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 ^Na_in , J ^Cl_in , 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 ^Na_in was unaffected by amiloride (10⁻⁴ M, NHE and Na⁺ channel blocker), but both J ^Na_in and J ^Cl_in were virtually eliminated (85–99 % blockade) by AgNO₃ (10⁻⁷ M). A time course study on cardinal tetras demonstrated that J ^Na_in 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 ^Na_{in .}. Additionally, J ^Na_in 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 ^Na_in (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 ^Na_in , 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.