Elevated Na+/K+-ATPase responses and its potential role in triggering ion reabsorption in kidneys for homeostasis of marine euryhaline milkfish (Chanos chanos) when acclimated to hypotonic fresh water

The milkfish (Chanos chanos) is an economic species in Southeast Asia. In Taiwan, the milkfish are commercially cultured in environments of various salinities. Na⁺/K⁺-ATPase (NKA) is a key enzyme for fish iono- and osmoregulation. When compared with gills, NKA and its potential role were less examined by different approaches in the other osmoregulatory organs (e.g., kidney) of euryhaline teleosts. The objective of this study was to investigate the correlation between osmoregulatory plasticity and renal NKA in this euryhaline species. Muscle water contents (MWC), plasma, and urine osmolality, kidney histology, as well as distribution, expression (mRNA and protein), and specific activity of renal NKA were examined in juvenile milkfish acclimated to fresh water (FW), seawater (SW 35‰), and hypersaline water (HSW 60‰) for at least two weeks before experiments. MWC showed no significant difference among all groups. Plasma osmolality was maintained within the range of physiological homeostasis in milkfish acclimated to different salinities, while, urine osmolality of FW-acclimated fish was evidently lower than SW- and HSW-acclimated individuals. The renal tubules were identified by staining with periodic acid Schiff’s reagent and hematoxylin. Moreover, immunohistochemical staining showed that NKA was distributed in the epithelial cells of proximal tubules, distal tubules, and collecting tubules, but not in glomeruli, of milkfish exposed to different ambient salinities. The highest abundance of relative NKA α subunit mRNA was found in FW-acclimated milkfish rather than SW- and HSW-acclimated individuals. Furthermore, relative protein amounts of renal NKA α and β subunits as well as NKA-specific activity were also found to be higher in the FW group than SW and the HSW groups. This study integrated diverse levels (i.e., histological distribution, gene, protein, and specific activity) of renal NKA expression and illustrated the potential role of NKA in triggering ion reabsorption in kidneys of the marine euryhaline milkfish when acclimated to a hypotonic FW environment.     

A scanning electron microscope study of the pseudobranchs of two marine teleosts

The pseudobranchs of bass, Dicentrarchus labrax L. , and grey mullet, Liza ramada (Risso) ( Mugil capito ), were examined in the scanning electron microscope (S.E.M.). Both pseudobranchs appeared gill-like in situ but the S.E.M. revealed their gross morphology to be different. The bass pseudobranch was a'free' pseudobranch, having separate lamellae along the filaments, with areas of fusion on the leading edges of some lamellae, whilst the mullet pseudobranch was 'semi-free' since the secondary lamellae were fused over a large area and only free along their trailing (opercular) edge where the chloride cells were situated. The surface of the epithelial cells presented different patterns of microridges and microvilli depending on their position in the pseudobranch. Three types of cell opening were found at the cell surface, belonging to chloride cells, mucous cells, and possibly rodlet cells. The S.E.M. observations were correlated with those of transmission electron microscope (T.E.M.) studies.     

The expression of gill Na,K-ATPase in milkfish,Chanos chanos,acclimated to seawater,brackish water and fresh water

Juvenile milkfish Chanos chanos (Forssk?l, 1775) were transferred from a local fish farm to fresh water (FW; 0 per thousand ), brackish water (BW; 10 per thousand, 20 per thousand ) and seawater (SW; 35 per thousand ) conditions in the laboratory and reared for at least two weeks. The blood and gill of the fish adapted to various salinities were analyzed to determine the osmoregulatory ability of this euryhaline species. No significant difference was found in plasma osmolality, sodium or chloride concentrations of milkfish adapted to various salinities. In FW, the fish exhibited the highest specific activity of Na, K-ATPase (NKA) in gills, while the SW group was found to have the lowest. Relative abundance of branchial NKA alpha-subunit revealed similar profiles. However, in contrary to other euryhaline teleosts, i.e. tilapia, salmon and eel, the naturally SW-dwelling milkfish expresses higher activity of NKA in BW and FW. Immunocytochemical staining has shown that most Na, K-ATPase immunoreactive (NKIR) cells in fish adapted to BW and SW were localized to the filaments with very few on the lamellae. Moreover, in FW-adapted milkfish, the number of NKIR cells found on the lamellae increased significantly. Such responses as elevated NKIR cell number and NKA activity are thought to improve the osmoregulatory capacity of the milkfish in hyposaline environments.     

