水通道蛋白8aa在斑馬魚仔魚上的功能性研究

Abstract

水通道蛋白(aquaporins, AQPs)是一群執行水分子通透的細胞膜蛋白。此外，有些AQPs也被發現具有二氧化碳、甘油、氨與尿素的通透性。最近研究將斑馬魚(Danio rerio) aqps基因表現於蛙卵會增加細胞膜對二氧化碳/NH3通透性。然而，目前仍沒有活體的實驗證實AQPs在動物體內參與二氧化碳(carbon dioxide, CO2)及NH3的通透能力。在本篇研究中，在原位雜交反應的結果中發現aqp8aa主要表現於斑馬魚仔魚的鰓上及皮膚上，而在利用免疫組織染色搭配原位雜交反應的結果發現AQP8AA主要在皮膚上表現於兩型的離子細胞上(HR cells and NaR cells)。而在高氨馴養(10 mM NH4+)的情況下aqp8aa的mRNA表現量有顯著提升的情況，而在高碳酸水馴養的情況下卻無此情況產生。利用反義核酸(morpholino oligonucleotides)抑制aqp8aa蛋白質的表現後，利用掃描式離子選擇性電極(scanning ion-selective technique, SIET)來分析H+及NH4+在斑馬魚仔魚皮膚及離子細胞上的運輸。在knockdown aqp8aa表現後，發現仔魚整體的H+及NH4+的排放量都有下降的情況，而在特定細胞也有相似的結果，而在CO2短暫灌流的結果中也發現魚體對於H+排放量都有下降的情況，在特定細胞也有相似的結果，由此結果推論AQP8AA在斑馬魚的仔魚上可能參與著此三物質的運輸。

Aquaporins (AQPs) are integral membrane proteins that facilitate water transport across cell membrane. In addition, some type of AQPs was also found to facilitate diffusion of CO2, glycerol, NH3, and urea. Recently, in vitro studies with Xenopus oocytes showed that AQP8 is able to facilitate CO2/NH3 diffusion. However, in vivo study was lacking to support AQP’s function in CO2/NH3 transport. In this study, AQP8AA were identified in the gills and larval skin of zebrafish. Triple in situ hybridization / immunohistochemistry localized AQP8AA in two subtypes of ionocytes (HR cells and NaR cells). The protein and mRNA expressions of AQP8aa were induced by high-ammonia acclimation (10 mM NH4+) but not hypercapnia water acclimation (1% CO2). Using morpholino oligonucleotides to knockdown AQP8AA protein expression and a scanning ion-selective technique (SIET) to analyze carbonic acid (H+) and NH4+ transport at the skin ionocytes of larvae, this study suggests that AQP8AA facilitates CO2/NH3 transport in the skin ionocytes.