National Repository of Grey Literature 38 records found  1 - 10nextend  jump to record: Search took 0.01 seconds. 
The molecular mechanisms and role of purinergic signaling in hypothalamus
Ivetic, Milorad ; Zemková, Hana (advisor) ; Mysliveček, Jaromír (referee) ; Smejkalová, Terézia (referee)
(EN): Extracellular ATP and purinergic P2 receptors (P2X and P2Y) are involved in a signaling network called "purinergic signaling" which is widely exploited in both somatic and neuronal tissues, and is also operative in endocrine system. The main focus of my thesis is on the role and expression of P2X and P2Y receptors in hypothalamic supraoptic nuclei (SON) producing hormones vasopressin and oxytocin, and the suprachiasmatic nuclei (SCN), the principal circadian pacemaker in mammals. In the first part of my thesis, we tested the hypothesis that P2X and P2Y receptors play a role in the enhanced release of hormones from magnocellular SON neurons stimulated through fasting/refeeding experimental protocol. We studied the effect of 2 h of refeeding after 48 h of fasting on hormone, P2X and P2Y mRNA expression in the SON tissue of 30-day-old rats, and the effect of ATP on electrophysiological properties of SON neurons in brain slices from control and fasted/refed rats. Quantitative real-time PCR revealed that the expression of P2X2 and arginine-vasopressin (AVP) mRNA was increased and P2Y1 mRNA expression was decreased in fasted/refed rats compared to controls, whereas P2X4, P2X7, P2Y2 and oxytocin mRNA levels were not significantly changed. Whole-cell patch clamp recordings showed that the amplitude...
Role of amino acid residues in the first transmembrane domain in deactivation of P2X7 receptor
Knězů, Michal ; Zemková, Hana (advisor) ; Vlachová, Viktorie (referee)
P2X7 receptor is trimeric ligand-gated ion channel activated by extracellular ATP. This receptor is expressed in various tissues and cell types, such as glial cells, immune system cells or Schwann cells, different epithelial tissues and sperm cells, and is involved in many cellular a physiological processes such as is metabolism regulation, phagocytosis or apoptosis. The P2X7 subunit is composed of intracellular N- and C-termini, two transmembrane domains (TM1 and TM2) and a large extracellular ligand-binding domain. P2X7 has low sensitivity for its natural agonist, but prolonged or repeated applications lead to its sensitization and increase in current amplitude. Activation of P2X7 receptor is also known to induce uptake of large organic ions, such as fluorescent dye ethidium bromide. Receptor sensitization is accompanied by prolongation deactivation, mechanism of which is still unkown, that can be measured as current decay evoked by washout of agonist. This work explores the role of individual TM1 residues in deactivation kinetics of the receptor both in naïve and sensitized states. Electrophysiological whole-cell patch clamp method was used to record agonist-stimulated membrane current and it's decay of wildtype P2X7 receptor (P2X7-WT) and TM1 alanine or leucine mutants. Mutations that were...
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...
Astrocyte volume changes during brain ischemic injury
Mikešová, Michaela ; Anděrová, Miroslava (advisor) ; Zemková, Hana (referee)
Brain ischemic injury is a complex of pathophysiological events following transient or permanent reduction of brain blood flow. It results in a disruption of neuronal and astrocytic physiological functions, long-term reduction of brain blood flow leads to the cell death. Number of recent studies is focused on astrocytes, which might play key roles in surviving cells, including neurons, during ischemic injury. Astrocytes provide many important functions, such as maintenance of ionic homeostasis, prevention of excitotoxicity, scavenging free radicals and others and thus astrocytes may dramatically swell during ischemic conditions and contribute notably to the development of cytotoxic edema. This thesis summarizes mechanisms possibly contributing to the astrocytic swelling during brain ischemic injury as well as methods used for studying astrocyte volume changes and their quantification. Since the brain edema dramatically complicates both course and treatment of ischemic injury, knowledge of mechanisms leading to astrocytic swelling and their volume regulation during ischemia/reperfusion might be used for developing new therapeutic approaches for the treatment of cerebral ischemia, mainly for reduction of negative impact of edema.
