腦部血清素5-HT1A自控受體和血清素運轉子(SERT)皆為可以調控細胞外血清素且都位於血清素核縫合(raphe nucleus)神經元樹突處。過去文獻證實，當5-HT1A受體活化會啟動下游PKC的活化，且已知PKC可影響SERT再回收血清素及表現在細胞膜上的能力。然而許多文獻已證實SERT可被其他受體所調節，然而對於一樣位於突觸前的5-HT1A受體是否調控SERT卻未有探討。基於以上理由，我們利用大白鼠血小板當作實驗的模型，因為過去文獻指出，血小板含有內生性的5-HT1A受體和SERT。首先，我們利用受體結合實驗及[3H] 5-HT回收實驗確認大白鼠血小板含有5-HT1A受體且也表現有功能性的SERT。之後，我們利用[3H] 5-HT回收實驗在大白鼠血小板處理5-HT1A受體致效劑 (8-OH-DPAT) 0.01~1μM，發現隨劑量增加有減少SERT再回收[3H] 5-HT的現象。此外當我們前處理5-HT1A受體拮抗劑 (WAY100135) 0.1~1μM則可以緩解8-OH-DPAT (0.1μM) 抑制SERT再回收的作用，因此可以確認5-HT1A受體會調控SERT使其減少再回收的功能。接著則進一步探討5-HT1A受體是藉由活化下游哪些訊號蛋白調控SERT的作用。在過去研究證實，PKC和p38 MAPK皆可調控SERT的功能，因此我們探討在大白鼠血小板中，PKC調控SERT情形和p38 MAPK表現及是否會調控SERT的作用。結果發現PKC活化劑 (β-PMA) 0.01~0.1μM及p38 MAPK抑制劑 (SB203580) 0.1~10μM，都會抑制SERT再回收[3H] 5-HT的能力，且p38 MAPK抑制劑會明顯減少p38 MAPK磷酸化的情形；另一方面，處理p38 MAPK活化劑 (anisomycin) 0.1~10μM，對於增加p38 MAPK磷酸化情形則不太明顯。而當不同時間點和濃度作用下活化5-HT1A受體 (1μM 8-OH-DPAT)，發現對於p38 MAPK磷酸化情形有些許的抑制作用，但會顯著增加PKCδ/T505及Akt/S473的磷酸化情形。若前處理PKC抑制劑 (GF109203X) 0.3μM或 PI3K抑制劑 (wortmmanin) 0.2μM都可以部分逆轉5-HT1A受體抑制SERT再回收的作用。最後，我們研究當5-HT1A受體抑制SERT再回收的作用，是否也會影響SERT在血小板細胞膜上的表現。利用surface biotinylation assay，當1μM 8-OH-DPAT處理10分鐘或0.1μM β-PMA反應30分鐘的時候，有明顯減少SERT在血小板表面表現的情形，且20μM WAY100135可以緩解8-OH-DPAT促使SERT進行內飲作用(endocytosis)。因此，我們推論，5-HT1A自控受體活化後，除了會抑制神經的活化而抑制血清素的生合成，另一方面也可以利用減少SERT再回收或表現來抑制SERT的功能，雙向調控以維持突觸間隙血清素的含量。

Presynaptic 5-HT1A autoreceptor and serotonin transporter (SERT) each plays an important role in regulation of extracellular 5-HT in the raphe and other brain regions. Based on the knowledge that 5-HT1A receptor positively linked with PKC pathway whiles SERT is one of the potential targets by PKC pathway, we intent to test if presynaptic 5-HT autoreceptors could function synergistically with SERT to fine-tune the 5-HT release. To this purpose, we isolated the rat platelets which bear endogenous 5-HT1A receptors and SERT and used them as a model system to explore the potential regulatory mechanism(s). First, we applied quick screening 5HT1AR binding and [3H] 5-HT uptake assay to confirm 5-HT1A receptor and SERT were both expressed in rat platelets. Second, administration of 5-HT1A receptor agonist, 8-OH-DPAT dose- (0.01-1μM) and time- (0-60 min)-dependently inhibited the [3H] 5-HT uptake in rat platelets. This inhibition could be reversed by pretreatment of 5-HT1A antagonist, WAY100135. To identify the signal pathway(s) that might underlie the 5-HT1A regulation on SERT, we found that 5-HT1A activation could trigger the PKCδ and Akt phsophorylation but appear not to functionally couple with p38 MAPK pathway. Both pretreatment of PKC inhibitor GF109203X or PI3K inhibitor wortmannin would partially reverse the inhibitory effect of 5-HT1A receptor–mediated [3H] 5-HT uptake. Further, both signals were demonstrated to be able to modulate the SERT uptake efficacy, since β-PMA (0.01-0.1μM), a PKC activator and SB203580 (0.1-10μM), a p38MAPK inhibitor, both dose-dependently inhibited the [3H]5-HT uptake in rat platelets. In addition, via surface biotinylation, we found both 8-OH-DPAT (1μM, 10 min) and β-PMA (0.1μM, 30 min) reduced SERT surface expression in rat platelets whiles 8-OH-DPAT-triggered SERT internalization could be reversed by the pretreatment of WAY100135 (20μM). In conclusion, for now, it is expected in the CNS, presynaptic 5-HT1A autoreceptor could exert similar regulation on SERT to fine-turn the transmitter release in the synapse.

