Browsing by Author "Feiteiro, Joana Rita Oliveira"
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- Efeito da testosterona na compartimentação do cGMP a nível vascularPublication . Feiteiro, Joana Rita Oliveira; Rodrigues, Maria Elisa Cairrão; Verde Lusquiños, José IgnacioOs canais de CNG são geralmente expressos em foto-recetores e em neurónio sensoriais oftativos e traduzem as alterações nas concentrações de cGMP e de cAMP, numa resposta elétrica e/ou num sinal de Ca2+ intracelular. Os CNG são ativados através da ligação direta a nucleótidos cíclicos, cAMP e cGMP. A síntese do monofosfato de guanosina cíclico (cGMP) é controlada por dois tipos de guanilato ciclase (GC) que diferem na sua ativação e localização celular: a CG membranar (pGC) presente na membrana plasmática, a qual é ativada pelo péptido natriuretico, como o péptido natriuretico auricular (ANP); e a GC solúvel (sGC) que esta localizada no citosol e a sua ativação é induzida pelo óxido nítrico (NO). O NO e o ANP usam o cGMP como segundo mensageiro. Existem muitos casos em que a ativação da pGC e da sGC conduz a diferentes efeitos funcionais, uma explicação para esses efeitos funcionais diferentes é o aumento do cGMP em locais subcelulares específicos, regulam diferentes alvos em distintas partes da célula. Estudos recentes realizados pelo nosso grupo em células musculares lisas da artéria umbilical humana demonstraram que a testosterona ativa os canais BKCa e KV devido à ativação da proteína cinase G (PKG). Além disso, o nosso grupo observou que a testosterona e o ANP, mas não SNP (nitroprussiato de sódio), estimula a atividade destes canais de potássio, o que sugere que a testosterona aumenta os níveis de cGMP por ativação pGC. Este estudo também sugeriu que esta hormona relaxa o musculo vascular através da ativação da pGC aumentado o nível intracelular do cGMP. Estes estudos também demonstraram que o ANP aumenta os níveis intracelulares de cGMP pela ativação da pGC e o NO aumenta os níveis intracelulares de cGMP através da ativação da sGC. Neste sentido o objetivo deste trabalho é analisar o efeito vasodilatador da testosterona e analisar a compartimentação do cGMP a nível vascular através da ativação dos CNG sensíveis a cGMP e a possível implicação das fosfodiesterases (PDE). A técnica patch clamp na configuração whole cell foi usada para medir o sinal de ativação dos canais de CNG. As células musculares lisas da artéria umbilical humana são infetadas com o adenovírus WT-CNGA2. Os compostos utilizados foram: ANP (0,1μM e 1μM), SNP (10μM e 100μM), IBMX (3-isobutil-1-metilxantina) (100μM, inibidor não selectivo das PDE), To-156 (1μM, inibidor especifico da PDE5), ciloestamida (10μM; inibidor especifico da PDE3) e Sp-8 ((8- (4-Clorofeniltio)- 3', 5'-monofosfato cíclico de guanosina) (100μM, análogo da molécula cGMP) e testosterona ( 0,1, 1 e 10μM). Analisando os resultados obtidos, observa-se que o ANP, SNP induzem um diferente aumento da ICNG. O sinal do cGMP induzido pelo ANP parece ser controlado pela PDE5 e pela PDE3. Contudo, a administração do SNP parece criar dois efeitos separados, um mais localizado junto à membrana plasmática que é controlado pela PDE3 e pela PDE5, e o outro efeito localizado no interior das células que é regulado apenas pela PDE3. Em relação ao efeito da testosterona, observa-se que esta hormona ativa os CNG e que esta ativação e dependente da sua concentração. Em suma, a distribuição temporal e espacial diferente do cGMP pode contribuir para efeitos específicos do ANP e de dadores do NO na função vascular. A testosterona age pelo aumento do cGMP que induz a ativação da PKG.
