Short-Term Activation by Low 17β-Estradiol Concentrations of the Na+/H+ Exchanger in Rat Aortic Smooth Muscle Cells: Physiopathological Implications

Low physiological concentrations of 17β-estradiol increased the intracellular pH of rat aortic smooth muscle cells by a rapid nongenomic mechanism. This effect was due to stimulation of the Na+/H+ exchanger activity, measured using the intracellular pH-sensitive fluorescent probe 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. The 17β-estradiol gave rise to a bell-shaped dose response, with a maximum at 10−12m and no significant effect at 10−9m. The specificity of the effect was verified by the use of the Na+/H+ exchanger inhibitor 5-(ethyl-N-isopropyl)amiloride and the lack of effect of the isomer 17α-estradiol. Inhibitors of the nuclear estrogen receptors, tamoxifen and ICI 182,780, completely prevented activation of the exchanger by 17β-estradiol. The effect of low estrogen concentrations on the intracellular pH was mimicked by both norepinephrine and phenylephrine, suggesting a connection between the increase of intracellular pH and the muscle contraction process. The transduction mechanism for this nongenomic effect of estrogens did not involve modulation of the cAMP content, whereas inositol 1,4,5-trisphosphate, protein kinase C and MAPK pathways appear to play a role, as indicated by both pharmacological approaches and immunoblot experiments on protein kinase C translocation and ERK phosphorylation. These results for the first time provide evidence for a nongenomic effect of low physiological concentrations of 17β-estradiol on intracellular pH that, together with other factors, may contribute to the development of hypertension and atherosclerosis in men and postmenopausal women and increase the risk of cardiovascular disease. Paradoxically, the lack of stimulation at high physiological estradiol levels could explain the protective effects found in premenopausal women

Sex differences in hepatic regulation of cholesterol homeostasis

Physiological sex differences may influence metabolic status and then alter the onset of some diseases. According to recent studies, it is now well established that females are more protected from hypercholesterolemia-related diseases, such as cardiovascular diseases until menopause. Female protection from hypercholesterolemia is mediated by the hypolipidemic properties of estrogens, even if mechanisms underlying this protection remain still debated. Even though the regulatory mechanisms of cholesterol homeostasis maintenance are well known, few data are available on the supposed differences between male and female in these processes. So, the aim of this work was to define, through an in vivo study, the putative sex-dependent regulation of the processes underlying cholesterol homeostasis maintenance. We examined 3-hydroxy 3-methylglutaryl coenzyme A reductase and its regulatory protein network as well as the amount of low-density lipoprotein receptor and cholesterol. The study was conducted in the liver and plasma of male and female rats, on adults and during postnatal development, and on 17-β-estradiol-treated male rats. Our data support that physiological differences in proteins involved in cholesterol balance are present between the sexes and, in particular, 3-hydroxy 3-methylglutaryl coenzyme A reductase shows lower activity and expression in female and 17-β-estradiol-treated male rats than in adult untreated male. Our data suggest that sex differences in enzyme expression depend on variation in regulatory proteins and seem to be related to estrogen presence. This work adds new evidence in the complicated picture of sex-dependent cellular physiology and establishes a new role for reductase regulatory proteins as a link between estrogen protective effects and cholesterol homeostasis

A comparative study of Sephadex, glass wool and Percoll separation techniques on sperm quality and IVF results for cryopreserved bovine semen

The aim of this study was to compare the effects of spermatozoa separation techniques on sperm quality and in-vitro fertilization (IVF) results for cryopreserved bovine semen. Sephadex, glass wool and Percoll gradient separation techniques were used for sperm separation and sperm motility, morphology and membrane integrity were evaluated before and after separation. Also, cleavage and blastocyst developmental rate were investigated after IVF with sperm recovered by each separation technique. The motility of samples obtained by the three separation techniques were greater compared to the control samples (p < 0.05). The percentage of spermatozoa with intact plasma-membrane integrity, identified by 6-carboxyfluoresceindiacetate/propidium iodide fluorescent staining and the hypo-osmotic swelling test, was highest in the glass wool filtration samples (p < 0.05). The cleavage and blastocyst rate of total oocytes produced from glass wool filtration samples were also higher than the control and Sephadex filtration samples (p < 0.05), but were not significantly different from Percoll separation samples. However, a significantly greater number of cleaved embryos produced by glass wool filtration developed to blastocyst stage than those produced by Percoll separation (p < 0.05). These results indicate that spermatozoa with good quality can be achieved by these three separation techniques and can be used for bovine IVF. In particular, it suggests that glass wool filtration would be the most effective method of the three for improving sperm quality and embryo production for cryopreserved bovine spermatozoa