Cellular and epithelial adjustments to altered salinity in the gill and opercular epithelium of a cichlid fish (Oreochromis mossambicus)

Morphological features of the gill and opercular epithelia of tilapia (Oreochromis mossambicus) have been compared in fish acclimated to either fresh water (FW) or hypersaline water (60 S) by scanning electron and fluorescence microscopy. In hyperosmoregulating, i.e., FW-acclimated, tilapia only those mitochondria-rich (MR) cells present on the filament epithelium of the gill were exposed to the external medium. After acclimation of fish to hypersaline water these cells become more numerous, hypertrophy extensively, and form apical crypts not only in the gill filament but also in the opercular epithelium. Regardless of salinity, MR cells were never found to be exposed to the external medium on the secondary lamellae. In addition, two types of pavement cells were identified having distinct morphologies, which were unaffected by salinity. The gill filaments and the inner operculum were generally found to be covered by pavement cells with microridges, whereas the secondary lamellae were covered exclusively by smooth pavement cells.     

Microtubule‐dependent changes in morphology and localization of chloride transport proteins in gill mitochondria‐rich cells of the tilapia,Oreochromis mossambicus

The tilapia (Oreochromis mossambicus) is a euryhaline fish exhibiting adaptive changes in cell size, phenotype, and ionoregulatory functions upon salinity challenge. Na⁺/Cl^? cotransporter (NCC) and Na⁺/K⁺/2Cl^? cotransporter (NKCC) are localized in the apical and basolateral membranes of mitochondria‐rich (MR) cells of the gills. These cells are responsible for chloride absorption (NCC) and secretion (NKCC), respectively, thus, the switch of gill NCC and NKCC expression is a crucial regulatory mechanism for salinity adaptation in tilapia. However, little is known about the interaction of cytoskeleton and these adaptive changes. In this study, we examined the time‐course of changes in the localization of NKCC/NCC in the gills of tilapia transferred from fresh water (FW) to brackish water (20‰) and from seawater (SW; 35‰) to FW. The results showed that basolateral NKCC disappeared and NCC was expressed in the apical membrane of MR cells. To further clarify the process of these adaptive changes, colchicine, a specific inhibitor of microtubule‐dependent cellular regulating processes was used. SW‐acclimated tilapia were transferred to SW, FW, and FW with colchicine (colchicine‐FW) for 96 h. Compared with the FW‐treatment group, in the MR cells of colchicine‐FW‐treatment group, (1) the average size was significantly larger, (2) only wavy‐convex‐subtype apical surfaces were found, and (3) the basolateral (cytoplasmic) NKCC signals were still exhibited. Taken together, our results suggest that changes in size, phenotype, as well as the expression of NCC and NKCC cotransporters of MR cells in the tilapia are microtubule‐dependent. J. Morphol. 277:1113–1122, 2016. © 2016 Wiley Periodicals, Inc.     

Changes to chloride and rodlet cells in gills, kidney and intestine of Dicentrarchus labrax (L.) exposed to reduced salinities

The effect of osmotic shock was investigated mainly in the chloride cells (CCs) and rodlet cells (RCs) of gills, and RCs of intestine and kidney of the European sea bass Dicentrarchus labrax obtained from a farm in the northern Adriatic Sea. During the experiment, fish were abruptly transferred from sea water to a salinity of 15 (15 SW) or to fresh water (FW). Numeric variation and ultrastructural changes of both cell types were evaluated at 24, 48 and 96 h after the transfer to lower salinity levels, using light and transmission electron microscopy (TEM). Exposure to FW produced a significant increase ( P < 0·05) in the number of branchial CCs and RCs within 96 and 24 h, respectively. Following osmotic challenge (either transfer to 15 SW or FW), kidney and intestine showed an evident increase in RC numbers. The cellular damage detected by TEM was the same for each sampling time (24, 48 and 96 h), but appeared more severe in fish exposed to FW (higher osmotic shock) than in those exposed to 15 SW. In RCs cytoplasmic vacuolizations, autophagosomes and autophagolysosomes with myelinoid bodies, dissolution and shrinkage of the typical inclusions were documented. Nevertheless, CCs showed vacuolization of endoplasmic reticulum and cytoplasmic dissolution and maintained the apical crypt typical of seawater acclimated fish. Renal tubular cells and intestinal epithelial cells showed similar changes to those reported for CCs and RCs.     

