臺灣博碩士論文加值系統

硬骨魚類能藉由鰓、腎臟、腸三種主要的滲透壓調節器官，調節體內的離子與滲透壓平衡。此外在海水馴養的日本鰻(Anguilla japonica)和美洲擬鰈(Pseudopleuronectes americanus)已經證實食道為滲透壓調節器官，能將喝入的海水進行去鹽作用(desalination)，其管壁的表皮細胞具調節鹽類通透性的能力。本論文從細胞組織的巨觀到微觀進行比較，並結合生理實驗，探討廣鹽性硬骨魚類虱目魚(Chanos chanos)食道參與滲透壓調節的功能。首先利用解剖學的方式觀察虱目魚食道的內部型態，結果發現食道前段顏色較深且具有螺旋狀的皺褶，而後段則是透明且呈現縱向的皺褶。食道的組織切片化學染色顯示，馴養於淡水和海水的虱目魚食道管腔表皮細胞型態無顯著變化，但是淡水虱目魚食道前段與後段肌肉層的切面高度均較海水個體顯著較厚。藉由飲水實驗發現海水個體相較於淡水個體，其食道後段因吞嚥含有亞甲基藍的海水而被深染，此結果驗證馴養在海水中的虱目魚具有喝海水以補充水分的行為。進一步建立食道離體的生理實驗分析其離子通透性，結果顯示海水虱目魚的食道比淡水個體的食道具有較好的離子通透性。離子通透性的差異意味著其表皮細胞具有不同的離子通道參與其中。使用Na+, K+-ATPase (NKA)抑制劑ouabain探討NKA是否參與海水食道對離子的通透性，結果發現加入抑制劑的實驗組，其食道的通透性顯著低於控制組。偵測海水與淡水虱目魚的食道上之NKA α-subunit表現之差異，比較海水及淡水虱目魚食道的前段及後段管腔內的NKA的基因表現量、蛋白質含量、活性以及細胞(NKA immunoreactive cells)數量，結果顯示海水虱目魚食道前段的各項數值皆顯著高於淡水虱目魚組；而在食道後段各項數值則兩組間無明顯變化。據此推論食道前段NKA的調控參與個體喝入海水的去鹽作用。此外本論文偵測到二種氯離子通道蛋白在虱目魚食道前段上具有環境鹽度依賴性表現，CLC-5-like protein表現量在海水魚食道顯著高於淡水個體的食道。另外，NKCC2-like protein的表現量則是在淡水個體中較高。兩者表現位置都是在靠近管腔內螺旋狀皺褶的表皮細胞上。綜和以上實驗結果，虱目魚在不同鹽度環境中馴養時，食道會藉由細胞組織型態的改變以及調整滲透壓相關蛋白的表現，在海水去鹽作用上扮演重要的角色，而在淡水適應則參與離子吸收的功能。因此，食道亦為虱目魚的滲透壓調節的重要器官，參與消化道系統的離子調節以達到滲透壓平衡的生理狀態。

To maintain homeostasis, marine teleosts drink seawater (SW) to supplement the internal water and excreted ions from gill, kidney and intestine. In esophagi of the eel (Anguilla japonica) and flounder (Pseudopleuronectes americanus), the epithelial cell exhibited desalination. In the present study, try to examine the osmoregulatory function of esophagus from marco to micro level in the milkfish (Chanos chanos). First, the internal morphologies indicated that there were many spiral valves in the anterior esophagus with deep color; while colorless posterior esophagus showed longitudinal wrinkles. These structures of esophagus are rarely described in other fishes. Histochemistry staining of esophageal sections revealed that no significant changes in epithelial cells between the freshwater (FW) and SW groups. Furthermore, the muscular layer of FW group is thicker than SW group. Our results indicated that posterior esophagus of SW group was dyed by methylene blue. The drinking experiment was designed to investigate the concept that SW-acclimated milkfish drank SW. On the other hand, the system of isolated milkfish esophagus were measured esophageal ion permeability in vitro. The ion permeabilities in the SW groups were more than FW groups. Difference of esophageal permeability implied that there were ion channels involved in the ion transport. Ouabain-inhibitor was performed to determine the effects of NKA whether on the ion permeability in the esophagi of the SW fish. The permeability of esophagus in ouabain group was lower than control group (ouabain-free). Therefore, the NKA expression of anterior esophagus is the SW fish was higher than that of the FW group including gene level, protein level, NKA activity and NKA immunoreactive cell (NKA-IR) number. However, there was no significant difference in the posterior esophagus. In the present study, two kind of Cl- channels are identified in the anterior esophagus with salinity dependent. Expression of CLC-5-like protein in the SW fish was higher than in the FW group, expression of NKCC2-like protein in the FW group was higher than the SW group. Both of the ion transporters to the epithelial cells of spiral valves in the anterior esophagus. Taken together, esophagus was an osmoregulatory organ involved in ion regulation for homeostasis in euryhaline fish milkfish.

