in reproductive transitions has been little tested in female vertebrates, especially in mammals. The present study sought to fill this gap, by first undertaking an experimental study, in which we identified DHT, androstenediol, and in particular etiocholanolone, as fecal androgen metabolites which reflect serum testosterone concentration in female rhesus macaques (Macaca mulatta). Using concentrations of fecal etiocholanolone as proxy for circulating testosterone, we then conducted a field study on 46 free-ranging rhesus macaques of Cayo Santiago, Puerto Rico, to test if testosterone mediates the trade-off between reproductive transition (a higher chance of reproducing in the next year) and current reproduction (providing more care to current offspring). While the evidence for testosterone was weak, the testing of fecal immunoreactive estrogen metabolites suggested a potential role of estrogen in reproductive trade-offs. We found large individual differences in fecal etiocholanolone concentrations during the early postpartum period that were unexplained even after accounting for sociodemographic factors such as age and dominance rank. Further investigation is needed to understand this variation. Our study suggests that the actions of testosterone in females may not have evolved to fulfil the same role in primate reproductive transitions as it does in males, and we encourage more studies to consider the function of testosterone in reproductive behaviors and life history transitions in females of mammalian taxa.

Investigations on the in vivo metabolism of 5α-androst-2-en-17-one.

RATIONALE: The anabolic steroid 5α-androst-2-en-17-one (2EN) is sold as a prohormone and has been investigated regarding its potential as a steroidal aromatase inhibitor. The administration of 2EN was detected in a doping control sample in 2015, and investigations into its metabolism allowed for the identification and characterization of three urinary metabolites. Unfortunately, the utility of the main metabolite 2ß,3α-dihydroxy-5α-androstan-17-one for doping control purposes was hampered under routine doping control conditions due to chromatographic issues, thus warranting further studies on the metabolism of the prohibited substance. METHODS: The metabolism of 2EN was reinvestigated after oral administration of twofold-deuterated 2EN employing hydrogen isotope ratio mass spectrometry (IRMS) in combination with high-accuracy/high-resolution mass spectrometry. After a single dose of 50 mg of doubly labeled 2EN, urine samples were collected for 9 days. All samples were processed using routine doping control methods for IRMS analysis, and all detected metabolites were further characterized by mass spectrometry-based investigations. RESULTS: More than 15 different metabolites still containing the deuterium label were detected after administration. The presence of steroids exhibiting a 5ß-configuration was unexpected as the administered 2EN features a 5α-configured pharmacophore. Further investigations corroborated a significant impact of the administered 2EN on etiocholanolone and 5ß-androstanediol. Seven metabolites of 2EN not present as endogenous compounds were identified as potential candidates for routine doping controls and could be detected for up to 9 days after administration. CONCLUSIONS: The new metabolites identified in this study enable the detection of the misuse of 2EN for up to 9 days. The conversion of a 5α-steroid to urinary metabolites with 5ß-configuration has not been reported so far and should be further investigated.

Characterizing the timing of yolk testosterone metabolism and the effects of etiocholanolone on development in avian eggs.

Maternal transfer of steroids to eggs can elicit permanent effects on offspring phenotype. Although testosterone was thought to be a key mediator of maternal effects in birds, we now know that vertebrate embryos actively regulate their exposure to maternal testosterone through steroid metabolism, suggesting testosterone metabolites, not testosterone, may elicit the observed phenotypic effects. To address the role steroid metabolism plays in mediating yolk testosterone effects, we used European starling (Sturnus vulgaris) eggs to characterize the timing of testosterone metabolism and determine whether etiocholanolone, a prominent metabolite of testosterone in avian embryos, is capable of affecting early embryonic development. Tritiated testosterone was injected into freshly laid eggs to characterize steroid movement and metabolism during early development. Varying levels of etiocholanolone were also injected into eggs, with incubation for either 3 or 5 days, to test whether etiocholanolone influences the early growth of embryonic tissues. The conversion of testosterone to etiocholanolone was initiated within 12 h of injection, but the increase in etiocholanolone was transient, indicating that etiocholanolone is also subject to metabolism, and that exposure to maternal etiocholanolone is limited to a short period during early development. Exogenous etiocholanolone manipulation had no significant effect on the growth rate of the embryos or extra-embryonic membranes early in development. Thus, the conversion of testosterone to etiocholanolone may be an inactivation pathway that buffers the embryo from maternal steroids, with any effects of yolk testosterone resulting from testosterone that escapes metabolism; alternatively, etiocholanolone may influence processes other than growth or take additional time to manifest.