Observations on the relationship between “chloride-type” and “pseudobranch-type” cells in the gills of a fish,Oligocottus maculosus

Summary Chloride cells in gill epithelium of Oligocottus maintained in sea water have a much branched system of agranular cytoplasmic tubules, numerous mitochondria, and a prominent apical crypt. The mitochondria are randomly dispersed and do not show preferential orientation with respect to the tubules.After brief exposure of fish to sea water diluted 1/100 with glass distilled water, the mitochondria and tubules become rearranged into parallel arrays and the apical crypts disappear. The appearance of these cells is similar to that of pseudobranch cells in Fundulus heteroclitus (Copeland and Dalton, 1959).These data suggest that chloride cells and pseudobranch cells represent different adaptive forms of a single cell type and that transformation from the chloride cell configuration to that of pseudobranch cells can be induced by osmotic stress.This work was performed in 1963 when the author was enrolled in the Fine Structure Training program of the Department of Biological Structure, University of Washington. Financial aid was provided by the United States Public Health Service (Grant No. 5T1 GM-136).     

Ion-deficient environment induces the expression of basolateral chloride channel, ClC-3-like protein, in gill mitochondrion-rich cells for chloride uptake of the tilapia Oreochromis mossambicus

Gill mitochondrion-rich (MR) cells contain different molecules to carry out functionally distinct mechanisms. To date, the putative mechanism of Cl(-) uptake through the basolateral chloride channel, however, is less understood. To clarify the Cl(-)-absorbing mechanism, this study explored the molecular and morphological alterations in branchial MR cells of tilapia acclimated to seawater (SW), freshwater (FW), and deionized water (DW). Scanning electron microscopic observations revealed that three subtypes of MR cells were exhibited in gill filament epithelia of tilapia. Furthermore, in DW-acclimated tilapia, the subtype I (ion-absorbing subtype) of MR cells predominantly occurred in gill filament as well as lamellar epithelia. Whole-mount double immunofluorescent staining revealed that branchial ClC-3-like protein and Na(+)/K(+)-ATPase (NKA), the basolateral marker of MR cells, were colocalized in tilapia. In SW-acclimated tilapia, all MR cells of gill filament epithelia exhibited faint fluorescence of ClC-3-like protein. In contrast, only some MR cells in gill filament epithelia of FW and DW tilapia expressed basolateral ClC-3-like protein; however, the fluorescence was more intense in FW and DW tilapia than in SW fish. In hyposmotic groups, the number of MR cells immunopositive for ClC-3-like protein was significantly higher in DW-exposed tilapia. Meanwhile, in gill lamellar epithelia of DW tilapia, all MR cells (subtype I) were ClC-3-like protein immunopositive. Double immunostaining of ClC-3-like protein and Na(+)/Cl(-) cotransporter (NCC) revealed that basolateral ClC-3-like protein and apical NCC were colocalized in some MR cells in FW and DW tilapia. Moreover, both mRNA and protein amounts of branchial ClC-3-like protein were significantly higher in DW-acclimated tilapia. To identify whether the expression of branchial ClC-3-like protein responded to changes in environmental [Cl(-)], tilapia were acclimated to artificial waters with normal [Na(+)]/[Cl(-)] (control), lower [Na(+)] (low Na), or lower [Cl(-)] (low Cl). Immunoblotting of crude membrane fractions for gill ClC-3-like protein showed that the protein abundance was evidently enhanced in tilapia acclimated to the low-Cl environment compared with the other groups. Our findings integrated morphological and functional classifications of ion-absorbing MR cells and indicated that ion-deficient water elevated the numbers of subtype I MR cells in both filament and lamellar epithelia of gills with positive ClC-3-like protein immunostaining and increased the expression levels of ClC-3-like protein. This study is the first to illustrate the exhibition of a basolateral chloride channel potentially responsible for Cl(-) absorption in the ion-absorbing subtype of gill MR cells of tilapia.     