目錄


中文摘要 i
Abstracts iii
前言 1
一、海水魚類喝水補償機制
二、海水魚類食道的去鹽功能
三、離子通道Na+, K+-ATPase (NKA)、Na+, K+,2Cl- cotransporter 2 (NKCC2)、chloride channel 5 (CLC-5)在表皮細胞中所扮演的角色
四、廣鹽性虱目魚
五、研究目的與假說
材料與方法 8
一、實驗動物
二、實驗環境
三、實驗設計
四、實驗方法
五、統計分析
結果 25
一、食道解剖與切片組織染色
二、食道外肌肉層厚度測定
三、飲水實驗
四、食道離體離子通透性實驗
五、Ouabain抑制實驗
六、食道上NKA的表現
七、食道上NKCC2-like protein與CLC-5-like protein的表現
討論 31
一、虱目魚食道組織學相關實驗
二、虱目魚食道飲水與離體離子通透性實驗
三、食道上NKA的表現
四、食道上NKCC2-like protein與CLC-5-like protein的表現
五、總結與未來展望
圖表 40
參考文獻 61
研討會發表紀錄 68
得獎獎狀 69

參考文獻
Abaurrea-Equisoain, M. A., Ostos-Garrido, M. V. (1996). Cell types in the esophageal epithelium of Anguilla anguilla (Pisces, Teleostei). Cytochemical and ultrastructural characteristics. Micron. 27, 419-429.
Adachi, S., Uchida, S., Ito, H., Hata, M., Hiroe, M., Marumo, F., Sasaki, S. (1994). Two isoforms of a chloride channel predominantly expressed in thick ascending limb of Henle's loop and collecting ducts of rat kidney. J. Biol. Chem. 269, 17677-17683.
Aguenaou, H., Boeuf, G., Colin, D. A. (1989). Na+ uptake through the brush border membranes of intestine from fresh water and sea-water adapted trout (Salmo gairdneri). J. Comp. Physiol. B. 159, 275-280.
Anderson, W. G., Takei, Y., Hazon, N. (2002). Osmotic and volaemic effects on drinking rate in elasmobranch fish. J. Exp. Biol. 205, 1115-1122.
Ando, M., Mukuda, T., Kozaka, T. (2003). Water metabolism in the eel acclimated to sea water: from mouth to intestine. Comp. Biochem. Physiol. B. 136, 621-633.
Ando, M., Nagashima, K. (1996). Intestinal Na+ and Cl- levels control drinking behavior in the seawater-adapted eel Anguilla Japonica. Exp. Biol. 199, 711-716.
Bagarinao, T. (1994). Systematics, distribution, genetics and life history of milkfish, Chanos chanos. Env. Biol. Fishes. 39, 23-41.
Blanco, G., Mercer, R. W. (1998). Isozymes of the Na+-K+-ATPase: heterogeneity in structure, diversity in function. Am. J. Physiol. 275, 633-650.
