鈣離子感應接受器在斑馬魚鈣離子吸收功能上之角色

Role of calcium-sensing receptor in Ca2+ uptake function of zebrafish (Danio rerio)

蘇哲賢(Che-Hsien Su)

指導教授 ： 黃鵬鵬

國立臺灣大學/生命科學院/漁業科學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.02232

摘要

鈣離子參與動物許多重要的生理作用，因此對脊椎動物而言如何維持鈣離子的恆定是相當重要的議題。淡水魚類生活在鈣離子濃度不斷變動的淡水環境中，因此對淡水魚類而言，適當的調控鈣離子流進與流出身體以維持魚體內鈣離子的恆定性更是相當的重要。
目前已知副甲狀腺素(Parathyroid hormone, PTH)在哺乳動物中扮演調節鈣離子吸收的重要角色，但在魚類仍不是很清楚，是否在魚類中就已存在PTH調控鈣離子吸收機制值得進一步探討。因此，本實驗利用斑馬魚為模式物種，想藉斑馬魚在生物資訊分析以及分子生理方法學上的優勢，來探討斑馬魚副甲狀腺素對斑馬魚胚胎鈣離子吸收機制的影響，結果發現斑馬魚pth1基因表現量會因低鈣環境的刺激而增加，而斑馬魚pth2基因表現量則不受環境鈣離子濃度的變化而改變。將斑馬魚胚胎以人類PTH (1-34)浸泡或過量表現斑馬魚PTH1 cRNA都會增加魚體總鈣含量與表皮鈣離子通道(epithelial calcium channel, ECaC)基因的表現量，但過量表現斑馬魚PTH2 cRNA則不影響魚體總鈣含量或ecac基因的表現量。以專一性反義核酸(morpholino)抑制斑馬魚PTH1蛋白質轉譯後，魚體總鈣含量、ecac、基側膜的鈣離子幫浦(basolateral plasma membrane Ca2+-ATPase, PMCA2)與鈉鈣交換蛋白(Na+/Ca2+ exchanger, NCX1b)基因的表現量都明顯下降，而抑制斑馬魚PTH2蛋白質轉譯後則不會影響魚體總鈣含量或與鈣離子運輸相關蛋白的基因表現。綜合上述結果推論，斑馬魚胚胎在適應鈣離子環境變化的過程中，斑馬魚PTH1能透過影響斑馬魚ecac基因的表現量來調控斑馬魚的鈣離子吸收功能，而PTH2在適應鈣離子環境變化上可能不具生理意義跟功能。
鈣離子感應接受器(calcium-sensing receptor, CaSR)是一種C型G蛋白偶合接受器。CaSR在哺乳動物中能感受體液鈣離子濃度，進而調控內分泌釋放調節鈣離子吸收以維持鈣離子濃度恆定。目前在一些魚類研究中雖推論CaSR對司坦尼氏降鈣素(stanniocalcin 1, STC-1)的調控關係，但是對CaSR在魚類鈣離子吸收調控上的功能仍不是很清楚。另一方面，雖然在哺乳類CaSR調控PTH的關係已被廣泛瞭解，但是關於魚類CaSR跟PTH之間的關係仍不明確，因此本研究以斑馬魚為實驗動物，探討CaSR在魚類吸收鈣離子功能中所扮演的角色。利用專一性反義核酸抑制斑馬魚CaSR蛋白質轉譯後，發現魚體總鈣含量顯著增加、ecac與pth1基因的表現量明顯上升，而stc-1基因表現量明顯下降。另一方面，浸泡人類PTH1 (1-34)或過量表現斑馬魚PTH1 cRNA會使斑馬魚stc-1表現量增加，但過量表現斑馬魚STC-1 cRNA卻不會影響斑馬魚pth1基因表現量，綜合上述之結果推論，斑馬魚CaSR能透過影響斑馬魚ecac基因的表現量來調控斑馬魚的鈣離子吸收功能，而此機制可能是透過影響斑馬魚stc-1和pth1基因的表現量，此外，斑馬魚可能存在著PTH1對STC-1的調控作用。

關鍵字

鈣離子感應接受器 ； 斑馬魚 ； 表皮鈣離子通道 ； 副甲狀腺素 ； 司坦尼氏降鈣素

並列摘要

Ca2+ is a vital element that serves various physiological roles in vertebrates. In vertebrates which include fish, Ca2+ homeostasis is an important issue. Freshwater fish normally face fluctuating Ca2+ conditions, and therefore it is critical for fish to control Ca2+ flow (into or out) of their bodies.
Parathyroid hormone has been well documented to be a vital endocrine regulating Ca2+ uptake in mammals, however, remains largely unclear in teleosts. In present study, zebrafish (Danio rerio) was used as a model to investigate the functions of parathyroid hormone on Ca2+ uptake. Zebrafish embryo acclimated to low-Ca2+ freshwater stimulated parathyroid hormone 1 (pth1) expression, but no effects in parathyroid hormone 2 (pth2) expression. The Ca2+ content and the expressions of epithelial calcium channel (ecac) were increased by exogenous human PTH (1-34) but no significant differences on other Ca2+ transporters expression. After the injection of PTH1 cRNA, the ecac expression was stimulated but this phenomenon was not found in PTH2 cRNA injection. Morpholino knockdown of the PTH1 showed Ca2+ uptake function decreased by inhibiting ecac expression. Taken together, PTH1 stimulates Ca2+ uptake by upregulating ecac, and PTH2 seems to be no effects on Ca2+ handling in zebrafish.
Extracellular calcium-sensing receptor (CaSR) could sense Ca2+ level of body fluid and regulate endocrine system to maintain Ca2+ homeostasis in mammals; however, the role of CaSR on Ca2+ handling of fish is still unclear. Morpholino knockdown of zebrafish CaSR was found to stimulate Ca2+ content and ecac expression. To further explore the mechanism behind this, we found gene expressions of stanniocalcin-1 (stc-1) and pth1 were modulated differentially in CaSR-deficient morphants. In conclusion, CaSR downregulates ecac expression decreasing Ca2+ uptake functions and this control pathway is suggested to mediate the secretion of STC-1 and PTH1.

並列關鍵字

CaSR ； zebrafish ； ECaC ； PTH1 ； STC-1

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