A mechanism for branchial acid excretion in marine fish: Identification of multiple Na⁺/H⁺ antiporter (NHE) isoforms in gills of two seawater teleosts

Both Na⁺/H⁺ exchange and the electrogenic extrusion of H⁺ via an H⁺-ATPase have been postulated to drive acid excretion across the branchial epithelium of fishes. While the H⁺-ATPase/Na⁺ channel system appears to be the predominant mechanism in some freshwater species, it may play a reduced role in seawater and brackish-water animals, where high external Na⁺ concentrations may thermodynamically favor Na⁺/H⁺ exchange driven by a Na⁺/H⁺ antiporter (NHE). In this study, we used molecular and immunological methods to assess the role of NHE isoforms in the branchial epithelium of the marine long-horned sculpin (Myoxocephalus octodecimspinosus) and the euryhaline killifish (Fundulus heteroclitus). Northern blot analysis of RNA probed with the human NHE-1 BamHI fragment suggested the presence of homologous gill NHE mRNA in sculpin. RT-PCR on gill RNA isolated from sculpin recovering from metabolic acidosis provided evidence for two distinct NHE isoforms; one with 76% amino acid homology to mammalian NHE-2, and another 92% homologous to trout erythrocytic β-NHE. Killifish also have transcripts with 91% homology to β-NHE. Immunological detection using monoclonal antibodies for mammalian NHE-1 revealed a protein antigenically similar to this isoform in the gills of both species. Metabolic acidosis caused an approximately 30-fold decrease in expression of the NHE-1-like protein in sculpin. We speculate that β-NHE in the gills plays the intracellular 'housekeeping' roles described for mammalian NHE-1. During systemic acidosis, apical gill NHE-2 (which is sensitive to external amiloride and low [Na⁺]) in parallel with a dramatic suppression of basolateral NHE-1 activity enhances net AH⁺ transfers to the water.