Differential design for hopping in two species of wallabies

We explored molecular and morphological alteration in gill mitochondria-rich (MR) cells of Mozambique tilapia, Oreochromis mossambicus, acclimated to deionized freshwater (DFW), freshwater (FW), 1/3-diluted seawater (1/3 SW) and seawater (SW). Scanning electron microscopic observations revealed that the apical membrane of MR cells appeared as a flat or slightly projecting disk in DFW and FW, being larger in DFW than in FW. In contrast, the apical membrane typically formed a pit structure in 1/3 SW and SW. The mRNA expression levels of Na(+)/H(+) exchanger-3 (NHE3) and Na(+)/Cl(-) cotransporter (NCC) in the gills were increased with decreasing environmental salinity, whereas Na(+)/K(+)/2Cl(-) cotransporter-1a (NKCC1a) expression was upregulated by increasing salinity. Immunofluorescence staining showed that the MR cell population of DFW- and FW-acclimated tilapia consisted mostly of MR cells with apical NHE3 and those with apical-NCC; MR cells with basolateral NKCC1a dominated in SW-acclimated tilapia. These results indicated that apical-NHE3 and apical-NCC MR cells were ion-absorbing cells, and that basolateral-NKCC1a MR cells were ion-secreting cells. In fish acclimated to 1/3 SW, both ion-absorbing and secreting cells existed in the gills, suggesting that fish in near-isotonic water were equipped with mechanisms of both hyper- and hypoosmoregulation to prepare for environmental salinity changes.     

Biochemical characterization of isolated branchial mitochondria-rich cells of Oreochromis mossambicus acclimated to fresh water or hyperhaline sea water

Mitochondria-rich cells have been separated from other epithelial cells of tilapia (Oreochromis mossambicus) gills by density gradient centrifugation on Percoll. During centrifugation two main bands of cells formed. The viability of the cells in both bands was high (>90%). In one band, 45–47% of the total cell number was mitochondria-rich cells. The other band contained at least 80% pavement cells, representing the majority of other gill epithelial cell types. A comparison of the activities of four enzymes involved in major metabolic and ion regulatory functions was made between these two different fractions of cells. Furthermore, the separation of gill epithelial cells and determination of enzymatic activity was carried out in tilapia after the fish were acclimated to fresh water or hyperhaline sea water (60 mg·ml^-1 S) to gain an indication of the relative contribution of mitochondria-rich cells and pavement cells to both NaCl excretion and absorption. Regardless of acclimation salinity, the activities of Na⁺/K⁺-ATPase, glutamate dehydrogenase and glucose-6-phosphate dehydrogenase were significantly higher in mitochondria-rich cells than in pavement cells. However, tilapia acclimated to hyperhaline sea water possessed significantly lower carbonic anhydrase activity in mitochondria-rich cells than in pavement cells. In contrast, no significant difference of carbonic anhydrase activity was observed between the two cell fractions in tilapia acclimated to fresh water.Abbreviations ATPase adenosine triphosphatase - CA carbonic anhydrase - DASPMI dimethylaminostyrylmethylpyridinium iodine - FW fresh-water - GIDH glutamate dehydrogenase - G6PDH glucose-6-phosphate dehydrogenase - HSW hyperhaline sea water (60 mg·ml^-1) - MR cells, mitochondria-rich cells - S salinity     

Distribution and development of rodlet cells in the gills and pseudobranch of the bass, Dicentrachus labrax (L)

Rodlet cells in various stages of development were found in large numbers in the bass gill and pseudobranch. In the gill, rodlet cells were found in the epithelium at the base of the secondary lamellae and on the filament between adjacent lamellae, whilst in the pseudobranch they were found over the whole area of the secondary lamellae as well as in the filament epithelium.
During early development, rodlet cells are characterised by their amorphous cell inclusions, prominent supranuclear Golgi complex and network of granular endoplasmic reticulum. Later, with formation of a fibrous border the arrangement of the cell organelles undergoes reorganisation; the endoplasmic reticulum becomes distended, numerous vesicles appear and the mitochondria aggregate in the apical region of the cell. One of the most striking features is the development of club-shaped sacs containing electron dense cores, which are orientated towards the open apex of the cell.
Various staining properties of rodlet cells for light and electron microscopy were compared with those of mucous cells found in the same tissues. Possible functions of the cell are discussed.     

Effects of acclimation salinity on the expression of selenoproteins in the tilapia,Oreochromis mossambicus

Selenoproteins are ubiquitously expressed, act on a variety of physiological redox-related processes, and are mostly regulated by selenium levels in animals. To date, the expression of most selenoproteins has not been verified in euryhaline fish models. The Mozambique tilapia, Oreochromis mossambicus, a euryhaline cichlid fish, has a high tolerance for changes in salinity and survives in fresh water (FW) and seawater (SW) environments which differ greatly in selenium availability. In the present study, we searched EST databases for cichlid selenoprotein mRNAs and screened for their differential expression in FW and SW-acclimated tilapia. The expression of mRNAs encoding iodothyronine deiodinases 1, 2 and 3 (Dio1, Dio2, Dio3), Fep15, glutathione peroxidase 2, selenoproteins J, K, L, M, P, S, and W, was measured in the brain, eye, gill, kidney, liver, pituitary, muscle, and intraperitoneal white adipose tissue. Gene expression of selenophosphate synthetase 1, Secp43, and selenocysteine lyase, factors involved in selenoprotein synthesis or in selenium metabolism, were also measured. The highest variation in selenoprotein and synthesis factor mRNA expression between FW- and SW-acclimated fish was found in gill and kidney. While the branchial expression of Dio3 was increased upon transferring tilapia from SW to FW, the inverse effect was observed when fish were transferred from FW to SW. Protein content of Dio3 was higher in fish acclimated to FW than in those acclimated to SW. Together, these results outline tissue distribution of selenoproteins in FW and SW-acclimated tilapia, and indicate that at least Dio3 expression is regulated by environmental salinity.     

