Estrogen mediates metabolic syndrome-induced erectile dysfunction: a study in the rabbit.

INTRODUCTION: Estrogen receptor (ER) α is critical in mediating the harmful effects of hyperestrogenism in fetal or neonatal life on the developing penis. In contrast, little is known on the impact of an excess of estrogens on penile function in adulthood. AIM: To investigate the effect of estrogens on metabolic syndrome (MetS)-associated erectile dysfunction (ED). METHODS: We employed a recently established animal model of high fat diet (HFD)-induced MetS. Subgroups of MetS rabbits were dosed with either testosterone (T) or tamoxifen. We evaluated penile responsiveness to acetylcholine (Ach) as well as the expression of genes related to penile smooth muscle relaxation and contractility. MAIN OUTCOME MEASURE: Associations between MetS-induced penile alterations and sex steroids were investigated in an animal model of HFD-induced MetS. To understand the role of either androgen deficiency or estrogen excess on ED, we treated subgroups of MetS rabbits with either T or tamoxifen, a classical ER antagonist. RESULTS: Feeding an HFD-induced MetS was associated to elevated estradiol (E2) and low T levels. E2, but not T, was independently and negatively associated with genes able to affect penile erection. Smooth muscle-related markers decreased as a function of E2 and were positively associated with all the variables investigated. Increasing concentrations of circulating E2 were negatively associated with Ach-induced relaxation. In HFD rabbits, in vivo T dosing significantly improved MetS and completely normalized circulating E2. Conversely, in vivo tamoxifen dosing reduced visceral adiposity and partially restored T level. Ach-induced relaxation was severely impaired by HFD and significantly restored, up to the control level, by both tamoxifen and T dosing. In rabbit smooth muscle cells cultures 17ß-E2 (1 nM) significantly reduced the expression of α-smooth muscle actin, transgelin, and phosphodiesterase type 5. The effects of 17ß-E2 were completely reverted by tamoxifen (100 nM). CONCLUSIONS: This study demonstrates, for the first time, that HFD-induced ED is more associated with a high E2, rather than to a low T, milieu. HFD-induced ED is partially restored by in vivo treatment not only with T but also with the nonsteroidal ER antagonist, tamoxifen.

Farnesoid X receptor activation improves erectile function in animal models of metabolic syndrome and diabetes.

INTRODUCTION: The farnesoid X receptor (FXR) is critically involved in the regulation of the hepato-biliary system. Recent data suggest a role for FXR in modulating other metabolic pathways and vascular function. AIM: To investigate whether long-term administration of the selective FXR agonist INT-747 ameliorates erectile function, we tested it in two animal models of metabolic derangements: a rabbit model of high-fat diet (HFD)-induced metabolic syndrome (MetS) and a rat model of streptozotocin (STZ)-induced type 1 diabetes. METHODS: HFD rabbit or STZ rats with or without chronic INT-747 dosing (10 mg/kg/day for 12 weeks). INT-747 addition to rabbit penile smooth muscle cells (rpSMCs). MAIN OUTCOME MEASURE: Effects of INT-747 on metabolic features and erectile function in animal models and clarification of mechanism of action in isolated cells. RESULTS: INT-747 dosing normalized visceral adiposity and glucose intolerance in HFD rabbits. INT-747 increased penile FXR expression and partially restored endothelial nitric oxide synthase and dimethylarginine dimethylaminohydrolase 1 expression as well as impaired nitric oxide (NO)-dependent relaxation (improved responsiveness to acetylcholine and electrical field stimulation). INT-747 was also effective in regulating NO downstream events, as shown by increased sodium nitroprusside-induced relaxation. Because phosphodiesterase type 5 and protein kinase G (PKG) were unaltered by INT-747, we analyzed the calcium-sensitizing RhoA/ROCK pathway. HFD increased, and INT-747 normalized, RhoA membrane translocation/activation. RhoA/ROCK signaling inhibition by INT-747 was confirmed in rpSMCs by confocal microscopy, MYPT1-phosphorylation, cytoskeleton remodeling, cell migration, and smooth muscle-related genes expression. In STZ rats, FXR penile expression was not altered but was significantly upregulated by INT-747 dosing. In this model, INT-747 improved penile erection induced by electrical stimulation of cavernous nerve and hypersensitivity to intracavernous injection of a ROCK-inhibitor, Y-27632, without improving hyperglycemia. CONCLUSION: In HFD rabbits, INT-747 dosing improved glucose sensitivity and MetS-associated erectile dysfunction, via upregulation of NO transmission and inhibition of RhoA/ROCK pathway. In STZ rats, INT-747 restored in vivo penile erection and sensitivity to ROCK inhibition, independently of effects on glycemia.