Isotope ratio mass spectrometry in antidoping analysis: The use of endogenous reference compounds.

RATIONALE: Isotope ratio mass spectrometry (IRMS) is an analytical technique required by the World Antidoping Agency (WADA) before releasing of an adverse finding for the abuse of pseudoendogenous steroids (i.e. testosterone). For every single individual, the delta 13 C values (‰) of the selected target compounds (TCs, i.e. testosterone and/or its precursors/metabolites) are compared with those of endogenous reference compounds (ERCs). The aim of this work is to investigate the individual variation in the delta values of four different commonly used ERCs to establish the maximum acceptable variation, in order to detect potential outliers. METHODS: Routine urine samples collected for antidoping purposes were submitted to IRMS confirmation. After a specific liquid chromatographic purification of the analytes of interest, the final extracts were analyzed by gas chromatography/combustion (GC/C)-IRMS. The selected ERCs monitored were pregnanediol, pregnanetriol, 11-keto-etiocholanolone and 11ß-hydroxyandrosterone. The obtained 13 C delta values were statistically analyzed to evaluate their inter- and intra-individual distribution. RESULTS: The delta values of the ERCs studied showed a normal distribution and no major differences among genders were observed. As expected, there are differences depending on the geographical origin of the samples, reflecting different dietary habits and food sources. The intra-individual dispersion, expressed as the standard deviation (SD) of the values of the studied ERCs, did not greatly exceed the instrumental error (0.5‰), demonstrating the good preservation of the delta values along the metabolic pathway. CONCLUSIONS: For the selected ERCs of non-sporting volunteers and the urinary specimens from more than 1000 sportsmen, we can propose a maximum SD of 0.54‰ and range of 1.2‰ for delta 13 C values as acceptance criteria to detect potential outliers. These cases can be caused by the external masking effect of the administration of a substance modifying the delta values or outliers due to unforeseen procedural artifacts.

Individual stress responses to Sarcoptes scabiei infestation in Iberian ibex, Capra pyrenaica.

In this study we have monitored the stress of Iberian ibex at individual level within the course of an experimental infection with Sarcoptes scabiei mites. For this purpose we have measured faecal 11-ketoetiocholanolone (11-k) using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). We used linear mixed models to explore the effects of host sex and age, clinic (mange status) and time (number of days post-infection) on the concentration of faecal 11-k. The most parsimonious model included clinic, time and host age, which explained 76.6% of the variance of the response variable. Moreover, the concentration of faecal 11-k varied greatly between individuals. Our results evidence the stressor nature of the disease and highlight the negative effects on hosts due to cortisol release and activity.

Inhibitory action of thymol on fecal microbial activity in Tamandua tetradactyla and its effect on glucocorticoid metabolite measurement.

