Acid-sensing ion channels are involved in epithelial Na⁺ uptake in the rainbow trout Oncorhynchus mykiss

Abstract

Dymowska AK, Schultz AG, Blair SD, Chamot D, Goss GG. Acidsensing ion channels are involved in epithelial Na ϩ uptake in the rainbow trout Oncorhynchus mykiss. Am J Physiol Cell Physiol 307: C255-C265, 2014. First published June 4, 2014 doi:10.1152/ajpcell.00398.2013.-A role for acid-sensing ion channels (ASICs) to serve as epithelial channels for Na ϩ uptake by the gill of freshwater rainbow trout was investigated. We found that the ASIC inhibitors 4=,6-diamidino-2-phenylindole and diminazene decreased Na ϩ uptake in adult rainbow trout in a dose-dependent manner, with IC 50 values of 0.12 and 0.96 M, respectively. Furthermore, we cloned the trout ASIC1 and ASIC4 homologs and demonstrated that they are expressed differentially in the tissues of the rainbow trout, including gills and isolated mitochondrion-rich cells. Immunohistochemical analysis using custom-made anti-zASIC4.2 antibody and the Na ϩ -K ϩ -ATPase (␣5-subunit) antibody demonstrated that the trout ASIC localizes to Na ϩ /K ϩ -ATPase-rich cells in the gill. Moreover, three-dimensional rendering of confocal micrographs demonstrated that ASIC is found in the apical region of mitochondrion-rich cells. We present a revised model whereby ASIC4 is proposed as one mechanism for Na ϩ uptake from dilute freshwater in the gill of rainbow trout. sodium uptake; gill; acid-sensing ion channels; mitochondrion-rich cells; fish; ionoregulation FISHES LIVING IN FRESHWATER (FW) must actively take up Na ϩ against a steep concentration gradient. Na ϩ uptake occurs via specialized mitochondrion-rich cells (MRCs), located on the fish gill epithelium (26, 29), and was initially proposed to be linked to NH 4 ϩ /H ϩ excretion (26). Subsequent studies have described two models of Na ϩ transport (for review see Refs. 8 and 18). In the first proposed model, Na ϩ is exchanged for H ϩ via an electroneutral Na ϩ /H ϩ exchanger (NHE) located on the apical membrane of MRCs in fish gill epithelia. The identification of multiple NHEs in the gills of zebrafish (Danio rerio) (51), Osorezan dace (Tribolodon hakonensis) (15), rainbow trout (Oncorhynchus mykiss) (7,19), and tilapia (Oreochromis mossambicus) (48) by immunocytochemistry, Western blot analysis, and RT-PCR, supports this model. However, significant thermodynamic constraints associated with the function of NHEs at very low ion concentrations (Na ϩ Ͻ0.1 mM) and low environmental pH (pH Ͻ5) (1, 34) suggest that fishes living in very soft and poorly buffered water would not be able to rely on a NHE-based mechanism for sufficient Na ϩ uptake. Recently, the NHE model was revised, whereby the ammonia transporter [rhesus (Rh) protein] present on the apical membrane of MRCs (30, 31) forms a functional metabolon with NHE2/3 (50). The revised model does alleviate the thermodynamic constraints associated with a low-pH environment, but not those imposed by low Na ϩ concentrations in the FW aquatic environment (6). Therefore, it is unlikely that this mechanism is the sole contributor to Na ϩ uptake by FW fishes living in low-ionic-strengt...

Interaction of osmoregulatory and acid–base compensation in white sturgeon (Acipenser transmontanus) during exposure to aquatic hypercarbia and elevated salinity

“…the first bout of pH e recovery) can be attributed to the HCSW sturgeon having accumulated relatively more Na + than Cl − during this initial salt-loading period of the SW acclimation. The apparent association between pH e recovery and a relative increase of Na + accumulation during this time may be the result of inward Na + transport being tied to outward H + transport via an apical NHE or an apical H + -ATPase with associated Na + channel (Claiborne and Heisler, 1984;Iwama and Heisler, 1991;Dymowska et al, 2014), or the co-transport of Na + and HCO 3 − into the extracellular compartment via a basolateral Na + /HCO 3 − co-transporter (NBC1) (Hirata et al, 2003;Perry et al, 2003).…”

Different mechanisms of Na+ uptake and ammonia excretion by the gill and yolk sac epithelium of early life stage rainbow trout

“…Given that these blockers have been demonstrated to inhibit J Na,in in several studies, we cannot discount the role of H + -ATPase and a putative Na + channel in overall branchial J Na,in by CYA trout. Moreover, recent evidence has suggested that this putative Na + channel might be an acid-sensing ion channel (ASIC; Dymowska et al, 2014Dymowska et al, , 2015 that is DAPI-sensitive and not affected by phenamil.…”

“…Based on the co-localization of Rhcg1 and NHEs (Fig. 4), PNA − ionocytes likely do not express apical Rhcg1 or Rhcg2 and might serve primarily for Na + /H + exchange via H + -ATPase and a putative Na + channel (Reid et al, 2003), which might be an ASIC (Dymowska et al, 2014). Finally, PVCs are likely important sites for J amm via Rhcg2, and H + -ATPase might be involved in active ammonia excretion by these cells (Nawata et al, 2007).…”

“…In this metabolon, Rh proteins transport ammonia from the cytosol to the apical boundary layer by binding NH 4 + , stripping off a proton, and conducting NH 3 Caner et al, 2015; see Wright and Wood, 2012, for review); these protons then drive Na + /H + exchange by NHE. H + -ATPase also plays a part in Na + /NH 4 + exchange by simultaneously driving electrogenic J Na,in via putative Na + channels, which are potentially acid-sensing ion channels (ASICs) (Dymowska et al, 2014), and promoting apical boundary layer acidification that facilitates apical acid-trapping of NH 3 (see Wright and Wood, 2012, for review). The current model of Na + /NH 4 + exchange in trout suggests that the NHE mechanism is localized to a subset of mitochondrion-rich cells or ionocytes that bind peanut agglutinin lectin (PNA + ionocytes), whereas the H + -ATPase mechanism is localized to ionocytes that do not bind PNA (PNA − ionocytes) (see Dymowska et al, 2012, for review).…”

Interaction of osmoregulatory and acid-base compensation in white sturgeon (Acipenser transmontanus) during exposure to aquatic hypercarbia and elevated salinity

“…the first bout of pH e recovery) can be attributed to the HCSW sturgeon having accumulated relatively more Na + than Cl − during this initial salt-loading period of the SW acclimation. The apparent association between pH e recovery and a relative increase of Na + accumulation during this time may be the result of inward Na + transport being tied to outward H + transport via an apical NHE or an apical H + -ATPase with associated Na + channel (Claiborne and Heisler, 1984;Iwama and Heisler, 1991;Dymowska et al, 2014), or the co-transport of Na + and HCO 3 − into the extracellular compartment via a basolateral Na + /HCO 3 − co-transporter (NBC1) (Hirata et al, 2003;Perry et al, 2003).Although a relative increase in Na + influx immediately following salinity exposure may have briefly aided in pH e recovery, such inward transport of Na + is counter-productive to the outward direction of Na + transport required for SW acclimation. As the sturgeon then made the physiological adjustments necessary to acclimate to SW and recover plasma osmolality ( presumably including a molecular reorganization of the gill epithelium in order to excrete Na + and Cl − ), this may have been to the detriment of any further pH e recovery.…”