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.

An animal homolog of plant Mep/Amt transporters promotes ammonia excretion by the anal papillae of the disease vector mosquito,Aedes aegypti

“…4D). In larvae of freshwater zebrafish, an Rh50 protein, Rhcg1, is required along with VA to actively excrete ammonia against a higher external concentration (Shih et al, 2013). The transcript abundance of Rhcg1 and VA increases with exposure to high environmental ammonia (HEA) and the capacity for ammonia excretion by ammonia-excreting cells increases (Braun et al, 2009).…”

“…First, NKA may transport ammonia as NH 4 + , which competes with K + or where there are separate binding sites for these two ions (Skou, 1960;Masui et al, 2002;Cruz et al, 2013). There is considerable evidence for this ammonia transport mechanism in a number of animal epithelia, such as crab gills and the antennal gland (see Weihrauch et al, 2004, for review), frog skin (Cruz et al, 2013), fish gills (Mallery, 1983), the epidermis of planaria (Weihrauch et al, 2012b) and potentially larval zebrafish skin (see Shih et al, 2013). Second, as NKA is co-localized with AeAmt1 on the basal side, it can provide a strong voltage gradient to drive NH 4 + through AeAmt1 from the hemolymph to the cytosol.…”

“…RhGP. Evidence for this mechanism of ammonia trapping at the apical membrane of epithelia in freshwater exists in a number of animals, such as zebrafish larval skin (Shih et al, 2013), the planarian epidermis (Weihrauch et al, 2012a), frog skin (Cruz et al, 2013) and fish gills (Wilson et al, 1994). The apical localization of VA in the anal papillae suggests that VA contributes to an ammonia-trapping transport mechanism; however, the specific putative ammonia transporter involved remains to be identified.…”

“…Members of the methylammonium permease/ammonium transporter (Mep/Amt) family of transporters play a critical role in ammonia uptake by plants and microorganisms (Thornton et al, 2006;Nygaard et al, 2006;Willmann et al, 2007;Gu et al, 2013;Wu et al, 2015). The annotated genomes of vertebrate animals lack Mep/Amt homologs and excretion of ammonia in animals has been linked to members of a related family of glycoproteins, the Rhesus glycoproteins (RhGPs) (Nawata et al, 2007;Braun et al, 2009;Shih et al, 2013). Interestingly, homologs of the Mep/Amt transporters are present in the invertebrates along with homologs of RhGPs (Weihrauch et al, 2012b; Fig.…”

Rh vs pH: the role of Rhesus glycoproteins in renal ammonia excretion during metabolic acidosis in a freshwater teleost fish

“…In freshwater teleosts, there is evidence that a 'Na + /NH 4 exchange complex' comprised of multiple transporters (Rhcg, V-type H + -ATPase, Na + /H + exchanger NHE2 and/or NHE3, Na + channel) operates as a metabolon Tsui et al, 2009;Weihrauch et al, 2009;Wright and Wood, 2009;Wu et al, 2010;Kumai and Perry 2011;Shih et al, 2013;Wright and Wood, 2012). It is thought that cytosolic NH 4 + ions entering the Rhcg channel are stripped of an H + before NH 3 moves by facilitated diffusion across the apical membrane down the partial pressure gradient (Nawata et al, 2010b).…”