斑馬魚之氯離子吸收機制與其功能方面之調控

王怡方

doi:10.6342/NTU201602000

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斑马鱼之氯离子吸收机制与其功能方面之调控

Chloride uptake mechanism and the functional regulation in zebrafish

王怡方 , 博士　　导师：黄鹏鹏　　

英文 DOI： 10.6342/NTU201602000

斑马鱼；氯离子；体液恒定；贺尔蒙； zebrafish ； chloride ； homeostasis ； hormone

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摘要 │ 参考文献 (191) │ 国际计量

摘要〈TOP〉

脊椎动物为了确保生理机能的正常运作必须维持体内氯离子的恒定，在由水生生物演化为陆生的过程中发展出一套复杂且精细的调控氯离子恒定的机制，以适应环境中氯离子浓度的变化。鱼类和陆生脊椎动物具有相似的调控氯离子机制，然而，非哺乳类的脊椎动物其调控氯离子的详细机制则尚不明确。之前的研究发现钠氯共同运输蛋白2b (NCC2b)负责斑马鱼在鳃上以及表皮的氯离子吸收，然而，详细的氯离子吸收的运输途径则仍属未知。
    内分泌系统的功能一直被视为在生物面对环境变化时负责维持正常的生理机能，然而，我们对於鱼类内分泌系统对於氯离子吸收的机制上所知甚少。因此，本研究旨在以斑马鱼为模式物种调查和表现NCC2b 的离子细胞相关的氯离子吸收途径，并进一步研究和氯离子吸收相关的贺尔蒙及其调控的机制与角色为何。
    第一章主要研究 CLC 氯离子通道在氯离子吸收扮演的角色。藉由原位杂和反应及免疫萤光染色实验发现CLC-2c 表现在NCC2b 离子细胞上，当clc-2c 基因表现缺失时，会造成斑马鱼胚胎体内氯离子含量下降，显示CLC-2c 和氯离子吸收相关。并且，当clc-2c 基因表现缺失时会刺激ncc2b mRNA 的表现，反之亦然。由分子与细胞生理的实验结果证明CLC-2c 和NCC2b 共同负责斑马鱼鳃上及表皮的氯离子吸收功能。综合演化上的分析，鱼类特有的NCC2b 和CLC-2c可能是共同参与鱼类鳃上的氯离子吸收机制而共同演化保留至今。
    第二章主要研究 CGRP 和其受器CRLR1 对於氯离子吸收机制的调控。适应在高氯的环境中会刺激cgrp 和crlr1 mRNA 的表现，当cgrp 基因缺失的时候会刺激ncc2b RNA 的表现同时造成表现NCC2b 的离子细胞的密度增高，反之，当CGRP过量表现时会抑制ncc2b mRNA的合成同时造成斑马鱼胚胎氯离子吸收的减少。本实验首次证实CGRP 会经由抑制NCC2b 的合成和降低表现NCC2b 离子细胞的分化来调控降低血液中的氯离子含量，了解CGRP 在调控氯离子吸收方面扮演的角色有助於我们更加了解脊椎动物相关的生理功能。
    第三章主要研究 AVT 对於氯离子吸收调控的机制。当avt 基因功能缺失时会造成NCC2b mRNA 和蛋白质的表现下降，同时造成表现NCC2b 的离子细胞的密度降低并且造成胚胎体内氯离子含量的下降，实验结果显示AVT 藉由调控离子运输蛋白的转抑或是转录机制进而达到调控氯离子吸收的功能。此外，avt基因功能缺失亦会造成cgrp 及crlr1 mRNA 表现量下降，显示了AVT 和CGRP之间有交互作用以便共同调控氯离子吸收的路径，维持生物体内的氯离子恒定。

