Active NH4+ excretion via Na+/NH4+(H+) exchange in the highly ammonia tolerant hagfish (Eptatretus stoutii)
Authors: Clifford Alexander Michael , Wilkie Michael P. , Edwards Susan L. , Weinrauch Alyssa , Goss Greg G. 
Published: June 2021

Hagfishes (Eptatretus stoutii) feed on carrion drops during which time they may be exposed to high concentrations of total ammonia (Tamm = NH3 + NH4+). Hagfish have extraordinary capacity to withstand and recover from exposure to high environmental ammonia (HEA), surviving at least 48h in 20 mM Tamm by limiting plasma Tamm accumulation to ~5 mM. This remarkable ability to maintain lower plasma [Tamm] relative to environmental [Tamm] is due to the hagfishes' capacity to excrete ammonia against large inwardly directed NH3 partial pressure (ΔPNH3; ~3800 μTorr) and NH4+ electrochemical (ENH4+; ~35 mV) gradients, suggesting that hagfish use active NH4+ transport to sustain ammonia excretion (Jamm) during exposure to HEA. To test this hypothesis, hagfish were exposed to sequentially higher concentrations of Tamm (0–20 mM) for 48 h. As predicted, hagfish consistently maintained a water:plasma [Tamm] ratio of ~2.5:1 after 24 h of exposure at each external Tamm concentration. The magnitude of these gradients and a resumption of outward Jamm during HEA further suggested that hagfish were actively transporting NH4+. We then tested the hypothesis that NH4+ excretion was facilitated by secondary active transport using Na+/NH4+(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 NH4+ transport. Plasma [Tamm] 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 14C‐methylamine (14C‐MA, a radiolabeled analogue of NH4+) in the perifusate. Hagfish were acclimated to 10 mM HEA for 24 h and afferent gill pouch arterioles were surgically cannulated and perfused with 14C‐MA/4 mM [Tamm] 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 14C‐MA flux (J14C‐MA as a surrogate for JNH4+) compared to control flux suggesting that there was appreciable Na+/NH4+ exchange. To further confirm this, we acclimated hagfish to either 10 mM HEA or control (no‐HEA) seawater for 24h before administering hagfish with 14C‐MA via caudal sinus injection. Hagfish were then transferred to HEA in either ASW or NFASW for 2.5h while measuring J14C‐MA against inwardly directed ENH4+ gradients. Control hagfish subsequently exposed to HEA‐spiked ASW and NFASW exhibited minimal J14C‐MA while HEA‐acclimated hagfish demonstrated a prominent ~60‐fold increase in J14C‐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+/NH4+(H+) exchange mechanism, likely mediated via NHE, is used by the hagfish to excrete ammonia against large inwardly directed ΔPNH3 and EMH4+ gradients, which may be encountered during feeding events.Support or Funding InformationNSERC (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.