博士 / 國防醫學院 / 生命科學研究所 / 91 / Mitochondria-rich (MR) cells are specialized epithelial cells for ion regulation in fishes. Morphologically different subtypes of MR cells were identified in freshwater fishes. In tilapia acclimated to freshwater, 3 subtypes of MR cells with different structure of apical openings, i.e., wavy convex, shallow basin, and deep hole, were identified. The object of the present study is to examine the structure-function relationship of these MR cells subtypes.
In chapter 1 of present study, correlations between the subtypes of MR cells and Cl- uptake capabilities were examined in yolk-sac tilapia larvae. I tested a hypothesis that these subtype cells represent MR cells equipped with variable efficiencies in Cl- uptake. Results showed that larvae acclimated to low-Cl- ([Cl-] = 0.001-0.007 mM) water developed higher densities of MR cells than those acclimated to high-Cl- ([Cl-] = 7.3-7.9 mM) water. The percentage of wavy-convex type in total MR cells was higher in low-Cl--acclimated larvae than in high-Cl--acclimated larvae, which displayed only deep-hole type. In addition, Cl- influx rates of whole larva measured with 36Cl- showed a coincident correlation with MR cell densities, i.e., low-Cl- larvae displayed higher Cl- influx rates than did high-Cl- larva, suggesting that tilapia larvae develop a higher density of MR cells with larger apical surfaces (wavy-convex type) to boost Cl- uptake in Cl--deficient water.
Based on the findings in the chapter 1, I proposed a model “activation and inactivation of MR cells” to elucidate the significance of the MR cell subtypes. In the chapter 2, evidences were proposed to support this model. Yolk-sac MR cells of the larvae were examined with immunocytochemistry and vital staining during acclimation to high, normal, or low ambient Cl- levels. Employing concanavalin-A (Con-A) and Na-K-ATPase double staining, MR cells were labeled and discriminated into active (Con-A positive) or inactive (Con-A negative) groups. The proportion of active cells gradually increased during 48-h acclimation to low-Cl- medium, but declined during acclimation to high-Cl- medium. However, the total densities of MR cells were not altered by ambient chloride. Furthermore, yolk-sac MR cells were vitally stained with DASPEI and traced in live larvae responding to ambient Cl- changes. The result consistently indicated that no significant turnover of MR cells was induced by ambient Cl- fluctuations, and the subtypes of MR cells represent various phases of MR cells, which are undergoing activation or inactivation.
Additionally, I was interested to know which Cl- transporter might be involved in Cl- uptake and regulation during activation and inactivation of MR cells. The Cl- transport protein, Na-K-2Cl cotransporter (NKCC), was chose as a candidate for the Cl- uptake function. In the chapter 3, the expression and cellular distribution of NKCC in branchial MR cells of adult tilapia were examined. Western blots and immunocytochemistry were used to determine the abundance and localization of NKCC in gills of tilapia. Western blots revealed 3 major bands centered at 282, 122, and 105 kDa in both SW- and FW-acclimated tilapia. Confocal images showed that NKCC was co-localized with Na-K-ATPase to basolateral membrane of SW MR cells. However, NKCC was concentrated at the apical membrane and “subapical” region (cytoplasm beneath the apical membrane) of MR cells in FW-acclimated tilapia. NKCC abundance was induced by ambient Cl- deficiency, and in contrast, suppressed by ambient high Cl-. This correlation indicates that NKCC might be involved in Cl- absorption of FW MR cells.
Finally, in order to determine the Cl- transport in MR cells, electrophysiological approach (ion-selective vibrating probe) was established to probe Cl- fluxes in intact tilapia larvae. The probe successfully detected Cl- fluxes at the tip of an artificial Cl- source and at skin surface of the larvae. However, the optical and probing resolutions of our system still need to be improved.
| Identifer | oai:union.ndltd.org:TW/091NDMC0105013 |
| Date | January 2003 |
| Creators | Li-Yih Lin, 林豊益 |
| Contributors | Pung-Pung Hwang, 黃鵬鵬 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 87 |
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