Salinity stress results in rapid cell cycle changes of tilapia (Oreochromis mossambicus) gill epithelial cells

We have developed a technique for immunocytochemistry of fish gill cells that we used to quantify tilapia (Oreochromis mossambicus) mitochondria-rich cells (MRC) and other gill cells (non-MRC) within different cell cycle phases by laser scanning cytometry. Gill cells fixed on coverslips were triple stained with propidium iodide to distinguish G1 vs. G2 phases, Ser10-phosphorylated histone H3 antibody to label mitotic cells, and Na(+)/K(+) ATPase antibody to label MRC. These parameters were measured at 0 (control), 4, 8, 16, 24, 48, 72, and 168 hr (1 week) following exposure of freshwater (FW) acclimated fish to 2/3 seawater (SW). MRC increased mitotic activity very rapidly peaking at 8 hr following SW exposure. This change in mitotic MRC is indicative of epithelial reorganization during SW acclimation. In contrast to MRC, the proportion of non-MRC (likely pavement cells (PVC)) in mitosis did not change significantly in response to SW exposure. Moreover, twice as many MRC were in mitosis compared with non-MRC, suggesting that MRC turn over faster than other cell types during SW acclimation. Following the mitosis peak, MRC accumulated in G2 phase over a period of 16-72 hr post-SW exposure. We also observed G2 arrest with similar kinetics following SW exposure in tilapia non-MRC (likely PVC). We interpret the G2 arrest that occurs after an initial wave of transient increase in MRC mitosis as a means for conserving energy for dealing with the osmotic stress imposed during the exposure of FW fish to SW.     

Morphology of reproductive accessory glands in female Sepia pharaonis (Cephalopoda: Sepiidae) sheds light on egg encapsulation

The pharaoh cuttlefish, Sepia pharaonis, is an important cephalopod fishery species in southeastern Asia, with understudied reproductive physiology. The present study aimed to investigate the cellular characteristics of epithelial cells found in the nidamental glands (NGs) and accessory NGs (ANGs), as well as the structural connections between these two glands in mature female S. pharaonis. A histological analysis revealed two types of epithelial cells in NGs: Alcian blue‐positive, PAS‐negative mucosubstance‐secreting cells and eosinophilic, PAS‐positive granule‐secreting cells. Using transmission electron microscopy, three types of epithelial cells were identified: cells with electron‐dense granules, cells with electron‐lucent granules, and cells with both cilia and microvilli in the apex. Mature ANGs contain an abundance of tubular units composed of epithelial cells resting on a thin layer of basal lamina. Innervated muscle cells are tightly adhered to the basal lamina. In addition, we observed epithelial canalization of ANG tubules penetrating through the connective tissue linking NGs and the walls of the tubules in ANGs, which allows the contents of the ANG tubules to be transported to the NGs. Our results suggest that ANGs participate in the encapsulation of the ova via the same pathway as NGs, which provides an important basis for future studies on the mechanism of protection provided by NGs and ANGs during embryonic development in S. pharaonis.     

Immunolocalization of Vacuolar-Type H(+)-ATPase in the Yolk-Sac Membrane of Tilapia (Oreochromis mossambicus) Larvae

