Subcellular localization of Na+/K+-ATPase isoforms resolved by in situ hybridization chain reaction in the gill of chum salmon at freshwater and seawater

The Na+/K+-ATPase (NKA) & alpha;1-isoforms were examined by in situ hybridization chain reaction (ISHCR) using short hairpin DNAs, and we showed triple staining of NKA & alpha;1a, & alpha;1b, and & alpha;1c transcripts in the gill of chum salmon acclimated to freshwater (FW) and seawater (SW). The NKA & alpha;1-isoforms have closely resembled nucleotide sequences, which could not be differentiated by conventional in situ hybridization. The ISHCR uses a split probe strategy to allow specific hybridization using regular oligo DNA, resulting in high specificity at low cost. The results showed that NKA & alpha;1c was expressed ubiquitously in gill tissue and no salinity effects were observed. FW lamellar ionocytes (type-I ionocytes) expressed cytoplasmic NKA & alpha;1a and nuclear NKA & alpha;1b transcripts. However, both transcripts of NKA & alpha;1a and & alpha;1b were present in the cytoplasm of immature type-I ionocytes. The developing type-I ionocytes increased the cytoplasmic volume and migrated to the distal region of the lamellae. SW filament ionocytes (type-II ionocytes) expressed cytoplasmic NKA & alpha;1b transcripts as the major isoform. Results from morphometric analysis and nonmetric multidimensional scaling indicated that a large portion of FW ionocytes was NKA & alpha;1b-rich, suggesting that isoform identity alone cannot mark the ionocyte types. Both immature or residual type-II ionocytes and type-I ionocytes were found on the FW and SW gills, suggesting that the chum salmon retains the potential to switch the ionocyte population to fit the ion-transporting demands, which contributes to their salinity tolerance and osmoregulatory plasticity.