青鱂魚仔魚體表離子細胞的排氨參與鈉離子吸收機制

Abstract

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.