Mechanisms of Na+ uptake, ammonia excretion, and their potential linkage in native Rio Negro tetras (Paracheirodon axelrodi, Hemigrammus rhodostomus, and Moenkhausia diktyota)

Abstract

Mechanisms of Na(+) uptake, ammonia excretion, and their potential linkage were investigated in three characids (cardinal, hemigrammus, moenkhausia tetras), using radiotracer flux techniques to study the unidirectional influx (J in), efflux (J out), and net flux rates (J net) of Na(+) and Cl(-), and the net excretion rate of ammonia (J Amm). The fish were collected directly from the Rio Negro, and studied in their native "blackwater" which is acidic (pH 4.5), ion-poor (Na(+), Cl(-) ~20 µM), and rich in dissolved organic matter (DOM 11.5 mg C l(-1)). J in (Na) , J in (Cl) , and J Amm were higher than in previous reports on tetras obtained from the North America aquarium trade and/or studied in low DOM water. In all three species, J in (Na) was unaffected by amiloride (10(-4) M, NHE and Na(+) channel blocker), but both J in (Na) and J in (Cl) were virtually eliminated (85-99 % blockade) by AgNO3 (10(-7) M). A time course study on cardinal tetras demonstrated that J in (Na) blockade by AgNO3 was very rapid (<5 min), suggesting inhibition of branchial carbonic anhydrase (CA), and exposure to the CA-blocker acetazolamide (10(-4) M) caused a 50 % reduction in J in (Na) .. Additionally, J in (Na) was unaffected by phenamil (10(-5) M, Na(+) channel blocker), bumetanide (10(-4) M, NKCC blocker), hydrochlorothiazide (5 × 10(-3) M, NCC blocker), and exposure to an acute 3 unit increase in water pH. None of these treatments, including partial or complete elimination of J in (Na) (by acetazolamide and AgNO3 respectively), had any inhibitory effect on J Amm. Therefore, Na(+) uptake in Rio Negro tetras depends on an internal supply of H(+) from CA, but does not fit any of the currently accepted H(+)-dependent models (NHE, Na(+) channel/V-type H(+)-ATPase), or co-transport schemes (NCC, NKCC), and ammonia excretion does not fit the current "Na(+)/NH4 (+) exchange metabolon" paradigm. Na(+), K(+)-ATPase and V-type H(+)-ATPase activities were present at similar levels in gill homogenates, Acute exposure to high environmental ammonia (NH4Cl, 10(-3) M) significantly increased J in (Na) , and NH4 (+) was equally or more effective than K(+) in activating branchial Na(+),(K(+)) ATPase activity in vitro. We propose that ammonia excretion does not depend on Na(+) uptake, but that Na(+) uptake (by an as yet unknown H(+)-dependent apical mechanism) depends on ammonia excretion, driven by active NH4 (+) entry via basolateral Na(+),(K(+))-ATPase.

Does dissolved organic carbon from Amazon black water (Brazil) help a native species, the tambaqui Colossoma macropomum to maintain ionic homeostasis in acidic water?

“…In three acidophilic characids of the Amazon Basin, tested in naturally acidic, DOC‐rich water, Na + uptake appeared dependent on ammonia excretion and the mechanism appeared to be strongly dependent on intracellular carbonic anhydrase and basolateral Na + , K + ATPase in the gill epithelium (Wood et al ., ). In the present study, the activities of both Na + , K + ATPase and v‐type H + , ATPase in the gills of C. macropomum were very similar in all four treatment groups (Table ), suggesting that neither acidic conditions nor the presence of SGC DOC affected these enzymes.…”

Toxicological perspective on the osmoregulation and ionoregulation physiology of major ions by freshwater animals: Teleost fish, crustacea, aquatic insects, and Mollusca

“…In rainbow trout and several tetra species acutely exposed to Ag + , Cl − uptake decreased, as did Na + uptake [113,290]. There was also a decrease in CA activity, which would limit the supply of HCO 3 − for exchange with Cl − .…”

Osmoregulation in freshwaters: Gene expression in the gills of a Neotropical cichlid in contrasting pH and ionic environments

“…Moreover, it remains to be resolved if this Na + /NH 4 + exchange model is effective in acidic, ion-poor environments, where the external [H + ] and [Na + ] are extremely unfavorable for exchange [62]. Some evidence from fish native to blackwater habitats suggests that these exchanges may be de-coupled, whereby Na + uptake depends on NH 3 excretion, but NH 3 excretion is independent of Na + uptake [15]. For example, Hirata et al [18] found that dace native to blackwater increased somatic NH 3 production (glutamate catalysis) to remediate Na + losses, as well as NHE, CA, NBC, and aquaporin (AQP3) transcription (but not HA), whereas non-adapted dace were not able to maintain plasma [Na + ] or pH.…”

“…Thus, our procedure captured only one of several dimensions important in adaptation to novel physicochemical environments, including population-level variation, developmental plasticity, and epigenetic effects [e.g., 5,6]. Physiological observation of Negro River fishes has indicated that some species possess higher affinity Na + uptake mechanisms and decoupling of NH 3 excretion from Na + import/H + export [e.g., 15]. In addition, the DOC found in Negro habitats may have unique chelating properties utilized by native fishes to minimize ion loss [78,79], and attempts to recreate this with other DOC sources (including Sigma humic acid) have produced mixed results [26,80,81].…”

“…Most research on osmoregulation in Neotropical fishes has focused on broadly or seasonally eurytopic fishes (those that occur in both water types) or contrasts among species endemic to a single water type (stenotopic). These studies have inferred that tolerance of blackwater requires greater resistance to ion loss and acidification, and that native blackwater fishes respond differently to acid challenges or ion supplementation than their whitewater or eurytopic counterparts [15,16]. Moreover, the observation that most species are not distributed across habitat types, a necessary intermediate stage for colonization of new habitats, implies that there are costs to eurytopy such that ionoregulatory strategies for blackwater versus whitewater are often evolutionarily mutually exclusive [e.g., 17].…”