Brandt, S., Jentsch, T. J. (1995). ClC-6 and ClC-7 are two novel broadly expressed members of the CLC chloride channel family. FEBS letters. 377, 15-20.
Chandy, M. (1956). On the oesophagus of milkfish Chanos chanos. J. Zool. Soc. 8, 79-84.
Chatchavalvanich, K., Marcos, R., Poonpirom, J., Thongpan, A., Rocha, E. (2006). Histology of the digestive tract of the freshwater stingray Himantura signifer Compagno and Roberts, 1982 (Elasmobranchii, Dasyatidae). Anat. Embryol. 211, 507-518.
Chow, D. C., Forte, J. G. (1995). Functional significance of the beta-subunit for
heterodimeric P-type ATPases. J. Exp. Biol. 198, 1-17.
Crear, D. (1980). Observations on the reproductive state of milkfish populations (Chanos chanos) from hypersaline ponds on Christmas Island (Pacific Ocean). Proc. World Maricul. Soc. 11, 548-556.
Cutler, C. P., Cramb, G. (2008). Differential expression of absorptive cation-chloride-cotransporters in the intestinal and renal tissues of the European eel (Anguilla anguilla). Comp. Biochem. Physiol. B. 149, 63-73.
Devuyst, O., Guggino, W. B. (2002). Chloride channels in the kidney: lessons learned from knockout animals. Am. J. Physiol. Renal Physiol. 283, 1176-1191.
Duan, D., Winter, C., Cowley, S., Hume, J. R., Horowitz, B. (1997). Molecular identification of a volume-regulated chloride channel. Nature. 390, 417-421.
Evans, D. H., Piermarini, P. M., Choe, K. P. (2005). The multifunctional fish gill: dominant site of gas exchange, osmoregulation, acidbase regulation, and excretion of nitrogenous waste. Physiol. Rev. 85, 97-177.
Feng, S. H., Leu, J. H., Yang, C. H., Fang, M. J., Huang, C. J., Hwang, P. P. (2002). Gene expression of Na+-K+-ATPase α1 and α3 subunits in gills of the teleosts Oreochromis mossambicus, adapted to different environmental salinities. Mar. Biotechnol. 4, 379–391.
Ferraris, R. P., Almendras, J. M., Jazul, A. P. (1988). Changes in plasma osmolality and chloride concentration during abrupt transfer of milkfish (Chanos chanos) from seawater to different test salinities. Aquaculture. 70, 145-157.
Fisher,,S. E., van Bakel, I., Lloyd, S. E., Pearce, S. H. S., Thakker, R.V., Craig, I. W. (1995). Cloning and characterization of CLCN5, the human kidney chloride channel gene implicated in Dent disease (an X-linked hereditary nephrolithiasis). Genomics, 29, 598–606.
Gamba, G. (2005). Molecular physiology and pathophysiology of electroneutral cationchloride cotransporters. Physiol. Rev. 85, 423-493.
Gilles-Baillien, M., Meister, M. F., Kirsch, R., & Gillard, C. (1984). pH profile along the gut of the silver eel and its modification during seawater acclimation. J. Exp. Biol. 113, 483-486.
Gunther, W., Luchow, A., Cluzeaud, F., Vandewalle, A., Jentsch, T. J. (1998). ClC-5, the chloride channel mutated in Dent's disease, colocalizes with the proton pump in endocytotically active kidney cells. Proc. Natl. Acad. Sci. 95, 8075–8080.
Hebert, S. C., Mount, D. B., Gamba, G. (2004). Molecular physiology of cationcoupled Cl– cotransport: the SLC12 family. Pflugers. Arch. 447, 580-593.
Hirano, T. (1974). Some factors regulating water intake by the eel, Anguilla japonica. J. Exp. Biol. 61, 737-747.
Hirano, T., Mayer-Gostan, N. (1976) Eel esophagus as an osmoregulatory organ. Proc. Natl. Acad. Sci. 73, 1348-1350.
