Na+/H+ Exchanger 3 Is Expressed in Two Distinct Types of Ionocyte, and Probably Augments Ammonia Excretion in One of Them, in the Gills of the Climbing Perch Exposed to Seawater

Abstract

The freshwater climbing perch, Anabas testudineus, is an euryhaline teleost and an obligate air-breather with the ability to actively excrete ammonia. Members of the Na+/H+ exchanger (NHE) family help maintain intracellular pH homeostasis and ionic balance through the electroneutral exchange of Na+ and H+. This study aimed to obtain, from the gills of A. testudineus, the full cDNA coding sequence of nhe3, and to determine the effects of exposure to seawater or 100 mmol l−1 of NH4Cl in fresh water on its mRNA and protein expression levels. Efforts were also made to elucidate the type of ionocyte that Nhe3 was associated with in the branchial epithelium of A. testudineus. The transcript level and protein abundance of nhe3/Nhe3 were very low in the gills of freshwater A. testudineus, but they increased significantly in the gills of fish acclimated to seawater. In the gills of fish exposed to seawater, Nhe3 was expressed in two distinct types of seawater-inducible Na+/K+-ATPase (Nka)-immunoreactive ionocytes. In Nkaα1b-immunoreactive ionocytes, Nhe3 had an apical localization. As these ionocytes also expressed apical Rhcg1 and basolateral Rhcg2, which are known to transport ammonia, they probably participated in proton-facilitated ammonia excretion in A. testudineus during seawater acclimation. In Nkaα1c-immunoreactive ionocytes, Nhe3 was atypically expressed in the basolateral membrane, and its physiological function is uncertain. For A. testudineus exposed to NH4Cl in fresh water, the transcript and protein expression levels of nhe3/Nhe3 remained low. In conclusion, the branchial Nhe3 of A. testudineus plays a greater physiological role in passive ammonia transport and acid-base balance during seawater acclimation than in active ammonia excretion during environmental ammonia exposure.

Ion uptake pathways in European sea bass Dicentrarchus labrax

“…Despite its very low expression, sea bass nhe2c is about 28 times more expressed in SW than in FW suggesting its role in acid secretion as reported previously for several nhe2/3 paralogs in SW (Chen et al, 2017;Claiborne et al, 1999;Liu et al, 2016). Contrary to trout nhe2, sea bass nhe2b and nhe2c…”

“…There is also evidence that NHE2 and NHE3 are expressed in gill ionocytes of teleosts maintained in seawater (SW), playing critical roles in acid secretion (Chen et al, 2017;Claiborne et al, 1999;Liu et al, 2016;Weihrauch et al, 2009). In many teleost species, nhe3 mRNA expression is high in FW compared to SW (Bollinger et al, 2016;Hiroi et al, 2008;Inokuchi et al, 2008;Watanabe et al, 2008) except in the climbing perch Anabas testudineus, where the opposite pattern has been shown with increased nhe3 mRNA and NHE3 protein expression in SW (Chen et al, 2017). In this latter species, NKA α1c-immunoreactive ionocytes express NHE3 and RHCG2 in basolateral membranes and lack apical RHCG1, the exact function of these cells remains to be determined.…”

A methodological evaluation of the determination of critical oxygen threshold in an estuarine teleost

“…2D) is curious; however, it seems likely that it relates to the increased H + excretion rates that will accompany anaerobic metabolism. The positive relationship between H + excretion and ammonia excretion is well documented and relates to the metabolon-style interactions of Na + H + exchanger 3 (NHE3) and the Rh ammonia transporters (Wright et al, 2016; Chen et al, 2017). Overall, our findings relating to ammonia build-up are in line with recent work by Regan and Richards (2017), which reported final total ammonia concentrations of ∼47 µmol l −1 in closed circuit trials.…”

Rapid blood acid–base regulation by European sea bass (Dicentrarchus labrax) in response to sudden exposure to high environmental CO2

“…The discrepancy between predicted and apparent NKA and NHE3 sizes may be explained by intrinsic limitations in Western blotting for precisely determining protein size ( Bass et al, 2017 ; Gwozdz and Dorey, 2017 ), together with the presence of post-translational modifications and isoforms (see Harter et al, 2021 , for a case of a fish NHE with an alternative start codon that results in a smaller-than-predicted protein). For example, the predicted size of NHE3 is 97 kDa in climbing perch Anabas testudineus , 94 kDa in killifish Fundulus heteroclitus and 93 kDa in rabbit Oryctolagus cuniculus , yet their apparent Western blot sizes are ∼85 kDa ( Chen et al, 2017 ), ∼77 kDa ( Edwards et al, 2005 ) and ∼80 kDa ( Biemesderfer et al, 1997 ), respectively. Although many studies have previously used the same anti-NHE3 antibodies as us for immunohistochemistry in other fish species ( Blondeau-Bidet et al, 2019 ; Christensen et al, 2012 ; Hiroi and McCormick, 2012 ; Seo et al, 2013 ; Wright et al, 2016 ), to our knowledge only Wright et al (2016) performed Western blots and did not report the predicted size of the immunoreactive band.…”