Browsing by Subject "asetyylikoliinin nikotiinireseptori"
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(2010)Neuronal nicotinic acetylcholine receptors are ligand-gated ion channel receptors that consist of five transmembral receptor subunits. They can form a functional receptor subtype solely out of alfa-subunits or out of a combination of alfa- and beta-subunits. The number of potential assembly of nicotinic receptor subunits is high because at least nine alfa-subunits (α2-α10) and three beta-subunits (β2-β4) have been recognized. The composition, location and pharmacological properties of different subtypes have not yet been fully characterized. However, it has been shown that the neuronal nicotinic receptors are involved in a wide range of physiological and pathofysiological processes especially in the central nervous system. Toxins from snakes and Conus -sea snails have proved to be important tools in neuropharmacological research, from which considerable information on structure and subunit combinations of the nicotinic receptors have been received. Receptors binding assays or autoradiographic experiments exploiting toxins have also been useful methods to locate the neuronal nicotinic receptors in mammalian brain. The aim of the experimental part of the Pro gradu was to characterize in vitro binding affinities of α-contoxin MII, α-conotoxin Vc1.1, neurotoxin II, α-cobratoxin and weak-toxin synthetized in Moscow and of well-known receptor ligands cytisine and methyllycaconitine to neuronal nicotinic receptors in SH-SY5Y- and SH-EP1-hα7-cell membrane preparations. SH-SY5Y-cells are known to express various neuronal receptor subtypes (α3* or α7) endogenously. For the SH-EP1-hα7-cells part, the cell line has been transfected with α7 nAChR-genes and it expresses only α7 receptor subtypes. Receptor competition studies were performed with [3H]-epibatidine (400 pM SH-SY5Y, 2000 pM SH-EP1-hα7) and the radioactivity was measured with a Microbeta- scintillation counter. [3H]-epibatidine was perceived to be displaced almost completely by cytisine and methyllycaconitine in both cell lines. In addition to this, the toxins were shown to bind to two distinct receptor-binding sites in SH-SY5Y-cells. Also α-contoxin MII and α-conotoxin Vc1.1 inhibited [3H]-epibatidine binding by biphasic manner, but the maximal displacement failed to be complete. From α-conotoxins only MII had affinity to α7 receptors in SH-EP1-hα7-cells. Neurotoxin II, α-cobratoxin and weak-toxin were not found to compete with [3H]-epibatidine for the same binding sites in SH-SY5Y-cells. The results confirm the assumption that cytisine and methyllycaconitine label various nAChR-subtypes. Instead, based on SH-SY5Y-cell assays α-conotoxins used in this study would seem to label spesifically only particular nAChR-subtypes. The receptor competition studies also confirm the prevalent conception that neurotoxin II, α-cobratoxin and weak-toxin do not bind to neuronal nicotinic receptor subtypes containing α3-subunits.
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(2017)The α5 subunit of nicotinic acetylcholine receptors forms functional receptors with other subunits as a structural subunit. It affects the structural and functional properties of the nicotinic receptor by increasing calcium permeability and accelerating desensitization. In the mammalian brain, the α5 mRNA is widely expressed, mostly in substantia nigra pars compacta, ventral tegmental area and interpeduncular nucleus. Its protein has been identified in various distinct brain areas, such as striatum, cortex and medial habenula. In the dorsal striatum partaking in motor functions, the α5 subunit modulates the release of dopamine, thus it is believed to have an impact on motor function. In the experimental part of the thesis mice lacking the α5 subunit were injected unilaterally with neurotoxin 6-hydroxydopamine (6-OHDA) in the striatum. The purpose was to determine the importance of the subunit with regard to the lesion extent and motor function. The motor functions of α5-deficient and wild type control mice were assessed in amphetamine- and apomorphine-induced rotametry. After the tests the mice were euthanized and their substantia nigra and striatal brain samples were collected for further analysis. The number of dopamine cells in the medial and dorsal tier of substantia nigra were determined, so as to quantify the extent of the lesions and to explain the research group's previous finding about the α5-deficient mice spinning less ipsilaterally in amphetamine induced rotametry. The α5-deficient mice were found to turn less ipsilaterally compared to the control mice in the amphetamine-induced rotametry and in the apomorphine-induced rotametry, first less contralaterally and subsequently more contralaterally than the control mice. The results of male mice, that were less in number, were excluded from the results as the difference between genders was significant in the wild type mice in the amphetamine-induced rotametry. There was no significant difference in the number of remaining dopamine cells between the genotypes after the lesioning in either of the areas of interest. However, the wild type mice tended to have less cells remaining in the medial tier of the substantia nigra. The observed differences between the genotypes in the rotametries could be accounted by differences in the amount of dopamine released from striatal neurons or differences in striatal dopamine receptor quantities or function. The results support the hypothesis about the contribution of the α5 subunit containing acetylcholine nicotinic receptors in motor function.
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