Hiroi, J., Yasumasu, S., McCormick, S. D., Hwang, P. P., Kaneko, T. (2008). Evidence for an apical Na–Cl cotransporter involved in ion uptake in a teleost fish. J. Exp. Biol. 211, 2584-2599.
Hwang, P. P., Lee T. H., Lin, L. Y. (2011) Ion regulation in fish gills: recent progress in the cellular and molecular mechanisms. Am. J. Physiol. Regul. Integr. Comp. Physiol. 301, 28-47.
Jentsch, T. J., Friedrich, T., Schriever, A., Yamada, H. (1999). The CLC chloride channel family. Pflugers. Arch. 437, 783-795.
Jentsch, T. J., Neagoe, I., Scheel, O. (2005). CLC chloride channels and transporters. Curr. Opin. Neurobiol. 15, 319-325.
Jentsch, T. J., Steinmeyer, K., Schwarz, G. (1990). Cloning in Xenopus oocytes. Nature. 348, 510-514.
Jentsch, T. J., Stein, V., Weinreich, F., Zdebik, A. A. (2002). Molecular structure and physiological function of chloride channels. Physiol. Rev. 82, 503–568.
Kawasaki, M., Uchida, S., Monkawa, T., Miyawaki, A., Mikoshiba, K., Marumo, F., Sasaki, S. (1994). Cloning and expression of a protein kinase C-regulated chloride channel abundantly expressed in rat brain neuronal cells. Neuron. 12, 597-604
Kieferle, S., Fong, P., Bens, M., Vandewalle, A., Jentsch, T. J. (1994). Two highly homologous members of the ClC chloride channel family in both rat and human kidney. Proc. Natl. Acad. Sci. 91, 6943-6947.
Kinoshita, I. (1981). Feeding habit and development of the digestive system in juvenile milkfish, Chanos chanos. MSc Thesis, Nagasaki University, Japan, 42 pp.
Laverty, G., Skadhauge, E. (2012). Adaptation of teleosts to very high salinity. Comp. Biochem. Physiol. A. 163, 1-6.
Lin, Y. M., Chen, C. N., Lee, T. H. (2003). The expression of gill Na, K-ATPase in milkfish,Chanos chanos, acclimated to seawater, brackish water and fresh water. Comp. Biochem. Physiol. B. 135, 489-497.
Lin, Y. M., Chen, C. N., Yoshinaga, T., Tsai, S. C., Shen, I. D., Lee, T. H. (2006). Short-term effects of hyposmotic shock on Na+/K+-ATPase expression in gills of the euryhaline milkfish, (Chanos chanos). Comp. Biochem. Physiol. A. 143, 406-415.
Lionetto, M. G., Schettino, T. (2006). The Na+–K+–2Cl− cotransporter and the osmotic stress response in a model salt transport epithelium. Acta. Physiol. 187, 115-124.
Lloyd, S. E., Gunther, W., Pearce, S. H., Thomson, A., Bianchi, M. L., Bosio, M., Thakker, R. V. (1997). Characterisation of renal chloride channel, CLCN5, mutations in hypercalciuric nephrolithiasis (kidney stones) disorders. Hum. Mol. Genet. 6, 1233-1239.
Lytle, C., Xu, J. C., Biemesderfer, D., Forbush III, B. (1995). Distribution and diversity of Na-K-Cl cotransport proteins: a study with monoclonal antibodies. Am. J. Physiol. 269, 1496-1505.
Meister, M. F., Humbert, W., Kirsch, R., Vivien-Roels, B. (1983). Structure and ultrastructure of the oesophagus in sea-water and fresh-water teleosts. Zoomorphology. 102, 33-51.
Miyazaki, H., Kaneko, T., Uchida, S., Sasaki, S., Takei, Y. (2002). Kidney-specific chloride channel, OmClC-K, predominantly expressed in the diluting segment of freshwater-adapted tilapia kidney. Proc. Natl. Acad. Sci. 99, 15782-15787.
