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 NH4+excretion, a scanning ion-selective electrode technique was applied to measure Na+and NH4+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 NH4+induced a larger inward Na+gradient. When measuring the ionic gradients, raising the external NH4+level (5 mM) blocked NH4+excretion and Na+uptake; in contrast, raising the external Na+level (10 mM) simultaneously enhanced Na+uptake and NH4+excretion. The addition of MOPS buffer (5 mM), which is known to block NH4+excretion, also suppressed Na+uptake. These results showed that Na+uptake and NH4+excretion by larval skin are associated when ambient Na+level is low. Knockdown of Rhcg1 translation with morpholino-oligonucleotides decreased both NH4+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 NH4+excretion and Na+uptake. This study provides loss-of-function evidence for the involvement of Rhcg1 and NHE3b in the ammonium-dependent Na+uptake mechanism in zebrafish larvae subjected to low-Na+water.