急遽鹽度改變對吳郭魚腦中能量代謝及細胞結構相關基因的影響

Abstract

廣鹽性硬骨魚類可直接從淡水馴化至海水，但在短時間內為因應急遽鹽度變化，導致腦部細胞膜通透性與能量代謝變化的機制仍不清楚。遂將廣鹽性莫三比克吳郭魚(Oreochromis mossambicus）由淡水直接轉移至25ppt的海水中，針對腦中CK-Mi、CK-B及cytochrome b等與能量代謝有關之基因，與PI-3kinase、β-catenin及β-spectrin等與細胞結構有關之基因，分別測定各基因RNA、蛋白質及其活性之表現。結果顯示轉移至25ppt海水後，CK-Mi及CK-B RNA表現在轉移至25ppt海水後1.5小時上升，至24小時仍維持很高。CK-B蛋白質表現量則隨轉移時間增加，在1小時一直維持至24小時。Cytochrome b RNA表現量在轉移至海水後1.5小時亦達到最高，之後下降至與淡水組相似。在細胞結構相關基因方面，PI-3 kinase RNA表現量增加，在轉移1.5小時達到最高，之後回復至與淡水組相近，而其酵素活性在1、2小時持續增加，至24小時則降回至與淡水組相近。β-catenin及β-spectrin RNA表現量在轉移0.5小時後急劇上升而之後下降，在2小時下降至與淡水組相近，而β-catetrin蛋白質表現量在1及2小時急遽下降，但在4小時又大量出現，並持續至24小時仍比淡水組高，β-spectrin蛋白質表現量在2小時達到最高，至24小時都比淡水組高。由以上結果可知PI-3 kinase、β-catenin及β-spectrin RNA表現量在海水轉移後的短時間內就明顯增加，且β-catenin RNA的表現比PI-3 kinase快，可知β-catenin可能幫助PI-3 kinase與cadherin-based adhesion complex結合，進行訊息傳遞以穩定細胞膜結構與通透性，此外β-spectrin蛋白質表現量增加，亦可證明其可能改變細胞骨架結構。綜合以上結果可推測氧化磷酸化與電子傳遞鏈的改變，再配合細胞結構的調節，進而提供更多的能量，以進行滲透壓調節而維持吳郭魚體內之恆定。

Freshwater (FW) teleosts are capable of acclimating to seawater (SW) following such a transfer from FW. However, their osmoregulating mechanisms are still unclear particularly those in the brain. Previous study shows that Na+-K+-ATPase activity is coincidental with creatine kinase (CK) activity during SW (25 ppt) transfer. The present study was made attempt to examine acute changes that occur in phophatidylinositol-3 kinase (PI-3 kinase), β-catenin, β-spectrin, CK-Mi, CK-B and cytochrome b expression of tilapia (Oreochromis mossambicus) brain in response to this transition. Total RNA and protein of tilapia brain after SW transfer at various times were isolated and applied for the RT-PCR, northern blotting, PI-3 kinase activity assay and western blotting. After RT-PCR, cloning and sequencing, partial sequences of PI-3 kinase, β-catenin, β-spectrin, CK-Mi, CK-B and cytochrome b were obtained. Northern blot was hybridized with homologous RNA probes labeled by digoxigenin. The RNA levels of CK-Mi express concomitantly with CK-B RNA after SW transfer. The CK-BB protein level is increased gradually after SW transfer. The increasing trend of Cytochrome b expression shows in tilapia brain. β-spectrin RNA in tilapia brain is increased at 0.5 h and theβ-spectrin protein level is increased gradually after SW transfer. The PI-3 kinase RNA is elevated and the PI-3 kinase (activity /immunoreactive) is increased within 2 h after SW transfer in tilapia brain. , β-catenin RNA is increased at 0.5 h and the β-catenin protein level is increased gradually in tilapia brain after SW transfer, indicating that , β-catenin facilitates the binding of PI-3 kinase with cadherin-based adhesion complex to maintain membrane structure of brain during osmotic shock. These data suggest that the oxidative processing provides more energy to meet the demand during SW transfer and might be through the signal transduction of PI-3 kinase to mediate the osmoregulation in brain.