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WATER AND ELECTROLYTE HOMEOSTASIS
1Institute of Cellular and Organismic Biology, Academia Sinica, Nankang, Taipei, Taiwan; 2Institute of Fishery Science, National Taiwan University, Taipei, Taiwan, Republic of China; and 3Department of Anatomy, St. Marianna University School of Medicine, Miyamae-ku, Kawasaki, Japan
Submitted 21 February 2009 ; accepted in final form 9 March 2009
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.
ionocytes; embryo; ion regulation; skin; gills
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