Sex steroid receptors in male human bladder: expression and biological function.

INTRODUCTION: In male, lower urinary tract symptoms (LUTS) have been associated, beside benign prostatic hyperplasia, to some unexpected comorbidities (hypogonadism, obesity, metabolic syndrome), which are essentially characterized by an unbalance between circulating androgens/estrogens. Within the bladder, LUTS are linked to RhoA/Rho-kinase (ROCK) pathway overactivity. AIM: To investigate the effects of changing sex steroids on bladder smooth muscle. METHODS: ER α, ER ß, GPR30/GPER1 and aromatase mRNA expression was analyzed in male genitourinary tract tissues, and cells isolated from bladder, prostate, and urethra. Estrogen and G1 effect on RhoA/ROCK signaling output like cell migration, gene expression, and cytoskeletal remodeling, and [Ca(2+) ](i) was also studied in hB cells. Contractile studies on bladder strips from castrated male rats supplemented with estradiol and testosterone was also performed. MAIN OUTCOME MEASURES: The effects of classical (ER α, ER ß) and nonclassical (GPR30/GPER1) estrogen receptor ligands (17 ß-estradiol and G1, respectively) and androgens on RhoA/ROCK-.mediated cell functions were studied in hB cells. Contractility studies were also performed in bladder strips from castrated male rats supplemented with testosterone or estradiol. RESULTS: Aromatase and sex steroid receptors, including GPR30, were expressed in human bladder and mediates several biological functions. Both 17 ß-estradiol and G1 activated calcium transients and induced RhoA/ROCK signaling (cell migration, cytoskeleton remodeling and smooth muscle gene expression). RhoA/ROCK inhibitors blunted these effects. Estrogen-, but not androgen-supplementation to castrated rats increased sensitivity to the ROCK inhibitor, Y-27632 in isolated bladder strips. In hB cells, testosterone elicited effects similar to estrogen, which were abrogated by blocking its aromatization through letrozole. CONCLUSION: Our data indicate for the first time that estrogen-more than androgen-receptors up-regulate RhoA/ROCK signaling. Since an altered estrogen/androgen ratio characterizes conditions, such as aging, obesity and metabolic syndrome, often associated to LUTS, we speculate that a relative hyperestrogenism may induce bladder overactivity through the up-regulation of RhoA/ROCK pathway.

Reduced expression of thyroid hormone receptors and beta-adrenergic receptors in human failing cardiomyocytes.

An altered thyroid hormone profile has been reported in patients with congestive heart failure. However, information regarding the status of thyroid hormone receptors in human failing cardiomyocytes is lacking. Therefore the expression of thyroid hormone and beta-adrenergic receptors was investigated in human ventricular cardiomyocytes isolated from patients with end-stage heart failure (FM, n=12), or from tentative donors (C, n=4). The expression of thyroid (TRalpha1, and TRbeta1) and beta-adrenergic receptors (ARB1 and ARB2) was measured at both the gene, and at the protein level. In FM the reduced mRNA expression of ARB1 (p<0.05, -37%) and ARB2 (p<0.05, -42%) was associated with a reduction of the messenger for TRalpha1 (p<0.05, -85%) and TRalpha2 (p<0.05, -73%). These findings were confirmed at the protein level for ARB1, ARB2 and TRalpha1. These data reveal that in human heart failure the reduction of beta-adrenergic receptors is associated with reduced expression of both TRalpha1 and TRalpha2 isoforms of thyroid hormone receptors.

Role of endothelin-1 in exposure to high altitude: Acute Mountain Sickness and Endothelin-1 (ACME-1) study.