- Effect of TBBPA on arterial contractile regulation and possible implications for the development of hypertensive diseasesPublication . Feiteiro, Joana Rita Oliveira; Oliveira, Maria Elisa Cairrão Rodrigues; Baptista, Cláudio Jorge MaiaThe endocrine disruptor (EDCs) is a compound that has been defined as “an exogenous agent that interferes with the production, release, transport, metabolism, binding, action or elimination of natural hormones in the body responsible for the maintenance of homeostasis and the regulation of developmental processes.” This compound can affect the endocrine function via interference with hormone pathways (e.g., oestrogen, androgen, or thyroid hormone). The constant human exposure to endocrine disruptors has raised some concerns. Some of these components are suspected of being harmful to human health. Brominated flame retardants (BFRs) are chemicals widely used in consumer products, including electronics, vehicles, plastics, and textiles, to reduce flammability. These compounds can interfere with hormone homeostasis, so they are considered endocrine disruptors. Tetrabromobisphenol A (TBBPA) is the most studied BFRs due to its toxicity and presence in a variety of environmental media and the human being. The exposure to this compound is associated with several health risks: thyroid disorders, diabetes, reproductive health, cancer, and neurobehavioral development disorders. In addition, TBBPA exposure can be correlated with some cardiovascular disorders, such as diabetes and obesity. This compound has also been detected in biological samples such as human serum, urine, and breast milk. Moreover, TBBPA has also been detected in the umbilical cord of Japanese pregnant women, proving a prenatal exposure to this compound. This observation suggests that TBBPA can cross the human placenta. In this scenario, it is important to understand how the TBBPA exposure effects the vascular tonus and if the endocrine disrupting effects from that exposure can be detected in future generations. In this project, organ bath and patch clamp techniques were developed and applied to achieve the main goal of this doctoral thesis: to study the effect of TBBPA on arterial contractility and analyse the mode of action of TBBPA as a human EDCs and understand its involvement in vascular disorders. This study was performed in two different study models: in the human umbilical artery (HUA) and in the rat aorta. Additionally, the cGMP compartmentation in human vascular smooth muscle was also analysed. Therefore, in the first research work presented, we infected smooth muscle cells with adenovirus containing mutants of the rat olfactory cyclic nucleotide-gated (CNG) channel-subunit to understand how the cGMP conveys different information and we recorded the associated cGMP-gated current (ICNG). The whole cell configuration of the patch clamp technique was used to measure the ICNG and the potassium current (IK) in human umbilical artery smooth muscle cells (HUASMC). Atrial Natriuretic Peptide (ANP) induced an activation of basal ICNG, whereas sodium nitroprusside (SNP) had a slight effect. IBMX (nonselective PDE inhibitor), T0-156 (PDE5 inhibitor), and cilostamide (PDE3 inhibitor) all had a small effect on the basal ICNG current. Concerning potassium channels, we observed that ANP and testosterone induced activation of IK and this effect is bigger than that induced by SNP, cilostamide and T0-156. Cilostamide and T0-156 decreased the ICNG stimulation induced by ANP and testosterone, suggesting that the pGC pool is controlled by PDE3 and PDE5. Thus, the effects of SNP show the presence of two separated pools, one next to the plasma membrane and controlled by the PDE5 and PDE3, and a second pool in the cytosol of the cells that is regulated mainly by PDE3. These findings show the existence of cGMP compartmentalization in human vascular smooth muscle cells, and this phenomenon is controlled by PDE3 and PDE5. The second research work evaluated the direct effects and the 24 h exposure of TBBPA on the HUA and also its mode of action (MOA). The viability of HUASMC was analysed using MTT assay and the cells exposed to high concentrations of TBBPA (500 and 1000 μM) showed a decrease in cell viability. Using the organ bath technique, endothelium-denuded HUA rings were contracted with serotonin (5-HT), histamine (His), and potassium chloride (KCl), and then the direct effects of TBBPA (0.01- 100 μM) were analysed. The effects of 24 hours TBBPA exposure (1, 10, and 50 μM) were also analysed on contractile responses of HUA to 5-HT, His, and KCl. Furthermore, the vascular MOA of TBBPA was studied through the analysis of cGMP and calcium (Ca2+) channels activity, these pathways are involved in the relaxation and contraction of HUA, respectively. Our results demonstrated that the direct effects of TBBPA induce a vasorelaxation of HUA. The 24h TBBPA exposure changed the vasoconstrictor response pattern of 5-HT, His and KCl and the vasorelaxant response pattern of SNP and nifedipine. This effect is due to the involvement of TBBPA with the NO/sGC/cGMP/PKG pathway and the interference in Ca2+ influx. Furthermore, using the real-time quantitative polymerase chain reaction (RT-qPCR), TBBPA clearly modulates L-type Ca2+ and large-conductance Ca2+ 1.1 α- and β1 subunit channels, and soluble guanylyl cyclase (sGC) and protein Kinase G. In this sense, our data demonstrated that TBBPA induces changes in the vascular homeostasis of HUA. In the last part of this work, the effect of TBBPA in rat aortic smooth muscle and the possible mechanisms involved were investigated and to achieve these goals, we started with the analysis of A7r5 cells viability. These cells were exposed to different TBBPA concentrations, and the results showed that the high concentrations of TBBPA (500 and 1000 μM) decreased the viability of the A7r5 cells. Then, using the organ bath technique, rat aorta rings without endothelium were contracted with Phenylephrine, Noradrenaline, and isosmotic KCl solution to evaluate the vascular effect of TBBPA (0.01–100 μM). Furthermore, MOA of TBBPA was studied through Nifedipine (specific blocker of L-type VGCC), tetraethylammonium (TEA), 4-aminopyridine (4-AP), and glybenclamide (Gly) (K+ channel inhibitors). Our results suggest that the direct effects of TBBPA induced vasorelaxation of rat aorta, involving the inhibition of Ca2+ channels and activation of potassium channels. Moreover, through RT-qPCR, it was demonstrated that TBBPA clearly modulates L-type Ca2+ and large-conductance Ca2+ 1.1 α- and β1 subunit channels, and sGC and protein Kinase G. Overall, it was shown that TBBPA exposure also interferes with vascular homeostasis of rat aorta through Ca2+ and K+ channels. In conclusion, the main findings of this thesis confirmed the crucial actions of TBBPA in vascular smooth muscle. These effects demonstrate that TBBPA induces smooth muscle relaxation through an endothelium-independent MOA. Due to sGC activation that increases the cGMP intracellular levels, inhibition of L-Type VGCC and activation of K+ channels were verified. Another innovative result of the present thesis was the identification of cGMP compartmentalization in human vascular smooth muscle cells. Further understanding and targeting of these results might be exploited in future studies to acknowledge the effects of TBBPA at the vascular level and its complexity in environmental and human exposure.