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Role of apoptosis in the mechanism of opioid effect
Moravcová, Simona ; Novotný, Jiří (advisor) ; Ostašov, Pavel (referee)
2. Abstract Opioids have been used as effective analgesics for many years. The worst problem is, however, their adverse effects and potential addiction. There are three known types of opioid receptors that differ mainly in their sensitivity to different opioids and their distribution in the CNS. This work also deals with apoptosis. Apoptosis is an important process not just in the embryonic development, e.g. for the right differentation of fingers and toes, but also in the adulthood, e.g. to suppress tumour formation. Whereas an apoptotic pathway can be initiated in many different ways, there is only one mechanism that actually causes the death of a cell. It is important for the cell death and division to be in balance. The breach of this balance may result in pathologic effects. This work summarizes the effects of opioids in connection with apoptosis. The main object of the research of this relationship was morphine which has both pro-apoptotic and anti-apoptotic effects. Another two frequently investigated opioids are methadone and heroin. Heroin is mainly used in the studies researching the impact of this highly addictive opioid on the apoptosis and embryonic development. Keywords: opioids, opioid receptors, G-proteins, apoptosis, morphine
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Myocardial beta-adrenergic signaling during adaptation of rats to chronic hypoxia
Hahnová, Klára ; Novotný, Jiří (advisor) ; Ostašov, Pavel (referee)
Endogenous cardiac protection against acute ischemia/reperfusion injury can by increased by cardiac adaptation to various forms of chronic hypoxia. Chronic hypoxia induces a large variety of adaptive changes in the myocardium that could be considered as protective, but the exact mechanism of increased ischemic tolerance is unknown. Different studies suggest that catecholamine release and their effect on -adrenergic signaling after adaptation to chronic hypoxia contributes to cardioprotection. In this study we focused on characterization of -adrenergic receptors ( -ARs) in the myocardium of rats after adaptation to three different hypoxic conditions: 1. intermittent normobaric hypoxia - INH/R (23 h hypoxia, 1 h reoxygenation), 2. intermittent normobaric hypoxia - INH (8 h hypoxia, 16 h normoxia), 3. continuous normobaric hypoxia - CNH (24 h hypoxia). We compared how each hypoxic model affects the total number of -adrenergic receptors and proportion of individual subtypes ( 1-and 2-ARs) in the left and right ventricles compared control normoxic rats. The INH model had apparently no effect on -ARs in either ventricles. On the other hand, adaptation to INH/R and CNH was accompanied by a significant decrease (by about 25%) in the total number of -adrenergic receptors in the right ventricles. Our present...
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Effect of cholesterol depletion on signalling cascade initiated with receptors coupled to G protein class Gq/G11
Ostašov, Pavel ; Svoboda, Petr (advisor) ; Teisinger, Jan (referee) ; Hof, Martin (referee)
Membrane domains are an important structure in plasamatic membrane. They concentrate various signaling molecules. Their main structural component is cholesterol and by its removal the membrane domains are disrupted. The aim of our work was to examine the effect of cholesterol depeletion on signaling initiated thyreothropin releasing hormone (TRH). Although its signaling cascade is located within membrane domains the receptor itself is not. We showed that cholesterol depletion by -cyclodextrin caused release of Gq/11 proteins and caveolin 2 from membrane domains. We also discovered that cholesterol depletion decreases potency of TRH to activate G proteins as well as induction of release of intracellular Ca2+ In the last part we investigated the effect of disruption of the cell membrane integrity by cholesterol depletion on thyrotropin-releasing hormone receptor (TRH-R) surface mobility and internalization in HEK293 cells stably expressing TRH-R-eGFP fusion protein. CLSM studies indicated that the internalization of receptor molecules initiated by TRH stimulation was significantly attenuated. The detailed analysis of recovery of TRH-R-eGFP fluorescence in bleached spots of different sizes indicated that cholesterol depletion results in an increase of overall receptor mobility. We suggest that migration of...
