- 學位論文
以大鼠腦血管內皮細胞 (ARBEC) 探討導致巴金森氏症之相關因子通透血腦屏障之機轉及其影響
Cellular uptake of MPTP/MPP+ and paraquat in adult rat brain endothelial cells and its implications to Parkinson’s disease
摘要
目前已經有許多化合物被報導可能導致巴金森氏症，包括了MPTP/MPP+、paraquat和rotenone都可造成實驗動物的神經壞死和巴金森氏症症狀；另一方面，nicotine則被發現對巴金森氏症有保護效果，可減少巴金森氏症的發生率。在本實驗室先前的研究中，發現MPP+可經由轉運蛋白運輸進入大腦微血管內皮細胞 (ARBEC)，且此運輸途徑可被nicotine競爭型抑制。在第一部分的實驗中，我們延續之前的研究，納入更多巴金森氏症相關的化合物進行研究，深入探討此機轉的可能性。另外，paraquat也是結構類似MPP+的致巴金森氏症環境因子，文獻中曾被報導可經由大型中性胺基酸轉運蛋白1 (LAT1) 轉運進入中樞，因此在第二部分的實驗中，我們同樣以ARBEC試驗腦血管內皮細胞攝取paraquat的機轉，以確認paraquat和LAT1的關係。 第一部分的研究結果指出，MPP+的結構類似物MPTP也能透過轉運蛋白運輸進入大鼠腦微血管內皮細胞，其進入細胞的km為35.7 ± 0.9 uM、Vmax為1228.4 ± 106.5 pmole/mg protein-30 sec，而nicotine也可以競爭型抑制的方式抑制細胞攝取MPTP，其抑制常數ki為329 uM。另一方面，我們發現其他結構上和MPTP/MPP+相關的巴金森氏症用藥—amantadine、selegiline也能顯著抑制MPTP/MPP+被腦血管內皮細胞攝取，並且計算出這兩個藥物對於細胞攝取MPTP的抑制常數分別為178 uM和246 uM，顯示這些巴金森氏症的治療藥物和nicotine一樣可能影響MPTP/MPP+進入中樞。由於RT-PCR的結果顯示大鼠腦微血管內皮細胞上有表現有機陽離子轉蛋白1 (OCT1)，卻偵測不到多巴胺轉運蛋白 (DAT) 的表現，由於細胞攝取MPP+的路徑可被OCT選擇性抑制劑—decynium22大幅度抑制，顯示MPP+是由OCT進入細胞，雖然細胞攝取MPTP以及nicotine的路徑也能被decynium22抑制，但由於此路徑不受典型有機陽離子存在的影響，因此MPTP以及nicotine可能除OCT外仍經由其他路徑進入腦血管內皮細胞。最後，其他結構類似MPTP/MPP+的isoquinoline類致巴金森物質在實驗中也能影響腦血管內皮細胞攝取MPTP/MPP+和nicotine，顯示此類物質可能也部份經由轉運蛋白進入中樞，但仍需進一步的實驗證明。 在第二部分的研究中，我們發現paraquat無法經由轉運蛋白轉介 (carrier-mediated) 的方式進入細胞，也無法影響LAT1在ARBEC上的功能，因此我們的實驗結果不支持paraquat可經由LAT1進入中樞的論點，另一方面，我們發現新生大鼠星狀細胞 (neonatal rat astrocyte，NRA) 可以轉運蛋白轉介的方式攝取paraquat，考慮到星狀細胞的偽足在血腦屏障上也有屏障的功能，這樣的現象是否和paraqaut進入中樞的路徑有關在未來可以進一步的探討。 總結以上的實驗結果，我們發現nicotine可以競爭型抑制的方式抑制MPTP進入腦血管內皮細胞，這部分地解釋了nicotine對於巴金森氏症的保護作用，而且除了nicotine之外，amantadine和selegiline也可能有相同的保護作用。另一方面，我們的實驗結果指出paraquat並不會經由LAT1運輸，然而其進入中樞的機轉仍需進一步的研究。
並列摘要
To date, compounds including MPTP/MPP+, paraquat and rotenone have been reported to be related to Parkinson’s disease (PD). On the other hand, nicotine may have neuroprotective effect on PD. Previously, using adult rat brain endothelial cell (ARBEC), we showed that nicotine competitively inhibited cellular uptake of MPP+. In this regard, the current study further examines the interaction between MPTP/MPP+ and several compounds, including nicotine, organic cations and MPTP/MPP+ analogues in ARBEC. In addition, since paraquat was reported to penetrate blood-brain barrier via large neutral amino acid transport 1 (LAT1), in the second part of this study, we used cell culture (ARBEC) model to further examine the interaction between LAT1 and paraquat. In the study of MPTP/MPP+, firstly, the results showed that cellular uptake of MPTP can be mediated by a carrier-mediated system, with Km and Vmax value of 35.7 ± 0.9 μM and 1228.4 ± 106.5 pmole/mg protein-30 sec, respectively. Secondly, the carrier system transported MPTP/MPP+ and nicotine was sensitive to decynium22, a selective organic cation transporter (OCT) inhibitor. RT-PCR showed that OCT1 mRNA was detectable in ARBEC. Thirdly, not only nicotine but also amantadine and selegiline can inhibit the cellular uptake of MPTP and MPP+, whereas caffeine and levodopa did not have any significant effect. Nicotine, amantadine and selegiline competitively inhibited cellular uptake of MPTP with inhibition constants (Ki) of 329 μM, 178 μM and 246 μM respectively. In addition, MPTP/MPP+ analogues such as tetrahydroisoquinoline (TIQ) and N-methyl-salsolinol showed significant inhibition on cellular uptake of MPTP, MPP+ and nicotine. These results indicated that nicotine, amantadine and selegiline may inhibit MPTP-like neurotoxins across the blood-brain barrier. Nonetheless, due to structural similarity, BBB transfer characteristics and structure-transport relationship for MPTP/MPP+ analogues need to be further elucidated. In the study of paraquat, different from the reports in literature, our result showed that LAT1 did not recognize and transport paraquat into the brain endothelial cell. Instead, paraquat seems to enter ARBEC by passive diffusion. On the other hand, neonatal rat astrocytes (NRA) can uptake paraquat via a carrier-mediated process. Since NRA was considered a secondary barrier in the BBB constitute, further study is required to investigate whether NRA plays a role in the transport of paraquat into the CNS. In conclusion, our results showed that nicotine could competitively inhibit cellular uptake of MPTP. It might partly explain the neuroprotective effect of nicotine in PD. In addition to nicotine, amantadine and selegiline may exhibit the same neuroprotective effect. On the other hand, our results showed that paraquat is not transported by LAT1. The transport mechanisms of paraquat through BBB should be further varified.
參考文獻
延伸閱讀
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