Miyazaki, H., Uchida, S., Takei, Y., Hirano, T., Marumo, F., Sasaki, S. (1999). Molecular Cloning of CLC Chloride Channels in (Oreochromis Mossambicus) and their functional complementation of yeast CLC gene mutant. Biochem. Biophys. Res. Commun. 255, 175-181.
Nagashima, K., Ando, M. (1994). Characterization of esophageal desalination in the seawater eel, Anguilla japonica. J. Comp. Physiol. 164, 47-54.
O'Grady, S. M., Musch, M. W., Field, M. (1986). Stoichiometry and ion affinities of the Na−K−Cl cotransport system in the intestine of the winter flounder (Pseudopleuronectes americanus). J. Membr. Biol. 91, 33-41.
Parmelee, J. T., Renfro, J. L. (1983). Esophageal desalination of seawater in flounder: role of active sodium transport. Am. J. Physiol. Renal Physiol. 245, 888-893.
Pham, P. C., Devuyst, O., Pham, P. T., Matsumoto, N., Shih, R. N., Jo, O. D., Yanagawa, N, Sun, A. M. (2004). Hypertonicity increases CLC-5 expression in mouse medullary thick ascending limb cells. Am. J. Physiol. Renal. Physiol. 287, 747-752.
Ross, J. A., Woo, P., Noordzij, P. (1996). Association of esophageal reflux and globus symptom: comparison of laryngoscopy and 24-hour pH manometry. Otolaryngol. Head Neck Surg. 115, 502–507.
Sakamoto, H., Kawasaki, M., Uchida, S., Sasaki, S., Marumo, F. (1996). Identification of a new outwardly rectifying Cl− channel that belongs to a subfamily of the ClC Cl− channels. J. Biol. Chem. 271, 10210–10216.
Sakamoto, H., Sado, Y., Naito, I., Kwon, T. H., Inoue, S., Endo, K., Marumo, F. (1999). Cellular and subcellular immunolocalization of ClC-5 channel in mouse kidney: colocalization with H+-ATPase. Am. J. Physiol. Renal Physiol. 277, 957-965.
Sardella, B. A., Matey, V., Cooper, J., Gonzalez, R. J., Brauner, C. J. (2004). Physiological,biochemical and morphological indicators of osmoregulatory stress in 'California' Mozambique tilapia (Oreochromis mossambicus x O. urolepis hornorum) exposed to hypersaline water. J. Exp. Biol. 207, 1399-1413.
Schettino, T., Lionetto, M. G. (2003). Cl− absorption in European eel intestine and its regulation. J. Exp. Zool. A. 300, 63-68.
Schmieder, S., Lindenthal, S., Ehrenfeld, J. (2002). Cloning and characterisation of amphibian ClC-3 and ClC-5 chloride channels. Biochim. Biophys. Acta. 1566, 55-66.
Slegtenhorst, M. A., Bassi, M. T., Borsani, G., Wapenar, M. C., Ferrero, G. B., De Conciliis, L., Grillo, A., Franco, B., Zoghbi, H. Y., Ballabio, A. (1994). A gene from the Xp22.3 regions shares homology with voltage-gated chloride channels. Hum. Mol. Genet. 3, 547–552.
Simonneaux, J., Barra, A., Humbert, W., Kirsch, R. (1987). The role of mucus in ion absorption by the oesophagus of the sea-water eel (Anguilla anguilla ). J. Comp. Physiol. B. 157, 187-199.
Skadhauge, E. (1969). The mechanism of salt and water absorption in the intestine of the eel (Anguilla anguilla) adapted to waters of various salinities. J. Physiol. 204, 135-158.
Steinmeyer, K., Ortland, C., Jentsch, T. J. (1991). Primary structure and functional expression of a developmentally regulated skeletal muscle chloride channel. Nature. 354, 301-304.
Tang, C. H., Chiu, Y. H., Tsai, S. C., Lee, T. H. (2009). Relative changes in the abundance of branchial Na+/K+‐ATPase α‐isoform‐like proteins in marine euryhaline milkfish (Chanos chanos) acclimated to environments of different salinities. J. Comp. Physiol. A. 311, 521-529.
