Rhcg1 and Rhbg mediate ammonia excretion by ionocytes and keratinocytes in the skin of zebrafish larvae: H+-ATPase-linked active ammonia excretion by ionocytes

Abstract

In zebrafish, Rhcg1 was found in apical membranes of skin ionocytes [H⁺-ATPase-rich (HR) cells], which are similar to α-type intercalated cells in mammalian collecting ducts. However, the cellular distribution and role of Rhbg in zebrafish larvae have not been well investigated. In addition, HR cells were hypothesized to excrete ammonia against concentration gradients. In this study, we attempted to compare the roles of Rhbg and Rhcg1 in ammonia excretion by larval skin and compare the capability of skin cells to excrete ammonia against concentration gradients. Using in situ hybridization and immunohistochemistry, Rhbg was localized to both apical and basolateral membranes of skin keratinocytes. A scanning ion-selective electrode technique (SIET) was applied to measure the NH₄⁺ flux at the apical surface of keratinocytes and HR cells. Knockdown of Rhbg with morpholino oligonucleotides suppressed ammonia excretion by keratinocytes and induced compensatory ammonia excretion by HR cells. To compare the capability of cells to excrete ammonia against gradients, NH₄⁺ flux of cells was determined in larvae exposed to serial concentrations of external NH₄⁺. Results showed that HR cells excreted NH₄⁺ against higher NH₄⁺ concentration than did keratinocytes. Knockdown of the expression of either Rhcg1 or H⁺ -ATPase in HR cells suppressed the capability of HR cells.