The mechanism of ammonia excretion in freshwater teleosts is not well understood. In this study, scanning ion-selective electrode technique was applied to measure H(+) and NH(4)(+) fluxes in specific cells on the skin of zebrafish larvae. NH(4)(+) extrusion was relatively high in H(+) pump-rich cells, which were identified as the H(+)-secreting ionocyte in zebrafish. Minor NH(4)(+) extrusion was also detected in keratinocytes and other types of ionocytes in larval skin. NH(4)(+) extrusion from the skin was tightly linked to acid secretion. Increases in the external pH and buffer concentration (5 mM MOPS) diminished H(+) and NH(4)(+) gradients at the larval surface. Moreover, coupled decreases in NH(4)(+) and H(+) extrusion were found in larvae treated with an H(+)-pump inhibitor (bafilomycin A1) or H(+)-pump gene (atp6v1a) knockdown. Knockdown of Rhcg1 with morpholino-oligonucleotides also decreased NH(4)(+) excretion. This study demonstrates ammonia excretion in epithelial cells of larval skin through an acid-trapping mechanism, and it provides direct evidence for the involvement of the H(+) pump and an Rh glycoprotein (Rhcg1) in ammonia excretion.
Shih TH, Horng JL, Liu ST, Hwang PP, Lin LY. Rhcg1 and NHE3b are involved in ammonium-dependent sodium uptake by zebrafish larvae acclimated to low-sodium water.
BackgroundRegulation of pH homeostasis is a central feature of all animals to cope with acid–base disturbances caused by respiratory CO2. Although a large body of knowledge is available for vertebrate and mammalian pH regulatory systems, the mechanisms of pH regulation in marine invertebrates remain largely unexplored.ResultsWe used squid (Sepioteuthis lessoniana), which are known as powerful acid–base regulators to investigate the pH regulatory machinery with a special focus on proton secretion pathways during environmental hypercapnia. We cloned a Rhesus protein (slRhP), V-type H+-ATPase (slVHA) and the Na+/H+ exchanger 3 (slNHE3) from S. lessoniana, which are hypothesized to represent key players in proton secretion pathways among different animal taxa. Specifically designed antibodies for S. lessoniana demonstrated the sub-cellular localization of NKA, VHA (basolateral) and NHE3 (apical) in epidermal ionocytes of early life stages. Gene expression analyses demonstrated that slNHE3, slVHA and slRhP are up regulated in response to environmental hypercapnia (pH 7.31; 0.46 kPa pCO2) in body and yolk tissues compared to control conditions (pH 8.1; 0.045 kPa pCO2). This observation is supported by H+ selective electrode measurements, which detected increased proton gradients in CO2 treated embryos. This compensatory proton secretion is EIPA sensitive and thus confirms the central role of NHE based proton secretion in cephalopods.ConclusionThe present work shows that in convergence to teleosts and mammalian pH regulatory systems, cephalopod early life stages have evolved a unique acid–base regulatory machinery located in epidermal ionocytes. Using cephalopod molluscs as an invertebrate model this work provides important insights regarding the unifying evolutionary principles of pH regulation in different animal taxa that enables them to cope with CO2 induced acid–base disturbances.
A noninvasive scanning ion-selective electrode technique (SIET) was applied to measure Cl- transport at individual mitochondrion-rich cells (MRCs) in the skin of euryhaline tilapia (Oreochromis mossambicus) larvae. In seawater (SW)-acclimated larvae, outward Cl- gradients (20-80 mM higher than the background) were measured at the surface, indicating a secretion of Cl- from the skin. By serial probing over the surface of MRCs and adjacent keratinocytes (KCs), a significant outward flux of Cl- was detected at the apical opening (membrane) of MRCs. Treatment with 100 microM ouabain or bumetanide inhibited the Cl- secretion by approximately 75%. In freshwater (FW)-acclimated larvae, a lower level of outward Cl- gradients (0.2-1 mM) was measured at the skin surface. Low-Cl- water (<0.005 mM) acclimation increased the apical Na+-Cl- cotransporter (NCC) immunoreactivity of MRCs in the larval skin. An inward flux of Cl- was detected when probing the exterior surface of a group of MRCs (convex-MRCs) that express the NCC. An NCC inhibitor (100 microM metolazone) reduced the flux by approximately 90%. This study provides direct and convincing evidence for Cl- transport by MRCs of SW- and FW-acclimated euryhaline tilapia and the involvement of an apical NCC in Cl- uptake of MRCs of FW-acclimated fish.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.