斑馬魚仔魚體表排氨功能與機制之研究

Abstract

淡水魚類移除體內含氮廢物最佳的方式，是直接將廢物以氨(ammonia，即NH3與NH4+)的形式排放到水體。具研究顯示，80%以上的氨會經由鰓排出。然而目前針對魚類鰓表皮細胞所作的研究仍未足以提供直接的證據說明排氨的運行方式。本實驗選用斑馬魚仔魚為模式動物，透過其體表的離子調節功能探討淡水魚類的排氨機制。 在本實驗中，利用掃描式離子選擇性電極技術（Scanning Ion-selective Electrode Technique, SIET）對仔魚體表離子作檢測。實驗發現在富含氫幫浦細胞( HRC）上的排氨的程度高於周遭的平舖細胞（PVC）與其它類型的離子細胞(Ionocyte)。以往的研究推論氫離子（H+）與排氨之間有密切的關係。在本實驗中，針對氫幫浦而使用的抑制劑bafilomycin A1與gene knockdown技術，會同時造成魚類H+與NH4+的梯度顯著降低。當給予水體高量緩衝溶液（5 mM 3-morpholinopropane sulfonic acid, MOPS）時，也發現H+與NH4+ 的排出量顯著下降。本實驗亦以SIET分析Rhcg1的功能，發現rhcg1 knockdown的仔魚其體表以及細胞排氨量明顯降低。綜合以上結果，本實驗證實仔魚體表細胞透過酸捕捉機制進行排氨功能，也為氫幫浦及Rhcg1提供參與排氨機制的直接證據。The most effective route for fishes to deal with toxic nitrogenous wastes is to excrete NH3/NH4+ into water directly. Over 80% of total ammonia in fish body is excreted into water via the gill epithelium. However, the mechanism for branchial ammonia transport of freshwater fish is not well understood. Using zebrafish larvae as a model, the present study investigated the mechanism of ammonia secretion by the skin of the larvae. Scanning ion-selective electrode technique (SIET) was applied to detect the NH4+ and H+ fluxes at specific cells of larval skin. NH4+ extrusion was relatively high in H+ pump-rich cells (HRCs), which were identified as the H+-secreting ionocyte in zebrafish. Minor NH4+ extrusion was also detected in keratinocytes and other types of ionocytes in larval skin. NH4+ secretion from the skin was tightly linked to acid secretion. Increases in the external pH and buffer concentration (5 mM MOPS, 3-morpholinopropane sulfonic acid) diminished H+ and NH4+ gradients at the larval surface. Moreover, coupled decreases in NH4+ and H+ extrusion were found in larvae treated with an H+-pump inhibitor (bafilomycin A1) or H+-pump gene (atp6v1a) knockdown. Knockdown of Rhcg1 (drrhcg1) also decreased NH4+ secretion at larval skin and HRCs. This study demonstrates ammonia secretion in epithelial cells of larval skin through an acid-trapping mechanism, and provides direct evidence for the involvement of the H+ pump and an Rh glycoprotein (Rhcg1) in ammonia secretion.