Immunolocalization of chloride transporters to gill epithelia of euryhaline teleosts with opposite salinity-induced Na+/K+-ATPase responses.

Opposite patterns of branchial Na(+)/K(+)-ATPase (NKA) responses were found in euryhaline milkfish (Chanos chanos) and pufferfish (Tetraodon nigroviridis) upon salinity challenge. Because the electrochemical gradient established by NKA is thought to be the driving force for transcellular Cl(-) transport in fish gills, the aim of this study was to explore whether the differential patterns of NKA responses found in milkfish and pufferfish would lead to distinct distribution of Cl(-) transporters in their gill epithelial cells indicating different Cl(-) transport mechanisms. In this study, immunolocalization of various Cl(-) transport proteins, including Na(+)/K(+)/2Cl(-) cotransporter (NKCC), cystic fibrosis transmembrane conductance regulator (CFTR), anion exchanger 1 (AE1), and chloride channel 3 (ClC-3), were double stained with NKA, the basolateral marker of branchial mitochondrion-rich cells (MRCs), to reveal the localization of these transporter proteins in gill MRC of FW- or SW-acclimated milkfish and pufferfish. Confocal microscopic observations showed that the localization of these transport proteins in the gill MRCs of the two studied species were similar. However, the number of gill NKA-immunoreactive (IR) cells in milkfish and pufferfish exhibited to vary with environmental salinities. An increase in the number of NKA-IR cells should lead to the elevation of NKA activity in FW milkfish and SW pufferfish. Taken together, the opposite branchial NKA responses observed in milkfish and pufferfish upon salinity challenge could be attributed to alterations in the number of NKA-IR cells. Furthermore, the localization of these Cl(-) transporters in gill MRCs of the two studied species was identical. It depicted the two studied euryhaline species possess the similar Cl(-) transport mechanisms in gills.

Chloride channel ClC-3 in gills of the euryhaline teleost, Tetraodon nigroviridis: expression, localization and the possible role of chloride absorption.

Previous studies have reported the mechanisms of ion absorption and secretion by diverse membrane transport proteins in gills of various teleostean species. To date, however, the chloride channel expressed in the basolateral membrane of mitochondrion-rich (MR) cells for Cl(-) uptake in freshwater (FW) fish is still unknown. In this study, the combination of bioinformatics tools [i.e. National Center for Biotechnology Information (NCBI) database, Tetraodon nigroviridis (spotted green pufferfish) genome database (Genoscope), BLAT and BLASTn] were used to identify the gene of ClC-3 (TnClC-3), a member of the CLC chloride channel family in the T. nigroviridis genome. RT-PCR analysis revealed that the gene encoding for the ClC-3 protein was widely expressed in diverse tissues (i.e. gill, kidney, intestine, liver and brain) of FW- and seawater (SW)-acclimated pufferfish. In whole-mount double immunofluorescent staining, branchial ClC-3-like immunoreactive protein was localized to the basolateral membrane of Na(+)/K(+)-ATPase (NKA) immunoreactive cells in both the FW- and SW-acclimated pufferfish. In response to salinity, the levels of transcript of branchial TnClC-3 were similar between FW and SW fish. Moreover, the membrane fraction of ClC-3-like protein in gills was 2.7-fold higher in FW compared with SW pufferfish. To identify whether the expression of branchial ClC-3-like protein specifically responded to lower environmental [Cl(-)], the pufferfish were acclimated to artificial waters either with a normal (control) or lower Cl(-) concentration (low-Cl). Immunoblotting of membrane fractions of gill ClC-3-like protein showed the expression was about 4.3-fold higher in pufferfish acclimated to the low-Cl environment than in the control group. Furthermore, branchial ClC-3-like protein was rapidly elevated in response to acute changes of environmental salinity or [Cl(-)]. Taken together, pufferfish ClC-3-like protein was expressed in the basolateral membrane of gill MR cells, and the protein amounts were stimulated by hyposmotic and low-Cl environments. The enhancement of ClC-3-like protein may trigger the step of basolateral Cl(-) absorption of the epithelium to carry out iono- and osmoregulatory functions of euryhaline pufferfish gills.

Differential responses in gills of euryhaline tilapia, Oreochromis mossambicus, to various hyperosmotic shocks.