Contribution of ten ectodomain cysteine residues to function of ATP-gated P2X4 receptor
Tvrdoňová, Vendula ; Zemková, Hana (advisor) ; Teisinger, Jan (referee)
Extracellular adenosine-5'-triphosphate (ATP), released from damaged cells or coreleased as a cotransmitter from synaptic vesicles, acts on its plasma membrane receptors termed purinergic. Purinergic P2X receptors are ATP-gated cation channels. To date seven P2X isoforms designated P2X1-7 have been cloned that are organized as trimeric homomers or heteromers. All P2X subunits share a similar structure consisting of a large extracellular loop, two transmembrane domains and intracellular N- and C- termini. An additional structural feature is conserved aminoacids, these include ten conserved cysteine residues in the extracellular loop. All ectodomain cysteines form disulfide bonds which are organized in two areas: three disulfide bridges are localized in the N-termini half and two in the C-termini half at P2X receptor. ATP binding pocket is apparently localized between two neighbouring subunits. The aim of this Diploma Thesis was to examine the relevance of ectodomain cysteine residue and/or disulfide bonds for the expression, function and ATP binding properties of the P2X receptor. All ten, one by one, ectodomain cysteines were substituted by alanines and ATP-induced currents was recorded in HEK293 cells expressing wild-type P2X4 receptor and its mutants. Low responsible or nonfunctional mutants...
The role of transmembrane domains in the structure and function of P2X receptors
Jindřichová, Marie ; Zemková, Hana (advisor) ; Langmeier, Miloš (referee) ; Martásek, Pavel (referee)
Purinergic P2X receptors represent a novel structural type of ligand-gated ion channels activated by extracellular ATP. So far, seven P2X receptor subunits have been found in excitable as well as non-excitable tissues. In the past ten years, the number of studies on P2X receptors has dramatically increased as investigators have begun to determine the physiological roles played by extracellular ATP and specific P2X receptor subtypes. It is already known that purinergic signaling is a key mechanism in pain sensation, brain injury, and immune processes. Little is known about their structure, mechanism of channel opening, localization and termination of ATP action by ectonucleotidases. Detailed knowledge about these events and the structure of purinergic receptor proteins evoke hope that new drugs will be developed that could prevent chronic pain and would be effective in protection against many diseases. The aim of this work is to summarize recent investigations and describe our contribution to elucidating the structure of P2X receptors. We examined the structure of transmembrane domains of the P2X4 receptor subtype, the main purinergic receptor-channel in the central nervous system, the mechanism of channel opening and closing and its sensitivity to agonists and allosteric modulator ivermectin. To...
Study of pharmacology and function of binding sites of nicotinic acetylcholine receptors
Kaniaková, Martina ; Krůšek, Jan (advisor) ; Chaloupka, Roman (referee) ; Zemková, Hana (referee)
Title: Study of pharmacology and function of binding sites of nicotinic acetylcholine receptors Author: Mgr. Martina Kaniaková Department: Institute of Physiology AS CR, v.v.i. Supervisor: RNDr. Jan Krůšek, CSc., Institute of Physiology AS CR, v.v.i. Abstract: Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels. We use the whole-cell patch-clamp technique to study functional and pharmacological properties of muscle and neuronal nicotinic receptors. Rat neuronal receptors were heterologously expressed in COS cells and human embryonic muscle receptors were studied in TE671 cells. Lobeline, a plant alkaloid with a long history of therapeutic use, interacts with the classical agonist-binding site of nAChRs. The final result of this interaction depends on the receptor subtype, lobeline and other agonists concentrations and the time schedule of application. Generally, lobeline is a very weak partial agonist eliciting deep desensitization at several subtypes of nAChRs. In combination with other agonists, lobeline acts as a competitive antagonist or coagonist. Using point mutation procedure we studied the functional role of negatively charged amino acids in the F-loop of β2 and β4 subunits of neuronal receptors. Neutralising mutations in β4 subunit led to up to eighteen-fold increase in the...
Spontaneous calcium permeability of ionic channel of P2X receptor after substitution ofconserved tyrosine in the 1st transmembrae domajn
Rupert, Marian ; Zemková, Hana (advisor) ; Balík, Aleš (referee)
Purinergic receptors are membrane ion channels that are activated by extracellular ATP. In vertebrates, seven genes encode subunits of P2X receptors. The subunits, designated P2X1-7, are 40 - 50% identical in amino acid sequences. P2X receptors are composed of three subunits and are found as homo- and heterotrimers in tissues of vertebrates. 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, at CNS injury and immune processes. P2X receptor subunit consists of two transmembrane domains, extracellular domain and intracellular N-and C-termini. Each transmembrane domain contains two amino acids conserved across all P2X subunits. In the first transmembrane domain receptor P2X2 are that Gly30 and Tyr43. In previous experiments performed on P2X2 receptor, electrophysiological measurements demonstrated that substitution of conserved Tyr43 in the first transmembrane domain with alanine prolongs the deactivation time of ion channel after agonist wash out. This work is focused on clarifying the role of conserved tyrosine in the process of opening and closing of ion channel of P2X...

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