吳郭魚鰓蓋膜氯離子吸收機制之研究

Abstract

本實驗之目的為探討環境中氯離子對淡水吳郭魚鰓蓋膜上 MR 細胞型態與蛋白質表現之影響。吳郭魚分別馴養於低鈉低氯、高鈉低氯或高鈉高氯人工水中，馴養結束後，以掃描式電子顯微鏡觀察鰓蓋膜上氯細胞型態變化並以西方墨點法偵測Na+，K+-ATPase 、 Cl-／ HCO3 - exchanger , H+-ATPase 與 carbonic anhydrase 蛋白質表現。實驗結果如下： ( 1 ）七天長期馴養實驗：突起型（ wavy 一 convex ）、淺盤型 ( shallow 一 basin ）與深洞型（ deep 一 hole ) MR 細胞皆出現於鰓蓋膜。鰓蓋膜上 MR 細胞密度於低氯環境中顯著高於高氯環境。突起型 MR 細胞只出現在低氯環境中，但在高鈉高氯人工水中並無發現此型細胞，顯示此型細胞與氯吸收有關而與鈉吸收無關。淺盤型 MR 細胞於三種人工水中數量最多。 ( 2 ）急性轉移實驗：將吳郭魚由高氯環境轉至低氯環境復，發現三種 MR 細胞數目在 6 小時增加（與0時相比），且在 96 小時內持續增加。淺盤型MR細胞在24小時開始增加且持續維持高密度。在 24小時內，淺盤型 MR 細胞增加且深洞型 MR 細胞減少，突起型 MR 細胞則在 24 小時開始增加。這些 MR 細胞之間的變化顯示深洞型 MR 細胞在低氯環境下會轉型為淺盤型 MR 細胞，進而轉變成突起型MR 細胞。 ( 3 ）運輸蛋白表現實驗：Na+，K+-ATPase 、 Cl-／HCO3-exchanger , H+-ATPase 與carbonic anhydrase 在淡水吳郭魚鰓蓋膜上皆有表現，然而在適應三種人工水一週後，四種蛋白質表現並無差異。 與鰓互相比較，鰓蓋膜 MR 細胞數量較少，且面對環境變化時敏感度較低，顯示鰓蓋膜在魚體內對於離子與滲透壓的調節，可能扮演著輔助的角色。但是鰓蓋膜與鰓對於氯吸收的機制是相似的，所以鰓蓋膜為代替鰓研究離子吸收的良好模式，但是在做實驗之前，須小心地將兩者的差異性納入考慮

The propose of the present study was to elucidate the Cl- effect on the functional morphology of opercular membrane. Effects of environmental Cl- concentrations on the morphology of MR cells and the protein expressions of ion transporters and enzymes in opercular membrane in freshwater tilapia were studied. Tilapia were acclimated to 3 artificial freshwaters: Low-Na-Low-Cl, High-Na-Low-Cl and High-Na-High-Cl. After acclimation, opercular membranes were subjected to the morphological observations of MR cells using scanning electron microscopy and to western blot for examination of the protein expression of Na+，K+-ATPase C1-/HCO3- exchanger, H+-ATPase and carbonic anhydrase. (1) One-week acclimation experiment: wavy-convex, shallow-basin and deep-hole MR cells were observed in opercular membrane. Cell density of MR cells was higher in Low-Na-Low-Cl and High-Na-Low-Cl media than those in High-Na-High-Cl medium. Wavy-convex MR cells appeared in Low-Cl media only but never in High-Na-High-Cl medium, indicating that wavy-convex type was associated with Cl- uptake but not for Na+ uptake. Shallow-basin MR cells were the dominant type in the 3 media. (2) Acute exposure experiment: tilapia were transferred from high Cl- to low Cl- environment, the numbers of 3 types MR cells increased at 6 hr (significantly different from that at 0 hr , p <0.05, t-test), and kept increasing within 96 hr. Shallow-basin MR cells started increasing within 24 hr and maintained a higher density. Within 24 hr exposure, shallow-basin MR cells started to increase while deep-hole MR cells decrease. Thereafter 24 hr, wavy-convex MR cells began to increase. These morphological changes suggested transformations from deep hole to shallow basin and finally to wavy-convex type of MR cells during acclimation to low Cl- exposure. (3) Transporter protein expression experiment: Na+，K+-ATPase, C1-/HCO3- exchanger, H+-ATPase and carbonic anhydrase were detcted in opercular membranes of freshwater tilapia. However there were no significant differences in the expressions of these 4 proteins among tilapia acclimated to the 3 media for 1 week. Comparing with gills, opercular membranes have much less MR cells and reveal a lower sensitivity and a slower regulation upon environmental challenges, indicating to play a minor or an accessory role in the ion- and osmo-regulation of a whole fish. However, the mechanism for Cl- uptake may be similar in both gill and opercular MR cells. Therefore, opercular membrane could be a suitable model for studying ion uptake mechanisms of gills if one can consider carefully the differences between these 2 tissues as mentioned above when the experiments are performed.