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研究生: 黃珮甄
Huang, Pei-Chen
論文名稱: 魚類表皮細胞麩胺酸-麩醯胺酸循環調控滲透恆定之研究
Exploring the existence of glutamate/glutamine cycle underlying coordination of maintaining osmotic balance in fish epithelium
指導教授: 曾庸哲
Tseng, Yung-Che
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2015
畢業學年度: 103
語文別: 英文
論文頁數: 61
中文關鍵詞: 廣鹽性魚類麩胺酸-麩醯胺酸循環尿素循環離子細胞
英文關鍵詞: euryhaline teleost, glutamate/glutamine cycle, urea cycle, ionocytes
論文種類: 學術論文
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  • 對廣鹽性硬骨魚類而言,維持魚體內滲透壓平衡以適應環境鹽度變化是相當重要的生理恆定機制,而最主要負責調節魚體滲透壓及離子恆定為魚鰓表皮的離子細胞,其可經由活化不同離子通道(例如:鈉鉀幫浦,Na+-K+-ATPase)或相關酵素,進行主動運輸模式進行生理調整,也因此需要消耗大量的能量。故適時的能量代謝啟動與即時的滲透度調節對魚類適應鹽度過程是極為必要的。
    從先前研究已得知,當環境鹽度變動時,廣鹽性硬骨魚類體內有麩胺酸累積和相關的代謝反應,然而其基本的細胞分子機制仍不明確。本研究運用廣鹽性青鱂魚作為實驗物種,證實了魚類表皮細胞中麩胺酸代謝及其運輸機制對於鹽度適應過程中之重要性。青鱂魚鰓中「麩胺酸家族」成員胺基酸含量會因環境鹽度波動有明顯上升;除此之外,魚鰓中麩胺酸及麩醯胺酸相關的運輸通道蛋白(EAATs, SAT1)及相對應合成酵素(GLS, GLUL)的基因表現相亦隨著環境鹽度增加而有顯著提升,且上述通道蛋白與酵素皆會表現在胚胎的上皮膜細胞。而鹽度刺激促使麩胺酸代謝產生的毒性氨(NH3),將進而促使尿素循環的速率決定酵素(CPS, OTC)的誘發反應,以合成尿素參與滲透調節。綜觀上述,環境鹽度改變可促進廣鹽性魚類鰓表皮中麩胺酸及麩醯胺酸進行代謝及運輸,代謝過程中產出的含氮廢物會藉由尿素循環啟動轉合成尿素。上述代謝途徑的誘發不僅有利於提供足夠的能量供給,且助於維持表皮細胞的滲透恆定。

    Maintenance of intact osmotic balance is one of the most important physiological processes for euryhaline teleosts under salinity challenges. In osmotic- or iono-regulation machinery, mitochondria rich (MR) cells in branchial epithelium are the major sites responsible for the active ion transport functions, which are conducted by the activations of various ion transporters (e.g. Na+-K+-ATPase, NKA) and enzymes; therefore, these processes are highly energy consuming. Consequently sufficient and immediate energy replenishment and osmolytes adjustment for fish epithelial osmo-balance are both necessary.
    Under ambient salinity perturbations, glutamate aggregation and correlative metabolisms have been observed in euryhaline teleosts. However, there is no substantial cellular and molecular evidence to support this notion. In this study, we propose that the glutamate metabolism and subsequent transepithelial transport may be vital for euryhaline Japanese medaka (Oryzias latipes) under salinity fluctuations. “Glutamate family” amino acids contents in gills were apparently increased by ambient salinity fluctuations. Moreover specific amino acid transporters (EAATs, SAT1) and glutamate (Glu)/glutamine (Gln) synthesis enzymes (GLS, GLUL) coding homologues in medaka gills were up-regulated in adult gills and found to be respectively expressed in larva yolk sac membrane as well. Concerning of NH4+ production due to deamination from glutamate in gill epithelium, NH4+-derived urea production cycle candidates, carbamoyl phosphate (CPS) and ornithine transcarbamylase (OTC), were up-regulated under ambient SW challenges. Those results inferred that Glu/Gln catabolism, subsequent transepithelial transport shuttle and further urea cycle activation from metabolic nitrogenous waste in epithelium of euryhaline teleosts may not only be vital for metabolites vitality supplying but also for cellular osmolality maintenance under hyperosmotic SW challenges.

    口試委員審定書 i 謝辭 ii 摘要 iii Abstract iv Table of content vi Introduction 1 Materials and Methods 6 Experimental animals 6 Seawater (SW) transferring experiments 6 Oxygen consumption 6 Examination of amino acids content 7 Examination of urea content and NH4+ excretion 8 Purification of total RNA 8 Reverse-transcription polymerase chain reaction (RT-PCR) analysis 9 Real-time quantitative PCR (qPCR) analysis 9 RNA probe synthesis 10 Whole mount in situ hybridization and immunocytochemistry (ICC) staining 10 Gill glutamate dehydrogenase (GDH) activity 11 Statistical analysis 12 Results 13 Effects of salinity challenges on intact metabolic responses 13 Acquisition for glutamate related amino acids constituents in gills after SW treatment 13 Glutamate /glutamine cycle and urea cycle related gene expressions in different tissues 14 Expressions of glutamate and glutamine transporters in fish epithelium under salinity challenges 14 Validation of glutamate and glutamine synthesis of in fish epithelium under salinity challenges 16 Spatial localization of Glu/Gln transporters and Glu/Gln synthesize enzymes in epithelium 17 Glutamate dehydrogenase activity, ammonia transport and urea production in gills 18 Discussion 20 Osmoregulation in fish 20 Glutamate and glutamine metabolism in gills of euryhaline teleost 21 The existence of Glu/Gln cycle in non-neuronal epithelium 24 Perspective and conclusion 25 References 27 Tables and figures 36 Figure 1. Effects of salinity transfer on metabolism and ammonia excretion. 38 Figure 2. Amino acid content (pmole/μg) in the gill of adult Japanese medaka Oryzias latipes acclimated to FW, 10 ‰ SW and 20 ‰ SW for 6, 24, 72 h. 40 Figure 3. Detection of the relevant gene of glutamate -glutamine cycle and urea cycle using RT-PCR analysis in adult medaka tissues. 42 Figure 4. Glutamate and glutamine transporters gene expression during salinity transfer. 44 Figure 5. Expressions of glutamate synthase genes (olgls) expressions during salinity transfer. 46 Figure 6. Expressions of glutamine synthetase genes (olglul) expressions during salinity transfer. 48 Figure 7. Double labeling utilizing in situ hybridization and immunochemistry of glutamate and glutamine transporters mRNA with Na+/ K+-ATPase (NKA; red) in yolk-sac skin of medaka larvae at 7 dpf. 50 Figure 8. Double labeling utilizing in situ hybridization and immunochemistry of glutamate synthase and glutamine synthetase mRNA with Na+/ K+-ATPase (NKA; red) in yolk-sac skin of medaka larvae at 7 dpf. 52 Figure 9. Gill glutamate dehydrogenase (GDH) activity 54 Figure 10. Expressions of ammonia transports genes expressions during salinity transfer. 56 Figure 11. Urea cycle enzyme gene expression and urea content during salinity transfer. 58 Figure 12. A schematic model of the glutamate-glutamine cycle existence in epithelium of euryhaline teleosts. 60

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