Faecal glucocorticoid measurement is a potentially important tool for improving wildlife conservation, but its use is still limited by methodological issues including the need to avoid modifications of steroids by faecal microorganisms during storage. The freezing of faeces is recommended as a means of avoiding such alterations, but this is costly under non-controlled environmental conditions. The present study was designed to determine whether the application of thymol reduced the proliferation of microorganisms in the faeces of Tamandua tetradactyla and whether it influenced faecal glucocorticoid metabolite (FGM) measurements. Tamandua tetradactyla faeces were individually collected after defaecation, divided into fractions (5.5 g each) and kept in sealed glass Petri dishes at 22 ±â€¯2 °C. A thymol solution (550 µL; 5 mg g-1 feces; 80% ethanol) or an 80% ethanol solution (550 µL, control) was added before storage of faeces. Negative controls for FGM consisted of samples without thymol or ethanol solutions. All samples were evaluated at 0, 24, 48 and 72 h post-defaecation. Thymol was first incubated with a glucocorticoid standard in a faeces-free tube or in a faecal sample in order to determine whether it interfered with FGM measurements. Data showed that thymol did not affect FGM measurements. Post-defaecation time caused a significant reduction in FGM measurements in the negative control, an increment at 48 h in the control, and no change in FGM measurements in thymol treatment. FGM measurements were significantly different between groups (negative control > control - treatment). Thymol caused a significant reduction of up to three orders of magnitude in total coliforms, total aerobic and anaerobic heterotrophic mesophilic bacteria, mold and yeast per gram of faeces at 24, 48 and 72 h. The reduction in microbial activity presumably contributed to the stability of FGM over time. Spore-forming bacteria (SFB) in faeces were not reduced by thymol. We propose thymol as an alternative to freezing since it stabilizes FGMs for at least 3 days after collection in the faeces of Tamandua tetradactyla.

Istaroxime, a positive inotropic agent devoid of proarrhythmic properties in sensitive chronic atrioventricular block dogs.

Current inotropic agents in heart failure therapy associate with low benefit and significant adverse effects, including ventricular arrhythmias. Istaroxime, a novel Na+/K+-transporting ATPase inhibitor, also stimulates SERCA2a activity, which would confer improved inotropic and lusitropic properties with less proarrhythmic effects. We investigated hemodynamic, electrophysiological and potential proarrhythmic and antiarrhythmic effects of istaroxime in control and chronic atrioventricular block (CAVB) dogs sensitive to drug-induced Torsades de Pointes arrhythmias (TdP). In isolated normal canine ventricular cardiomyocytes, istaroxime (0.3-10 µM) evoked no afterdepolarizations and significantly shortened action potential duration (APD) at 3 and 10 µM. Istaroxime at 3 µg/kg/min significantly increased left ventricular (LV) contractility (dP/dt+) and relaxation (dP/dt-) respectively by 81 and 94% in anesthetized control dogs (n = 6) and by 61 and 49% in anesthetized CAVB dogs (n = 7) sensitive to dofetilide-induced TdP. While istaroxime induced no ventricular arrhythmias in control conditions, only single ectopic beats occurred in 2/7 CAVB dogs, which were preceded by increase of short-term variability of repolarization (STV) and T wave alternans in LV unipolar electrograms. Istaroxime pre-treatment (3 µg/kg/min for 60 min) did not alleviate dofetilide-induced increase in repolarization and STV, and mildly reduced incidence of TdP from 6/6 to 4/6 CAVB dogs. In six CAVB dogs with dofetilide-induced TdP, administration of istaroxime (90 µg/kg/5 min) suppressed arrhythmic episodes in two animals. Taken together, inotropic and lusitropic properties of istaroxime in CAVB dogs were devoid of significant proarrhythmic effects in sensitive CAVB dogs, and istaroxime provides a moderate antiarrhythmic efficacy in prevention and suppression of dofetilide-induced TdP.

Istaroxime Inhibits Motility and Down-Regulates Orai1 Expression, SOCE and FAK Phosphorylation in Prostate Cancer Cells.

