Effetti degli ormoni steroidei nell'omeostasi intracellulare del calcio e nella regolazione della crescita cellulare in cellule derivate da cervelletto di feto bovino

Autor: Suman, Matteo
Jazyk: italština
Rok vydání: 2013
Předmět:
Popis: Steroid hormones differently affect various physiological processes by genomic or non-genomic mechanisms, in both excitable and non-excitable cells. Neurosteroids play a key role in the regulation of many cellular processes, such as cell growth, cell differentiation and apoptosis, exerting their effect in different ways (i.e. promoting or inhibiting a particular event) depending on the brain region of action. In this work two different themes were investigated: 1) the slow genomic effects exerted by estradiol (E2) and testosterone (T) in the regulation of intracellular calcium (Ca2+) homeostasis in a non-excitable cellular model; 2) the trophic effects promoted by estrogens in neuronal cells and in their dendritic branches. Recent experimental evidences highlight the involvement of estrogens and androgens in the regulation of intracellular homeostasis of calcium ion (Ca2+), one of the most important second messengers mediating steroid hormones action. The effects of neurosteroids on excitable cells are well documented; however, these steroids also influence numerous physiological events in non-excitable cells, such as fibroblasts or vascular endothelial cells. In the first part of this work I have focused my attention on an immortalized endothelial-like cell line derived from fetal bovine cerebellum. E2 (10nM) and T (10nM) effects on intracellular Ca2+ homeostasis were tested by varying the exposure time to the hormones (8, 24, 48 h). Ca2+ measurements were performed with genetically encoded Ca2+ probes (Cameleons) targeted to the main subcellular compartments involved in intracellular Ca2+ homeostasis (cytosol, endoplasmic reticulum, mitochondria). While T treatment had not any effect on intracellular Ca2+ fluxes, mitochondrial Ca2+ uptake significantly decreased after 48-h exposure to E2, whereas cytosolic and endoplasmic reticulum responses were unaffected. The effect of E2 on mitochondrial Ca2+ handling was blocked by ICI 182,780 (10nM), a pure estrogen receptor antagonist, suggesting that the effect was estrogen-receptor–mediated. To investigate further the effect of E2 on mitochondria, we tested two other concentrations of E2 (1 nM and 100 nM) and we observed a similar effect on the Ca2+ peak decrease indicating that saturation was possibly reached at 1 nM. To evaluate whether the decrease of Ca2+ affected mitochondrial membrane potential (ΔΨm), cells were monitored in the presence of tetra-methyl-rhodamine-methylester (TMRM): no significant changes were seen between cells treated with E2 and controls. To investigate a mechanism of action, we assessed the possibile involvement of the permeability transition pore (PTP), an inner mitochondrial membrane channel influencing energy metabolism and cell viability. We treated cells with CyclosporinA (CsA) (0.8μM), which binds to the matrix chaperone cyclophilin-D and regulates PTP opening. CsA reversed the effects of a 48-h treatment with E2, suggesting a possible transcriptional modulation of proteins involved in the mitochondrial permeability transition process. Taken together, these data reveal that, in this immortalized endothelial-like cell line, the genomic effect exerted by E2 can affect PTP opening without causing a collapse in ΔΨm. In the second part of this study I examined the effects of estradiol in the regulation of the growth of neuronal cells and of their dendritic branches. Dendrites are the principal cellular sites where neurons receive, process, and integrate inputs from their multiple pre-synaptic partners. It is widely known that, in early developmental stages, estrogens modulate neuronal morphology through a regulation of dendritic development. Estrogens regulate neuronal plasticity, synaptogenesis and cellular growth in many brain regions, such as cortex and hypothalamus. Few papers focused on what estrogens cause in the morphology of cerebellum, a region of the brain with a crucial role in controlling balance, posture, motor coordination as well as many other cognitive processes. To characterize the trophic effects of E2, primary neuronal cells from bovine cerebellum of both male and female fetuses were analyzed. To highlight differences in the morphological organization of neuronal cells we performed fluorescent-immunocytochemical analysis using a specific marker of immature neurons (βIII-tubulin). The whole area and the whole perimeter of neuronal cells, the number, the length and the diameter of dendritic branches were estimated and comparisons between E100nM samples and control cells were performed; differences intra sex were also considered. Results indicate that E100nM increases all the parameters in cells from females, while in males the trophic effect is restricted only to some values. Moreover, females have greater values than males in the soma dimensions, in the total number of branches and in the length of dendritic branches. On the contrary, males show superior values in the diameter of dendrites. Taken together, these preliminary data suggest that E100nM produces a trophic effect in both male and female samples; since several differences between males and females emerged, it could be supposed that neurons of the cerebellum in this embryonic stage exhibit dimorphic properties.
Databáze: OpenAIRE
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