Effects of nicotine on the activity of glutamate transporter type 3 and the modulation mechanism

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의과대학 의학과
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서울대학교 대학원
학위논문 (박사)-- 서울대학교 대학원 : 의학과, 2014. 2. 임영진.
Introduction: Nicotine, main ingredient of tobacco elicits seizure in animal model and cigarette smoking is regarded as behavioral risk factors associated with epilepsy or seizure. In hippocampus, origin of nicotine-induced seizures, most of the glutamate uptake could be performed primarily by excitatory amino acid transporter type 3 (EAAT3). It has been reported that dysfunction of EAAT3 is related to temporal lobe epilepsy. Reduction of EAAT3 activity may be mediated by PKC and PI3K, two intracellular signaling molecules. Therefore, I hypothesized that nicotine may attenuate EAAT3 activity by mediation of PKC and PI3K.
Methods: I investigated the effects of nicotine on the activity of EAAT3 by using Xenopus oocyte expression system and the involvement of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K). Rat EAAT3 was expressed in Xenopus oocytes by injecting EAAT3 mRNA. L-Glutamate (30 μM)-induced inward currents were recorded via the two-electrode voltage clamp method. Responses were quantified by integration of the current trace and reported in microCoulombs (μC).
Results: Nicotine decreased EAAT3 activity in a dose-dependent manner when oocyte was exposed to nicotine for 72 h. Since the inhibition reached maximal at nicotine concentrations at 0.03 μM, I used 0.03 μM for further experiments. Nicotine (0.03 μM for 72 h) significantly reduced Vmax, but did not alter Km value of EAAT3 for glutamate. When nicotine treated oocytes were incubated with phorbol-12-myrisate-13-acetate, a protein kinase C (PKC) activator, PMA-induced increase in EAAT3 activity was abolished. PKC inhibitors (staurosporine, chelerythrine and calphostin C) significantly reduced basal EAAT3 activity. Whereas, there were no significant differences among the PKC inhibitors, nicotine, and PKC inhibitors + nicotine groups. In similar fashion, PI3K inhibitors (wortmannin and LY294002) significantly decreased EAAT3 activity, however no statistical differences were observed among PI3K inhibitors, nicotine, and PI3K inhibitors plus nicotine groups.
Conclusions: This study demonstrates that nicotine decreases EAAT3 activity and this effect seems to be mediated by PKC and PI3K. This study may provide an additional mechanism for nicotine induced seizure.
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College of Medicine/School of Medicine (의과대학/대학원)Dept. of Medicine (의학과)Theses (Ph.D. / Sc.D._의학과)
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