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Am J Physiol Regul Integr Comp Physiol (June 24, 2004). doi:10.1152/ajpregu.00513.2003
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Submitted on September 4, 2003
Accepted on June 21, 2004

A putative H+/K+-ATPase in the Atlantic stingray, Dasyatis sabina: primary sequence and expression in gills

Keith P Choe1*, Jill W Verlander2, Charles S Wingo3, and David H Evans1

1 Department of Zoology, University of Florida, Gainesville, FL, USA
2 College of Medicine, University of Florida, Gainesville, FL, USA
3 College of Medicine, University of Florida, Gainesville, FL, USA; Research Service, Veterans Affairs Medical Center, Gainesville, FL, USA

* To whom correspondence should be addressed. E-mail: kchoe{at}zoo.ufl.edu.

In mammals, the gastric H+/K+-ATPase (HK{alpha}1) mediates acid secretion in the stomach and kidneys. Like mammals, elasmobranchs also secrete acid from their stomachs, but unlike mammals they primarily use their gills for systemic acid excretion instead of their kidneys. The purpose of this study was to determine if an HK{alpha}1 orthologue exists in an elasmobranch (Atlantic stingray, Dasyatis sabina), to determine if it is expressed in gills, and if so, to localize its expression and determine if its expression is regulated during hypercapnia or freshwater acclimation. A polyclonal antibody made against a HK{alpha}1 peptide detected HK{alpha}1 immunoreactivity in protein isolates and tissue sections of stingray stomachs and gills. Immunohistochemistry demonstrated that HK{alpha}1 immunoreactivity was present in a subpopulation of epithelial cells in both organs. Double-labeling experiments in the gills showed that HK{alpha}1 immunoreactivity occurred in Na+/K+-ATPase-rich cells and not in V-type H+-ATPase-rich cells. Reverse-transcription polymerase chain reactions (RT-PCRs) were 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 HK{alpha}1 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, and demonstrate that its expression increased during freshwater acclimation but not exposure to hypercapnia.




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