Tang, C. H., Wu, W. Y., Tsai, S. C., Yoshinaga, T., Lee, T. H. (2010). Elevated Na+/K+-ATPase responses and its potential role in triggering ion reabsorption in kidneys for homeostasis of marine euryhaline milkfish (Chanos chanos) when acclimated to hypotonic fresh water. J. Comp. Physiol. B. 180, 813-824.
Therien, A. G., Blostein, R. (2000). Mechanisms of sodium pump regulation. Am. J. Physiol. Cell Physiol. 279, 541-566.
Therien, A. G., Goldshleger, R., Karlish, S. J., Blostein, R. (1997). Tissue-specific distribution and modulatory role of the γ subunit of the Na, K-ATPase. J. Biol. Chem. 272, 32628-32634.
Thiemann, A., Grunder, S., Pusch, M., Jentsch, T. J. (1992). A chloride channel widely expressed in epithelial and non-epithelial cells. Nature. 356, 57-60.
Uchida, S. (2000). In vivo role of CLC chloride channels in the kidney. Am. J. Physiol. Renal Physiol. 279, 802-808.
Uchida, S., Sasaki, S., Furukawa, T., Hiraoka, M., Imai, T., Hirata, Y., Marumo, F. (1993). Molecular cloning of a chloride channel that is regulated by dehydration and expressed predominantly in kidney medulla. J. Biol. Chem. 268, 3821-3824.
Verrey, F., Schaerer, E., Zoerkler, P., Paccolat, M. P., Geering, K., Kraehenbuhl, J. P., Rossier, B. C. (1987). Regulation by aldosterone of Na+, K+-ATPase mRNAs, protein synthesis, and sodium transport in cultured kidney cells. J. Cell Biol. 104, 1231-1237.
Wang, J., Schwinger, R. H. G., Frank, K., Muller-Ehmsen, J., Martin-Vasallo, P., Pressley, T. A., Xiang, A., Erdmann, E., McDonough, A. A. (1996). Regional expression of sodium pump subunits isoforms and Na+-Ca2+-exchanger in the human heart. J. Clin. Invest. 98, 1650–1658
Watanabe, S., Mekuchi, M., Ideuchi, H., Kim, Y. K., Kaneko, T. (2011). Electroneutral cation-Cl- cotransporters NKCC2β and NCCβ expressed in the intestinal tract of Japanese eel Anguilla japonica. J. Comp. Physiol. A. 159, 427-435.
Whittamore, J. M. (2012). Osmoregulation and epithelial water transport: lessons from the intestine of marine teleost fish. J. Comp. Physiol. B. 182,1-39.
Wilson, R., Gilmour, K., Henry, R., Wood, C. (1996). Intestinal base excretion in the seawater-adapted rainbow trout: a role in acid-base balance? J. Exp. Biol. 199, 2331-2343.
Wollnik, B., Kubisch, C., Steinmeyer, K., Pusch, M. (1997). Identification of functionally important regions of the muscular chloride channel CIC-1 by analysis of recessive and dominant myotonic mutations. Hum. Mol. Genet. 6, 805–811.
Xue, H., Liu, S., Ji, T., Ren, W., Zhang, X. H., Zheng, L. F., Zhu, J. X. (2009). Expression of NKCC2 in the rat gastrointestinal tract. Neurogastroenterol. Motil. 21, 1068-1089.
Yamamoto, M., Hirano, T. (1978). Morphological changes in the esophageal epithelium of the eel, Anguilla japonica, during adaptation to seawater. Cell Tissue Res. 192, 25-38.
Yamamoto, K., Cox, J. P., Friedrich, T., Christie, P. T., Bald, M., Houtman, P. N., Thakker, R. V. (2000). Characterization of renal chloride channel (CLCN5) mutations in Dent's disease. J. Am. Soc. Nephrol. 11, 1460-1468.