環境中鈣離子濃度對魚類鈣離子吸收之影響

Abstract

淡水魚主要由水體中吸收鈣離子，超過百分之九十五的鈣吸收是由鰓上的富含粒腺體細胞（mitochondria-rich cells, MRCs）主動運輸所達成。根據目前魚類鈣吸收模式，鈣離子首先被動地透過一個未知的非膜電位依賴性鈣離子通道進入細胞質中，並在底側膜端由鈉鈣交換蛋白以及細胞膜鈣幫浦主動地送入血液中。然而現今並沒有足夠的分子證據可以支持這個模式。本論文之目的為利用環境中鈣離子的變化，來探討鈣離子運輸蛋白在魚類鈣離子吸收機制的角色。 首先，莫三比克吳郭魚（Oreochromis mossambicus）鰓在原位雜合反應及細胞免疫螢光染色的結果，發現上皮細胞鈣離子通道（tECaC）表現在MRCs（鰓上最主要的離子運輸細胞）中，但只有一部份的MRCs表現tECaC，顯示這一型MRCs主要負責調控鈣離子之吸收，細胞膜鈣幫浦第2型（tPMCA2）及鈉鈣交換蛋白第1型b（tNCX1b）表現位置結果與tECaC結果相同。在斑馬魚（Danio rerio）的實驗結果，zECaC、zPMCA2及zNCX1b皆表現在鰓離子運輸細胞。 進一步，以即時定量聚合酶鏈反應(qRT-PCR) 比較適應高、低鈣淡水的吳郭魚稚魚鰓mRNA的表現，結果發現tECaC及tNCX1b mRNA表現量在低鈣處理組顯著高於高鈣處理組，tPMCA2 mRNA則兩者處理無顯著的差異。適應高、低鈣淡水的斑馬魚，鰓zECaC mRNA表現量與吳郭魚有相似的結果，而zPMCA2及zNCX1b mRNA則兩者處理無顯著的差異。比較吳郭魚稚魚適應淡水和海水其鰓上mRNA的表現，結果發現tECaC mRNA表現量在海水組高於淡水組，tPMCA2 mRNA表現量在海水組顯著低於淡水組，而tNCX1b mRNA則兩者處理無顯著的差異。從不同魚種及水環境的差異，可得知ECaC在魚類的鈣離子吸收中扮演最重要角色之一。 綜合以上結果，魚類鈣吸收機制主要由特定一型的MRCs，經由ECaC、PMCA2或NCX1b來進行，但是ECaC扮演著關鍵的受調控之角色。

Freshwater fish absorb Ca2+ mainly from environment, and over 95% of Ca2+ is actively absorbed via gill mitochondria-rich cells (MRCs). In the current model for Ca2+ in fish gill MRCs, Ca2+ is passively diffused into the cytosol through the apical unidentified voltage-independent Ca2+ channels, and then is transported out of the cells via the basolateral plasma Ca2+-ATPase (PMCA) and Na+/Ca2+ exchanger (NCX). However, so far there is only very few convincing molecular and cellular evidence to support this model. In the present study, we aimed: (1) to provide molecular evidence for the current model for Ca2+ uptake in fish gill MR cells; (2) to examine how environmental Ca2+ concentration regulates gene expressions of Ca2+ transporters in fish gills. Experiments of in situ hybridization and immunocytochemistry showed that Na+/K+-ATPase (a marker for MRCs) and the tilapia epithelial Ca2+ channel (ECaC) mRNA were colocalized in tilapia (Oreochromis mossambicus) gills, and only part of MRCs express the tECaC, the expression patterns of the tPMCA2 and tNCX1b were similar to the pattern of tECaC. The zECaC, zPMCA2 and zNCX1b were also found specifically expressed in zebrafish (Danio rerio) gills. These indicate that a subtype of MRCs is specifically responsible for Ca2+ uptake. Quantitative-PCR（qRT-PCR） results showed that mRNA expression of the tECaC and NCX1b in gills was higher in tilapia acclimated to low-Ca2+ freshwater (FW) than that acclimated to high-Ca2+ FW. However, tPMCA2 mRNA expression did not show any significant change between the 2 groups. In the experiments on the model animal, zebrafish, the expression patterns of the zECaC was similar to that in tilapia, but zPMCA2 and zNCX1b mRNA expression did not show any significant change between the 2 groups. In another experiments, the expression level of tECaC was higher in SW acclimated tilapia gill than that in FW group. The expression of tPMCA2 was higher in FW gill than that in SW ones. However, tNCX1b mRNA did not show any significant changes in all the conditions. For conclusions, ECaC, PMCA and NCX1b, co-expressed in a subtype of MRCs, may be the major players in Ca2+ uptake function of fish gills, and ECaC is probably the main regulatory transporter in this mechanism.