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Določanje privzema dopamina v astrocite novorojene podgane
ID Velimirović, Eva (Author), ID Kržan, Mojca (Mentor) More about this mentor... This link opens in a new window

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Abstract
Astrociti so poleg presinaptične in postsinaptične živčne celice enakovredni del sinapse (tridelne sinapse). Tesno ovijajo sinapse in skrbijo za odstranjevanje živčnih prenašalcev iz sinaptične špranje ter preprečujejo, da bi ti v visokih koncentracijah delovali nevrotoksično. Veliko je že bilo raziskanega o privzemu histamina in glutamata v astrocite, manj pa je znanega o prenašalcih, ki sodelujejo pri privzemu dopamina. Dopamin se privzema iz zunajceličnega prostora in iz sinaptične špranje skozi membrano v notranjost nevrona z aktivnim prenosom ali z olajšano difuzijo. V magistrski nalogi smo na kulturah astrocitov, ki smo jih izolirali iz možganske skorje in striatuma novorojene podgane, raziskovali lastnosti prenašalcev dopamina. Želeli smo preveriti, ali se prenašalci in način prenosa dopamina v astrocite iz različnih delov možganov razlikujejo in ali v posameznih delih prevladuje aktivni prenos ali olajšana difuzija. Privzem radioaktivno označenega [3H]-dopamina v astrocite možganske skorje je časovno, koncentracijsko in temperaturno odvisen proces. Kinetika privzema se je v naših poskusih razlikovala glede na del možganov, iz katerega smo izolirali astrocite. V fizioloških pogojih (pH 7,4) privzem ni najoptimalnejši. Dopamin se učinkoviteje prenaša pri nižjem pH. Analizirali smo kinetične lastnosti privzema dopamina v astrocite možganske skorje in striatuma. Ugotovili smo, da se dopamin privzema prek nizkoafinitetnega prenašalnega sistema s konstanto po Michaelisu in Maud Menten (Km) 1063 nM ter maksimalno hitrostjo privzema (Vmax) 13,06 fmol/mg proteina/min. Privzem je skozi celoten koncentracijski interval neodvisen od Na^+. Prav tako privzema niso zmanjšali selektivni zaviralci dopaminskega prenašalca (DAT) in noradrenalinskega prenašalca NET. Lastnosti prenašalca ustrezajo prenosu olajšane difuzije. Dopamin se v astrocite striatuma skozi skoraj celoten koncentracijski interval privzema z visokoafinitetnim prenašalcema s konstanto po Michaelisu in Maud Menten (Km) 195,3 nM in maksimalno hitrostjo privzema (Vmax) 25,76 fmol/mg proteina/min. Prenašalec je bil skoraj celoten interval odvisen od prisotnosti Na^+ in privzem dopamina se je zmanjšal ob prisotnosti zaviralcev aktivnih prenašalcev DAT in NET. Vse to so lastnosti aktivnega prenosa. V višjih koncentracijah inkubacije (150 nM) se je kinetika privzema spremenila. Privzem je postal neodvisen od prisotnosti Na^+ in v manjši meri občutljiv na prisotnost zaviralcev aktivnega prenosa. Sklepamo, da se pri višjih koncentracijah aktivni prenašalci zasitijo in večjo vlogo pri privzemu dobijo prenašalci olajšane difuzije.

Language:Slovenian
Keywords:Dopamin, astrociti, privzem dopamina, olajšana difuzija, aktivni prenos
Work type:Master's thesis/paper
Organization:FFA - Faculty of Pharmacy
Year:2019
PID:20.500.12556/RUL-109914 This link opens in a new window
Publication date in RUL:10.09.2019
Views:1134
Downloads:237
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Secondary language

Language:English
Title:Determination of dopamine uptake into neonatal rat astrocytes
Abstract:
Astrocytes are in addition to the presynaptic and postsynaptic nerve cells, an equivalent part of the synapse (tripartite synapse). They tightly wrap the synapses and are responsible for the removal of neurotransmitters from the synaptic cleft and prevent them from working neurotoxic. Much has already been explored about the removal of glutamate, histamine and other neurotransmitters. Far less studies have been done to clarify the mechanism of astrocytes dopamine uptake. Dopamine can be transported into the neuron via active uptake or facilitated diffusion. In the thesis experiments, we investigated the properties of the dopamine uptake in astrocytes that were isolated from the cortex and striatum of the newborn rat. We wanted to check whether the uptake differs in different parts of the brain and which uptake (active transport or facilitated diffusion) is dominant in individual areas of the brain. The dopamine uptake in astrocytes is time, concentration and pH dependent. The kinetics of the transport was different in astrocytes that were isolated from different brain regions. Under physiological conditions (pH 7,4) the uptake is not optimal. It gets much higher at lower pH. We found that dopamine uptake in astrocytes from the cerebral cortex is transmitted through a low-affinity transport system with a Michaelis and Maud Menten (Km) constant 1063 and a maximum uptake rate 13,06 fmol/mg protein/min. The uptake was ?Na?^+ independent and it wasn’t inhibited by selective DAT and NET inhibitors. The characteristics of the uptake correspond to the facilitated diffusion. The uptake of [3H]-dopamine in the astrocytes of the striatum is transmitted with a high-affinity transporter with a Michaelis and Maud Menten constant (Km) 195.3 nM, and maximum rate of uptake (V max) of 25,76 fmol/mg protein/min. The transporter was dependent on the presence of ?Na?^+ almost the entire concertation interval and the uptake of dopamine decreased in the presence of DAT and NET inhibitors. The transporter characteristics correspond to the active transport. At higher concentrations (higher than 150 nM), the kinetics of the uptake changed and low-affinity transporters and facilitate diffusion became the main uptake mechanism. The uptake became ?Na?^+ independent and insensitive to the presence of selective DAT and NET inhibitors.

Keywords:Dopamine, astrocytes, dopamine uptake, facilitated diffusion, active transport

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