BACKGROUND/AIMS: Istaroxime is a validated inotropic Na+/K+ ATPase inhibitor currently in development for the treatment of various cardiac conditions. Recent findings established that this steroidal drug exhibits potent apoptotic responses in prostate tumors in vitro and in vivo, by affecting key signaling orchestrating proliferation and apoptosis, such as c-Myc and caspase 3, Rho GTPases and actin cytoskeleton dynamics. In the present study we examined whether istaroxime is affecting cell motility and analyzed the underlying mechanism in prostate tumor cells. METHODS: Migration was assessed by transwell and wound healing assays, Orai1 and Stim1 abundance by RT-PCR and confocal immunofluorescence microscopy, Fura-2 fluorescence was utilized to determine intracellular Ca2+ and Western blotting for FAK/pFAK measurements. RESULTS: We observed strong inhibition of cell migration in istaroxime treated DU-145 prostate cancer cells. Istaroxime further decreased Orai1 and Stim1 transcript levels and downregulated Orai1 protein expression. Moreover, SOCE was significantly decreased upon istaroxime treatment. Furthermore, istaroxime strikingly diminished phosphorylated FAK levels. Interestingly, the efficacy of istaroxime on the inhibition of DU-145 cell migration was further enhanced by blocking Orai1 with 2-APB and FAK with the specific inhibitor PF-00562271. These results provide strong evidence that istaroxime prevents cell migration and motility of DU-145 prostate tumor cells, an effect at least partially attributed to Orai1 downregulation and FAK de-activation. CONCLUSION: Collectively our results indicate that this enzyme inhibitor, besides its pro-apoptotic action, affects motility of cancer cells, supporting its potential role as a strong candidate for further clinical cancer drug development.

Potential Antiosteoporotic Natural Product Lead Compounds That Inhibit 17ß-Hydroxysteroid Dehydrogenase Type 2.

17ß-Hydroxysteroid dehydrogenase type 2 (17ß-HSD2) converts the active steroid hormones estradiol, testosterone, and 5α-dihydrotestosterone into their weakly active forms estrone, Δ4-androstene-3,17-dione, and 5α-androstane-3,17-dione, respectively, thereby regulating cell- and tissue-specific steroid action. As reduced levels of active steroids are associated with compromised bone health and onset of osteoporosis, 17ß-HSD2 is considered a target for antiosteoporotic treatment. In this study, a pharmacophore model based on 17ß-HSD2 inhibitors was applied to a virtual screening of various databases containing natural products in order to discover new lead structures from nature. In total, 36 hit molecules were selected for biological evaluation. Of these compounds, 12 inhibited 17ß-HSD2 with nanomolar to low micromolar IC50 values. The most potent compounds, nordihydroguaiaretic acid (1), IC50 0.38 ± 0.04 µM, (-)-dihydroguaiaretic acid (4), IC50 0.94 ± 0.02 µM, isoliquiritigenin (6), IC50 0.36 ± 0.08 µM, and ethyl vanillate (12), IC50 1.28 ± 0.26 µM, showed 8-fold or higher selectivity over 17ß-HSD1. As some of the identified compounds belong to the same structural class, structure-activity relationships were derived for these molecules. Thus, this study describes new 17ß-HSD2 inhibitors from nature and provides insights into the binding pocket of 17ß-HSD2, offering a promising starting point for further research in this area.

Sarcoendoplasmic reticulum Ca(2+) ATPase. A critical target in chlorine inhalation-induced cardiotoxicity.

Autopsy specimens from human victims or experimental animals that die due to acute chlorine gas exposure present features of cardiovascular pathology. We demonstrate acute chlorine inhalation-induced reduction in heart rate and oxygen saturation in rats. Chlorine inhalation elevated chlorine reactants, such as chlorotyrosine and chloramine, in blood plasma. Using heart tissue and primary cardiomyocytes, we demonstrated that acute high-concentration chlorine exposure in vivo (500 ppm for 30 min) caused decreased total ATP content and loss of sarcoendoplasmic reticulum calcium ATPase (SERCA) activity. Loss of SERCA activity was attributed to chlorination of tyrosine residues and oxidation of an important cysteine residue, cysteine-674, in SERCA, as demonstrated by immunoblots and mass spectrometry. Using cardiomyocytes, we found that chlorine-induced cell death and damage to SERCA could be decreased by thiocyanate, an important biological antioxidant, and by genetic SERCA2 overexpression. We also investigated a U.S. Food and Drug Administration-approved drug, ranolazine, used in treatment of cardiac diseases, and previously shown to stabilize SERCA in animal models of ischemia-reperfusion. Pretreatment with ranolazine or istaroxime, another SERCA activator, prevented chlorine-induced cardiomyocyte death. Further investigation of responsible mechanisms showed that ranolazine- and istaroxime-treated cells preserved mitochondrial membrane potential and ATP after chlorine exposure. Thus, these studies demonstrate a novel critical target for chlorine in the heart and identify potentially useful therapies to mitigate toxicity of acute chlorine exposure.

