The thiazide-sensitive Na⁺-Cl⁻cotransporter (NCC), a member of the SLC12 family, is mainly expressed in the apical membrane of the mammalian distal convoluted tubule (DCT) cells, is responsible for cotransporting Na⁺and Cl⁻from the lumen into DCT cells and plays a major role in the mammalian renal NaCl reabsorption. The NCC has also been reported in fish, but the functional role in fish ion regulation is yet unclear. The present study used zebrafish as an in vivo model to test the hypothesis of whether the NCC plays a role in Na⁺and/or Cl⁻uptake mechanisms. Four NCCs were cloned, and only one of them, zebrafish (z) slc12a10.2 was found to predominately and specifically be expressed in gills. Double in situ hybridization/immunocytochemistry in zebrafish skin/gills demonstrated that the specific expression of zslc12a10.2 mRNA in a novel group of ionocytes differed from those of the previously-reported H⁺-ATPase-rich (HR) cells and Na⁺-K⁺-ATPase-rich (NaR) cells. Gill mRNA expression of zslc12a10.2 was induced by a low-Cl environment that stimulated fish Cl⁻influx, while a low-Na environment suppressed this expression. Incubation with metolazone, a specific inhibitor of the NCC, impaired both Na⁺and Cl⁻influx in 5-day postfertilization (dpf) zebrafish embryos. Translational knockdown of zslc12a10.2 with a specific morpholino caused significant decreases in both Cl⁻influx and Cl⁻content of 5-dpf zebrafish embryos, suggesting that the operation of zNCC-like 2 results in a net uptake of Cl⁻in zebrafish. On the contrary, zslc12a10.2 morphants showed increased Na⁺influx and content that resulted from upregulation of mRNA expressions of Na⁺-H⁺exchanger 3b and carbonic anhydrase 15a in HR cells. These results for the first time provide in vivo molecular physiological evidence for the possible role of the NCC in the Cl⁻uptake mechanism in zebrafish skin/gills.

Exposure to fluctuating temperatures accelerates the mitochondrial respiration and increases the formation of mitochondrial reactive oxygen species (ROS) in ectothermic vertebrates including fish. To date, little is known on potential oxidative damage and on protective antioxidative defense mechanisms in the brain of fish under cold shock. In this study, the concentration of cellular protein carbonyls in brain was significantly increased by 38% within 1 h after cold exposure (from 28°C to 18°C) of zebrafish (Danio rerio). In addition, the specific activity of superoxide dismutase (SOD) and the mRNA level of catalase (CAT) were increased after cold exposure by about 60% (6 h) and by 60%–90% (1 and 24 h), respectively, while the specific glutathione content as well as the ratio of glutathione disulfide to glutathione remained constant and at a very low level. In addition, cold exposure increased the protein level of hypoxia-inducible factor (HIF) by about 50% and the mRNA level of the glucose transporter zglut3 in brain by 50%–100%. To test for an involvement of uncoupling proteins (UCPs) in the cold adaptation of zebrafish, five UCP members were annotated and identified (zucp1-5). With the exception of zucp1, the mRNA levels of the other four zucps were significantly increased after cold exposure. In addition, the mRNA levels of four of the fish homologs (zppar) of the peroxisome proliferator-activated receptor (PPAR) were increased after cold exposure. These data suggest that PPARs and UCPs are involved in the alterations observed in zebrafish brain after exposure to 18°C. The observed stimulation of the PPAR-UCP axis may help to prevent oxidative damage and to maintain metabolic balance and cellular homeostasis in the brains of ectothermic zebrafish upon cold exposure.

Glucose, which plays a central role in providing energy for metabolism, is primarily stored as glycogen. The synthesis and degradation of glycogen are mainly initialized by glycogen synthase (GS) and glycogen phosphorylase (GP), respectively. The present study aimed to examine the glycogen metabolism in fish liver and gills during acute exposure to seawater. In tilapia (Oreochromis mossambicus) gill, GP, GS and glycogen were immunocytochemically colocalized in a specific group of glycogen-rich (GR) cells, which are adjacent to the gill's main ionocytes, mitochondrion-rich (MR) cells. Na⁺/K⁺-ATPase activity in the gills, protein expression and/or activity of GP and GS and the glycogen content of the gills and liver were examined in tilapia after their acute transfer from freshwater (FW) to 25‰ seawater (SW). Gill Na⁺/K⁺-ATPase activity rapidly increased immediately after SW transfer. Glycogen content in both the gills and liver were significantly depleted after SW transfer, but the depletion occurred earlier in gills than in the liver. Gill GP activity and protein expression were upregulated 1–3 h post-transfer and eventually recovered to the normal level as determined in the control group. At the same time, GS protein expression was downregulated. Similar changes in liver GP and GS protein expression were also observed but they occurred later at 6–12 h post-transfer. In conclusion, GR cells are initially stimulated to provide prompt energy for neighboring MR cells that trigger ion-secretion mechanisms. Several hours later, the liver begins to degrade its glycogen stores for the subsequent energy supply.