拉氏清溪蟹（Candidiopotamonrathbunae）鰓部上皮細胞之Na+,K+-ATPase與V-typeH+-ATPase調節模式的探討

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Title:	拉氏清溪蟹（Candidiopotamonrathbunae）鰓部上皮細胞之Na+,K+-ATPase與V-typeH+-ATPase調節模式的探討
Other Titles:	The osmoregulatory pattern of Na+, K+-ATPase and V-type H+-ATPase in gill epithelia of freshwater crab Candidiopotamon rathbunae
Authors:	曾姿萍 Tseng, Tzu-Ping
Contributors:	林惠真 Lin, Hui-Chen 東海大學生命科學系
Keywords:	拉氏清溪蟹;鰓;離子調節 gill;ionoregulation
Date:	2008
Issue Date:	2011-03-24T05:35:07Z (UTC)
Abstract:	過去對於螃蟹生理適應的研究顯示，進行氣體交換、離子調節與含氮廢物排除功能的主要器官為鰓，因此，研究螃蟹鰓部結構與功能，可以了解環境與螃蟹生理機制的關係。在離子調節方面，大部分螃蟹靠著Na+, K+-ATPase (NKA)和V-type H+-ATPase (HA)進行主動調節，需消耗能量以維持體液滲透壓與環境滲透壓呈一梯度差。NKA大量位於鰓上上皮細胞的基底膜，做為驅動頂膜離子運輸的原動力。在淡水環境，螃蟹利用頂膜的HA將H+離子打出細胞外，造成電位梯度差，與使用NKA驅動離子調節相比，為較有效率的方式。本實驗室過去研究十三種螃蟹，也支持在鰓部上皮細胞頂膜的HA是螃蟹能否適應淡水環境的重要假說。至此，關於滲透壓調節機制的研究對海生螃蟹較多，純淡水蟹的鰓部結構及調節機制，仍需更多實驗證據支持。我的實驗利用純淡水生的拉氏清溪蟹 (Candidiopotamon rathbunae)，進行鰓部結構觀察，以釐清淡水蟹鰓部結構特化的爭議。結果發現，拉氏清溪蟹具有九對鰓；靠近出鰓血管端和出鰓與入鰓血管之間的鰓薄板邊緣通道，各有一處隆起，入鰓血管側的特徵是具有瘤狀突起，出鰓血管側則是有兩排刺。在上皮細胞厚度比較方面，螃蟹馴養於淡水環境，第一、二和四至六對鰓上皮細胞厚度顯著大於第七至九對鰓，不同厚度的上皮細胞，可能具有不同的生理功能。不同對鰓之間NKA活性比較顯示，其中第四至六對鰓活性最高。當螃蟹轉移至去離子水，其中第二和四至七對鰓在轉移一天後NKA活性升高，第七天活性降低，顯示第四、五和六對鰓可能為拉氏清溪蟹的離子調節鰓。比較第五和第九對鰓之HA活性，以及比較淡水與轉移至去離子水四天處理組之活性，結果皆沒有顯著差異。西方點墨法所測得蛋白質的含量，轉移至去離子水環境第一、四，和七天後，無論是NKA或HA蛋白質量皆沒有增加；推測轉移七天內，NKA利用提高現有蛋白質的交換效率幫助吸收離子，而不是增加蛋白質的量。以上結果與過去螃蟹研究，前鰓氣體交換，後鰓進行離子調節的研究結果不同。過去研究已知與離子吸收有關的HA位在細胞膜上，於是第二部分實驗觀察HA在細胞中分布的位置。在免疫組織染色與免疫金粒子標定結果顯示，NKA與HA主要位在基底膜。而只有少數HA分布在上皮細胞的頂膜。標定其他與離子運輸有關的膜蛋白質發現，Na+/K+/2Cl- cotransporter分布在基底膜。Na+/H+ exchanger主要分布在頂膜，而柱狀細胞的基底膜也有分布。此二次級主動離子運輸蛋白質為提供上皮細胞幫助Na+和Cl-離子運輸的膜蛋白質。本實驗以拉氏清溪蟹為研究材料，首次提出淡水螃蟹具有前鰓為主要離子調節生理功能，而後鰓主要具有氣體交換功能的分工現象。鰓上NKA為其驅動離子運輸的動力來源；位在基底膜上的HA可能具有離子調節以外的功能，推測為幫助酸鹼平衡，這個部分須更進一步釐清。 For those studies on the physiological adaptation of crabs, the gill is the main organ of the gas exchange, ion regulation, and ammonia excretion functions. A detailed investigation on the structures and functions of crab gills is crucial for the discussion of environmental physiology of the crabs. In the context of ion regulation, Na+, K+-ATPase (NKA) and V-type H+-ATPase (HA) are important for maintaining an ion gradient between the body and the external environment, and the activities of both enzymes are energy consuming. In general, NKA is distributed in the basolateral membranes of gill epithelia and provides the driving force for ion flux. In fresh water, crabs use apically located HA to pump H+ ion out of the cell, creating an higher electrogenic difference across the membrane than the basolaterally located NKA created. This powers further transport of ions. In our laboratory, we conducted a survey on 13 species of crabs and our results supported the hypothesis that the apical distribution of HA is important for freshwater adaptation. Until now, most discussions on osmoregulatory mechanisms are based on marine crabs, but very few were on freshwater crabs. In my study, by examining the structures of gills of the freshwater crab Candidiopotamon rathbunae, I intended to clarify debate of structural specialization of gill epithelia of freshwater crabs. My results indicated that C. rathbunae has nine pairs of gills and the marginal canals of the lamellae near the efferent side and the middle of marginal canals are swollen. There are two rows of sharp spikes underneath the efferent side and knobs on the surface of the afferent vessels. Among the nine pairs of gills, the epithelia from the first, second and the fourth to sixth gills are thicker than those from the seventh to ninth gills, suggesting different physiological functions. The NKA activities of the fourth, fifth, and sixth pairs of gills are higher than those of other gills. On the first day after transferal to deionized water, NKA activity of the second and the fourth to seventh pairs of gills increased. By day 7, the activities of the second and the fourth to seventh pairs of gills decreased. The results indicated that the fourth to sixth pairs of gills are the ion-regulatory gills. The HA activities of the fifth and ninth gills did not differ significantly between the freshwater treatments and the fourth day in deionized water. The proteins of both NKA and HA did not differ significantly among days after transferal. It is the increase in the NKA activities of proteins, but not in the abundance, that would lead to the rise of the ion exchange efficiency. My results were different from the previous studies in respect to the anterior gills being for gas exchanger and the posterior ones for ion regulation. The relative importance of HA in ionoregulation deserves further investigation. Since only cell membrane located HA is involved in ion uptake, I need to clarify the location of the HA. In the second part of my study, by applying immuno-histochemical staining and immuno-gold labeling, both NKA and HA were found in basolateral membrane while there was a few HA in the apical membrane. The other two ion-uptake membrane proteins were also located by immuno-fluorescence staining and only basolateral Na+/K+/2Cl- cotransporter and Na+/H+ exchanger in the apical membrane were found in the gill epithelia. The Na+/H+ exchanger in the basolateral membrane were only found in the pillar cell. Using C. rathbunae as the study subject, this is the first study to describe that the anterior gills of freshwater crabs is for ion regulation, and the posterior gills is for gas exchange. Gill NKA is the driving forces for ion transport. More investigation is needed on the basolaterally located HA before its function, possibly acid-base balance, can be concluded.
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