Physiological and molecular responses of the spiny dogfish shark (Squalus acanthias) to high environmental ammonia: scavenging for nitrogen

Abstract

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...

Feeding through your gills and turning a toxicant into a resource: how the dogfish shark scavenges ammonia from its environment

“…2) at the highest seawater ammonia-N concentration (nominally1600 µmol l −1 ) was previously reported in S. acanthias suckleyi exposed to 1000 µmol l −1 (Nawata et al, 2015a), and has also been seen in sharks infused directly with comparable ammonia-N loads (Wood et al, 1995;Nawata et al, 2015b). Very likely, it results from a stimulation of urea-N production by the ornithine-urea cycle in excess of the requirements for ureosmotic homeostasis, perhaps as a strategy to avoid ammonia intoxication (Walsh et al, 2007).…”

“…The data of Nawata et al (2015a) showing ammonia-N uptake against both P NH3 diffusion gradients and NH 4 + electrochemical gradients at the gills suggest that this is an active transport process. Under normal conditions, the effective permeability of the branchial epithelium to ammonia efflux is even lower than that to urea efflux (Wood et al, 1995), for which there is considerable evidence of active retention (Fines et al, 2001;Wood et al, 2013).…”

“…Dogfish (N=5) were fitted with indwelling caudal artery catheters for TEP measurements, exactly as previously described (Nawata et al, 2015a). After overnight recovery of the animals in the standard wooden boxes, the TEP was measured first in seawater containing 200 µmol l −1 NH 4 HCO 3 , and then KCl was added to raise the K + concentration to 100 mmol l −1 , and the TEP was measured again after 3 h. A high impedance voltmeter (PHM 84 meter, Radiometer, Copenhagen, Denmark) was used to record TEP, using 3 mol l −1 KCl-agar bridges connected to Ag/AgCl electrodes (WPI, Sarasota, FL, USA).…”

“…Given the resulting high rates of urea-N loss across the gills and the opportunistic, irregular feeding habits of many of these predators (reviewed by Wood et al, 2007b), it is not surprising that they appear to be N limited in nature (Haywood, 1973;Wood et al, 2007a;Kajimura et al, 2006Kajimura et al, , 2008. Recently, based on experiments in which Pacific dogfish sharks (Squalus acanthias suckleyi) were exposed to a very high environmental ammonia concentration (1000 µmol l −1 ), Nawata et al (2015a) presented evidence that elasmobranchs may actively take up ammonia from the environment and use it to synthesize the essential osmolyte urea. The goals of the present study were to determine whether this phenomenon occurs at more environmentally realistic levels of external ammonia, to quantitatively assess whether it promotes net N retention and to characterize the mechanism(s) involved in terms of concentration dependence kinetics, sensitivity to pH and pharmacology.…”

Ventilatory sensitivity to ammonia in the Pacific hagfish (Eptatretus stoutii), a representative of the oldest extant connection to the ancestral vertebrates

“…In our previous studies on responses to HEA in seawater fish, we had found that NH 4 HCO 3 was preferable to NH 4 Cl because, unlike the latter, it did not acidify the water (Wood and Nawata, 2011;Nawata et al, 2015;De Boeck and Wood, 2015). However, these studies had involved concentrations only up to 1.5 mmol l −1 , whereas in the present study we were using much higher concentrations, so we thought it prudent to investigate both salts.…”