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A study of signaling and cytoprotective potential of cannabinoid GPR55 receptors in PC12 cells
Pavluch, Vojtěch ; Novotný, Jiří (advisor) ; Ostašov, Pavel (referee)
At the end of the 20th century it was known that cannabinoid drugs interact with two receptors, CB1 and CB2. Subsequent pharmacological studies have confirmed that there are other receptors interacting with cannabinoids. GPR55 is a transmembrane G protein coupled receptor, which together with the receptor GPR18 and GPR119 belong to a group of new cannabinoid receptors and is involved in the function of the endocannabinoid system. In addition to some of cannabinoid substances, it is stimulated primarily phospholipid lysofosfatidylinositolem. LPI-dependent signaling GPR55 plays an important role in the regulation of many physiological and pathological processes, such as pain, inflammation, cell proliferation, or endothelial function. It was found that LPI confers tolerance to ischemic brain damage and has a cytoprotective effect on the pyramidal cells. The aim of the study was to determine whether the application of five ligands induce phosphorylation of protein kinase ERK 1/2, Akt and activate the GTPase RhoA and whether activation of the receptor GPR55 has cytoprotective effect in model cell line PC12, in which hypoxic conditions were simulated by adding CoCl2. For working methods were used SDS-PAGE, Western bloting and colorimetric measurement. Pharmacological studies in recent years have shown...
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Expression of G-protein Dexras1 in the rat suprachiasmatic nucleus.
Marschnerová, Tereza ; Bendová, Zdeňka (advisor) ; Ostašov, Pavel (referee)
The main mammalian circadian oscillators, suprachiasmatic nuclei (SCN), are stored in the hypothalamus along the third ventricle. They are responsible for time synchronization of behavioral and physiological processes in the body. The endogenous period and phase are controlled by rhythmic changes in the external environment, especially by changing of light and darkness. The light pulse during the night activates NMDA type glutamate receptors, neuronal NO synthase kinase and MAPK ERK1 / 2. This signaling pathway affects the transcription of clock genes and thus the mechanism generating circadian oscillations. Protein Dexras1 acts as a nucleotide exchanger monomer and heterotrimer Gi/o protein and is considered a connecting link between the activation of NMDA glutamate receptor agonist, nNOS signaling and phosforylation of ERK1 / 2. It is rhythmically expressed in the suprachiasmatic nucleus and its genetic deletion leads to abnormal light synchronization.This may indicate its role in the signaling cascade that results in the information concerning the light pulse from the retina to the SCN. The aim of this thesis was to describe the expression of mRNA and protein Dexras1 in the development of the rat suprachiasmatic nuclei and correlate it with the maturation mechanism of light synchronization. Our...
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Apoptosis in rat myocardium: effect of morphine
Moravcová, Simona ; Novotný, Jiří (advisor) ; Ostašov, Pavel (referee)
2. Abstract The aim of this dissertation was to study the expression of proteins that participate in apoptosis in myocardium of rats treated with low (0,1 mg/kg; 1 mg/kg) and high (10 mg/kg) doses of morphine. Low doses of morphine were administered daily for 28 days and high doses for 10 days. In addition, the effect of one week of drug withdrawal was studied in animals administered 1 mg/kg of morphine. Another aim was to determine activity of caspase-3 in samples of rat myocardium prepared from control rats and those exposed to high dose (10 mg/kg) of morphine for 10 days. The balance between cell division and cell death is crucial for the maintenance of homeostasis. Apoptosis is controled by proteins from the Bcl-2 family. These proteins are able to regulate permeability of outer mitochondrial membrane and may facilitate the release of cytochrom c and other proteins from mitochondria under certain conditions. The release of these proteins then leads to activation of executive enzymes of apoptosis. In this thesis we investigated the expression of proapoptotic proteins AIF, Bax, Bak, Bid and antiapoptotic protein Bcl-2 in samples from left myocardial ventricles of rats that was treated with different doses of morphine. Except for a significant decrease in the expression of AIF after short-term treatment...
