Browsing by Author "Rocha, Duarte Nuno de Almeida Santos e Lopes"
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- Are polyamines detected by the TAAR receptor family in the human Choroid Plexus?Publication . Rocha, Duarte Nuno de Almeida Santos e Lopes; Santos, Cecília Reis Alves dos; Gonçalves, Isabel Maria Theriaga Mendes VarandaChoroid plexuses (CPs) are highly vascularized structures located at the ventricular system of the brain constituted by a monolayer of choroid plexus epithelial cells (CPEC) located on a basement membrane which form a barrier between the blood and the cerebrospinal fluid (CSF) (BCSFB). CPECs through their interconnections restrict the passage of different substances and pathogens between the blood and the CSF. Moreover, the microvilli at the apical side and an extensive infolding at the basolateral side enhance the contact between the CP epithelium and CSF, and between the epithelium and stroma interstitial fluid, respectively, thus allowing one of its main functions which is to maintain cerebral homeostasis. Among other features, there is the chemical vigilance which is performed by different mechanisms as the taste signalling, the clearance of xenobiotics, the clearance of amyloid beta and finally but with more relevance to this work, the olfactory signalling. This olfactory cascade is activated when an odorant binds to an olfactory receptor (OR) which is a G protein-coupled receptor (GPCR) that exists in different tissues in mammalian´s body. The trace amine-associated receptors (TAARs) are a family of rhodopsin-like GPCRs which act as olfactory receptors with TAAR1 exception and are expressed in different tissues of different species. In humans there are 6 different types of TAARs and 3 pseudogenes. TAAR 13c, identified in zebrafish, can bind to cadaverine, a polyamine. Polyamines are polycations which appear naturally in cells and are closely related with neuronal cell biochemical activity at different points. Cadaverine is a diamine compound produced by the putrefaction of animal tissue and enable an aversive response to this odour. A previous data base research showed that TAAR1, 2 and 5 are the human receptors with more homology with TAAR13c and one of the more abundant TAARs in humans, which probably can trigger a response to a polyamine stimulus. TAAR1 was selected for this study because we could not amplify TAAR2 and 5 by RT-PCR in HIBCPP cell line. The HIBCPP cell line from choroid plexus carcinoma was used to the experiments and the expression of the mRNAs to the TAAR1 was demonstrated by the RT-PCR technique. In respect to polyamine´s biosynthesis and catabolic pathway enzymes identification, the RT-PCR technique enabled the confirmation of their mRNA expression after Sanger sequencing. The expression of key enzymes to these pathways was also demonstrated by immunocytochemistry and Western blot, in human CP cell line. To test the cell´s response to polyamines, calcium imaging (Ca2+ imaging) assays were performed and showed clear responses to cadaverine. After these tests, silencing of TAAR1 with a specific small interfering RNA (siRNA) was done and preliminary Ca2+ imaging responses to cadaverine enable to see a decrease in CP cells response, highlighting the potential role of TAAR1 in this polyamine response. In the closer future, more silencing assays will be done and Ca2+ imaging with transfected cells will be done too with cadaverine and other polyamines already tested as spermine, spermidine and putrescine. Western blot and immunocytochemistry techniques will enable the TAAR1 protein characterization and localization in the HIBCPP cells.