S-PALMITOYLATION CONTROLS ESTROGEN RECEPTOR a ASSOCIATION WITH CAVEOLIN-1 AND CELL CYCLE PROGRESSION

Estrogen receptor (ER)a-mediated rapid events are now defined as necessary and sufficient for 17b-estradiol (E2)-induced cell cycle-regulating genes (e.g., cyclin D1) and G1/S phase transition. These actions are thought to require a plasma membrane ERa. We postulated that S-palmitoylation of the Cys447 residue may explain the ability of ERa to associate to plasma membrane making possible E2-dependent rapid functions. Cell lines expressing transfected or endogenous human ERa (HeLa and HepG2, respectively) or the ERa non-palmitoylable Cys447Ala mutant transfected in HeLa cells have been used as experimental models. Here, we report direct evidence that ERa is a palmitoylated protein. The mutation of the Cys447 residue to Ala impairs ERa palmitoylation and results in the loss of ERa plasma membrane localization and interaction with caveolin-1. In turn, E2-induced rapid non-genomic signals (i.e., ERK and AKT activation, cyclin D1 promoter activity and DNA synthesis) are also prevented. On the other hand, the Cys447Ala mutation significantly decreases the E2-induced transactivation of an estrogen responsive element construct probe. Remarkably, both ERa palmitoylation and its interaction with caveolin-1 are reduced by E2 in time and dose dependent fashion. Similar results have been obtained in HepG2 cells. As a whole, these data indicate that palmitoylation can be regarded as a regulatory device enabling ERa to initiate non genomic signal transduction pathways that lead cell to proliferate

Biphasic Estradiol-induced AKT Phosphorylation Is Modulated by PTEN via MAP Kinase in HepG2 Cells

We reported previously in HepG2 cells that estradiol induces cell cycle progression throughout the G(1)–S transition by the parallel stimulation of both PKC-α and ERK signaling molecules. The analysis of the cyclin D(1) gene expression showed that only the MAP kinase pathway was involved. Here, the presence of rapid/nongenomic, estradiol-regulated, PI3K/AKT signal transduction pathway, its modulation by the levels of the tumor suppressor PTEN, its cross-talk with the ERK pathway, and its involvement in DNA synthesis and cyclin D(1) gene promoter activity have all been studied in HepG2 cells. 17β-Estradiol induced the rapid and biphasic phosphorylation of AKT. These phosphorylations were independent of each other, being the first wave of activation independent of the estrogen receptor (ER), whereas the second was dependent on ER. Both activations were dependent on PI3K activity; furthermore, the ERK pathway modulated AKT phosphorylation by acting on the PTEN levels. The results showed that the PI3K pathway, as well as ER, were strongly involved in both G(1)–S progression and cyclin D(1) promoter activity by acting on its proximal region (-254 base pairs). These data indicate that in HepG2 cells, different rapid/nongenomic estradiol-induced signal transduction pathways modulate the multiple steps of G(1)–S phase transition

ROS increase is the cause of the age related 3-hydroxy-3-methylglutaryl coenzyme A reductase deregulation

Aim: It has been observed that the age-related total activation of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoAR) is due to a lack of regulation by phosphorylation/dephosphorylation. Such fully activation has been related to ROS increase but the responsible mechanism is still unknown. So, aim of this work was to study the mechanism underlying ROS induced impaired regulation of HMG-CoAR using a cellular model, the HepG2 cell line. In particular, in this system with high ROS content induced and in aged rat liver, the involvement of AMP dependent kinase (AMPK) and protein phosphatase 2A (PP2A), the main modulators of reductase short term regulation, has been studied.Methods: To induce ROS content as high as in aged rat liver, HepG2 cell line were treated with 200µM H2O2. ROS content was measured by fluorescence analysis. HMG-CoAR activation state was evaluated by a radioisotopic method. The activation state of the AMPK and the level of PP2A was measured by western blotting. H2O2-induced signal transduction pathway was evaluated by western blotting and the downstream effects by using specific inhibitors. Protein association was evaluated by co-immunoprecipitation.Results: In HepG2 cell line the HMG-CoAR results completely activated by ROS. AMPK activation state is high even if the enzyme isnt able to phosphorylate HMG-CoAR; PP2A level doesnt change. The p38/MAPK phosphorylation rises and the use of a p38 inhibitor or some phosphatase inhibitors prevents ROS-induced HMG-CoAR full activation. Both in H2O2 treated cell and aged liver the association of PP2A with HMG-CoAR is well detectable.Conclusion: The presented data show that ROS increase is able to induce HMG-CoAR full activation enhancing the reductase-PP2A association through p38 activation and subsequent dephosphorylation of the enzyme. On the contrary the modified activity of the kinases seem to be not involved in this deregulative process