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Modulatory effect of monovalent ions on δ-opioid receptors
Vošahlíková, Miroslava ; Svoboda, Petr (advisor) ; Jakubík, Jan (referee) ; Kršiak, Miloslav (referee)
The exact role of opioid receptors in drug addiction and modulatory mechanism of action of monovalent cations on these receptors are still not fully understood. Our results support the view that the mechanism of addiction to morphine is primarily based on desensitization of μ- and δ-opioid receptors. Desenzitization of agonist response proceeds already at the level of G protein functional activity. Long-term exposure of rats to morphine resulted in increase of number of δ-opioid receptors and change of their sensitivity to sodium ions. Analysis of the effect of different monovalent ions on agonist binding in δ-OR- Gi1α (Cys351 -Ile351 )-HEK293 cell line confirmed the preferential sensitivity of δ-opioid receptor to sodium ions. We have distinguished the high- and low-affinity Na+ sites. Biophysical analysis of interaction of lithium, sodium, potassium and cesium ions with plasma membranes isolated from HEK293 cells with the help of fluorescent probes indicated that monovalent ions interact, in low-affinity manner, with the polar, membrane-water interface of membrane bilayer. Key words: morphine, forebrain cortex, opioid receptors, G proteins, monovalent ions, plasma membrane, fluorescence spectroscopy.
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Evaluation of opioid and TLR-4 receptors in the mechanism of opioid effects on heart muscle cells
Biriczová, Lilla ; Novotný, Jiří (advisor) ; Alánová, Petra (referee)
It has been reported that opioid receptor activation mimics ischemic preconditioning, which may protect the heart from the development of infarction. Toll-like receptor 4 (TLR-4) during infarction stimulates cytokine production leading to inflammation and injury of the heart tissue. Our aim was to study the effect of morphine in vitro on the viability and oxidative state of H9c2 cells (rat cardiomyoblasts) and the role of TLR-4 during oxidative stress. Our experiments showed that pretreatment with morphine before tert-butylhydroperoxide (t-BHP)-, 2,2'-bipyridyl (BP)- and lipopolysaccharide (LPS)-induced oxidative stess had protective effect on the viability of H9c2 cells and markedly reduced the production of reactive oxygen species (ROS). The protective effect of morphine was diminished after naloxone treatment, which confirms the role of opioid receptors in preconditioning. TLR-4 inhibition by TAK-242 pretreatment and silencing TLR-4 by RNA interference resulted in a partial increase in cell viability but significant attenuation of ROS production after t-BHP and BP treatment. The action of LPS was reduced in response to TLR-4 silencing. Interestingly, naloxone pretreatment and suppression of TLR-4 markedly alleviated oxidative stress and resulted in a significant improvement of cell viability. We...
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Effect of chronic morphine on cell survival after oxidative stress in the SH-SY5Y neuroblastoma cell line
Moutelíková, Karolína ; Hejnová, Lucie (advisor) ; Musílková, Jana (referee)
Morphine is a natural opioid which is used in medicine due to his potent analgesic and sedative effects. In the forefront of scientific interest is a chronic usage of opioids which can lead to a development of drug addiction. Morphine role in oxidative stress was described in last years. It was revealed its protective potencial by many studies. However, some studies described its pro-oxidative effect. The aim of this study was to determinate effect of chronic morphine on cell survival after oxidative stress caused by H202 analog - tBHP in the SH-SY5Y neuroblastoma cell line. The results verified morphine protective effect against oxidative stress. The highest protective effect of morphine was achieved in a concetration of 10 µM. It was desribed that morphine can induce activation of mu-opioid (MOR) and Toll-like 4 (TLR4) receptors signalling pathway on molecular level. The aim of this thesis was to evaluate the role of MOR a TLR4 in protective effect of morphine against oxidative stress by two methods. Firstly, it was used tests of oxidative stress on cell viability. The obtained results demonstrated majority role of TLR4 and minory role of MOR. Afterwards, we assesed changes in the expression of MOR a TLR4 after chronic morphine by SDS-PAGE electrophoresis. Results of these experiments did not...
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Methadone and its usage in treatment of opioid drug addiction
Chona, Kembe ; Svoboda, Petr (advisor) ; Hejnová, Lucie (referee)
Methadone, a synthetic opioid created in the 1940s is a potent mu opioid receptor agonist. Opioid receptors form a sub-group of the GPCR super-family. Their most significant role is the inhibition of neural pathways by regulating the activity of ionic channels and effector proteins. µ-opioid receptors are the site of action of heroin, methadone and other classical opioid agonists. Due to the opioid receptors distribution in both the central nervous system and peripheral tissues, methadone affects a wide variety of functions in the organism. Methadone induces many of the effects of classical opioids including analgesia, respiratory suppression, sedation, euphoria. While originally being developed as an analgesic it had soon shown potential for other therapeutic methods. Methadone maintenance therapy was introduced in 1963, by professor Vincent P. Dole and his team. It quickly became clear that methadone substitution therapy is indeed very effective and shows the highest ability to retain patients. Thanks to its high oral bioavailability, higher intrinsic efficacy and long terminal half-life methadone is the first choice drug for opioid substitution therapy. Methadone, used in appropriate doses produces only mild adverse effects and has the ability to normalize physiological homeostasis disrupted by...
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Influence of opioid receptor genetic polymorphism on drug addiction.
Syrová, Kateřina ; Hejnová, Lucie (advisor) ; Brejchová, Jana (referee)
Opioid substances have been used by mankind for several millenia not only for their ability to abolish even very intensive states of pain, but also for their ability to stimulate the brain reward circuit. However, a strong addiction can be formed to opioids quickly. This thesis focuses on collecting knowledge about the most abundant polymorphisms of these genes, their physiological impact and potential influence on drug addiction. Key words: opioid receptors, drug addiction, genetic polymorphism
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Role of glutamatergic transmission in mechanisms of addiction to morphine.
