CHOE, K.P.; VERLANDER, J.W.; WINGO, C.S.; EVANS, D.H.; Univ. of Florida; Univ. of Florida; Veterans Affairs Medical Center; Univ. of Florida: H⁺/K⁺-ATPase in an elasmobranch (Atlantic stingray, Dasyatis sabina): primary sequence and evidence for expression in gills

The SCH28080-sensitive H⁺/K⁺-ATPase (HKa1) secretes acid in the stomach and kidneys of mammals. Like mammals, elasmobranchs also secrete gastric acid, but unlike mammals they primarily use their gills for systemic acid excretion instead of their kidneys. This study was conducted to determine if an HKa1 orthologue exists in an elasmobranch (Atlantic stingray, Dasyatis sabina), to determine if it is expressed in gills, and if so, to localize its expression in gills. A polyclonal antibody made against a HKa1 peptide detected HKa1 immunoreactivity in western blots and tissue sections of stingray stomachs and gills. Immunohistochemistry demonstrated that HKa1 immunoreactivity was present in epithelial cells of both organs. Double-labeling experiments in the gills showed that HKa1 immunoreactivity occurred in Na⁺/K⁺-ATPase-rich cells and not in vacuolar H⁺-ATPase-rich cells. Reverse-transcription polymerase chain reactions (RT-PCRs) were then used to deduce the primary sequence of a putative H⁺/K⁺-ATPase from the stomach of Atlantic stingrays. The 3421 base pair cDNA includes a coding region for a 1,025 amino acid protein that is over 80% identical to HKa1 of mammals. RT-PCRs were then used to demonstrate that this transcript is also expressed in the gills. To our knowledge, this is the first H⁺/K⁺-ATPase sequence reported for any elasmobranch, and the first full-length sequence for any fish. We also provide the first evidence for its expression in gills of any fish, setting the groundwork for future studies on the possible role of this putative HKa1 in branchial acid-base transport.