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Effect of stress on corticosteroid metabolism in peripheral tissues
Makal, Jakub ; Pácha, Jiří (advisor) ; Vybíral, Stanislav (referee)
Stressor influence can lead to homeostatic disruption. To eliminate this threat, mechanism which compensates negative effects of stressor was evolved by organisms. It's called stress response. One of two major systems that moderate stress response of organism is hypothalamic- pituitary-adrenal axis (HPA axis). Effectors of the HPA axis are glucocorticoids, steroid hormones secreted from adrenal glands. Enzymes which metabolize glucocorticoids are located in target tissues for these hormones. They convert active glucocorticoids into their inactive forms, or vice versa. Untill now, two such enzymes have been described - 11beta-hydroxysteroid dehydrogenases type 1 and type 2. It was proved that expression and activity of these enzymes can change under the influence of stressor. These changes are tissue-specific and dependent on type of applied stressor.
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Influence of cytokines on peripheral metabolism of glucocorticoids
Vitáčková, Kateřina ; Pácha, Jiří (advisor) ; Liberda, Jiří (referee)
11β-hydroxysteroid dehydrogenase (11βHSD) participates in prereceptor metabolism of glucocorticoids by conversion of their active forms (cortisol or corticosterone) to inactive forms (cortisone or 11-dehydrocorticosterone), thereby regulating concentration and activity of glucocorticoids in organism. 11βHSD type 1 (11βHSD1) activates glucocorticoids from their biologically inactive keto-forms, whereas the role of 11βHSD type 2 (11βHSD2) is their biological reinactivation. Previous in vivo studies showed that mRNA expression and enzyme activity of 11βHSD1 or 2 can be modified by influence of these cytokines, for example IL-1β and TNF-α. The aim of this study was to determine whether these cytokines play a role in regulation of 11βHSD expression in distal colon organ cultures and whether the changes of 11βHSD2 expression are induced by a direct effect of cytokines on epithelial cells. Total RNA was isolated from all samples and RNA was transformed to cDNA by reverse transcription. Subsequently mRNA of 11βHSD1 or 2 and the given housekeeping gene was measured by Q-PCR. Incubation of distal colon organ cultures with TNF-α (10 ng/ml; 48 hours) caused significant increase of 11βHSD1 expression, but no influence on 11βHSD2 expression was observed. In contrast, the expression of 11βHSD2 but not 11βHSD1 was...
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Cholinergic regulation of ion transport in the large intestine
Hock, Miroslav ; Pácha, Jiří (advisor) ; Moravec, Jan (referee) ; Kolínská, Jiřina (referee)
anglicky Acetylcholine is one of the most important mediators of enteric nervous system involved in the regulation of ion transport in the large intestine. Although, recently, plenty of new evidences of various expression of ion transport proteins in distal and proximal colon was published, there still lacks an electrophysiological study comparing these parts of colon considering all that new findings. The aim of this study was thus to compare cholinergic regulation of ion transport in distal and proximal colon. We measured responses of distal and proximal colon in Ussing chambers by voltage-clamp method. The colonic epithelium was clamped to 0 mV and responses were recorded as changes of short-circuit current (SCC). Instead of acetylcholine we used its stable analogue carbachol. Data were processed and analyzed using a VBA code I wrote for this purpose for MS Excel 2007. We confirmed that carbachol acts directly on epithelial cells via muscarinic acetylcholine receptors in both, distal and proximal colon. These responses to carbachol were not influenced by inhibitors of Cl- channels situated in apical membrane (CFTRinh-172 and niflumic acid). Inhibition of basolateral influx of Cl- by serosal Ba2+ and furosemid reduced responses to carbachol in both, distal and proximal colon. Inhibitors of K+...
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Regulation of calcium influx via voltage-dependent calcium channels (L-VDCC) into the vascular smooth muscle of genetically hypertensive rats
Surovcová, Mária ; Kuneš, Jaroslav (advisor) ; Hampl, Václav (referee) ; Pácha, Jiří (referee)
The present in vivo study was focused on better understanding of the pathophysiological mechanisms contributing to high blood pressure maintenance in spontaneously hypertensive rats (SHR). Genetic hypertension is characterised by abnormally elevated activity of sympathetic nervous system (SNS) and increased sensitivity to catecholamine-induced vasoconstriction comprising both enhanced calcium influx to vascular smooth muscle cells via L-type of voltage-dependent calcium channels (L-VDCC) and altered vascular sensitivity to intracellular calcium mediated by RhoA/Rho-kinase pathway. Thus, our aims were to study the regulation of L-VDCC channels, to determine the role of "calcium sensitization" in hypertension and finally to evaluate which of these pathways is important in the maintenance of high blood pressure. Using conscious SHR rats and their normotensive controls, WKY, we have confirmed that high sympathetic tone is responsible for increased calcium influx via L-VDCC in hypertension. The experiments based on the pertussis toxin-induced inactivation of inhibitory G proteins (Gi) have revealed that the control of L-VDCC by SNS is mediated by Gi protein- coupled pathway, the elimination of which leads to the attenuation of sympathetic vasoconstriction and to the decrease of blood pressure response to...
