魚類表皮細胞麩胺酸-麩醯胺酸循環調控滲透恆定之研究

Abstract

對廣鹽性硬骨魚類而言，維持魚體內滲透壓平衡以適應環境鹽度變化是相當重要的生理恆定機制，而最主要負責調節魚體滲透壓及離子恆定為魚鰓表皮的離子細胞，其可經由活化不同離子通道(例如：鈉鉀幫浦，Na+-K+-ATPase)或相關酵素，進行主動運輸模式進行生理調整，也因此需要消耗大量的能量。故適時的能量代謝啟動與即時的滲透度調節對魚類適應鹽度過程是極為必要的。 從先前研究已得知，當環境鹽度變動時，廣鹽性硬骨魚類體內有麩胺酸累積和相關的代謝反應，然而其基本的細胞分子機制仍不明確。本研究運用廣鹽性青鱂魚作為實驗物種，證實了魚類表皮細胞中麩胺酸代謝及其運輸機制對於鹽度適應過程中之重要性。青鱂魚鰓中「麩胺酸家族」成員胺基酸含量會因環境鹽度波動有明顯上升；除此之外，魚鰓中麩胺酸及麩醯胺酸相關的運輸通道蛋白(EAATs, SAT1)及相對應合成酵素(GLS, GLUL)的基因表現相亦隨著環境鹽度增加而有顯著提升，且上述通道蛋白與酵素皆會表現在胚胎的上皮膜細胞。而鹽度刺激促使麩胺酸代謝產生的毒性氨(NH3)，將進而促使尿素循環的速率決定酵素(CPS, OTC)的誘發反應，以合成尿素參與滲透調節。綜觀上述，環境鹽度改變可促進廣鹽性魚類鰓表皮中麩胺酸及麩醯胺酸進行代謝及運輸，代謝過程中產出的含氮廢物會藉由尿素循環啟動轉合成尿素。上述代謝途徑的誘發不僅有利於提供足夠的能量供給，且助於維持表皮細胞的滲透恆定。Maintenance of intact osmotic balance is one of the most important physiological processes for euryhaline teleosts under salinity challenges. In osmotic- or iono-regulation machinery, mitochondria rich (MR) cells in branchial epithelium are the major sites responsible for the active ion transport functions, which are conducted by the activations of various ion transporters (e.g. Na+-K+-ATPase, NKA) and enzymes; therefore, these processes are highly energy consuming. Consequently sufficient and immediate energy replenishment and osmolytes adjustment for fish epithelial osmo-balance are both necessary. Under ambient salinity perturbations, glutamate aggregation and correlative metabolisms have been observed in euryhaline teleosts. However, there is no substantial cellular and molecular evidence to support this notion. In this study, we propose that the glutamate metabolism and subsequent transepithelial transport may be vital for euryhaline Japanese medaka (Oryzias latipes) under salinity fluctuations. “Glutamate family” amino acids contents in gills were apparently increased by ambient salinity fluctuations. Moreover specific amino acid transporters (EAATs, SAT1) and glutamate (Glu)/glutamine (Gln) synthesis enzymes (GLS, GLUL) coding homologues in medaka gills were up-regulated in adult gills and found to be respectively expressed in larva yolk sac membrane as well. Concerning of NH4+ production due to deamination from glutamate in gill epithelium, NH4+-derived urea production cycle candidates, carbamoyl phosphate (CPS) and ornithine transcarbamylase (OTC), were up-regulated under ambient SW challenges. Those results inferred that Glu/Gln catabolism, subsequent transepithelial transport shuttle and further urea cycle activation from metabolic nitrogenous waste in epithelium of euryhaline teleosts may not only be vital for metabolites vitality supplying but also for cellular osmolality maintenance under hyperosmotic SW challenges.