青鱂魚在高碳酸環境中的排酸調節

Abstract

鰓上的離子細胞為魚類酸鹼調節重要的場所，在過去研究中已經發現在青鱂魚離子細胞頂膜上的NHE3以及Rhcg1參與了排酸以及排氨的機制。為了探討魚類面臨高碳酸環境如何進行排酸調節，本研究以青鱂魚為模式動物，利用掃描式離子選擇電極技術(SIET)偵測仔魚皮膚上特定細胞表面的H+、Na+、NH4+等離子濃度梯度，以分析離子細胞(MRC)以及角質化細胞(KC)在排酸調節的功能。結果顯示，短期高碳酸環境處理(2.08% CO2，10分鐘)會造成仔魚體表H+累積(梯度)增加195%，MRC的H+累積增加108%，KC則未顯著差異。在長期高碳酸環境處理後(2.08% CO2，5天)，仔魚體表H+累積量增加257%，但是體表NH4+梯度卻下降44%。而在長期高碳酸環境，MRC以及KC細胞表面卻呈現鹼化的現象(H+梯度為負值)。使用鈉氫交換蛋白(NHE)的抑制劑EIPA處理高碳酸組的仔魚後，皮膚的H+梯度顯著的降低(短期高碳酸環境降60.1%，長期高碳酸環境降52.5%)扮演排酸重要角色。為了更進一步探討離子細胞表面鹼化的原因，利用碳酸酐酶(Carbonic anhydrase, CA)的抑制劑乙醯唑胺(Acetazolamide, AZ)處理。結果發現AZ能夠抑制此鹼化現象(下降52.3%)，推測細胞表面的鹼化與CO2(HCO3-)形成有關。利用定量PCR分析mRNA的變化，發現鰓上NHE3、CA15以及Rh蛋白的表現量在高碳酸處理後皆顯著提升(NHE3增加107%, Rhcg1增加68%, Rhcg2 136%, Rhbg 41%, CA15 256%)。利用Rhcg1原位雜交反應以及NHE3和NKA的雙重免疫染色，發現仔魚皮膚上離子細胞密度在高碳酸處理後顯著的增加34%。本研究推論青鱂魚在高碳酸環境中，主要利用離子細胞上的NHE3進行排酸，同時利用細胞表面的CA與Rh蛋白將CO2(HCO3-)回收到離子細胞內。Epithelial Mitochondria-rich cells (MRCs) in fish gills play a critical role in acid-base regulation. In our previous studies, we have found Na+/H+ exchanger (NHE3) and rhesus glycoprotein (Rhcg1) were involved in the acid and base excretion at the apical membrane of MRCs in medaka (Oryzias latipes). To date, little information is known on the acid-base regulation as fish faced the hypercapnia environment. In the present study, a scanning ion-selective electrode technique (SIET) was applied to measure H+, Na+ and NH4+gradient at the specific cell on the skin of medaka larvae. Under acute hypercapnia, the H+ gradient of larval skin and MRCs was increased 195% and 108% respectively. Keratinocytes (KC) were not affected by hypercapnia. After chronic hypercapnia, the H+ gradient of larval skin show 2.57-fold higher than that of control. But the NH4+gradient reduced 44%. Further, the proton excretion of ionocytes and keratinocytes was decreasing which meant the alkalization occurred at the surface of MRCs and KCs. Treatment with 5-ethylisopropyl amiloride (EIPA) and acetazolamide (AZ) was suppressed the proton excretion and sodium absorption which represent carbonic anhydrase (CA) and NHE were involved in the alkalization at the surface of larval skin. RT-PCR results denoted that mRNA levels of nhe3, rhbg, rhcg1, rhcg2, ca15 were up-regulated during chronic hypercapnia. In situ hybridization and immunocytochemistry shown the mRNA of rhcg1 was localized with NHE3 at MRCs of medaka. In addition, the cell density of MRCs was elevated 34% after hypercapnia treatment. Taken together, we suggested that NHE3 was responsible for the acid excretion during hypercapnia. At the same time, CA (CA15) and Rh proteins (Rhcg1) played an important part in the CO2 (HCO3-) resorption at the surface of MRCs in medaka.