BACKGROUND: The degree of pulmonary hypertension in healthy subjects exposed to acute hypobaric hypoxia at high altitude was found to be related to increased plasma endothelin (ET)-1. The aim of the present study was to investigate the effects of ET-1 antagonism on pulmonary hypertension, renal water, and sodium balance under acute and prolonged exposure to high-altitude-associated hypoxia. METHODS AND RESULTS: In a double-blind fashion, healthy volunteers were randomly assigned to receive bosentan (62.5 mg for 1 day and 125 mg for the following 2 days; n=10) or placebo (n=10) at sea level and after rapid ascent to high altitude (4559 m). At sea level, bosentan did not induce any significant changes in hemodynamic or renal parameters. At altitude, bosentan induced a significant reduction of systolic pulmonary artery pressure (21+/-7 versus 31+/-7 mm Hg, P<0.03) and a mild increase in arterial oxygen saturation versus placebo after just 1 day of treatment. However, both urinary volume and free water clearance (H2OCl/glomerular filtration rate) were significantly reduced versus placebo after 2 days of ET-1 antagonism (1100+/-200 versus 1610+/-590 mL; -6.7+/-3.5 versus -1.8+/-4.8 mL/min, P<0.05 versus placebo for both). Sodium clearance and segmental tubular function were not significantly affected by bosentan administration. CONCLUSIONS: The present results indicate that the early beneficial effect of ET-1 antagonism on pulmonary blood pressure is followed by an impairment in volume adaptation. These findings must be considered for the prevention and treatment of acute mountain sickness.

Hyperglycemia activates JAK2 signaling pathway in human failing myocytes via angiotensin II-mediated oxidative stress.

Hyperglycemia was reported to enhance angiotensin (Ang) II generation in rat cardiomyocytes, and Ang II inhibition reduces cardiovascular morbidity and mortality in diabetic patients. In diabetic patients, the enhanced activation of intracellular pathways related with myocyte hypertrophy and gene expression might enhance the progression of cardiac damage. Therefore, we investigated the effects of glucose on Ang II-mediated activation of Janus-activated kinase (JAK)-2, a tyrosine kinase related with myocyte hypertrophy and cytokine and fibrogenetic growth factor overexpression, in ventricular myocytes isolated from nonfailing human hearts (n = 5) and failing human hearts (n = 8). In nonfailing myocytes, JAK2 phosphorylation was enhanced by Ang II only in the presence of high glucose (25 mmol/l) via Ang II type I (AT1) receptors (+79% vs. normal glucose, P < 0.05). JAK2 activation was prevented by inhibitors of reactive oxygen species (ROS) generation (diphenyleneiodonium [DPI], tiron, and apocynin). In myocytes isolated from failing hearts, JAK2 phosphorylation was enhanced by high glucose alone (+107%, P < 0.05). High glucose-induced JAK2 activation was blunted by both ACE inhibition (100 nmol/l ramipril) and AT1 antagonism (1 mumol/l valsartan), thus revealing that the effects are mediated by autocrine Ang II production. Inhibition of ROS generation also prevented high glucose-induced JAK2 phosphorylation. In conclusion, in human nonfailing myocytes, high glucose allows Ang II to activate JAK2 signaling, whereas in failing myocytes, hyperglycemia alone is able to induce Ang II generation, which in turn activates JAK2 via enhanced oxidative stress.

The in vivo tyrosine phosphorylation level of yeast immunophilin Fpr3 is influenced by the LMW-PTP Ltp1.

Tyr-phosphorylation in Saccharomyces cerevisiae is essential in controlling the activity of MAP kinase regulating mating, pseudohyphal growth, and cell wall biosynthesis. Yeast serves as a model system for studying the biological function of many protein kinases and PTPs. Two LMW-PTP from yeast have been cloned, namely, Ltp1 from S. cerevisiae and Stp1 from Schizosaccharomyces pombe. The sequences of both enzymes are relatively similar to those of the mammalian LMW-PTP. Recently we showed that the yeast immunophilin Fpr3 interacts with Stp1 and its dephosphorylated state induces a growth defective phenotype. Here we show the phosphatase activity of Ltp1 on Fpr3 and we demonstrated that Tyr 184 is the residue phosphorylated on in vivo Fpr3. We also described the marked activation of Ltp1 by adenine in S. cerevisiae proteome and determined in vivo the influence of tyrosine phosphorylation on Fpr3 localization.