The mammalian kidney excretes its metabolic acid load through the proton-transporting cells, intercalated cells, in the distal nephron and collecting duct. Fish excrete acid through external organs, gill, or skin; however, the cellular function is still controversial. In this study, molecular and electrophysiological approaches were used to identify a novel cell type secreting acid in skin of zebrafish (Danio rerio) larvae. Among keratinocytes covering the larval surface, novel proton-secreting ionocytes, proton pump (H(+)-ATPase)-rich cells, were identified to generate strong outward H(+) flux. The present work demonstrates for the first time, with a noninvasive technique, H(+)-secreting cells in an intact animal model, the zebrafish, showing it to be a suitable model in which to study the functions of vertebrate transporting epithelia in vivo.

LY. Ammonium-dependent sodium uptake in mitochondrion-rich cells of medaka (Oryzias latipes) larvae. In this study, a scanning ion-selective electrode technique (SIET) was applied to measure H ϩ , Na ϩ , and NH 4 ϩ gradients and apparent fluxes at specific cells on the skin of medaka larvae. Na ϩ uptake and NH3/NH 4 ϩ excretion were detected at most mitochondrion-rich cells (MRCs). H ϩ probing at MRCs revealed two group of MRCs, i.e., acid-secreting and base-secreting MRCs. Treatment with EIPA (100 M) blocked 35% of the NH3/ NH 4 ϩ secretion and 54% of the Na ϩ uptake, suggesting that the Na ϩ /H ϩ exchanger (NHE) is involved in Na ϩ and NH3/NH 4 ϩ transport. Low-Na ϩ water (Ͻ0.001 mM) or high-NH 4 ϩ (5 mM) acclimation simultaneously increased Na ϩ uptake and NH3/NH 4 ϩ excretion but decreased or even reversed the H ϩ gradient at the skin and MRCs. The correlation between NH 4 ϩ production and H ϩ consumption at the skin surface suggests that MRCs excrete nonionic NH3 (base) by an acid-trapping mechanism. Raising the external NH 4 ϩ significantly blocked NH 3/NH4 ϩ excretion and Na ϩ uptake. In contrast, raising the acidity of the water (pH 7 to pH 6) enhanced NH3/NH 4 ϩ excretion and Na ϩ uptake by MRCs. In situ hybridization and real-time PCR showed that the mRNAs of the Na ϩ /H ϩ exchanger (slc9a3) and Rhesus glycoproteins (Rhcg1 and Rhbg) were colocalized in MRCs of medaka, and their expressions were induced by low-Na ϩ acclimation. This study suggests a novel Na ϩ /NH 4 ϩ exchange pathway in apical membranes of MRCs, in which a coupled NHE and Rh glycoprotein is involved and the Rh glycoprotein may drive the NHE by generating H ϩ gradients across apical membranes of MRCs.

The mechanism of ammonia excretion in freshwater teleosts is not well understood. In this study, scanning ion-selective electrode technique was applied to measure H(+) and NH(4)(+) fluxes in specific cells on the skin of zebrafish larvae. NH(4)(+) extrusion was relatively high in H(+) pump-rich cells, which were identified as the H(+)-secreting ionocyte in zebrafish. Minor NH(4)(+) extrusion was also detected in keratinocytes and other types of ionocytes in larval skin. NH(4)(+) extrusion from the skin was tightly linked to acid secretion. Increases in the external pH and buffer concentration (5 mM MOPS) diminished H(+) and NH(4)(+) gradients at the larval surface. Moreover, coupled decreases in NH(4)(+) and H(+) extrusion were found in larvae treated with an H(+)-pump inhibitor (bafilomycin A1) or H(+)-pump gene (atp6v1a) knockdown. Knockdown of Rhcg1 with morpholino-oligonucleotides also decreased NH(4)(+) excretion. This study demonstrates ammonia excretion in epithelial cells of larval skin through an acid-trapping mechanism, and it provides direct evidence for the involvement of the H(+) pump and an Rh glycoprotein (Rhcg1) in ammonia excretion.

Shih TH, Horng JL, Liu ST, Hwang PP, Lin LY. Rhcg1 and NHE3b are involved in ammonium-dependent sodium uptake by zebrafish larvae acclimated to low-sodium water.

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