指導教授推薦書…………………………………………………………
口試委員會審定書………………………………………………………
授權書…………………………………………………………………...iii
誌謝……………………………………………………………………...iv
中文摘要…………………………………………………………………v
英文摘要………………………………………………………………..vii
目錄……………………………………………………………………...ix
A. Background……………………………………………………….. 1
1. Serotoninergic system ………………………………………... 1
2. Classification of Serotonin Receptor………………………… 2
2.1 Characters of 5-HT1A receptor……………………………..2
2.2 5-HT1A autoreceptor………………………………………3
2.3 5-HT1A receptor-mediated cellular signaling………………4
3. Platelets serotonin……………………………………………...5
4.1 Serotonin transporter(SERT)………………………………….. 6
4.2 Regulation of SERT………………………………...…………7
5. Serotonin-dependent disorder: Depression………………..……8
B. Specific aim…………………………………………………………9
C. Experimental designs………………………………………………10
D. Materials and Methods……………………………………………11
1. Materials…………………………………………………….11
2. Animals………………………………………………………….12
3. Isolation of rat platelets……………………………………….13
4. Platelet 5-HT1A receptor binding assay………………………13
5. [3H] 5-HT uptake assay………………………………………….14
6. Western immunoblots…………………………………………...14
7. Surface biotinylation…………………………………………….15
8. Data analysis and statistics………………………………………15
E. Results………………………………………………………………..15
1. Characterization of 5-HT1A receptor binding and SERT uptake in
human and rat platelets…………………………………….……16
2. Activation of 5-HT1A receptors decreased [3H] 5-HT uptake in the
rat platelets………………………………………………….16
3. Regulation of p38 MAPK on SERT activity and activation of
5-HT1A receptors appeared not to functionally couple with p38
MAPK pathway in the rat platelets……..……………………….17
4. The effect of 5-HT1A receptor-mediated SERT inhibition via
PKCδ/Thr505 in the rat platelets……..………………………..18
5. The effect of 5-HT1A receptor-mediated SERT inhibition via
Akt/Ser473 in the rat platelets…………….…………………....18
6. Activation of 5-HT1A receptor enhanced SERT internalization in
the rat platelets……………………………………………….19
F. Discussion………………………………………………………..20
G. Reference…………………………………………………………...25
Table……………………………………………………………………..34
Figure…………………………………………………………………35



Table 1. Characterization of 5-HT1A receptor binding and SERT uptake in rat platelets………………………………………………………………34
Figure 1. The effect of 5-HT1A agonist, 8-OH-DPAT on [3H] 5-HT uptake in rat platelets……………………………………………………………35
Figure 2. Characterization of basal p38 MAPK phosphorylation in rat platelets………………………………………………………………….38
Figure 3. The effect of SB203580 on [3H] 5-HT uptake in rat platelets………………………………………………………………….40
Figure 4 . The effect of 8-OH-DPAT on p38 MAPK phosphorylation in rat platelets………………………………………………….…………41
Figure 5 . Dose-dependent effect of PKC activator, β-PMA on [3H] 5-HT uptake in rat platelets……………………………...…………………….42
Figure 6 . The effect of 8-OH-DPAT on PKCδ T505 phosphorylation in rat platelets………………………………………………………………43
Figure 7. The effect of 8-OH-DPAT on PKCα/βII and PKCδ/θ (S643/676) phosphorylation in rat platelets…………………………………………44 Figure 8. Inhibitors of PKCδ block 8-OH-DPAT reduction of SERT activity in rat platelets…………………………………...........................45
Figure 9 . The effect of 8-OH-DPAT on Akt s473 phosphorylation in rat platelets………………………………………………………………….46
Figure 10. Inhibitors of PI3K block 8-OH-DPAT reduction of SERT activity in rat platelets…………………………………………………47
Fiqure 11. The effect of 8-OH-DPAT on surface expression of SERT in rat platelets…………………………………………………...………….48

Fiqure 12. Schematic diagram illustrates the regulation of serotonin
5-HT1A receptor on SERT in rat platelets………………………………49

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