Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22981
標題: 氫離子幫浦在廣鹽性墨綠凹鼻魨鰓上的表現與可能的功能
Expression and putative roles of V-type H+-ATPase in gills of the euryhaline teleost, Tetraodon nigroviridis
作者: 周書堃
Chou, Shu-Kun
關鍵字: V-type H+-ATPase
氫離子幫浦
spotted green pufferfish
Cl--uptake
墨綠凹鼻魨
氯離子吸收
出版社: 生命科學系所
引用: Adachi I, Puopolo K, Marquez-Sterling N, Arai H, and Forgac M. Dissociation, cross-linking, and glycosylation of the coated vesicle proton pump. J Biol Chem 265: 967-973, 1990. Alper SL. Genetic diseases of acid-base transporters. Annu Rev Physiol 64: 899-923, 2002. Beyenbach KW and Wieczorek H. The V-type H+ ATPase: molecular structure and function, physiological roles and regulation. J Exp Biol 209: 577-589, 2006. Boesch ST, Eller B, and Pelster B. Expression of two isoforms of the vacuolar-type ATPase subunit B in the zebrafish Danio rerio. J Exp Biol 206: 1907-1915, 2003. Catches JS, Burns JM, Edwards SL, and Claiborne JB. Na+/H+ antiporter, V-H+-ATPase and Na+/K+-ATPase immunolocalization in a marine teleost (Myoxocephalus octodecemspinosus). J Exp Biol 209: 3440-3447, 2006. Chang IC and Hwang PP. Cl- uptake mechanism in freshwater-adapted tilapia (Oreochromis mossambicus). Physiol Biochem Zool 77: 406-414, 2004. Chang IC, Wei YY, Chou FI, and Hwang PP. Stimulation of Cl- uptake and morphological changes in gill mitochondria-rich cells in freshwater tilapia (Oreochromis mossambicus). Physiol Biochem Zool 76: 544-552, 2003. Choe KP, O''Brien S, Evans DH, Toop T, and Edwards SL. Immunolocalization of Na+/K+-ATPase, carbonic anhydrase II, and vacuolar H+-ATPase in the gills of freshwater adult lampreys, Geotria australis. J Exp Zoolog A Comp Exp Biol 301: 654-665, 2004. Esaki M, Hoshijima K, Kobayashi S, Fukuda H, Kawakami K, and Hirose S. Visualization in zebrafish larvae of Na+ uptake in mitochondria-rich cells whose differentiation is dependent on foxi3a. Am J Physiol Regul Integr Comp Physiol 292: R470-480, 2007. Evans DH. Teleost fish osmoregulation: what have we learned since August Krogh, Homer Smith, and Ancel Keys. Am J Physiol Regul Integr Comp Physiol 295: R704-713, 2008. Evans DH, Piermarini PM, and Choe KP. The multifunctional fish gill: dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste. Physiol Rev 85: 97-177, 2005. Filippova M, Ross LS, and Gill SS. Cloning of the V-ATPase B subunit cDNA from Culex quinquefasciatus and expression of the B and C subunits in mosquitoes. Insect Mol Biol 7: 223-232, 1998. Finberg KE, Wagner CA, Stehberger PA, Geibel JP, and Lifton RP. Molecular cloning and characterization of Atp6v1b1, the murine vacuolar H+ -ATPase B1-subunit. Gene 318: 25-34, 2003. Gillespie J, Ozanne S, Tugal B, Percy J, Warren M, Haywood J, and Apps D. The vacuolar H+-translocating ATPase of renal tubules contains a 115-kDa glycosylated subunit. FEBS Lett 282: 69-72, 1991. Hawkings GS, Galvez F, and Goss GG. Seawater acclimation causes independent alterations in Na+/K+- and H+-ATPase activity in isolated mitochondria-rich cell subtypes of the rainbow trout gill. J Exp Biol 207: 905-912, 2004. Hiroi J, Kaneko T, Uchida K, Hasegawa S, and Tanaka M. Immunolocalization of Vacuolar-Type H+-ATPase in the Yolk-Sac Membrane of Tilapia (Oreochromis mossambicus) Larvae. Zoolog Sci 15: 447-453, 1998. Hirose S, Kaneko T, Naito N, and Takei Y. Molecular biology of major components of chloride cells. Comp Biochem Physiol B Biochem Mol Biol 136: 593-620, 2003. Hootman SR and Philpott CW. Accessory cells in teleost branchial epithelium. Am J Physiol 238: R199-206, 1980. Horng JL, Lin LY, and Hwang PP. Functional