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First published online May 15, 2009
Journal of Experimental Biology 212, 1716-1730 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.024851
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Ammonia and urea transporters in gills of fish and aquatic crustaceans

Dirk Weihrauch1, Michael P. Wilkie2 and Patrick J. Walsh3,*

1 Department of Biological Sciences, University of Manitoba, 190 Dysart Road, Winnipeg, MB, R3T 2N2 Canada
2 Department of Biology, Wilfrid Laurier University, 75 University Avenue West, Waterloo, ON, N2L 3C5 Canada
3 Department of Biology, Centre for Advanced Research in Environmental Genomics, University of Ottawa, 30 Marie Curie, Ottawa, ON, K1N 6N5 Canada


Figure 1
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  		Fig. 1. An updated model of ammonia excretion by typical freshwater fishes. As CO2 is excreted across the gills it is hydrated in the gill water (unstirred boundary layers) to generate H+ and HCO3–. The resulting H+ generated by CO2 hydration, and probably apical H+-ATPase activity, traps NH3 as NH4+, as it passively diffuses into the gill water, maintaining the transcellular PNH3 gradient. Emerging genomic and physiological evidence suggests that ammonia transport across the plasma membrane of gill cells depends upon the presence of Rhesus glycoproteins (see text for references). Based on this evidence it is speculated that Rhcg or Rhbg glycoproteins on the basolateral membrane act as the conduit for NH3 transport (but see discussion about whether Rh glycoproteins are also NH4+ permeable) into the gill cell cytosol, followed by outward NH3 diffusion via apical Rhcg glycoproteins. The possibility also remains that a unique Na+-dependent NH4+-ATPase, as yet uncharacterized, also contributes to basolateral ammonia transport (Salama et al., 1999Go). Owing to the presence of deep tight junctions between adjacent cells in the freshwater gill, it seems unlikely that there is appreciable paracellular NH4+ diffusion in freshwater fishes. CA, carbonic anhydrase. See text for further details. (Modified from Wilkie, 2002Go.)

 

Figure 2
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  		Fig. 2. Updated model of ammonia excretion by marine fishes. Ammonia excretion in sea water probably occurs by both passive NH3 and NH4+ diffusion via transcellular pathways, and `leakier' paracellular routes. Owing to the higher buffering capacity of sea water, gill water acidification is probably not involved in the ammonia excretion process. As the predominant cells found in the gill epithelium, pavement cells (PV cells) are probably the major site of ammonia excretion in marine fishes. Convincing evidence from pufferfish suggests that Rhbg and Rhcg2 glycoproteins are restricted to the basolateral and apical membranes of PV cells, respectively (Nakada et al., 2007bGo). Such an arrangement supports a model in which NH3 enters the cytosol via a basolateral Rhbg, and exits via the apical Rhcg2. The convincing evidence that NHE2 is expressed in the gills of many marine fishes supports the hypothesis that apical Na+/NH4+ exchange also contributes to branchial ammonia excretion. However, as NHE2 proteins are mainly restricted to mitochondria rich (MR) cells, which cover a small proportion of the gill epithelium, their contribution to total ammonia excretion may be minor. Ammonia may incidentally enter the MR cells by displacing K+ on the branchial Na+/2Cl–/K+ co-transporter and/or the Na+/K+-ATPase. Apical Rhcg1 and/or apical Na+/NH4+ exchange may therefore serve as `relief valves' that promote the removal of ammonia that enters the MR cell via these basolateral transport systems. See text for further details. (Modified from Wilkie, 2002Go.)

 

Figure 3
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  		Fig. 3. (A) Ammonia excretion across the gills of the giant mudskipper P. schlosseri. NH4+ has a similar hydrated radius to K+. Extracellular NH4+ may therefore enter the cytosol of MR cells, which are abundant on the lamellae of the gill, via either ouabain-sensitive Na+/K+-ATPases and/or Na+/2Cl–/K+ co-transporters which are expressed in the MR cells at high levels. Ammonia may also enter the cytosol by passive NH3 diffusion, and subsequently be excreted to the water when favorable PNH3 gradients are present. It is not known if Rh glycoproteins play any role in NH3 diffusion in the mudskipper. Under conditions of high environmental ammonia, or when ammonia accumulates in water chambers formed by fused lamellae, NH4+ appears to be extruded via an amiloride-sensitive Na+/NH4+ (H+) antiporter on the MR cell apical membrane. Base excretion probably takes place via the apical Cl–/HCO3– exchange, with Cl– returning to the water via an apical cystic fibrosis transmembrane conductance regulator (CFTR) channel (not shown) (modified from Wilson et al., 2000Go). See text for further details. (B) Possible mode of ammonia volatilization by the Mangrove killifish (K. marmoratus). Alkalinization of the cutaneous surface moves the pH of this region nearer the pK' of ammonia, generating high NH3 partial pressures. The NH3 is subsequently volatilized as air currents move across the skin surface. Based on molecular evidence, it seems logical to suggest that at least some NH3 enters the cytosolic compartment via Rhbg, but during air exposure basolateral NH4+ transport would also probably be needed to generate the high cytosolic total ammonia and PNH3 needed to facilitate the transfer of the NH3 to the surface of the skin. Both Rhcg1 and/or Rhcg2 mRNA expression increases during air exposure in K. marmoratus, suggesting that outward transfer of NH3 across the apical membrane of cutaneous cells is via these glycoproteins. The mechanism of cutaneous surface alkalinization in air-exposed K. marmoratus has not yet been resolved. Alkalinization could involve apical Cl–/HCO3– exchange, which would depend upon carbonic anhydrase-mediated CO2 hydration in the gill cytosol leading to the generation of the required HCO3–. However, the simultaneously generated H+ would tend to acidify the intracellular space, unless it was removed via a basolateral transport system (not shown), as has been suggested in fish gut epithelia (Grosell, 2006Go). The model depicted is based on original studies by Frick and Wright (Frick and Wright, 2002aGo, Frick and Wright, 2002bGo), Littwiller et al. (Littwiller et al., 2006) and Hung et al. (Hung et al., 2007Go). See text for further details.

 

Figure 4
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  		Fig. 4. Proposed hypothetical model of active ammonia excretion across gills of the shore crab Carcinus maenas. According to this model, NH4+ is pumped across the basolateral membrane by Na+/K+-ATPase or traverses the membrane via Cs+-sensitive channels. Dissociation of cytosolic NH4+ to H+ and NH3 is accompanied by diffusion of NH3 into vesicles acidified by a H+-ATPase. The ammonia-loaded vesicles then are moved via microtubules to the apical membrane where vesicles fuse with the external membrane, releasing NH4+ into the subcuticular space. Then the NH4+ is believed to diffuse across the cuticle, via amiloride-sensitive structures. The role and location of the crustacean ammonia transporter RhCM, identified in Carcinus maenas gill epithelium (GenBank accession: AF364404), are presently uncharacterized. Paracellular ammonia diffusion and non-ionic transcellular diffusion of NH3 might also occur under physiologically meaningful transepithelial ammonia gradients (modified from Weihrauch et al., 2004Go).

 

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© The Company of Biologists Ltd 2009