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研究生: 吳淑貞
Shu-Chen Wu
論文名稱: 青鱂魚仔魚體表離子細胞的排氨參與鈉離子吸收機制
Ammonia-dependent Na+ uptake in mitochondria-rich cells of medaka (Oryzias latipes) larvae
指導教授: 林豊益
Li-Yih Lin
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: 2009
畢業學年度: 97
語文別: 英文
論文頁數: 51
中文關鍵詞: 離子細胞富含粒線體細胞鈉氫交換蛋白鈉離子吸收青將魚掃瞄式離子選擇電極技術
英文關鍵詞: ionocyte, mitochondria-rich cell, Na+/H+ exchanger, Na+ uptake, Rhesus glycoprotein, ammonia, medaka, scanning ion-selective electrode technique
論文種類: 學術論文
相關次數: 點閱:328下載:4
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  • 鈉氫交換蛋白(Na+/H+ exchanger,NHE)主要分佈於富含粒線體細胞(MR細胞)頂膜,是淡水魚類鰓上皮執行Na+吸收的重要機制,過程中同時發生排酸現象。然而早期文獻從排氨量與Na+吸收量呈現相關的結果推論Na+吸收過程協同發生排氨,並認為此現象是因NHE執行Na+/NH4+的交換所致。然而近年來發現排氨主要以非離子態NH3經由Rh蛋白排除,否定了NHE執行Na+/NH4+交換的可能。因此,NHE如何在淡水環境中驅動Na+/H+交換,及排氨量與Na+吸收呈現相關的原因至今仍未明瞭。本研究以青鱂魚仔魚為模式動物,利用掃瞄式離子選擇電極技術(SIET)進行非侵入性量測,探討其體表細胞的Na+吸收機制與排H+、排NH4+間的關連性,並試圖推論NHE如何參與Na+吸收機制。結果發現,NHE抑制劑(100 uM EIPA)浸泡會顯著抑制仔魚排酸、排氨及Na+吸收,顯示NHE參與此三種離子的調節機制。低鈉水(<0.001 mM)馴養個體會增加體表Na+吸收與排NH4+,但降低了體表H+濃度;高氨水(5 mM NH4+)馴養也造成類似結果。而在測量環境中給予短時間高氨處理(5 mM NH4+)可同時抑制排NH4+與Na+吸收並增加體表H+累積濃度。以上結果顯示魚體排氨機制可能驅動NHE進行Na+吸收。從仔魚體表單一細胞離子流測量結果發現,Na+吸收與排NH4+主要發生在MR細胞。以H+電極測量後發現體表MR細胞有排酸(MRC+)和排鹼(MRC-)二型,高氨與與低鈉水馴養都會增加MRC-的比例。在測量環境中給予短時間高氨處理(5 mM NH4+),排鹼型MR細胞會轉變為排酸型,而同時抑制Na+吸收。顯示MRC-可能排除大量NH3造成細胞外H+被結合成NH4+而形成排鹼現象。此外,酸性水體(pH6)理論上不利於NHE的驅動,然而結果顯示短期酸處理促進Na+吸收與排NH4+。由此推論Rh蛋白在輔助NH3排放的過程中,會造成細胞膜內外H+梯度的增加進而有利推動NHE進行Na+吸收。

    The mechanisms of Na+ uptake and NH4+ excretion at gills of freshwater fish have been studied for decades but the detail remains unclear. To investigate the mechanisms, a scanning ion-selective electrode technique (SIET) was applied to detect the H+, Na+, and NH4+ activities and fluxes at the skin surface of newly-hatched medaka larvae. By probing the ionic fluxes at specific cells in the skin, MRCs were found to be the major sites for Na+ uptake and NH4+ excretion. However, H+ probing at MRCs revealed two groups of MRCs: acid-secreting MRCs (MRC+) and base-secreting (probably NH3) MRCs (MRC-). Treatment with EIPA (100 μM) respectively blocked H+ excretion, NH4+ excretion, and Na+ uptake by 22%, 35%, and 54 %, suggesting that the Na+/H+ exchanger (NHE) is involved in H+, Na+, and NH4+ transport. Low-Na+ water (< 0.001 mM) or high-NH4+ water (5 mM) acclimation caused more MRC- appearing in skin surface, and simultaneously increased Na+ uptake and NH4+ excretion but decreased or even reversed the H+ gradient at the skin and the H+ flux at MRCs. Raising the external NH4+ significantly blocked NH4+ excretion and Na+ uptake, but increased the H+ gradient at the skin. In contrast, raising the acidity of the water (pH 7 to pH 6) enhanced NH4+ excretion and Na+ uptake by MRCs while the H+ activity at the apical surface of MRCs was reduced. The correlation between NH4+ production and H+ consumption suggests that MRCs excrete non-ionic NH3 (base) by an acid-trapping mechanism. The present study suggests a Na+/NH4+ exchange pathway in apical membranes of MRCs, in which a coupled NHE and Rhesus (Rh) glycoprotein is involved, and the Rh glycoprotein may drive the NHE by generating H+ gradients across apical membranes of MRCs.

    Table of Contents----------------------------1 誌謝------------------------------------------2 摘要------------------------------------------3 Abstract-------------------------------------4 Introduction---------------------------------5 Materials and Methods------------------------12 Illustrations of Experimental Designs--------16 Results--------------------------------------19 Discussion-----------------------------------26 References-----------------------------------33 Figures--------------------------------------39

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