regulation of H+-ATPase-rich cells in zebrafish embryos acclimated to an acidic environment. Am J Physiol Cell Physiol 296: C682-692, 2009. Hwang PP and Lee TH. New insights into fish ion regulation and mitochondrion-rich cells. Comp Biochem Physiol A Mol Integr Physiol 148: 479-497, 2007. Inokuchi M, Hiroi J, Watanabe S, Hwang PP, and Kaneko T. Morphological and functional classification of ion-absorbing mitochondria-rich cells in the gills of Mozambique tilapia. J Exp Biol 212: 1003-1010, 2009. Kaneko T, Watanabe S, Lee KM. Functional Morphology of Mitochondrion-Rich Cells in Euryhaline and Stenohaline Teleosts. Aqua-BioScience Monographs Vol. 1,pp. 1-62, 2008. Katoh F, Hyodo S, and Kaneko T. Vacuolar-type proton pump in the basolateral plasma membrane energizes ion uptake in branchial mitochondria-rich cells of killifish Fundulus heteroclitus, adapted to a low ion environment. J Exp Biol 206: 793-803, 2003. Lin CH and Lee TH. Sodium or potassium ions activate different kinetics of gill Na, K-ATPase in three seawater- and freshwater-acclimated euryhaline teleosts. J Exp Zoolog A Comp Exp Biol 303: 57-65, 2005. Lin CH, Tsai RS, and Lee TH. Expression and distribution of Na, K-ATPase in gill and kidney of the spotted green pufferfish, Tetraodon nigroviridis, in response to salinity challenge. Comp Biochem Physiol A Mol Integr Physiol 138: 287-295, 2004. Lin H, Pfeiffer D, Vogl A, Pan J, and Randall D. Immunolocalization of H+-ATPase in the gill epithelia of rainbow trout. J Exp Biol 195: 169-183, 1994. Lin H, Randall D. Proton pumps in fish gills. In: Wood CM, Shuttleworth TJ. (Eds), Cellular and Molecular Approaches To Fish Ionic Regulation. Academic Press, New York, pp. 233-236, 1995. Lin LY, Horng JL, Kunkel JG, and Hwang PP. Proton pump-rich cell secretes acid in skin of zebrafish larvae. Am J Physiol Cell Physiol 290: C371-378, 2006. Marshall MS, Grosell M. Ion transport, osmoregulation, and acid-base balance. In: Evans DH, Claiborne JB. (Eds), The Physiology of Fishes, CRC Press, Boca Raton, FL, pp. 179-214, 2006. McCormick, SD. Hormonal contral of gill Na+,K+-ATPase and chloride cell function. In: Wood CM, Shuttleworth TJ. (Eds), Cellular and Molecular Approaches To Fish Ionic Regulation. Academic Press, New York, pp. 285-315, 1995. Nanda A, Brumell JH, Nordstrom T, Kjeldsen L, Sengelov H, Borregaard N, Rotstein OD, and Grinstein S. Activation of proton pumping in human neutrophils occurs by exocytosis of vesicles bearing vacuolar-type H+-ATPases. J Biol Chem 271: 15963-15970, 1996. Nelson RD, Guo XL, Masood K, Brown D, Kalkbrenner M, and Gluck S. Selectively amplified expression of an isoform of the vacuolar H+-ATPase 56-kilodalton subunit in renal intercalated cells. Proc Natl Acad Sci U S A 89: 3541-3545, 1992. Parks SK, Tresguerres M, and Goss GG. Theoretical considerations underlying Na+ uptake mechanisms in freshwater fishes. Comp Biochem Physiol C Toxicol Pharmacol 148: 411-418, 2008. Perry SF, Beyers ML, and Johnson DA. Cloning and molecular characterisation of the trout (Oncorhynchus mykiss) vacuolar H+-ATPase B subunit. J Exp Biol 203: 459-470, 2000. Perry SF, Furimsky M, Bayaa M, Georgalis T, Shahsavarani A, Nickerson JG, and Moon TW. Integrated responses of Na+/HCO3- cotransporters and V-type H+-ATPases in the fish gill and kidney during respiratory acidosis. Biochim Biophys Acta 1618: 175-184, 2003. Piermarini PM and Evans DH. Immunochemical analysis of the vacuolar proton-ATPase B-subunit in the gills of a euryhaline stingray (Dasyatis sabina): effects of salinity and relation to Na+/K+-ATPase. J Exp Biol 