In teleosts, a branchial metabolon links ammonia excretion to Na + uptake via Rh glycoproteins and other transporters. Ureotelic elasmobranchs are thought to have low branchial ammonia permeability, and little is known about Rh function in this ancient group. We cloned Rh cDNAs (Rhag, Rhbg and Rhp2) and evaluated gill ammonia handling in Squalus acanthias. Control ammonia excretion was <5% of urea-N excretion. Sharks exposed to high environmental ammonia (HEA; 1 mmol −1 NH 4 HCO 3 ) for 48 h exhibited active ammonia uptake against partial pressure and electrochemical gradients for 36 h before net excretion was reestablished. Plasma total ammonia rose to seawater levels by 2 h, but dropped significantly below them by 24-48 h. Control ΔP NH3 (the partial pressure gradient of NH 3 ) across the gills became even more negative (outwardly directed) during HEA. Transepithelial potential increased by 30 mV, negating a parallel rise in the Nernst potential, such that the outwardly directed NH 4 + electrochemical gradient remained unchanged. Urea-N excretion was enhanced by 90% from 12 to 48 h, more than compensating for ammonia-N uptake. Expression of Rhp2 (gills, kidney) and Rhbg (kidney) did not change, but branchial Rhbg and erythrocytic Rhag declined during HEA. mRNA expression of branchial Na + /K + -ATPase (NKA) increased at 24 h and that of H + -ATPase decreased at 48 h, while expression of the potential metabolon components Na + /H + exchanger2 (NHE2) and carbonic anhydrase IV (CA-IV) remained unchanged. We propose that the gill of this nitrogen-limited predator is poised not only to minimize nitrogen loss by low efflux permeability to urea and ammonia but also to scavenge ammonia-N from the environment during HEA to enhance urea-N synthesis.KEY WORDS: Elasmobranchs, Rh glycoproteins, Gene expression, Urea, Gills, Ornithine-urea cycle, Transepithelial potential, P NH3 gradient INTRODUCTIONThe discovery that Rhesus (Rh) glycoproteins are expressed in the gills (Nakada et al., 2007;Nawata et al., 2007) has created a new understanding of the mechanisms of ammonia excretion in teleosts RESEARCH ARTICLE 1 Bamfield Marine Sciences Centre, 100 Pachena Road, Bamfield, BC, Canada V0R 1B0. (reviewed by Wright and Wood, 2009;Wright and Wood, 2012;Weihrauch et al., 2009). These channel proteins appear to facilitate the diffusion of NH 3 along P NH 3 (partial pressure of NH 3 ) gradients (Nawata et al., 2010b) and in the gills they function as part of a metabolon which flexibly couples ammonia excretion to Na + uptake (Tsui et al., 2009;Ito et al., 2013). The theory proposes that the deprotonation of NH 4 + as NH 3 enters the apical Rh channel creates a source of protons to fuel Na + uptake via Na + /H + exchangers (NHEs) and/or via a coupled Na + channel/V-type H + -ATPase system, while at the same time the NH 3 leaving the Rh channel traps protons, thereby creating an external microenvironment in which [H + ] is less concentrated. This mechanism becomes particularly prominent during chronic exposure to high enviro...