鹽度改變初期肌酸激?供應鈉-幫浦能量維持吳郭魚體內滲透壓之恆定

Abstract

當外界環境的鹽度改變時，某些硬骨魚類，可從淡水直接馴化至海水，但是在短期間內主動運輸與能量代謝之關係仍不甚清楚，為更進一步瞭解廣鹽性吳郭魚( Oreochromis mossambicus )在急遽鹽度變化過程中的生理反應，遂將吳郭魚分別從淡水直接轉移至 25 ppt 及 35 ppt 的海水中，以觀察腦及鰓中Na+, K+-ATPase 與creatine kinase（肌酸激?）之活性及其蛋白質表現量，並針對腦中 ATP、creatine（肌酸）、creatinine（肌氨酸酐）的含量進行檢測。結果顯示轉移至 25 ppt 海水組（和淡水組比較），鰓中Na+,K+-ATPase 活性急速的升高，轉移4小時後鰓Na+, K+-ATPase活性有稍微下降的趨勢，而8小時後漸趨穩定，但 24 小時仍高於淡水組；反觀 35 ppt 海水組，在轉移1.5小時後，鰓中Na+, K+-ATPase活性才急速的增加，但和 25ptt 海水組之間並無顯著的差異，而Na+, K+- ATPase α次單元的蛋白質表現量，在 25 ppt 海水組，其變化的趨勢與Na+, K+- ATPase活性的變化一致，至於 35ppt 海水組則維持較高的表現量，且明顯的高於 25 ppt 海水組。在肌酸激?活性方面， 25 ppt 海水組在轉移後 2 小時顯著高於淡水組，轉移 4 小時後逐漸的下降，而 16 小時降至最低點，但 24 小時後再升高；而 35 ppt 海水組，鰓中肌酸激?活性剛開始明顯地下降（和淡水組比較），直到 2 小時後驟然升高，而 25 ppt 海水組 MM 型態的肌酸激?蛋白質表現量和肌酸激?活性的變化較吻合，由此推測 MM 型態的肌酸激?可能受到鹽度因數的影響，至於 35 ppt 海水組則持續維持較高表現量，反觀 BB 型態的肌酸激?蛋白質表現量，則無顯著變化。在 25 ppt 海水組以鰓中Na+,K+-ATPase 及肌酸激?活性做相關係數檢定，發現轉移至海水的前 4 小時相關係數高達 0.89 ，而前 8 小時的相關係數亦達 0.81，由此可推測磷酸肌酸迴路 (phosphocreatine circuit) 供給鰓中Na+,K+-ATPase進行滲透壓調節所需要的緊急能量來源之一。另一方面，腦中Na+,K+-ATPase活性轉移至 25 ppt 及 35 ppt 海水後，前2小時即遠高於淡水組，約4小時後 35 ppt 海水組，則因承受不了急遽的鹽度變化，導致魚體內滲透壓無法保持恆定而死亡，反觀 25 ppt 海水組在轉移 8 小時後，Na+, K+-ATPase活性仍持續增高，並達到較平穩的狀態，而Na+, K+- ATPase α次單元的蛋白質表現量，無論 25 ppt 或 35 ppt 海水組均維持有較淡水組高的表現量。腦中肌酸激?活性，轉移至海水 2 小時後，無論是 25ppt 或 35ppt 海水組均顯著的高於淡水組，而 25 ppt 海水組至 16 小時後才和淡水組無顯著的差異，而腦中 BB 型態的肌酸激?蛋白質表現量，在 25 ppt 海水組，和肌酸激?活性的變化趨勢相似，至於35ppt 海水組自 0.5 小時即持續的增加，直到2小時突然急遽的降低，且明顯的低於淡水組及 25ppt 海水組。腦中ATP 的含量，在25ppt 海水組，維持穩定的量，而 35 ppt 海水組，在轉移入海水 0.5 小時，急速升高，之後即持續下降。腦中肌酸的含量，和肌酸激?活性的變化趨勢相似。此外肌酸的代謝產物肌氨酸酐，無論在25 ppt 或 35 ppt 海水組，均遠比淡水組低。腦Na+,K+-ATPase活性及肌酸激?活性相關係數前 4 小時相關係數高達 0.97，而前8小時的相關係數亦達 0.87。由以上可知當環境中鹽度產生急遽的變化時，腦中調節滲透壓的生理變化比鰓靈敏，且 phosphocreatine circuit 供給腦及鰓所需要的能量。

Some freshwater teleosts are capable of adapting to seawater when challenged; however the related energetic and physiological consequences are still unclear. The present study was conducted to examine changes in brain and gills parameters (Na+,K+-ATPase, creatine kinase, creatinine and ATP) and expression of Na+,K+-ATPase and creatine kinase (CK) in tilapia (Oreochromis mossambicus) during acute responses to transfer from fresh water (FW) to seawater (SW). After transfer to SW, gill Na+,K+-ATPase activities of 25 ppt SW fish maintained at higher levels within 24 h than did those of FW fish. Fish in 35 ppt SW did not show increased gill Na+,K+-ATPase activities until 1.5 h after transfer, and then the activities did not significantly differ from those of fish in 25 ppt SW. Protein levels of the Na+,K+-ATPase α subunit (by Western blot) increased in concert with gill Na+,K+-ATPase activity in SW (25 ppt), but the protein levels in 35 ppt SW were maintained at a higher level than those in the FW control and 25-ppt-SW groups right after transfer. Compared to FW, gill CK activities in 35 ppt SW declined within 1.5 h, and afterwards were dramatically elevated at 2 h as was the case in 25 ppt SW, but the levels in 35 ppt SW were lower than those in 25 ppt SW. The Western blot of muscle-type CK (MM form) was highly associated with the salinity change, showing a pattern of changes similar to that in CK activity, however levels in 35 ppt SW were higher than those in 25 ppt SW. Expression and activity of Na+,K+- ATPase were highly correlated with those of CK in fish gills after transfer from FW to SW, suggesting that phosphocreatine acts as the energy source to meet the osmoregulatory demand during acute transfer. On the other hand, the brain Na+,K+-ATPase activity of fish in SW (25 ppt) maintained at higher levels than that of fish in FW. Subsequently, brain Na+,K+-ATPase remained at a higher level for 8 h. In contrast, brain Na+,K+-ATPase activity of fish in 35 ppt SW maintained at a higher level within 2 h compared to that of fish in FW. Interestingly, brain CK activity in both 25 and 35 ppt SW groups were significantly elevated within 2 h after transfer. The brain ATP contents of fish in 35 ppt SW increased abruptly within 0.5 h and gradually decreased within 2 h. Compared to these of fish in 35 ppt SW, changes in brain ATP contents of fish in 25 ppt SW had leveled off within 24 h. The Na+,K+-ATPase activity and alpha subunit expression of tilapia (Oreochromis mossambicus) brain and gills have a high correlation in the early stage after transfer to SW, suggesting that different tissues are coordinate for the responses to external environment. Specific types of creatine kinase, MM-type in gills and BB-type in brain, are regulated by salinity. Association Na+,K+-ATPase activity with CK activity in brain and gills of fish after transfer from FW to SW, implies that brain and gills phosphocreatine/creatine kinase is with respect to abrupt salinity changes in early stage of crisis period.