204: 3251-3259, 2001. Piermarini PM, Verlander JW, Royaux IE, and Evans DH. Pendrin immunoreactivity in the gill epithelium of a euryhaline elasmobranch. Am J Physiol Regul Integr Comp Physiol 283: R983-992, 2002. Post RL and Jolly PC. The linkage of sodium, potassium, and ammonium active transport across the human erythrocyte membrane. Biochim Biophys Acta 25: 118-128, 1957. Puopolo K, Kumamoto C, Adachi I, Magner R, and Forgac M. Differential expression of the "B" subunit of the vacuolar H+-ATPase in bovine tissues. J Biol Chem 267: 3696-3706, 1992. Rainboth WJ. Fishes of the Cambodian Mekong. FAO Species Identification Field Guide for Fishery Purposes. FAO, Rome. 265 pp, 1996. Smith AN, Borthwick KJ, and Karet FE. Molecular cloning and characterization of novel tissue-specific isoforms of the human vacuolar H+-ATPase C, G and d subunits, and their evaluation in autosomal recessive distal renal tubular acidosis. Gene 297: 169-177, 2002. Stevens TH and Forgac M. Structure, function and regulation of the vacuolar H+-ATPase. Annu Rev Cell Dev Biol 13: 779-808, 1997. Sudhof TC, Fried VA, Stone DK, Johnston PA, and Xie XS. Human endomembrane H+ pump strongly resembles the ATP-synthetase of Archaebacteria. Proc Natl Acad Sci U S A 86: 6067-6071, 1989. Sullivan G, Fryer J, and Perry S. Immunolocalization of proton pumps (H+-ATPase) in pavement cells of rainbow trout gill. J Exp Biol 198: 2619-2629, 1995. Sun-Wada GH, Yoshimizu T, Imai-Senga Y, Wada Y, and Futai M. Diversity of mouse proton-translocating ATPase: presence of multiple isoforms of the C, d and G subunits. Gene 302: 147-153, 2003. Tang CH and Lee TH. The effect of environmental salinity on the protein expression of Na+/K+-ATPase, Na+/K+/2Cl- cotransporter, cystic fibrosis transmembrane conductance regulator, anion exchanger 1, and chloride channel 3 in gills of a euryhaline teleost, Tetraodon nigroviridis. Comp Biochem Physiol A Mol Integr Physiol 147: 521-528, 2007a. Tang CH and Lee TH. The novel correlation of carbonic anhydrase II and anion exchanger 1 in gills of the spotted green pufferfish, Tetraodon nigrovirids. J Exp Zool Part A Ecol Genet Physiol 307: 411-418, 2007b. Tresguerres M, Katoh F, Fenton H, Jasinska E, and Goss GG. Regulation of branchial V-H+-ATPase, Na+/K+-ATPase and NHE2 in response to acid and base infusions in the Pacific spiny dogfish (Squalus acanthias). J Exp Biol 208: 345-354, 2005. Tresguerres M, Katoh F, Orr E, Parks SK, and Goss GG. Chloride uptake and base secretion in freshwater fish: a transepithelial ion-transport metabolon? Physiol Biochem Zool 79: 981-996, 2006. Tresguerres M, Parks SK, Wood CM, and Goss GG. V-H+ -ATPase translocation during blood alkalosis in dogfish gills: interaction with carbonic anhydrase and involvement in the postfeeding alkaline tide. Am J Physiol Regul Integr Comp Physiol 292: R2012-2019, 2007. Wilson JM, Laurent P, Tufts BL, Benos DJ, Donowitz M, Vogl AW, and Randall DJ. NaCl uptake by the branchial epithelium in freshwater teleost fish: an immunological approach to ion-transport protein localization. J Exp Biol 203: 2279-2296, 2000. Wilson JM, Leitão A, Gonçalves AF, Ferreira C, Reis-Santos R, Fonseca A, Silva JMD, Antunes JC, Pereira-Wilson C, and Coimbra J. Modulation of branchial ion transport protein expression by salinity in glass eels (Anguilla anguilla L.). Mar Biol 151:1633-1645, 2007. Yan JJ, Chou MY, Kaneko T, and Hwang PP. Gene expression of Na+/H+ exchanger in zebrafish H+-ATPase-rich cells during acclimation to low-Na+ and acidic environments. Am J Physiol Cell Physiol 293: C1814-1823, 2007.
