Tsui T K N, Hung C Y C, Nawata C M, Wilson J M, Wright P A, Wood C M
Department of Biology, McMaster University, Hamilton, Ontario, Canada L8S 4K1.
J Exp Biol. 2009 Mar;212(Pt 6):878-92. doi: 10.1242/jeb.021899.
The mechanisms of ammonia excretion at fish gills have been studied for decades but details remain unclear, with continuing debate on the relative importance of non-ionic NH(3) or ionic NH(4)(+) permeation by various mechanisms. The presence of an apical Na(+)/NH(4)(+) exchanger has also been controversial. The present study utilized an in vitro cultured gill epithelium (double seeded insert, DSI) of freshwater rainbow trout as a model to investigate these issues. The relationship between basolateral ammonia concentration and efflux to apical freshwater was curvilinear, indicative of a saturable carrier-mediated component (K(m)=66 micromol l(-1)) superimposed on a large diffusive linear component. Pre-exposure to elevated ammonia (2000 micromol l(-1)) and cortisol (1000 ng ml(-1)) had synergistic effects on the ammonia permeability of DSI, with significantly increased Na(+) influx and positive correlations between ammonia efflux and Na(+) uptake. This increase in ammonia permeability was bidirectional. It could not be explained by changes in paracellular permeability as measured by [(3)H]PEG-4000 flux. The mRNA expressions of Rhbg, Rhcg2, H(+)-ATPase and Na(+)/H(+) exchanger-2 (NHE-2) were up-regulated in DSI pre-exposed to ammonia and cortisol, CA-2 mRNA was down-regulated, and transepithelial potential became more negative. Bafilomycin (1 micromol l(-1)), phenamil (10 micromol l(-1)) and 5-(N,N-hexamethylene)amiloride (HMA, 10 micromol l(-1)) applied to the apical solution significantly inhibited ammonia efflux, indicating that H(+)-ATPase, Na(+) channel and NHE-2 pathways on the apical surface were involved in ammonia excretion. Apical amiloride (100 micromol l(-1)) was similarly effective, while basolateral HMA was ineffective. Pre-treatment with apical freshwater low in [Na(+)] caused increases in both Rhcg2 mRNA expression and ammonia efflux without change in paracellular permeability. These data suggest that Rhesus glycoproteins are important for ammonia transport in the freshwater trout gill, and may help to explain in vivo data where plasma ammonia stabilized at 50% below water levels during exposure to high environmental ammonia ( approximately 2300 micromol l(-1)). We propose an apical ;Na(+)/NH(4)(+) exchange complex' consisting of several membrane transporters, while affirming the importance of non-ionic NH(3) diffusion in ammonia excretion across freshwater fish gills.
几十年来，人们一直在研究鱼类鳃部氨排泄的机制，但细节仍不清楚，关于非离子态NH₃或离子态NH₄⁺通过各种机制渗透的相对重要性仍存在争议。顶端Na⁺/NH₄⁺交换体的存在也存在争议。本研究利用淡水虹鳟鱼的体外培养鳃上皮（双层接种插入物，DSI）作为模型来研究这些问题。基底外侧氨浓度与向顶端淡水的外流之间的关系呈曲线，表明在一个大的扩散线性成分之上叠加了一个可饱和的载体介导成分（Kₘ = 66 μmol·l⁻¹）。预先暴露于高氨（2000 μmol·l⁻¹）和皮质醇（1000 ng/ml）对DSI的氨通透性有协同作用，Na⁺内流显著增加，氨外流与Na⁺摄取之间呈正相关。这种氨通透性的增加是双向的。它不能用通过[³H]PEG - 4000通量测量的细胞旁通透性变化来解释。预先暴露于氨和皮质醇的DSI中，Rhbg、Rhcg2、H⁺ - ATP酶和Na⁺/H⁺交换体 - 2（NHE - 2）的mRNA表达上调，CA - 2 mRNA下调，跨上皮电位变得更负。应用于顶端溶液的巴弗洛霉素（1 μmol·l⁻¹）、非那利得（10 μmol·l⁻¹）和5 -（N，N - 六亚甲基）氨氯吡脒（HMA，10 μmol·l⁻¹）显著抑制氨外流，表明顶端表面的H⁺ - ATP酶、Na⁺通道和NHE - 2途径参与氨排泄。顶端氨氯吡脒（100 μmol·l⁻¹）同样有效，而基底外侧HMA无效。用[Na⁺]低的顶端淡水预处理导致Rhcg2 mRNA表达和氨外流均增加，而细胞旁通透性无变化。这些数据表明，恒河猴糖蛋白对淡水鳟鱼鳃部的氨转运很重要，并且可能有助于解释体内数据，即在暴露于高环境氨（约2300 μmol·l⁻¹）期间，血浆氨稳定在低于水体水平50%的情况。我们提出一个由几种膜转运体组成的顶端“Na⁺/NH₄⁺交换复合体”，同时肯定非离子态NH₃扩散在淡水鱼鳃氨排泄中的重要性。
