Evidence for the involvement of branchial Vacuolar-type H+-ATPase in the acidification of the external medium by the West African lungfish, Protopterus annectens, exposed to ammonia-loading conditions

Abstract

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−1 NH4Cl 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−1 NH4Cl for six days. Correspondingly, the fish exposed to ammonia had a greater capacity to acidify the external medium, presumably to decrease the ratio of [NH3] to [NH4+] in order to reduce the influx of exogenous NH3.