摘要: 氫離子幫浦 (V-type H+-ATPase) 是一級主動運輸的蛋白同時也提供驅動力來驅動二級主動運輸系統。因此,氫離子幫浦在上皮細胞中的離子調控或酸鹼平衡上扮演著重要的角色。氫離子幫浦是由負責水解ATP的V1複合體和負責運輸氫離子的V0複合體所組成。V1複合體裡的A次單元為ATP結合進行催化的位置;B次單元則負責與ATP結合。本次實驗魚種墨綠凹鼻魨 (Tetraodon nigroviridis)為廣鹽性硬骨魚類,並且在不同的實驗環境中被證實是個高效率的滲透壓調節者,可以忍受從淡水直接轉移到半淡鹹水;也由於墨綠凹鼻魨的基因已經被完全解序,有完整的基因體資料庫,所以在研究廣鹽性硬骨魚類的離子調節方面上墨綠凹鼻魨是一個良好的模式魚種。在本研究中,我們運用了分子選殖技術將V1複合體上的兩個A次單元體的isoform部份序列定序,且將此序列轉譯出來的胺基酸序列與不同的物種比較,結果顯示墨綠凹鼻魨和其他物種都有高度的相似性。由於鈉鉀幫浦 (Na+/K+-ATPase)被認知為鰓上富含粒線體細胞 (Mitochondrion-rich cells)的標定蛋白,所以我們進一步藉由免疫螢光染色及共軛焦顯微鏡觀察,將氫離子幫浦的A和B次單元與鈉鉀幫浦進行對照,實驗結果發現氫離子幫浦的A和B次單元和鈉鉀幫浦都共同表現於富含粒線體細胞的基底膜上 (basolateral membrane)。接著利用西方墨點法偵測氫離子幫浦A次單元在不同器官的蛋白質表現量,實驗結果發現在腦、鰓、肝臟、小腸都偵測到有兩個isoform的表現,分子量分別約為110和120 kDa。此外,同時也偵測B次單元不同器官的氫離子幫浦的蛋白質表現,包括腦、鰓、肝臟、小腸、腎臟,實驗結果發現只有在肝臟上表現兩個B次單元的isoform,分子量約為55和65 kDa,其餘器官則只單獨表現55 kDa的B次單元isoform。但是在mRNA基因表現方面,B次單元的基因在所偵測的器官上 (腦、鰓、肝臟、小腸、腎臟)卻都有氫離子幫浦的兩個isoform基因表現。此外,馴養在淡水環境中的墨綠凹鼻魨的氫離子幫浦B次單元的蛋白質表現量顯著地高於適應於半淡鹹水環境的魚。而在適應低濃度離子的人工水實驗中,我們發現氫離子幫浦B次單元的蛋白質表現量在低氯人工水的環境下明顯大於低鈉組以及控制組。綜合以上實驗結果指出,墨綠凹鼻魨鰓上富含粒線體細胞上的氫離子幫浦對於氯離子吸收可能扮演相當重要的角色。
V-type H+-ATPase (V-HA) is a primary active enzyme which provides the driving force for secondary active transport systems and plays an important role on ionregulator or acid-base regulation in epithelial cells. The V-HA consists of two large multisubunit subcomplexes which are designated as the V1 for ATP hydrolysis and the V0 composes of diverse proteins necessary for proton transport. In the V1 subcomplex, the A subunit is important for ATP-binding and catalysis. The B subunit is also responsible for ATP-binding. The spotted green pufferfish (Tetraodon nigroviridis) is an advanced euryhaline teleost and this species is demonstrated to be an efficient osmoregulator in experimental conditions. Since pufferfish can tolerate acute transfer from fresh water (FW) to brackish water (BW) or vice versa and has the complete genome database, the pufferfish is a good animal model to study ionoregulatory mechanisms in euryhaline teleost. The V-HA A subunit were cloned and sequenced in gills of the pufferfish. In addition, the alignment analysis of amino acid sequence of pufferfish V-HA A subunit indicated that V-HA A exhibited high identity with other vertebrates V-HA A subunit. Localization of V-HA A and B subunit in pufferfish gills was revealed by double-immunofluorescent staining with Na+/K+-ATPase (NKA) and observed by the confocal microscopy. The results showed that the V-HA A and B subunit were co-localized with NKA in the basolateral membrane of the mitochondrion-rich (MR) cells in FW. Immunoblotting of the membrane fractions to detect the V-HA A subunit protein expression showed that the pufferfish HA A subunit exhibited two isoforms with molecular weight (MW) 110 and 120 kDa, respectively. Furthermore, the protein expression of V-HA B subunit showed that the B1 isoform was ubiquitously expressed in various organs (i.e. brain, gill, liver, intestine and kidney), but the B2 isoform was detected only in liver. However, the mRNA of V-HA B subunit isoforms were expressed in all detected organs. The relative protein abundance of B subunit was significantly higher in gills of FW-acclimated pufferfish than BW-acclimated individuals. In artificial water-acclimated experiment, however, we found that the protein expression of V-HA B subunit increased significantly in low-Cl- environment rather than low-Na+ and control groups. These results indicated that branchial V-HA might play a role on Cl--uptake mechanism of pufferfish.
URI: http://hdl.handle.net/11455/22981
其他識別: U0005-2906200904342600
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2906200904342600
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