Moutelíková, Karolína ; Hejnová, Lucie (advisor) ; Červená, Kateřina (referee)
Drugs are used by mankind since ancient times. One group of these substances are opioids. Opioids have antinociceptive effects and can induce euphoria and relaxation as well. A chronic usage of opioids can lead to a development of drug addiction and phenomens like tolerance and sensitization. One of the most used opioids in medicine is morphine. Morphine is isolated from opium of poppy (Papaver somniferum). Direct effect of morphine is mediated via activation of μ- opioid receptors and their signal cascade. It was implicated that the usage of morphine affects other neurotransmmiter systems in the brain and these neurotransmmiter systems play a signifikant role in the development of addiction and other phenomena. One of these systems is an important excitatory brain system - glutamergic system. This bacherol thesis focuses on interrelationship between opioid and glutamatergic systems during addiction.There were described changes in a composition of glutame ionotropic receptors and changes in their expression as well as in expression of metabotropic glutamate receptors. These changes differ in various parts of the brain and in diverse stages of addiction on morphine too. In spite of all diferences, the results of studies indicate that glutamatergic receptors play a significant role in the development...
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Study of opioid receptors
Cechová, Kristína ; Hudeček, Jiří (advisor) ; Holáň, Vladimír (referee)
1 ABSTRACT In this Thesis, we studied properties of μ-, δ-, and κ-opioid receptors in lymphocytes isolated from rat spleen. This splenocytes were exposed to mitogen concanavalin A or opiate morphine and cultivated for 48 hours. Under physiological conditions, level of opioid receptors in immune cells is very low. Due to various factors such as presence of opioids, mitogens, long-term exposition to stress, expression of these receptors can be amplified. In this study we demonstrated, that concanavalin A causes up-regulation of μ-, δ- and κ-opioid receptors in lymphocytes isolated from rat spleen. In control cells no significant signal of μ- or δ-receptors was observed. In contrast, κ-opioid receptors were detected already in control cells. Concanavalin A stimulation caused a 2.4 - fold increase of these receptors. In lymphocytes treated with morphine only μ-opioid receptors were up-regulated, whereas in control cells, there was no signal for these receptor type. δ-opioid receptors were not detected in control or morphine treated cells. κ-opioid receptors were determined in control and also in morphine affected lymphocytes but the amount of these receptors wasn't changed by morphine. Detection of μ-, δ- and κ-opioid receptors using Western blot technique in lymphocytes isolated from rat spleen, that were...
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Modulatory effect of monovalent ions on δ-opioid receptors
Vošahlíková, Miroslava ; Svoboda, Petr (advisor) ; Jakubík, Jan (referee) ; Kršiak, Miloslav (referee)
The exact role of opioid receptors in drug addiction and modulatory mechanism of action of monovalent cations on these receptors are still not fully understood. Our results support the view that the mechanism of addiction to morphine is primarily based on desensitization of μ- and δ-opioid receptors. Desenzitization of agonist response proceeds already at the level of G protein functional activity. Long-term exposure of rats to morphine resulted in increase of number of δ-opioid receptors and change of their sensitivity to sodium ions. Analysis of the effect of different monovalent ions on agonist binding in δ-OR- Gi1α (Cys351 -Ile351 )-HEK293 cell line confirmed the preferential sensitivity of δ-opioid receptor to sodium ions. We have distinguished the high- and low-affinity Na+ sites. Biophysical analysis of interaction of lithium, sodium, potassium and cesium ions with plasma membranes isolated from HEK293 cells with the help of fluorescent probes indicated that monovalent ions interact, in low-affinity manner, with the polar, membrane-water interface of membrane bilayer. Key words: morphine, forebrain cortex, opioid receptors, G proteins, monovalent ions, plasma membrane, fluorescence spectroscopy.
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Changes in opioid signalisation during the ontogenesis.
Hoľuková, Martina ; Hejnová, Lucie (advisor) ; Škrabalová, Jitka (referee)
Opioid receptors interact with opiate compounds, causing the inhibition of neuroexcitability. The activation of signaling pathway of opioid receptors plays crucial role in the treatment of chronic and cancer pain. Summary of the general knowledge about opioid receptors and about the development of tolerance is in the first part of this work. Next part of the thesis concerns on ontogenesis of opioid receptors and other components related to the opioid signaling pathways as age seems to have an immense influence on molecules within and related to the opioid signaling. Finally, last part of this work collects data about the influence of morphine during ontogenesis as morphine is one of the most used opiate compound used in clinical treatment. Key words Opioid receptor, signalization, ontogenesis, addiction, tolerance, morphine
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Conformational states of the opiod receptors
Provazníková, Adéla ; Hudeček, Jiří (advisor) ; Entlicher, Gustav (referee)
This Thesis elucidates the relationships between structure and function of opioid receptors. The mechanism of opioid receptor function could be better understood on the basis of the recent knowledge of their three-dimensional structure. In the first part of the Thesis, methods for membrane protein crystallization and X-ray structural analysis are reviewed with a special focus on in meso crystallization. Next, the three opioid receptor subtype crystal structures are described and analysed, starting with their general architecture and proceeding to the detailed description including the binding pocket for opioid ligands with the help of visualization with the PyMol software. In its third part, this Thesis re-examines the structure-function correlations predicted on the basis of site-directed mutagenesis (Décaillot F.M. et al, Nature Structural Biology 10, 629, 2003) in the light of now available crystal structure. This analysis is pointing to the potentially important contacts of aspartate D128, suggesting that ligand binding and receptor activation might involve changes in its interaction with tyrosine Y308.
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