Assessing the role of the acid-sensing ion channel ASIC4b in sodium uptake by larval zebrafish.

Na⁺ uptake in larval zebrafish (Danio rerio) is coordinated by three mechanisms: Na⁺/H⁺-exchanger 3b (NHE3b) expressed in H⁺-ATPase-rich (HR) cells, an unidentified Na⁺ channel coupled to electrogenic H⁺-ATPase expressed in HR cells, and Na⁺-Cl^--cotransporter (NCC) expressed in NCC cells. Recently, acid-sensing ion channels (ASICs) were proposed to be the putative Na⁺ channel involved in H⁺-ATPase-mediated Na⁺ uptake in adult zebrafish and rainbow trout. In the present study, we hypothesized that ASICs also play this role in Na⁺ uptake in larval zebrafish. In support of this hypothesis, immunohistochemical analyses revealed that ASIC4b was expressed in HR cells on the yolk sac skin at 4 days post-fertilization (dpf). However, neither treatment with the ASIC-specific blocker 4,6-diamidino-2-phenylindole (DAPI) nor morpholino knockdown of ASIC4b reduced Na⁺ uptake in circumneutral conditions at 4 dpf. However, because ASIC4b knockdown led to significant increases in the mRNA expression of nhe3b and ncc and a significant increase in HR cell density, it is possible that Na⁺ influx was sustained by increased participation of non-ASIC4b pathways. Moreover, when fish were reared in acidic water (pH = 4), ASIC4b knockdown led to a stimulation of Na⁺ uptake at 3 and 4 dpf, results which also were inconsistent with an essential role for ASIC-mediated Na⁺ uptake, even under conditions known to constrain Na⁺ uptake via NHE3b. Thus, while ASIC4b clearly is expressed in HR cells, the current functional experiments cannot confirm its involvement in Na⁺ uptake in larval zebrafish.

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