Branchial ammonia excretion in the Asian weatherloach Misgurnus anguillicaudatus.

The weatherloach, Misgurnus anguillicaudatus, is a freshwater, facultative air-breathing fish that lives in streams and rice paddy fields, where it may experience drought and/or high environmental ammonia (HEA) conditions. The aim of this study was to determine what roles branchial Na(+)/K(+)-ATPase, H(+)-ATPase, and Rhcg have in ammonia tolerance and how the weatherloach copes with ammonia loading conditions. The loach's high ammonia tolerance was confirmed as was evident from its high 96 h LC(50) value and high tissue tolerance to ammonia. The weatherloach does not appear to make use of Na(+)/NH(4)(+)-ATPase facilitated transport to excrete ammonia when exposed to HEA or to high environmental pH since no changes in activity were observed. Using immunofluorescence microscopy, distinct populations of vacuolar (V)-type H(+)-ATPase and Na(+)/K(+)-ATPase immunoreactive cells were identified in branchial epithelia, with apical and basolateral staining patterns, respectively. Rhesus C glycoprotein (Rhcg1), an ammonia transport protein, immunoreactivity was also found in a similar pattern as H(+)-ATPase. Rhcg1 (Slc42a3) mRNA expression also increased significantly during aerial exposure, although not significantly under ammonia loading conditions. The colocalization of H(+)-ATPase and Rhcg1 to the similar non-Na(+)/K(+)-ATPase immunoreactive cell type would support a role for H(+)-ATPase in ammonia excretion via Rhcg by NH(4)(+) trapping. The importance of gill boundary layer acidification in net ammonia excretion was confirmed in this fish; however, it was not associated with an increase in H(+)-ATPase expression, since tissue activity and protein levels did not increase with high environmental pH and/or HEA. However the V-ATPase inhibitor, bafilomycin, did decrease net ammonia flux whereas other ion transport inhibitors (amiloride, SITS) had no effect. H(+)-ATPase inhibition also resulted in a consequent elevation in plasma ammonia levels and a decrease in the net acid flux. In gill, aerial exposure was also associated with a significant increase in membrane fluidity (or increase in permeability) which would presumably enhance NH(3) permeation through the plasma membrane. Taken together, these results indicate the gill of the weatherloach is responsive to aerial conditions that would aid ammonia excretion.

Ammonia transport in cultured gill epithelium of freshwater rainbow trout: the importance of Rhesus glycoproteins and the presence of an apical Na+/NH4+ exchange complex.

The mechanisms of ammonia excretion at fish gills have been studied for decades but details remain unclear, with continuing debate on the relative importance of non-ionic NH(3) or ionic NH(4)(+) permeation by various mechanisms. The presence of an apical Na(+)/NH(4)(+) exchanger has also been controversial. The present study utilized an in vitro cultured gill epithelium (double seeded insert, DSI) of freshwater rainbow trout as a model to investigate these issues. The relationship between basolateral ammonia concentration and efflux to apical freshwater was curvilinear, indicative of a saturable carrier-mediated component (K(m)=66 micromol l(-1)) superimposed on a large diffusive linear component. Pre-exposure to elevated ammonia (2000 micromol l(-1)) and cortisol (1000 ng ml(-1)) had synergistic effects on the ammonia permeability of DSI, with significantly increased Na(+) influx and positive correlations between ammonia efflux and Na(+) uptake. This increase in ammonia permeability was bidirectional. It could not be explained by changes in paracellular permeability as measured by [(3)H]PEG-4000 flux. The mRNA expressions of Rhbg, Rhcg2, H(+)-ATPase and Na(+)/H(+) exchanger-2 (NHE-2) were up-regulated in DSI pre-exposed to ammonia and cortisol, CA-2 mRNA was down-regulated, and transepithelial potential became more negative. Bafilomycin (1 micromol l(-1)), phenamil (10 micromol l(-1)) and 5-(N,N-hexamethylene)amiloride (HMA, 10 micromol l(-1)) applied to the apical solution significantly inhibited ammonia efflux, indicating that H(+)-ATPase, Na(+) channel and NHE-2 pathways on the apical surface were involved in ammonia excretion. Apical amiloride (100 micromol l(-1)) was similarly effective, while basolateral HMA was ineffective. Pre-treatment with apical freshwater low in [Na(+)] caused increases in both Rhcg2 mRNA expression and ammonia efflux without change in paracellular permeability. These data suggest that Rhesus glycoproteins are important for ammonia transport in the freshwater trout gill, and may help to explain in vivo data where plasma ammonia stabilized at 50% below water levels during exposure to high environmental ammonia ( approximately 2300 micromol l(-1)). We propose an apical ;Na(+)/NH(4)(+) exchange complex' consisting of several membrane transporters, while affirming the importance of non-ionic NH(3) diffusion in ammonia excretion across freshwater fish gills.

