青鱂魚仔魚在鹽度適應過程之離子細胞功能研究–離子細胞功能之可塑性

The functional study of Mitochondrion-Rich Cells in medaka larvae subjected to salinity changes – Functional plasticity of Mitochondrion-Rich Cells

沈婉萍(Wan-Ping Shen)

指導教授 ： 林豊益

國立臺灣師範大學/理學院/生命科學研究所/碩士(2010年)

摘要

魚類鰓上富含粒線體的細胞 ( mitochondrion-rich cells , MRCs ) 被認為是具有離子調節功能的細胞，在海水的環境中，這類細胞負責將過多的離子由魚體內排出至外界環境中，在淡水下則是負責離子吸收的功能。在海水中，Cl-主要是由MRCs的頂膜排出 ; 而Na+被認為由MRCs與輔助細胞 ( accessory cells , ACs ) 間形成的不緊密的細胞間隙擴散排出。前人研究發現在鹽度適應過程中，MRCs能持續存在於鹽度適應的過程，顯示MRCs可能進行功能轉變以因應鹽度轉變。然而，這些MRCs是否能夠直接在淡水型與海水型功能間轉換仍缺乏直接的證據。因此本研究目的是提出細胞功能轉變的直接證據，並探討在淡、海水轉移中MRCs型態與功能轉變間的相互關係。利用掃描式離子選擇電極(SIET)在青鱂魚仔魚表皮上分析MRCs的Na, Cl運送與型態變化。結果發現，在海水馴養下的青鱂魚仔魚體表可觀察到兩類型的MRCs : ( 一 ) 單一存在具有明顯對外開口的MRCs ( single-mitochondrion-rich cells, s-MRCs ) ; ( 二 ) 由AC與1~2顆 MRCs組成的多細胞複合體型態 (multicellular complex-mitochondrion-rich cells, mc-MRCs)。在有AC伴隨的MRCs的開口上，有明顯較高的Na+、Cl-排出; 無AC伴隨的s-MRCs開口上只有明顯的Cl-排出，無明顯的Na+排出，證明AC確實對排Na+扮演重要功能。此外，隨著適應的鹽度提昇，mc-MRCs出現比例會提高。利用SIET測量仔魚體表的Na+, Cl-濃度梯度變化，發現仔魚由淡水轉移至海水過程中，在轉移3小時內Na+呈現吸收，隨後逐漸轉為排出，至第5小時後達到與海水控制組相同的量，而Cl-排放在轉移後2小時即快速達到與海水適應組相同的量，顯示海水適應過程中Cl- 調節比Na+ 調節快。此外，由海水轉移至淡水過程中，在轉移30分鐘內，體表Na+, Cl-排出即快速降低至與淡水適應組相同的程度。在單一MRCs上測量Na+, Cl-離子流(ionic flux)發現，由海水轉移至淡水3-5分鐘內，MRCs 上Na+的排出量快速降低至原本的2-3 %以下，顯示出MRCs快速的調節能力。而利用活體的連續觀測方式，追蹤mc-MRCs及s-MRCs在海水轉移至淡水環境後，發現MRCs的開口有逐漸變大的現象。此外，mc-MRCs的AC會逐漸遠離MRC開口。本實驗直接證明海水型MRCs能夠在淡水適應過程中，直接轉變為淡水型的MRCs。

關鍵字

青鱂魚 ； 離子細胞 ； 鹽度適應 ； 離子調節 ； 掃描式離子選擇電極 ； 富含粒線體的細胞

並列摘要

Mitochondrion-rich (MR) cells (also called chloride cells) are specialized ionocytes in fish gill. These cells are involved in the ion secretion in seawater (SW) and ion uptake in fresh water (FW). It is believed that Cl− is secreted through the apical membrane of MRCs in SW, meanwhile, Na+ secretion occurs down its electrochemical gradient via a paracellular pathway between MRCs and accessory cells (ACs). Previous studies suggested that MRCs can change their morphology and function during extremely salinity changes such as FW to SW or vice versa, implying that MRCs posses a functional plasticity in ion regulation, i.e. taking up ions in FW and secreting ions in SW. However, evidence that can support this hypothesis is few and not convincing. In this study, a non-invasive technique, scanning ion-selective electrode technique (SIET) was applied to sequentially detect the Na+ and Cl- fluxes at the same MRCs in medaka larvae subjected to salinity changes. In vivo observation of SW-acclimated larvae revealed two types of MRCs: (1) single-MRCs (s-MRCs) which do not have a associated accessory cells (ACs) (2) multicellular complex- MRCs (mc-MRCs) which are consisted of MRCs and companied ACs. Using SIET, significant outward fluxes of Na+ and Cl- were detected at the apical surface of mc-MRCs, whereas only an outward flux of Cl- but not Na+ was detected at s-MRCs, indicating that ACs is required for Na+ secretion in SW fish. The presence of ACs increased with the salinity of medium to which the larvae were acclimated. During the transfer from FW to SW or vice versa, time-course changes of Na+ and Cl- gradients at the skin of the larvae were measured with SIET. An inward Na+ gradient was detected at the skin within 3 hrs after the FW to SW transfer, however, an outward Na+ gradient was detected after 3 hrs and the gradients gradually increased with time and reached a relatively steady value after 5 hrs. In addition, an outward Cl- gradient was also detected during the transfer and the value increased with time and reached a steady value at 2 hrs after the transfer, suggesting that Cl- regulation is faster than Na+ regulation in medaka larvae. In contrast, when the larvae were transferred from SW to FW, an instantaneous drop of outward Na+ and Cl- gradients was detected within 30 mins. At individual MRCs, a rapid decline of Na+ and Cl- fluxes also occurred after the SW to FW transfer. At the same time, both s-MRCs and mc-MRCs increased their apical opening sizes, and the ACs were found to depart from mc-MRCs during the transfer. Most importantly, a dramatic alteration of Na+ fluxes was found by sequentially measuring the same MRCs subjected to the transfer. Our data demonstrated that MRCs posses duel functions (ion uptake and ion secretion) and most importantly they can change their function from an ion secreting to an ion absorbing MRCs within 5 hrs.

並列關鍵字

Mitochondrion-Rich cells ； scanning ion-selective electrode technique ； salinity changes ； accessory cells ； ion regulation ； Japanese medaka

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