Rhesus Glycoprotein P2 (Rhp2) Is a Novel Member of the Rh Family of Ammonia Transporters Highly Expressed in Shark Kidney

The soil-dwelling nematode Caenorhabditis elegans is a bacteriovorous animal, excreting the vast majority of its nitrogenous waste as ammonia (25.3±1.2 µmol gFW −1 day ). Although these roundworms have been used for decades as genetic model systems, very little is known about their strategy to eliminate the toxic waste product ammonia from their bodies into the environment. The current study provides evidence that ammonia is at least partially excreted via the hypodermis. Starvation reduced the ammonia excretion rates by more than half, whereas mRNA expression levels of the Rhesus protein CeRhr-2, V-type H + -ATPase (subunit A) and Na + /K + -ATPase (α-subunit) decreased correspondingly. Moreover, ammonia excretion rates were enhanced in media buffered to pH 5 and decreased at pH 9.5. Inhibitor experiments, combined with enzyme activity measurements and mRNA expression analyses, further suggested that the excretion mechanism involves the participation of the V-type H +

Section: Introductionmentioning

confidence: 99%

Ammonia excretion inCaenorhabditis elegans: mechanism and evidence of ammonia transport of the Rhesus protein CeRhr-1

Adlimoghaddam

Boeckstaens

Marini

et al. 2015

“…In elasmobranchs, there are only two previous reports of Rh proteins -Rhbg in the intestine, rectal gland and kidney (the only tissues examined) of the little skate, Leucoraja erinacea (Anderson et al, 2010), and Rhp2 in the kidney of the houndshark, T. scyllium (Nakada et al, 2010). In contrast to the present findings, Nakada et al (Nakada et al, 2010) reported that Rhp2 was present only in the kidney, but this conclusion was based on northern blot screening, which is less sensitive than the qPCR employed here. As branchial Rhp2 expression did not respond to HEA exposure (Fig.…”

mentioning

confidence: 99%

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

Walsh

Wood

2015

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...

“…This result suggests that the two molecules share a common transport system, and is again consistent with a mechanism mediated by Rh proteins. For trout Rh proteins expressed in oocytes, concentrations of ammonia approximately 2-fold greater than those used here are needed to achieve 50% inhibition of [ 14 C]MA uptake (Nawata et al, 2010a), and in the only comparable study on an elasmobranch Rh protein, a 10-fold higher concentration was needed to cause 100% inhibition (Nakada et al, 2010). In our experiments, we wished to avoid such high concentrations as they would probably have been fatal to the intact animal.…”

Section: Environmental and Physiological Relevance Of Branchial Ammonmentioning

confidence: 99%

“…Together with basolaterally located V-type H + -ATPase (Tresguerres et al, 2005(Tresguerres et al, , 2006, these Rh proteins could function as a metabolon to pump ammonia-N from gill cells to blood, analogous to the apical Rhcg metabolon that pumps ammonia-N from gill cells to the external water in teleosts (Wright andWood, 2009, 2012). In the kidney of another elasmobranch, Triakis scyllium, Nakada et al (2010) have similarly proposed that basolateral Rhp2 serves to recover ammonia-N from the urine. Unfortunately, as yet, there are no pharmacological blockers of Rh proteins to test these ideas.…”

Section: Perspectivesmentioning

confidence: 99%

“…Water pH was monitored at hourly intervals using a Symphony SP70P probe and meter (VWR, Radner, PA, USA) and adjusted as necessary to maintain pH within ±0.05 units of the target values. (Nakhoul et al, 2010;Caner et al, 2015) and fish systems (Nakada et al, 2007(Nakada et al, , 2010Nawata et al, 2007;Nawata et al, 2010a). In light of the recent discovery of Rh glycoprotein mRNA expression in the gills of S. acanthias suckleyi (Nawata et al, 2015a,b) In light of debate on whether ammonia excretion is normally coupled to Na + uptake in the gills of marine elasmobranchs (reviewed by Wright and Wood, 2016), we employed the broadspectrum Na + transport blocker amiloride (Benos, 1982;Kleyman and Cragoe, 1988) to evaluate whether the uptake of ammonia (and also of [ Experiment iv: effect of high seawater K + concentration on ammonia-N uptake NH 4 + may substitute for K + through ionic mimicry in some biological transporters (Martinelle and Häggström, 1993;Good, 1988).…”

Section: Experimental Seriesmentioning

confidence: 99%

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

Wood

Giacomin

2016

Nitrogen (N) appears to be a limiting dietary resource for elasmobranchs, required not only for protein growth but also for urea-based osmoregulation. Building on recent evidence that the toxicant ammonia can be taken up actively at the gills of the shark and made into the valuable osmolyte urea, we demonstrate that the uptake exhibits classic Michaelis-Menten saturation kinetics with an affinity constant (K m ) of 379 µmol l −1 , resulting in net N retention at environmentally realistic ammonia concentrations (100-400 µmol l −1 ) and net N loss through stimulated urea-N excretion at higher levels. Ammonia-N uptake rate increased or decreased with alterations in seawater pH, but the changes were much less than predicted by the associated changes in seawater P NH3 , and more closely paralleled changes in seawater NH 4 + concentration. Ammonia-N uptake rate was insensitive to amiloride (0.1 mmol l ), suggesting that the mechanism does not directly involve Na + or K + transporters, but was inhibited by blockade of glutamine synthetase, the enzyme that traps ammonia-N to fuel the ornithine-urea cycle. High seawater ammonia inhibited uptake of the ammonia analogue [ 14 C]methylamine. The results suggest that branchial ammonia-N uptake may significantly supplement dietary N intake, amounting to about 31% of the nitrogen acquired from the diet. They further indicate the involvement of Rh glycoproteins (ammonia channels), which are expressed in dogfish gills, in normal ammonia-N uptake and retention.