Intestinal FXYD12 and sodium-potassium ATPase: A comparative study on two euryhaline medakas in response to salinity changes

Abstract

FXYD proteins are the regulators of sodium-potassium ATPase (Na+/K+-ATPase, NKA). In teleosts, NKA is a primary driving force for the operation of many ion transport systems in the osmoregulatory organs (e.g. intestines). Hence, the purpose of this study was to determine the expression of FXYD proteins and NKA α-subunit in the intestines of two closely related medakas (Oryzias dancena and O. latipes), which came from different salinity habitats and have diverse osmoregulatory capabilities, to illustrate the association between NKA and FXYD proteins of two medaka species in response to salinity changes. The results showed that the fxyd12 mRNA was the most predominant in the intestines of both medakas. The association of FXYD12 and NKA in the intestines of the two medaka species was demonstrated via double immunofluorescent staining and co-immunoprecipitation. Upon salinity challenge, the localization of FXYD12 and NKA was similar in the intestines of the two medaka species. However, the expression profiles of intestinal FXYD12 and NKA (mRNA and protein levels), as well as NKA activity differed between the medakas. These results showed that FXYD12 may play a role in modulating NKA activity in the intestines of the two medakas following salinity changes in the maintenance of internal homeostasis. These findings contributed to knowledge of the expression and potential role of vertebrate FXYD12, the regulators of NKA, upon salinity challenge.

Fig 1

Levels of intestinal fxyd mRNA in the Indian medaka (Od; A) and the Japanese medaka (Ol; B). The values are means ± SEM (total N = 12; N = 4 in the freshwater, brackish water, and seawater group, respectively). Different letters indicate significant differences among fxyd genes, excluding fxyd12 (P < 0.05). A.u., arbitrary units.

Fig 2

Immunohistochemical localization of NKA α-subunit (NKA) and FXYD12 in paraffin cross sections of intestines of the brackish water-acclimated Indian medaka (Od; A-C) and fresh water-acclimated Japanese medaka (Ol; D-E). Immunosignals of NKA (B, E) and FXYD12 (C, F) were both detected in the basolateral membrane of intestinal epithelium, compared with the negative control (A, D). V, villus; *, lumen. Scale bar: 20 μm.

Fig 3

Double immunofluorescence staining of NKA α-subunit (NKA; green; A-C) and FXYD12 (red; D-F) in intestinal cross-cryosections of the Indian medaka (Od). The merged images (yellow; G, H, I) revealed that FXYD12 colocalised in the basolateral membrane of NKA-immunoreactive cells in the fresh water- (FW; A, D, G), brackish water- (BW; B, E, H), and seawater- (SW; C, F, I) acclimated fish. V, villus; *, lumen. Scale bar: 20 μm.

Fig 4

Double immunofluorescence staining of NKA α-subunit (NKA; green; A-C) and FXYD12 (red; D-F) in intestinal cross-cryosections of the Japanese medaka (Ol). The merged images (yellow; G, H, I) revealed that FXYD12 colocalised to the basolateral membrane of NKA-immunoreactive cells in the fresh water- (FW; A, D, G), brackish water- (BW; B, E, H), and seawater- (SW; C, F, I) acclimated fish. V, villus; *, lumen. Scale bar: 20 μm.

Fig 5

Confocal 3D micrographs of double immunofluorescence staining of NKA α-subunit (NKA; green; A, D) and FXYD12 (red; B, E) in the cryosection of the brackish water-acclimated Indian medaka intestines. Immunosignals of NKA and FXYD12 were both detected in epithelial cell of intestinal villi. The results were similar between the cross section (A-C) and longitudinal section (D-F) of the epithelial cells. The merged image (yellow; C, F) revealed that FXYD12 colocalised to the basolateral membrane of NKA-immunoreactive cells. *, lumen. Scale bar: 10 μm.

Fig 6

Co-immunoprecipitation of FXYD12 and NKA α-subunit (NKA α) in the intestines (Int.) of the brackish water-acclimated Indian medaka (Od; A, C) and the fresh water-acclimated Japanese medaka (Ol; B, D). Immunoreactive bands of NKA α (A, B) or FXYD12 (C, D) were detected at 100 or 10 kDa, respectively. Lane 1, positive control (total intestinal lysates); lane 2, negative immunoblot control using pre-immune serum for immunoprecipitation; lanes 3 and 4, experimental group using antibodies (FXYD12 and NKA α, respectively) for immunoprecipitation. The 55 kDa bands in lanes 2–4 are the IgG heavy chains of the FXYD12 or NKA α antibody. M, marker (kDa).

Fig 7

Effects of salinity on mRNA levels (A-D) and protein abundance (E-H) of intestinal FXYD12 and NKA α-subunit (NKA α) in the Indian medaka (Od) and the Japanese medaka (Ol). The values are means ± SEM (N = 6 or 10 in A-D,H or E-G, respectively). Dissimilar letters indicate significant differences among various salinity groups (P < 0.05). Actin was used as an internal control for the immunoblots. FW, fresh water; BW, brackish water; SW, seawater.

Fig 8

Effects of salinity on intestinal NKA activity in the Indian medaka (Od; A) and the Japanese medaka (Ol; B). The values are means ± SEM (N = 6). Dissimilar letters indicate significant differences among various salinity groups (P < 0.05). FW, fresh water; BW, brackish water; SW, seawater.