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Membrane Properties of Glial Cells after Ischemia in vivo
Pivoňková, Helena ; Anděrová, Miroslava (advisor) ; Vyklický, Ladislav (referee) ; Vlachová, Viktorie (referee)
In my PhD thesis, I aimed to characterize the membrane properties of astrocytes and NG2 glia during the acute and chronic phases of the ischemic injury in the central nervous system with a focus on the expression and activity of K+ ion channels. Two ischemia models in rats were used for the experiments - global cerebral ischemia 5 causing selective neuronal loss and astrogliosis in the CA1 region of the hippocampus, and incubation of spinal cord segments in a solution with high K+ concentration. We found that hippocampal astrocytes depolarize starting 3 days after ischemia, and demonstrate an increase in the inward rectification of their whole cell currents 1 month after ischemia. The Kir4.1 channel expression in the hippocampal tissue was downregulated starting 3 days after ischemia, while the expression of Kir2.1, Kir5.1 and TREK1 channels was strongly upregulated in reactive astrocytes 1 month after ischemia. NG2 glia displayed a significant increase in the outwardly rectifying delayed and A-type K+ (KDR and KA) currents 2 hours and 3 days after ischemia, and a decrease in the inwardly rectifying K+ currents 3 days after ischemia, a typical current pattern of proliferating cells. Complex astrocytes in the spinal cord pre-incubated in a solution with high [K+] showed the largest changes in their membrane...
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Modulation of nociceptive synaptic transmission in the spinal cord dorsal horn
Špicarová, Diana ; Paleček, Jiří (advisor) ; Vlachová, Viktorie (referee) ; Rokyta, Richard (referee)
7 ABSTRACT Pathological pain states linked to several diseases or tissue damage are often associated with increased sensitivity to stimuli. The main underlying mechanisms of this hypersensitivity are peripheral sensitization of nociceptors and central sensitization in the spinal cord. One of the crucial processes of central sensitization is the modulation of synaptic transmission at the dorsal horn of the spinal cord. Studies included in my doctoral thesis investigate the possibilities of regulation of synaptic strength by cytokine TNFα, insulin and TRPV1 receptors agonist N-oleoyldopamine (OLDA). These three compounds are synthesized in the CNS, while TNFα is produced in the spinal cord notably during neuropathy. TNFα and insulin have a potential to modulate synaptic transmission. Endogenous TRPV1 receptors agonist OLDA can activate spinal TRPV1 receptors, which are highly expressed on central endings of nociceptive dorsal root ganglion (DRG) neurons. TRPV1 receptors are known as integrators of nociceptive stimuli particularly from the studies of peripheral receptors on nociceptors, which could be sensitized by inflammatory mediators and activated by temperature increase or decrease pH that is unlike in the spinal cord. In our experiments miniature excitatory postsynaptic currents (mEPSCs) or evoked EPSCs...
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The role of conserved residues in the S4/S4-S5 region of vanilloid TRP channels
Boukalová, Štěpána ; Vlachová, Viktorie (advisor) ; Doležal, Vladimír (referee)
The vanilloid transient receptor potential channel TRPV1 is a tetrameric six-transmembrane segment (S1-S6) channel that can be synergistically activated by various proalgesic agents such as capsaicin, protons, heat or highly depolarizing voltages. In TRPV1 channel, the charged residues in the S4 region and intracellular S4-S5 linker have been proposed to constitute a part of a voltage sensor that acts in concert with other stimuli to regulate channel activation. Molecular basis of this gating event are poorly understood. We mutated charged residues all along the S4 and the S4-S5 linker of TRPV1 and related vanilloid receptors to identify potential voltage-sensing residues. The functionality of most of the TRPV1 mutants was altered with respect to voltage, capsaicin, heat and/or their interactions. We identified two amino acid residues (R557 and D576) that could potentially constitute part of TRPV1 voltage sensor. The non-functional charge-reversing mutations R557E and R579E were partially rescued by charge-swapping mutations R557E/E570R and D576R/R579E, indicating that electrostatic interactions contribute to allosteric coupling between the voltage-, temperature- and capsaicin-dependent activation mechanisms. The mutant K571E was normal in all aspects of TRPV1 activation except for 2aminoethoxydiphenyl...