Euryhaline tilapia (Oreochromis mossambicus) survived in brackish water (BW; 20 per thousand) but died in seawater (SW; 35 per thousand) within 6 h when transferred directly from fresh water (FW). The purpose of this study was to clarify responses in gills of FW tilapia to various hyperosmotic shocks induced by BW or SW. In FW-acclimated tilapia, scanning electron micrographs of gills revealed three subtypes of MR cell apical surfaces: wavy-convex (subtype I), shallow-basin (subtype II), and deep-hole (subtype III). Density of apical surfaces of mitochondrion-rich (MR) cell in gills of the BW-transfer tilapia decreased significantly within 3 h post-transfer due to disappearance of subtype I cells, but increased from 48 h post-transfer because of increasing density of subtype III cells. SW-transfer individuals, however, showed decreased density of MR cell openings after 1 h post-transfer because subtype I MR cell disappeared. On the other hand, relative branchial Na+/K+-ATPase (NKA) alpha1-subunit mRNA levels, protein abundance, and NKA activity of the BW-transfer group increased significantly at 6, 12, and 12 h post-transfer, respectively. In the SW-transfer group, relative mRNA and protein abundance of gill NKA alpha1-subunit did not change while NKA activity declined before dying in 5 h. Upon SW transfer, dramatic increases (nearly 2-fold) of plasma osmolality, [Na+], and [Cl(-)] were found prior to death. For the BW-transfer group, plasma osmolality was eventually controlled by 96 h post-transfer by enhancement of NKA expression and subtype III MR cell. The success or failure of NKA activation from gene to functional protein as well as the development of specific SW subtype in gills were crucial for the survival of euryhaline tilapia to various hyperosmotic shocks.

Cortisol regulation of Na+, K+-ATPase ß1 subunit transcription via the pre-receptor 11ß-hydroxysteroid dehydrogenase 1-like (11ß-Hsd1L) in gills of hypothermal freshwater milkfish, Chanos chanos.

Hypothermal stress changes the balance of osmoregulation by affecting Na+, K+-ATPase (Na-K-ATPase) activity or inducing modulation to epithelium permeability in fish. Meanwhile, cellular concentrations of cortisol can be modulated by the pre-receptor enzymes 11ß-hydroxysteroid dehydrogenase 1 and 2 (11ß-Hsd1 and 2). In fish, increasing levels of exogenous cortisol stimulate Na+ uptake via specific interaction with cortisol. This study investigated cortisol effects on expression of Na-K-ATPase subunit proteins and activity in gills of milkfish under hypothermal stress and revealed that the plasma cortisol contents as well as gill 11ß-hsd1l and na-k-atpase ß1 mRNA abundance were decreased in fresh water (FW) milkfish. Meanwhile, in the seawater (SW) milkfish, the plasma cortisol contents and gill 11ß-hsd1l and na-k-atpase ß1 mRNA abundance was increased under hypothermal stress. On the other hand, the abundance of 11ß-hsd2 mRNA increased in both FW and SW. In addition, 11ß-hsd1l expression increased in FW milkfish but decreased in SW milkfish after cortisol injection. Accordingly, the results that gill Na-K-ATPase activity of FW milkfish was affected by environmental temperatures as well as cortisol-dependent Na-K-ATPase ß1-subunit levels might be due to increased expression of 11ß-hsd1l that elevated intracellular cortisol contents. In hypothermal SW milkfish, decreasing abundance of Na-K-ATPase ß1 protein due to reduced expression of 11ß-hsd1l was found after cortisol injection. Thus, under hypothermal stress, 11ß-HSD1L in FW milkfish gills was used to modulate cortisol and the following effects on increasing the transcription of Na-K-ATPase ß1 protein.

Salinity Effects on Strategies of Glycogen Utilization in Livers of Euryhaline Milkfish (Chanos chanos) under Hypothermal Stress.

The fluctuation of temperature affects many physiological responses in ectothermic organisms, including feed intake, growth, reproduction, and behavior. Changes in environmental temperatures affect the acquisition of energy, whereas hepatic glycogen plays a central role in energy supply for the homeostasis of the entire body. Glycogen phosphorylase (GP), which catalyzes the rate-limiting step in glycogenolysis, is also an indicator of environmental stress. Here, we examined the effects of salinity on glycogen metabolism in milkfish livers under cold stress. A reduction of feed intake was observed in both freshwater (FW) and seawater (SW) milkfish under cold adaptation. At normal temperature (28°C), compared to the FW milkfish, the SW milkfish exhibited greater mRNA abundance of the liver isoform of GP (Ccpygl), higher GP activity, and less glycogen content in the livers. Upon hypothermal (18°C) stress, hepatic Ccpygl mRNA expression of FW milkfish surged at 3 h, declined at 6 and 12 h, increased again at 24 h, and increased significantly after 96 h. Increases in GP protein, GP activity, and the phosphorylation state and the breakdown of glycogen were also found in FW milkfish livers after 12 h of exposure at 18°C. Conversely, the Ccpygl transcript levels in SW milkfish were downregulated after 1 h of exposure at 18°C, whereas the protein abundance of GP, GP activity, and glycogen content were not significantly altered. Taken together, under 18°C cold stress, FW milkfish exhibited an acute response with the breakdown of hepatic glycogen for maintaining energy homeostasis of the entire body, whereas no change was observed in the hepatic glycogen content and GP activity of SW milkfish because of their greater tolerance to cold conditions.