并列摘要〈TOP〉

Vertebrates need to maintain body fluid Cl- homeostasis to ensure normal operation of physiological process; the transition from aquatic to terrestrial environments necessitated the development of sophisticated mechanisms to ensure Cl- homeostasis in the face of fluctuating Cl-levels. The homeostatic mechanism of Cl- in aquatic fish appears to be similar to that of terrestrial vertebrates; however, the mechanism in non-mammalian vertebrates is poorly understood. Previous studies in zebrafish identified Na+-Cl- cotransporter (NCC) 2b-expressing cells in the gills and skin as the major ionocytes responsible for Cl- uptake. However, the mechanism by which basolateral ions exit from NCC2b-expressing cells is still unclear. Endocrine system is considered as the primary system to response to the environmental changes and maintain the normal physiological functions in vertebrates. However, less is known about the endocrine control of Cl- uptake mechanism in fish. The aims of this study are to use zebrafish as a model to investigate the Cl- uptake pathway of NCC2b-expressing ionocytes and identify hormones involved in the Cl- uptake mechanisms and to elucidate the mechanism in the regulation of Cl- uptake function.
    In the first chapter, the role of CLC Cl- channels in the Cl- uptake mechanism were examined. Doubled in situ hybridization/immunocytochemistry indicated colocalization of apical NCC2b with basolateral CLC-2c. Loss-of-function of clc-2c resulted in a significant decrease in whole body Cl- content in zebrafish embryos, which suggests a role of CLC-2c in Cl- uptake. Translational knockdown of clc-2c stimulated ncc2b mRNA expression and vice versa, revealing cooperation between these two transporters in the context of zebrafish Cl- homeostasis. Several lines of molecular and cellular physiological evidences demonstrated the cofunctional role of apical NCC2b and basolateral CLC-2c in the gill/skin Cl- uptake pathway. Taking the phylogenetic evidence into consideration, fish-specific NCC2b and CLC-2c may have coevolved to perform extra-renal Cl- uptake during the evolution of vertebrates in an aquatic environment.
    In the second chapter, the roles of calcitonin gene-related peptide (CGRP) and its receptor, calcitonin receptor-like receptor (CRLR1), in the regulation of Cl- uptake mechanism were examined. Acclimation to high-Cl− artificial water stimulated the mRNA expression of cgrp and crlr1 when compared with low-Cl−. CGRP knockdown induced upregulation of the ncc2b, while overexpression of CGRP resulted in the downregulation of ncc2b mRNA synthesis and a simultaneous decrease in Cl− uptake in embryos. Consistent with these findings, knockdown of either cgrp or crlr1 was found to increase the density of NCC2b-expressing cells in embryos. This is the first demonstration that CGRP acts as a hypochloremic hormone through suppressing NCC2b expression and the differentiation of NCC2b-expressing cells. Elucidation of this novel function of CGRP in fish body fluid Cl− homeostasis promises to enhance our understanding of the related physiology in vertebrates.
    In the third chapter, the role of arginine vasotocin (AVT) on Cl- uptake regulation was examined. The loss-of-function of avt significantly downregulated the mRNA and protein expressions of NCC2b. Moreover, NCC2b expressing cells were significantly decreased in avt morphants. The whole body Cl- content was also declined in avt morphants. These results suggest that AVT exerts its actions on Cl uptake pathway in zebrafish embryos through regulating the transcriptional and/or translational levels of the ion transporters. Notably, the mRNA expression of cgrp and crlr1 were downregulated in avt morphants, suggesting that the crosstalk between AVT and CGRP mediated the regulation signaling of Cl-uptake mechanisms in zebrafish.

参考文献 ( 191 ) 〈TOP〉

Hirose, S., Kaneko, T., Naito, N., Takei, Y., 2003. Molecular biology of major components of chloride cells. Comp Biochem Physiol B Biochem Mol Biol 136, 593-620.
连结：
Horng, J.L., Hwang, P.P., Shih, T.H., Wen, Z.H., Lin, C.S., Lin, L.Y., 2009. Chloride transport in mitochondrion-rich cells of euryhaline tilapia (Oreochromis mossambicus) larvae. Am J Physiol Cell Physiol 297, C845-854.
连结：
Inokuchi, M., Hiroi, J., Watanabe, S., Hwang, P.P., Kaneko, T., 2009. Morphological and functional classification of ion-absorbing mitochondria-rich cells in the gills of Mozambique tilapia. J Exp Biol 212, 1003-1010.
连结：
Inokuchi, M., Hiroi, J., Watanabe, S., Lee, K.M., Kaneko, T., 2008. Gene expression and morphological localization of NHE3, NCC and NKCC1a in branchial mitochondria-rich cells of Mozambique tilapia (Oreochromis mossambicus) acclimated to a wide range of salinities. Comp Biochem Physiol A Mol Integr Physiol 151, 151-158.
连结：
Jentsch, T.J., Poet, M., Fuhrmann, J.C., Zdebik, A.A., 2005. Physiological functions of CLC Cl- channels gleaned from human genetic disease and mouse models. Annu Rev Physiol 67, 779-807.
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