Molecular detection and immunological localization of gill Na+/H+ exchanger in the dogfish (Squalus acanthias).

The dogfish (Squalus acanthias) can make rapid adjustments to gill acid-base transfers to compensate for internal acidosis/alkalosis. Branchial Na⁺/H⁺ exchange (NHE) has been postulated as one mechanism driving the excretion of H⁺ following acidosis. We have cloned gill cDNA that includes an open reading frame coding for a 770-residue protein most homologous (~71%) to mammalian NHE2. RT-PCR revealed NHE2 transcripts predominantly in gill, stomach, rectal gland, intestine, and kidney. In situ hybridization with an antisense probe against NHE2 in gill sections revealed a strong mRNA signal from a subset of interlamellar and lamellae cells. We developed dogfish-specific polyclonal antibodies against NHE2 that detected a ⁺70-kDa protein in Western blots and immunologically recognized branchial cells having two patterns of protein expression. Cytoplasmic and apical NHE2 immunoreactivity were observed in cells coexpressing basolateral Na⁺-K⁺-ATPase. Other large ovoid cells more generally staining for NHE2 also were strongly positive for basolateral H⁺-ATPase. Gill mRNA levels for NHE2 and H⁺-ATPase did not change following systemic acidosis (as measured by quantitative PCR 2 h after a1- or 2-meq/kg acid infusion). These data indicate that posttranslational adjustments of NHE2 and other transport systems (e.g., NHE3) following acidosis may be of importance in the short-term pH adjustment and net branchial H⁺ effiux observed in vivo. NHE2 may play multiple roles in the gills, involved with H⁺ efflux from acid-secreting cells, basolateral H⁺ reabsorption for pH⁺ regulation, and in parallel with H⁺-ATPase for the generation of HCO₃^- in base-secreting cells. [ABSTRACT FROM AUTHOR]