CLAIBORNE, J.B.*; GUNNING, D.L.; WALL, B.P.; MORRISON-SHETLAR, A.I.: Gill Na⁺/H⁺ antiporters (NHE) in marine fish

The branchial epithelium in marine fish is the main site of acid-base transfers between the extracellular space and the environment. Na⁺/H⁺ exchange proteins (NHE) are thought to contribute to net acid excretion in these animals. We have used molecular and biochemical methods to detect these antiporters in the branchial epithelium of two species of fish. 3’/5′-RACE reactions allowed us to clone and sequence for the first time a full length cDNA transcript for an NHE-2-like isoform from the gills of the marine long-horned sculpin (Myoxocephalus octodecimspinosus). The open reading frame is approximately 2890 bp in length and has a ~57% amino acid homology to the rat NHE-2 isoform with much lower homology at the 3′ end. We have previously shown NHE-1 like immunoreactivity in the gills of the sculpin and the seawater adapted mummichog (Fundulus heteroclitus). Using Western analysis on gill membrane preparations from fresh and seawater adapted mummichogs, we have now observed proteins approximately 80 kDa and 77 kDa in size that are immunoreactive to antibodies made against mammalian NHE-1 (aa 514-818) and NHE-3 (aa 528-648), respectively. Following exposure of this species to 1% hypercapnia for one hour, an increase in expression was detected for both NHE-like protein isoforms in seawater adapted animals, while no difference in expression of NHE-1-like proteins could be detected in freshwater adapted fish. We hypothesize that during acidosis, apical gill NHE activity enhances net H⁺ transfers to the water in these marine species. Supported by NSF-IBN-9808141 to JBC and AIMS.