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Effect of stress on expression of glucocorticoid receptors and enzymes of glucocorticoid metabolism in specific structures of rat brain
Kvapilová, Pavlína ; Pácha, Jiří (advisor) ; Telenský, Petr (referee)
Stress response is trigerred by a number of factors, which, depending on the type of response they generate, involve different brain structures. These structures then relay the information to the paraventricular nucleus of hypothalamus (PVN), which is the main integration center for information about the imbalance of homeostasis induced by stressors. If it meets a sufficient number of excitatory signals, the PVN activates the hypothalamic pituitary adrenal axis, which ultimately triggers the secretion of stress hormones glucocorticoids, which then act back on the brain. This action is influenced by several factors, mainly the presence of local metabolism of glucocorticoids. Local metabolism is provided by the enzymes 11-hydroxysteroid dehydrogenases, which can locally activate or deactivate the hormone molecules and thus amplify or attenuate their effects.
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Endogenous steroid dehydroepiandrosterone and its role in modulation of local metabolism of glucocorticoids
Imrichová, Terezie ; Pácha, Jiří (advisor) ; Kůs, Vladimír (referee)
The anti-glucocorticoid effect of dehydroepiandrosterone (DHEA) have been known for many years. However, its molecular basis have not been elucidated yet. The results of certain experiments suggest that not DHEA but its 7-oxygenated metabolites 7-OH-DHEA, 7-OH-DHEA a 7-oxo-DHEA are the antiglucocorticoid molecules. Various hypothesis about how these steroids exert their antiglucocorticoid action have been tested during the last several years. Some of them were reliably disproved (e.g. the competitive inhibition of glucocorticoid receptors), others were validated (e.g. the DHEA-mediated change in expression of certain enzymes participating in glucocorticoid metabolism), and yet others are still being considered. Nevertheless, clarifying the nature of the anti-glucocorticoid effect of DHEA or its metabolites is crucial for its possible use as a therapeutic drug.
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Analysis of the involvement of α2 - AMPK in the beneficial effects of n-3 polyunsaturated fatty acids on obesity - associated metabolic derangements
Jeleník, Tomáš ; Rossmeisl, Martin (advisor) ; Cahová, Monika (referee) ; Pácha, Jiří (referee)
It is well established that n-3 polyunsaturated fatty acids with long chain (n-3 LC-PUFA) have beneficial effects on the obesity-induced metabolic disorders in mice. However, in obese humans, the potency of these fatty acids to positively affect obesity and insulin resistance has been shown to be lower. The aim of the studies described in this thesis was to verify various approaches aimed at increasing efficiency of n-3 LC-PUFA and to study the involvement of 2 subunit of AMP-activated protein kinase (2-AMPK) in the mechanisms of action of these compounds. Firstly, various chemical derivatives of DHA were tested in mice. Substance-2, the -ethyl ester of DHA, completely prevented and even partially reversed the development of obesity, fat accumulation, impaired glucose tolerance, dyslipidemia and white adipose tissue inflammation, even though the dose was only 10 % of that normally used in mice for the treatment with n-3 LC-PUFA. Secondly, the combination of n-3 LC-PUFA and a low-dose of anti-diabetic rosiglitazone prevented, in additive manner, development of dyslipidemia and insulin resistance, reduced the accumulation of body fat and adipocyte hypertrophy, while inducing adiponectin in mice fed a high-fat diet. This treatment also reversed impaired glucose tolerance in obese mice. The major part...
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Local metabolism of glucocorticoids in female Prague hereditary hypertriglyceridemic rats
Klusoňová, Petra ; Pácha, Jiří (advisor) ; Kopecký, Jan (referee) ; Haluzík, Martin (referee)
11-hydroxysteroid dehydrogenase (11HSD1) is an oxidoreductase which catalyzes conversion of inactive 11-oxo steroid derivatives into active 11-hydroxy forms. 11HSD1 elevates intracellular level of active glucocorticoid (GC) hormones: cortisol in human tissues and corticosterone in rodents, therefore local level of active GCs can be set independently from systemic secretion driven by hypothalamo-pituitary-adrenal axis (HPA axis). Chronic systemic excess of GCs results in development of Cushing's syndrome which is characterised by central obesity and other metabolic disturbances. Despite normal serum levels of GCs, the patients with idiopathic obesity also develop metabolic syndrome. It was suggested that GCs could be elevated locally in target tissues due to enhanced 11HSD1 activity. This hypothesis was confirmed in transgenic rodent models. Prague hereditary hypertriglyceridemic (HHTg) rats represent a non-obese model of metabolic syndrome without genetic manipulations or specific mutations. The strain was bred by cross-mating of Wistar rat individuals with elevated serum levels of triglycerides (TGs). The strain exhibit hypertriglyceridemia and hypertension. When kept on high carbohydrate diet HHTg rats exhibit alterations in glucose homeostasis. Since there are no data that would describe...
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