Ip Yuen K, Leong Charmaine W Q, Boo Mel V, Wong Wai P, Lam Siew H, Chew Shit F
Department of Biological Sciences, National University of Singapore, Kent Ridge, Singapore 117543, Republic of Singapore.
Department of Biological Sciences, National University of Singapore, Kent Ridge, Singapore 117543, Republic of Singapore.
Comp Biochem Physiol A Mol Integr Physiol. 2022 Nov;273:111297. doi: 10.1016/j.cbpa.2022.111297. Epub 2022 Aug 17.
African lungfishes are obligatory air-breathers with exceptionally high environmental ammonia tolerance. They can lower the pH of the external medium during exposure to ammonia-loading conditions. This study aimed to demonstrate the possible involvement of branchial vacuolar-type H-ATPase (Vha) in the ammonia-induced acidification of the external medium by the West African lungfish, Protopterus annectens, and to examine whether its capacity to acidify the medium could be augmented after exposure to 100 mmol l NHCl for six days. Two full coding cDNA sequences of Vha subunit B (atp6v1b), atp6v1b1 and atp6v1b2, were obtained from the internal gills of P. annectens. The sequence of atp6v1b1 comprised 1548 bp, encoding 515 amino acids (57.4 kDa), while that of atp6v1b2 comprised 1536 bp, encoding 511 amino acids (56.6 kDa). Using a custom-made antibody reactive to both isoforms, immunofluorescence microscopy revealed the collective localization of Atp6v1b (atp6v1b1 and atp6v1b2) at the apical or the basolateral membrane of two different types of branchial Na/K-ATPase-immunoreactive ionocyte. The ionocytes labelled apically with Atp6v1b presumably expressed Atp6v1b1 containing a PDZ-binding domain, indicating that the apical Vha was positioned to transport H to the external medium. The expression of Atp6v1b was regulated post-transcriptionally, as the protein abundance of Atp6v1b and Vha activity increased significantly in the gills of fish exposed to 100 mmol l NHCl for six days. Correspondingly, the fish exposed to ammonia had a greater capacity to acidify the external medium, presumably to decrease the ratio of [NH] to [NH] in order to reduce the influx of exogenous NH.
非洲肺鱼是必须进行空气呼吸的鱼类，对环境氨具有极高的耐受性。在暴露于氨负荷条件下时，它们能够降低外部介质的pH值。本研究旨在证明西非肺鱼（Protopterus annectens）鳃部液泡型H-ATP酶（Vha）可能参与氨诱导的外部介质酸化过程，并研究在暴露于100 mmol·l NH₄Cl六天后，其酸化介质的能力是否会增强。从P. annectens的内鳃中获得了Vha亚基B（atp6v1b）的两个完整编码cDNA序列，即atp6v1b1和atp6v1b2。atp6v1b1的序列由1548 bp组成，编码515个氨基酸（57.4 kDa），而atp6v1b2的序列由1536 bp组成，编码511个氨基酸（56.6 kDa）。使用对两种同工型均有反应的定制抗体，免疫荧光显微镜显示Atp6v1b（atp6v1b1和atp6v1b2）在两种不同类型的鳃Na/K-ATP酶免疫反应性离子细胞的顶端或基底外侧膜上共同定位。顶端标记有Atp6v1b的离子细胞可能表达含有PDZ结合结构域的Atp6v1b1，这表明顶端Vha的位置可将H转运到外部介质中。Atp6v1b的表达在转录后受到调节，因为在暴露于100 mmol·l NH₄Cl六天的鱼的鳃中，Atp6v1b的蛋白质丰度和Vha活性显著增加。相应地，暴露于氨的鱼具有更强的酸化外部介质的能力，推测是为了降低[NH₄⁺]与[NH₃]的比例，以减少外源NH₃的内流。