To investigate the involvement of the yolk-sac membrane in ion absorption, developmental changes in whole-body cation contents, cellular localization of vacuolar-type H(+)-ATPase (V-ATPase), and size and density of pavement and chloride cells in the yolk-sac membrane were examined in tilapia (Oreochromis mossambicus) larvae in fresh water (FW) and those transferred to seawater (SW) at 2 days before hatching (day-2). The whole-body content of Na(+) in embryos and larvae adapted to both FW and SW increased constantly from day-2 to day 10, although they were not fed through the experiment. The yolk-sac membrane of FW larvae at days 0 and 2 showed V-ATPase immunoreactivity in pavement cells, but not in chloride cells. No positive immunoreactivity was detected in SW larvae. Whole-mount immunocytochemistry showed that some pavement cells were intensively immunoreactive, whereas others were less or not immunoreactive. Electron-microscopic immunocytochemistry revealed that V-ATPase immunoreactivity was present in the apical regions of pavement cells in FW larvae, especially in their ridges. The pavement cells in FW larvae were significantly smaller in size but higher in density than those in SW. These results suggest that pavement cells are the site of active Na(+) uptake in exchange for H(+) secretion through V-ATPase in FW-adapted tilapia during early life stages.     

Fine structure of the pseudobranch of the flounder Paralichthys lethostigma

Summary The free or non-glandular pseudobranch of the flounder Paralichthys lethostigma was studied with the electron microscope. Cells typical of glandular type pseudobranchs are found. This indicates that, at least in the flounder, the free pseudobranch should be called glandular. In addition, the chloride-type cells, commonly found in the gill, buccal epithelium, and surface epithelium of other fish, have been found in the pseudobranch, where they have not been described previously. The fine structure of both the chloride-type and the pseudobranch-type cell has been characterized and contrasted. We have not been able to confirm previous reports that the specific cells in both pseudobranch and gill are identical in the flounder.Supported by grant-in-aid from the USPHS (General Medical Sciences Institute, GM-06836) and the National Science Foundation (GB-676).     

Sodium or potassium ions activate different kinetics of gill Na, K-ATPase in three seawater- and freshwater-acclimated euryhaline teleosts

The effects of [Na(+)] or [K(+)] on Na, K-ATPase activity of FW-acclimated and SW-acclimated tilapia, puffer and milkfish were examined in gill homogenates. [Na(+)] or [K(+)] stimulated Na, K-ATPase hydrolyzing ATP in all experimental groups. ATP hydrolysis stimulated by [Na(+)] or [K(+)] followed Michaelian-Menten kinetics. Km values for [K(+)] (i.e., Km(K)), were lower in SW- than FW-acclimated tilapia and puffer fishes (tilapia: 8.69+/-0.22 vs. 11.93+/-1.17 mM; puffer: 13.51+/-1.39 vs. 30.52+/-2.66 mM). Km values for [Na(+)] (i.e., Km(Na)), were lower in FW- than SW-acclimated milkfish (3.76+/-0.54 vs. 7.55+/-1.08 mM). These data suggest that [K(+)] stimulates ATP hydrolysis to rates higher in SW- than FW-acclimated tilapia and puffer fishes, while [Na(+)] stimulated ATP hydrolysis at rates higher in FW- than SW-acclimated milkfish. This is the first demonstration that Na, K-ATPase activity of euryhaline tilapia, puffer, and milkfish modulated by [Na(+)] or [K(+)] have different effects between FW- and SW-acclimated groups. Such responses as changes in properties of branchial Na, K-ATPase may contribute to improve the osmoregulatory capacity of tilapia, puffer and milkfish to acclimate in seawater and fresh water.     

Ambient salinity modulates the expression of sodium pumps in branchial mitochondria-rich cells of Mozambique tilapia,Oreochromis mossambicus

Na,K-ATPase (sodium pumps) provide the primitive driving force for ion transport in branchial epithelial cells. Immunoblots of epithelial homogenates of both seawater (SW)- and freshwater (FW)-adapted tilapia gills as well as rat brain homogenate, a positive control, revealed one major band with a molecular weight of about 100 kDa. SW-adapted tilapia gills possessed larger (about 2-fold) amounts of sodium pumps compared with FW-adapted tilapia gills. (3)H-ouabain binding representing functional binding sites of Na,K-ATPase was also higher (about 3.5-fold) in gills of SW-adapted tilapia compared to that of FW-adapted fish. Moreover, specific activities of SW fish were higher (about 2-fold) than those of FW fish. Double labeling of Na,K-ATPase and Con-A, a fluorescent marker of MR cells, in tilapia gills followed by analysis with confocal microscopy showed that sodium pumps were localized mainly in MR cells, including the SW type and different FW types. Although more-active expression of Na,K-ATPase was demonstrated in gills of SW-adapted tilapia, no significant differences in densities of apical openings of MR cells were found between SW- and FW-adapted fish. These results indicate that, during salinity challenge, tilapia develop more "functional" Na,K-ATPase in SW-type MR cells to meet physiological demands.