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Identification of new neuroactive steroids that are able to interact with allosteric binding sites on purinergic P2X receptors
Sivčev, Sonja ; Zemková, Hana (advisor) ; Vyklický, Vojtěch (referee) ; Stojilkovic, Stanko S. (referee)
(EN) Purinergic P2X receptors are ATP-gated cation channels with multiple physiological roles and are emerging as important therapeutic targets in a range of diseases. P2X subunit consists of two transmembrane helices (TM1 and TM2), an extracellular ATP-binding domain, and intracellular N- and C- termini. Seven different P2X subunits (P2X1-7) can assemble to form homotrimeric or heterotrimeric ion channels permeable for monovalent cations and calcium. P2X are ubiquitously expressed. Among them, P2X2, P2X4, and P2X7 are the most abundant within the brain. The activity of P2X depends not only on the presence of ATP but also on allosteric modulators that may inhibit or potentiate the activity of these channels. Our aim was to identify new molecules that could interact with allosteric binding sites on P2X receptors, design and synthesize new analogues of neurosteroids, and define crucial receptor domains and amino acids important for neurosteroid binding. By using a patch-clamp electrophysiology technique we recorded ATP-induced currents in HEK293T cells transfected with rat P2X2, P2X4, and P2X7, as well as in the rat anterior pituitary cells and hypothalamic neurons endogenously expressing these receptors. We found that 17β-ester derivatives of testosterone, namely testosterone butyrate and...
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Expression of purinergic P2 receptors and their role in intracellular calcium signaling of neonatal pituitary cells
Šprláková, Katarína ; Stiborová, Marie (advisor) ; Ryšlavá, Helena (referee)
This diploma thesis focuses on study of the expression of purinergic receptors P2X and P2Y in the cells of neonatal adenohypofysis. Purinergic receptors are activated by binding of extracellular ATP and their activation leads to an increase in intracellular concentrations of calcium ions. The aim of this thesis was to determine by microfluorimetry method whether neonatal pituitary cells contain purinergic receptors. In addition, the influence of various agonists and antagonists of purinergic receptors on intracellular concentrations of ions Ca2+ in neonatal pituitary cells was monitored. The influence of extracellular ATP on secretion of a luteinizing hormone was observed by radioimmunoassay and it was compared with the effect of a hormone GnRH. It was found that neonatal pituitary cells are less sensitive to extracellular ATP than adult pituitary cells, but the relative sensitivity to other agonists of purinergic receptors is similar to the one of adult pituitary cells. In the mixed population of neonatal pituitary cells there were identified ATP- and 2 MeSATP- sensitive purinergic receptors P2X, as well as ADP-sensitive receptors P2Y. Further, the BzATP-sensitive receptors P2X7, PPADS-sensitive receptors P2X2 and BDBD-sensitive receptors P2X4 were identified. Finally, it was proved that...
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Trafficking of purinergic P2X receptor in eukaryotic cell
Pražák, Šimon ; Tvrdoňová, Vendula (advisor) ; Kolář, David (referee)
Purinergic receptors are membrane ion channels activated by extracellular ATP. In vertebrates, seven genes encoding P2X subunits was found. These subunits are designated as P2X1 - 7. Every P2X receptor subunit consists of two transmembrane domains, extracellular domain and intracellular N- and C- termini. P2X receptors fold to homo- or heterotrimers. P2X receptors have a wide distribution in the organism, functional receptors are found in neurons, glial cells, muscle cells and also in nonexcitable tissues as epithelial, endothelial, and in hemopoietic tissue. Purinergic signalling plays an important role in pain transmission, CNS injury and immune processes. P2X receptors are synthesized on the rough endoplasmic reticulum and are transported to the plasma membrane after post-translational modifications in the Golgi apparatus. The distribution and transport of P2X receptors is subunit specific and dependent on the cell type in which they are expressed. P2X receptors can be divided into three groups according to the way they are moved in the cell, which differ in transport speed, plasma membrane accumulation rate and rate of internalization.
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Expression of purinergic P2 receptors and their role in intracellular calcium signaling of neonatal pituitary cells
Šprláková, Katarína ; Stiborová, Marie (advisor) ; Ryšlavá, Helena (referee)
This diploma thesis focuses on study of the expression of purinergic receptors P2X and P2Y in the cells of neonatal adenohypofysis. Purinergic receptors are activated by binding of extracellular ATP and their activation leads to an increase in intracellular concentrations of calcium ions. The aim of this thesis was to determine by microfluorimetry method whether neonatal pituitary cells contain purinergic receptors. In addition, the influence of various agonists and antagonists of purinergic receptors on intracellular concentrations of ions Ca2+ in neonatal pituitary cells was monitored. The influence of extracellular ATP on secretion of a luteinizing hormone was observed by radioimmunoassay and it was compared with the effect of a hormone GnRH. It was found that neonatal pituitary cells are less sensitive to extracellular ATP than adult pituitary cells, but the relative sensitivity to other agonists of purinergic receptors is similar to the one of adult pituitary cells. In the mixed population of neonatal pituitary cells there were identified ATP- and 2 MeSATP- sensitive purinergic receptors P2X, as well as ADP-sensitive receptors P2Y. Further, the BzATP-sensitive receptors P2X7, PPADS-sensitive receptors P2X2 and BDBD-sensitive receptors P2X4 were identified. Finally, it was proved that...
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