Rhcg1 and NHE3b are involved in ammonium-dependent sodium uptake by zebrafish larvae acclimated to low-sodium water

To investigate whether Na ⁺ uptake by zebrafish is dependent on NH ₄ ⁺ excretion, a scanning ion-selective electrode technique was applied to measure Na ⁺ and NH ₄ ⁺ gradients at the yolk-sac surface of zebrafish larvae. Low-Na ⁺ acclimation induced an inward Na ⁺ gradient (uptake), and a combination of low Na ⁺ and high NH ₄ ⁺ induced a larger inward Na ⁺ gradient. When measuring the ionic gradients, raising the external NH ₄ ⁺ level (5 mM) blocked NH ₄ ⁺ excretion and Na ⁺ uptake; in contrast, raising the external Na ⁺ level (10 mM) simultaneously enhanced Na ⁺ uptake and NH ₄ ⁺ excretion. The addition of MOPS buffer (5 mM), which is known to block NH ₄ ⁺ excretion, also suppressed Na ⁺ uptake. These results showed that Na ⁺ uptake and NH ₄ ⁺ excretion by larval skin are associated when ambient Na ⁺ level is low. Knockdown of Rhcg1 translation with morpholino-oligonucleotides decreased both NH ₄ ⁺ excretion and Na ⁺ uptake by the skin and Na ⁺ content of whole larvae. Knockdown of nhe3b translation or inhibitor (5-ethylisopropyl amiloride) treatment also decreased both the NH ₄ ⁺ excretion and Na ⁺ uptake. This study provides loss-offunction evidence for the involvement of Rhcg1 and NHE3b in the ammonium-dependent Na ⁺ uptake mechanism in zebrafish larvae subjected to low-Na ⁺ water.