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Mechanisms of activation and modulation of the TRPV1 receptor: relationship between structure and function
Touška, Filip ; Vlachová, Viktorie (advisor) ; Teisinger, Jan (referee)
Vanilloid Receptor Subtype 1 - TRPV1 - is an important member of the TRP ion channel family. The physiological role of this ion channel is to integrate the nociceptive signals from the peripheral nervous system. TRPV1 participates in the detection of noxious stimuli on the primary afferent sensory neurons: of the chemical stimuli, such as capsaicin, resiniferatoxin, inflammatory mediators, endogenous vanilloids, and of the low pH (<6.5), and of the noxious heat stimuli (>43řC). Apart from the high expression on the dorsal root ganglion neurons and trigeminal ganglions, the functional expression of this receptor has been also found in the central nervous system, where its role remains elusive, however. Structural-functional studies which have been carried out since the receptor was first cloned, that is 13 years ago, have revealed several specific characteristics. Nonetheless, a number of questions remain to be answered, particularly those concerning cellular mechanisms and regulation of TRPV1's activation, modulation, and its complex function in the nervous system. The aim of this diploma thesis was to characterize, using electrophysiological and fluorescence techniques, the activation and desensitization kinetics of recombinant TRPV1 receptor in relation to the dynamic changes of intracellular calcium....
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Vanilloid receptor TRPV1 in mechanisms of acute pain
Nováková, Karolina ; Vlachová, Viktorie (advisor) ; Kozák, Jiří (referee) ; Rokyta, Richard (referee) ; Kršiak, Miloslav (referee)
2. SUMMARY Tbe vanilloid receptor (also callcd 'capsaicin receptor') ISa nonselective catian chaLUlcl that IS predominantly exprcssed by nociceptive primary sensory neurons. This receptor was tenned vaniUoid receptor subtype 1 (TRPV I) because it became the founding member of the vanilloid receptor subfamily ortransient receptor potential (TRP) channels, a large supeďamily af nonselective cation channels that play important roles in many sensory functions. The TRPV I channel cao be activated by pungcnt vanilJoid compounds like capsaicin ar resiniferatoxln, acid (pH < 6.5), noxious heat (> 43řC), phorbol csters, endogcnous arachidonic acid derivatives, ar depolarizing voltages. Upon activation by capsaicin, TRPV 1 can regulate cellular calcium levels via direct penneatioD (PCJPN. - lO), wbich concomitantly downregulates its own activity and activates different G-protein- and phospholipase C-coupled signaling cascades. Understanding tbese underlying mechanisms is one af the key strategies that offer a way to alleviatr;: neuropathic and inflammatory pain. Although tbe molecular identificatian of tbe TRPV l channels bas greatly facilitated understanding tbc mechanisms ofthermal hyperalgesia associated with inflammation and thase by which vanilloids increase and subsequendy modulate nociceptor activity, there is...
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Study of Structure-Function Relationship of Temperature-Gated TRP Channels
Benedikt, Jan ; Vlachová, Viktorie (advisor) ; Kršiak, Miloslav (referee) ; Blahoš, Jaroslav (referee)
Sensory physiology research was heavily influenced by molecular identification of transient receptor potential (TRP) ion channel family. Discovery of these unique family of membrane receptors allowed detailed study of their structure-function relationship. TRP channel expression in sensory neurons, but also apparently in keratinocytes provides living organisms with the ability to fast and accurately detect noxious thermal and chemical stimuli and to transmit this noxious signaling to higher nervous system structures. Despite recent efforts to elucidate molecular mechanisms of temperature or chemical activation of these non-selective cation channels, there is still no unifying hypothesis that is able to explain complex behaviour of these receptors. This dissertation aims to investigate three aspects of the TRP channel function: 1. Molecular characterization of acute desensitization of vanilloid receptor TRPV1 and investigation of the role of phosphorylation sites for calmodulin kinase II. 2. To characterize mechanisms of etanol-induced inhibition of menthol receptor TRPM8 and to find out possible physiological consequences of this inhibition. 3. To explore the role of inner pore region in activation gating of ankyrin receptor TRPA1 and identify amino acids involved in this process. Our findings contribute to...
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Structure and function of the vanilloid receptor TRPV1
Sušánková, Klára ; Vlachová, Viktorie (advisor) ; Kršiak, Miloslav (referee) ; Chvátal, Alexandr (referee)
2 11.. SSuummmmaarryy The vanilloid receptor (TRPV1) plays a role of the polymodal detector of nociceptive stimuli in a subset of small and medium sized primary sensory neurons in mammals. This channel can be activated by vanilloid compounds, low pH, and noxious heat, and its function is modulated by a wide range of endogenous and exogenous agents. Our results demonstrate that the reducing agent dithiothreitol (DTT) strongly potentiates both the native and recombinant rat TRPV1 channel when applied at millimolar concentrations to the external solution under intact whole-cell conditions. Since the effects of DTT were almost immediate, dose-dependent, and reversible, the contribution of extracellular cysteine residues within the putative pore-loop region of TRPV1, Cys616, Cys621, and Cys634 has been proposed. We show that the chemical modification of TRPV1 by both reducing and oxidizing agents leads to an increased response to heat. In addition, we identify the extracellularly located cysteine at position 621 which contributes to the DTT-induced potentiation of heat-activated currents mediated by TRPV1. Our data also indicate that capsaicin concentration is radically altered by the presence of oxidizing agents. More generally, the redox-active substances can substantially affect the activity of TRPV1 channels...
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