青鱂魚海水型離子細胞之排氨、排鹽以及酸鹼平衡機制研究

Abstract

海水硬骨魚皮膚或鰓上的離子細胞主要負責排氨、排酸以及排鹽等功能。過去研究認為酸促進氨排放的機制對於淡水魚排氨相當重要。然而，酸促進氨排放的機制對於海水魚排氨的重要性至今仍未清楚。除此之外，離子細胞的排酸機制在海水魚的研究中也尚未釐清。本研究的目的是利用廣鹽性物種青鱂魚為模式，用來釐清海水魚離子細胞的排氨、排鹽以及酸鹼平衡機制。 在第一章利用選擇性離子掃秒電極的電生理技術來偵測仔魚體表的氫離子濃度梯度，發現海水青鱂魚卵黃囊體表的區域有酸性層的存在。卵黃囊體表的離子細胞其排氫的能力比角質細胞佳。在Tricine buffer和EIPA藥物處理後發現仔魚體表的排酸和排銨皆明顯的下降。藉由原位雜交反應以及化學免疫染色的方式標定出Na+/H+ exchanger 2 (NHE2) mRNA和NHE3蛋白皆表現在同一型海水離子細胞上。經定量即時聚合酶鏈鎖反應分析中發現青鱂魚鰓上的NHE3、Rhesus B glycoprotein (Rhbg)、Rhcg1和Rhcg2的mRNA在海水馴養後表現量下降，NHE2則是表現量上升。然而，在高銨海水馴養後青鱂魚鰓上的NHE3、Rhbg、Rhcg1和Rhcg2的mRNA表現量皆上升。這些發現證實海水型離子細胞同時具有排酸以及排氨的能力，藉由離子細胞的NHE和Rh蛋白來參與酸促進氨排放的排氨機制。 在第二章將利用電生理技術測量海水青鱂魚仔魚體表離子細胞的氫和氯離子排放量。在NHE、Carbonic anhydrase (CA)、anion exchanger (AE)蛋白的抑制劑處理後發現離子細胞的排氫和排氯皆明顯下降。在短期CO2馴養後會同時刺激離子細胞酸和氯的排放量。透過原位雜交反應和化學免疫染色的方式定位CA2和AE1皆表現在同一型海水離子細胞上。青鱂魚鰓上的Na+/K+/2Cl- cotransporter (NKCC1a)、CA2和AE1 的mRNA在海水馴養後皆上升。另外，在酸化的海水馴養後鰓上的AE1a和AE1b 的mRNA皆上升；而NKCC1a則是在鹼化的海水馴養後上升。這些結果解釋海水魚離子細胞的排酸機制並重新定義過去所建立的排氯機制。 在第三章我們將近一步的探討NHE2蛋白在海水型離子細胞的酸鹼平衡和排鹽功能。在抑制NHE2蛋白質表現後離子細胞的排酸和排氯皆明顯的下降，說明NHE2參與了海水型離子細胞的酸鹼平衡和排鹽功能。綜合這些結果，我們解釋海水型離子細胞的排氨、排鹽以及酸鹼平衡機制並且發現這些機制彼此間的是有關聯性的。Ionocytes in the skin and gills of seawater (SW) teleosts are responsible for ammonia, acid and salt secretion. It is known that the proton-facilitated ammonia excretion is critical for a fish's ability to excrete ammonia in freshwater. However, it remains unclear whether that mechanism is also critical for ammonia excretion in SW. In addition, the mechanism through which ionocytes secrete acid in SW is still unclear. The aim of this study is to use euryhaline species “medaka” as a model to clarify the mechanisms of ammonia excretion, salt secretion and acid-base balance of ionocytes in SW fishes. In chapter 1, using a scanning ion-selective electrode technique (SIET) to measure H+ gradients, an acidic boundary layer was detected at the yolk-sac surface of SW-acclimated medaka (Oryzias latipes) larvae. The H+ gradient detected at the surface of ionocytes was higher than that of keratinocytes in the yolk sac. Treatment with Tricine buffer or EIPA (a NHE inhibitor) reduced the H+ gradient and ammonia excretion of larvae. In situ hybridization and immunochemistry showed that slc9a2 (Na+/H+ exchanger 2; NHE2) and slc9a3 (NHE3) were expressed in the same SW-type ionocytes. A real-time PCR analysis showed that transfer to SW downregulated branchial mRNA expressions of slc9a3 and Rhesus glycoproteins (rhcg1, rhcg2, and rhbg) but upregulated that of slc9a2. However, slc9a3, rhcg1, rhcg2, and rhbg expressions were induced by high ammonia in SW. These findings suggest that SW-type ionocytes play a role in acid and ammonia excretion and that the Na+/ H+ exchanger and Rh glycoproteins are involved in the proton-facilitated ammonia excretion mechanism. In chapter 2, the SIET was used to measure H+ and Cl- secretion by ionocytes in the skin of medaka larvae acclimated to SW. Treatment with inhibitors of NHE, Carbonic anhydrase (CA), and anion exchanger (AE) suppressed both H+ and Cl- secretion by ionocytes. Short-term exposure to hypercapnic SW stimulated both H+ and Cl- secretion. Protein and mRNA of CA2 and AE1 were localized to ionocytes in the skin. Branchial mRNA levels of Na+/K+/2Cl- cotransporter (NKCC1a), CA2, and AE1a increased together with the salinity to which fish were acclimated. In addition, both AE1a and AE1b mRNA increased in fish acclimated to acidified (pH 7) SW; NKCC1a mRNA increased in fish acclimated to pH 9 SW. This finding reveals the mechanism of H+ secretion by ionocytes, and refines our understanding of the well-established mechanism of Cl- secretion by ionocytes of SW fish. In chapter 3, we further examined the role of NHE2 on acid-base regulation and slat secretion of ionocytes. Knockdown of NHE2 impairs both H+ and Cl- secretion of SW-type ionocytes. These results show that the NHE2 is involved in both H+ and Cl- secretion of SW-type ionocytes. Taken those data together, we identify the mechanisms of ammonia excretion, salt secretion and acid-base balance in SW-type ionocytes and observe that those mechanisms are associated with each other.