Active NH ₄ ⁺ excretion via Na ⁺ /NH ₄ ⁺ (H ⁺ ) exchange in the highly ammonia tolerant hagfish (Eptatretus stoutii)

Hagfishes ( Eptatretus stoutii ) feed on carrion drops during which time they may be exposed to high concentrations of total ammonia (T amm = NH 3 + NH 4 + ). Hagfish have extraordinary capacity to withstand and recover from exposure to high environmental ammonia (HEA), surviving at least 48h in 20 mM T amm by limiting plasma T amm accumulation to ~5 mM. This remarkable ability to maintain lower plasma [T amm ] relative to environmental [T amm ] is due to the hagfishes' capacity to excrete ammonia against large inwardly directed NH 3 partial pressure (Δ P NH 3 ; ~3800 μTorr) and NH 4 + electrochemical (E NH4+ ; ~35 mV) gradients, suggesting that hagfish use active NH 4 + transport to sustain ammonia excretion ( J amm ) during exposure to HEA. To test this hypothesis, hagfish were exposed to sequentially higher concentrations of T amm (0–20 mM) for 48 h. As predicted, hagfish consistently maintained a water:plasma [T amm ] ratio of ~2.5:1 after 24 h of exposure at each external T amm concentration. The magnitude of these gradients and a resumption of outward J amm during HEA further suggested that hagfish were actively transporting NH 4 + . We then tested the hypothesis that NH 4 + excretion was facilitated by secondary active transport using Na + /NH 4+ (H + ) exchange via apical NHE (Na + /H + Exchanger). Hagfish were acclimated to 10 mM HEA for 24h and then transferred to HEA in either artificial seawater (ASW) or Na + ‐free artificial seawater (NFASW) to determine if Na + acted as a counter‐substrate for outward NH 4 + transport. Plasma [T amm ] increased by ~40% when hagfish were transferred to HEA‐NFASW, but was unaffected when transferred to HEA‐ASW. Next, we utilized a newly established in situ hagfish dual gill perfusion/perifusion (extracellular aspect/water duct) technique with pharmacological inhibition of NHE activity while measuring appearance of 14 C‐methylamine ( 14 C‐MA, a radiolabeled analogue of NH 4 + ) in the perifusate. Hagfish were acclimated to 10 mM HEA for 24 h and afferent gill pouch arterioles were surgically cannulated and perfused with 14 C‐MA/4 mM [T amm ] hagfish saline while the water ducts were cannulated and perifused with 10 mM HEA‐ASW spiked with either amiloride (500 μM) or DMSO (vehicle control). Amiloride application resulted in a 52% reduction of 14 C‐MA flux ( J 14C‐MA as a surrogate for J NH4 + ) compared to control flux suggesting that there was appreciable Na + /NH 4 + exchange. To further confirm this, we acclimated hagfish to either 10 mM HEA or control (no‐HEA) seawater for 24h before administering hagfish with 14 C‐MA via caudal sinus injection. Hagfish were then transferred to HEA in either ASW or NFASW for 2.5h while measuring J 14C‐MA against inwardly directed E NH4 + gradients. Control hagfish subsequently exposed to HEA‐spiked ASW and NFASW exhibited minimal J 14C‐MA while HEA‐acclimated hagfish demonstrated a prominent ~60‐fold increase in J 14C‐MA when placed in HEA‐ASW, but only a 14‐fold increase was observed in animals transferred HEA‐NFASW, indicating the necessity of Na + for this mechanism. Expression analysis on two hagfish NHEs was conducted on gill tissue from control and HEA‐exposed hagfish. Our results demonstrate that an active Na + /NH 4 + (H + ) exchange mechanism, likely mediated via NHE, is used by the hagfish to excrete ammonia against large inwardly directed Δ P NH3 and E MH4 + gradients, which may be encountered during feeding events. Support or Funding Information NSERC (GGG: 203736) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

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