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Myocardial beta-adrenergic signaling during adaptation of rats to chronic hypoxia
Hahnová, Klára ; Novotný, Jiří (advisor) ; Ostašov, Pavel (referee)
Endogenous cardiac protection against acute ischemia/reperfusion injury can by increased by cardiac adaptation to various forms of chronic hypoxia. Chronic hypoxia induces a large variety of adaptive changes in the myocardium that could be considered as protective, but the exact mechanism of increased ischemic tolerance is unknown. Different studies suggest that catecholamine release and their effect on -adrenergic signaling after adaptation to chronic hypoxia contributes to cardioprotection. In this study we focused on characterization of -adrenergic receptors ( -ARs) in the myocardium of rats after adaptation to three different hypoxic conditions: 1. intermittent normobaric hypoxia - INH/R (23 h hypoxia, 1 h reoxygenation), 2. intermittent normobaric hypoxia - INH (8 h hypoxia, 16 h normoxia), 3. continuous normobaric hypoxia - CNH (24 h hypoxia). We compared how each hypoxic model affects the total number of -adrenergic receptors and proportion of individual subtypes ( 1-and 2-ARs) in the left and right ventricles compared control normoxic rats. The INH model had apparently no effect on -ARs in either ventricles. On the other hand, adaptation to INH/R and CNH was accompanied by a significant decrease (by about 25%) in the total number of -adrenergic receptors in the right ventricles. Our present...
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Role of apoptosis in the mechanism of opioid effect
Moravcová, Simona ; Novotný, Jiří (advisor) ; Ostašov, Pavel (referee)
2. Abstract Opioids have been used as effective analgesics for many years. The worst problem is, however, their adverse effects and potential addiction. There are three known types of opioid receptors that differ mainly in their sensitivity to different opioids and their distribution in the CNS. This work also deals with apoptosis. Apoptosis is an important process not just in the embryonic development, e.g. for the right differentation of fingers and toes, but also in the adulthood, e.g. to suppress tumour formation. Whereas an apoptotic pathway can be initiated in many different ways, there is only one mechanism that actually causes the death of a cell. It is important for the cell death and division to be in balance. The breach of this balance may result in pathologic effects. This work summarizes the effects of opioids in connection with apoptosis. The main object of the research of this relationship was morphine which has both pro-apoptotic and anti-apoptotic effects. Another two frequently investigated opioids are methadone and heroin. Heroin is mainly used in the studies researching the impact of this highly addictive opioid on the apoptosis and embryonic development. Keywords: opioids, opioid receptors, G-proteins, apoptosis, morphine
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Effect of cholesterol depletion on signalling cascade initiated with receptors coupled to G protein class Gq/G11
Ostašov, Pavel ; Svoboda, Petr (advisor) ; Teisinger, Jan (referee) ; Hof, Martin (referee)
Membrane domains are an important structure in plasamatic membrane. They concentrate various signaling molecules. Their main structural component is cholesterol and by its removal the membrane domains are disrupted. The aim of our work was to examine the effect of cholesterol depeletion on signaling initiated thyreothropin releasing hormone (TRH). Although its signaling cascade is located within membrane domains the receptor itself is not. We showed that cholesterol depletion by -cyclodextrin caused release of Gq/11 proteins and caveolin 2 from membrane domains. We also discovered that cholesterol depletion decreases potency of TRH to activate G proteins as well as induction of release of intracellular Ca2+ In the last part we investigated the effect of disruption of the cell membrane integrity by cholesterol depletion on thyrotropin-releasing hormone receptor (TRH-R) surface mobility and internalization in HEK293 cells stably expressing TRH-R-eGFP fusion protein. CLSM studies indicated that the internalization of receptor molecules initiated by TRH stimulation was significantly attenuated. The detailed analysis of recovery of TRH-R-eGFP fluorescence in bleached spots of different sizes indicated that cholesterol depletion results in an increase of overall receptor mobility. We suggest that migration of...
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