In this study, a scanning ion-selective electrode technique (SIET) was applied to measure H+, Na+, and NH4+ gradients and apparent fluxes at specific cells on the skin of medaka larvae. Na+ uptake and NH3/NH4+ excretion were detected at most mitochondrion-rich cells (MRCs). H+ probing at MRCs revealed two group of MRCs, i.e., acid-secreting and base-secreting MRCs. Treatment with EIPA (100 μM) blocked 35% of the NH3/NH 4+ secretion and 54% of the Na+ uptake, suggesting that the Na+/H+ exchanger (NHE) is involved in Na+ and NH3/NH4+ transport. Low-Na+ water (<0.001 mM) or high-NH4+ (5 mM) acclimation simultaneously increased Na+ uptake and NH 3/NH4+ excretion but decreased or even reversed the H+ gradient at the skin and MRCs. The correlation between NH4+ production and H+ consumption at the skin surface suggests that MRCs excrete nonionic NH3 (base) by an acid-trapping mechanism. Raising the external NH4+ significantly blocked NH3/NH4+ excretion and Na+ uptake. In contrast, raising the acidity of the water (pH 7 to pH 6) enhanced NH3/NH4+ excretion and Na + uptake by MRCs. In situ hybridization and real-time PCR showed that the mRNAs of the Na+/H+ exchanger (slc9a3) and Rhesus glycoproteins (Rhcg1 and Rhbg) were colocalized in MRCs of medaka, and their expressions were induced by low-Na+ acclimation. This study suggests a novel Na+/NH4+ 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+ gradients across apical membranes of MRCs.
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