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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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Characterization of CART peptide analogs in vitro and in vivo
Nagelová, Veronika ; Maletínská, Lenka (advisor) ; Vybíral, Stanislav (referee)
Peptide CART (cocaine- and amphetamine- regulated transcript) is a neuropeptide acting in the hypothalamus to reduce food intake (anorexigenic peptide). Despite all efforts the receptor and the mechanism of action is still unknown. This peptide has two biologically active forms, CART(55-102) and CART(61-102). Peptide CART is able to bind to pheochromocytoma cells PC12. PC12 cells differentiated in neuronal phenotype with NGF (nerve growth factor) showed a higher number of binding sites (11250 ± 2520 binding sites/cell) compared to undifferentiated cells (3600 ± 570 binding sites/cell). PC12 cells differentiated by dexamethasone to chromaffin cells showed high non-specific binding. Peptide CART contains three disulfide bridges. To clarify the importance of each disulfide bridge to maintain biological activity, analogues with one (analogue 3, 4 and 5) or two (2, 6, 7 and 8) disulfide bridges and a peptide analogue of CART (61-102), which has methionin at position 67 replaced with norleucine were synthesized. We showed that biological activity was unchanged at analogue 1 and analogue 7 containing disulfide bridges in positions 74-94 and 88-101. When investigating cell signaling in PC12 cells, we tested if peptide CART activate of c-Fos, c-Jun, phosphorylated ERK1/2, CREB, JNK and p38. CART peptide...
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