Intracellular dyssynchrony of diastolic cytosolic [Ca²âº] decay in ventricular cardiomyocytes in cardiac remodeling and human heart failure.

RATIONALE: Synchronized release of Ca²âº into the cytosol during each cardiac cycle determines cardiomyocyte contraction. OBJECTIVE: We investigated synchrony of cytosolic [Ca²âº] decay during diastole and the impact of cardiac remodeling. METHODS AND RESULTS: Local cytosolic [Ca²âº] transients (1-µm intervals) were recorded in murine, porcine, and human ventricular single cardiomyocytes. We identified intracellular regions of slow (slowCaR) and fast (fastCaR) [Ca²âº] decay based on the local time constants of decay (TAUlocal). The SD of TAUlocal as a measure of dyssynchrony was not related to the amplitude or the timing of local Ca²âº release. Stimulation of sarcoplasmic reticulum Ca²âº ATPase with forskolin or istaroxime accelerated and its inhibition with cyclopiazonic acid slowed TAUlocal significantly more in slowCaR, thus altering the relationship between SD of TAUlocal and global [Ca²âº] decay (TAUglobal). Na⁺/Ca²âº exchanger inhibitor SEA0400 prolonged TAUlocal similarly in slowCaR and fastCaR. FastCaR were associated with increased mitochondrial density and were more sensitive to the mitochondrial Ca²âº uniporter blocker Ru360. Variation in TAUlocal was higher in pig and human cardiomyocytes and higher with increased stimulation frequency (2 Hz). TAUlocal correlated with local sarcomere relengthening. In mice with myocardial hypertrophy after transverse aortic constriction, in pigs with chronic myocardial ischemia, and in end-stage human heart failure, variation in TAUlocal was increased and related to cardiomyocyte hypertrophy and increased mitochondrial density. CONCLUSIONS: In cardiomyocytes, cytosolic [Ca²âº] decay is regulated locally and related to local sarcomere relengthening. Dyssynchronous intracellular [Ca²âº] decay in cardiac remodeling and end-stage heart failure suggests a novel mechanism of cellular contractile dysfunction.

Evaluating capture stress in wild gray mouse lemurs via repeated fecal sampling: method validation and the influence of prior experience and handling protocols on stress responses.

Reliable measurements of physiological stress are increasingly needed for eco-physiological research and for species conservation or management. Stress can be estimated by quantifying plasma glucocorticoid levels, but when this is not feasible, glucocorticoid metabolites are often measured from feces (FGCM). However, evidence is accumulating on the sensitivity of FGCM measurements to various nuisance factors. Careful species- and context-specific validations are therefore necessary to confirm the biological relevance and specificity of the method. The goals of this study were to: (1) establish and validate sampling methods and an enzymeimmunoassay to measure FGCM in the gray mouse lemur (Microcebus murinus); (2) explore causes of variability in the FGCM measurements, and; (3) assess the consequences of capturing and handling for free-living individuals by quantifying their stress responses via repeated fecal sampling within capture sessions. We further assessed the influence of different handling protocols and the animals' previous capture experience on the magnitude of the physiological response. Our validations identified the group-specific measurement of 11ß-hydroxyetiocholanolone as the most suitable assay for monitoring adrenocortical activity. The sample water content and the animal's age were found to significantly influence baseline FGCM-levels. Most captured animals exhibited a post-capture FGCM-elevation but its magnitude was not related to the handling protocol or capture experience. We found no evidence for long-term consequences of routine capturing on the animals' stress physiology. Hence the described methods can be employed to measure physiological stress in mouse lemurs in an effective and relatively non-invasive way.