Tribute to R. G. Boutilier: acid-base transfer across fish gills.

The gills are the major site of acid-base regulation in most fish. Acid-base transfer across fish gills is dominated by carbon dioxide and ammonia excretion, especially the former. Bicarbonate buffering in the blood is less than that found in mammals; regulation of ventilation has little effect on CO(2) levels in the blood and control of ventilation is not used to regulate body pH in fish. Proton ATPase (freshwater fish), Na(+)/H(+) exchangers (marine fish) and anion exchangers (marine and freshwater fish) are located in the gills. These transporters contribute to the regulation of internal pH, but little is known about how this is done in fish. Fish kept in confined water volumes acidify their environment, largely due to CO(2). This acidification augments ammonia excretion and reduces ammonia toxicity. The possible involvement of ammonia recycling in acid excretion is also discussed.

Dogmas and controversies in the handling of nitrogenous wastes: ammonia tolerance in the oriental weatherloach Misgurnus anguillicaudatus.

The oriental weatherloach Misgurnus anguillicaudatus is an extremely ammonia-tolerant fish. Many ammonia-protection mechanisms have been reported in this fish. Six strategies used by this fish to deal with the problem of excess ammonia are described. The fish can (1) reduce ammonia production through reduction in protein and/or amino acid catabolism; (2) reduce ammonia production and obtain energy through partial amino acid catabolism leading to alanine formation; (3) detoxify ammonia to glutamine; (4) tolerate very high ammonia levels in its tissues; (5) get rid of ammonia as NH(3) gas and, probably, (6) possesses background K(+) channels that are impermeable to NH(4)(+). The effects of extracellular ammonia on the contraction performance of the heart from this fish were found to be the same as in rainbow trout, an ammonia-sensitive fish. It suggests that the hearts of most, if not all, fish species are protected against ammonia. MK-801, an NMDA receptor blocker, was found to have a protective effect against ammonia intoxication in the oriental weatherloach, which suggests that the NMDA receptor, as in mammals, is involved in ammonia toxicity.

Ammonia toxicity in fish.

Ammonia is present in the aquatic environment due to agricultural run-off and decomposition of biological waste. Ammonia is toxic to all vertebrates causing convulsions, coma and death, probably because elevated NH4+ displaces K+ and depolarizes neurons, causing activation of NMDA type glutamate receptor, which leads to an influx of excessive Ca2+ and subsequent cell death in the central nervous system. Present ammonia criteria for aquatic systems are based on toxicity tests carried out on, starved, resting, non-stressed fish. This is doubly inappropriate. During exhaustive exercise and stress, fish increase ammonia production and are more sensitive to external ammonia. Present criteria do not protect swimming fish. Fish have strategies to protect them from the ammonia pulse following feeding, and this also protects them from increases in external ammonia, as a result starved fish are more sensitive to external ammonia than fed fish. There are a number of fish species that can tolerate high environmental ammonia. Glutamine formation is an important ammonia detoxification strategy in the brain of fish, especially after feeding. Detoxification of ammonia to urea has also been observed in elasmobranches and some teleosts. Reduction in the rate of proteolysis and the rate of amino acid catabolism, which results in a decrease in ammonia production, may be another strategy to reduce ammonia toxicity. The weather loach volatilizes NH3, and the mudskipper, P. schlosseri, utilizes yet another unique strategy, it actively pumps NH4+ out of the body.

Accumulation of ammonia in the body and NH(3) volatilization from alkaline regions of the body surface during ammonia loading and exposure to air in the weather loach Misgurnus anguillicaudatus.

The weather loach Misgurnus anguillicaudatus inhabits rice fields that experience drought in summer and ammonia loading during agricultural fertilisation. Exposure of specimens to ammonia led to the accumulation of ammonia in muscle, liver and blood. The level of ammonia reached in the plasma was the highest reported among fishes. Ammonia was not detoxified to urea, and urea excretion rate was unaffected by ammonia exposure. Fish acidified the water to reduce ammonia loading. Ammonia loading, unlike aerial exposure, did not induce glutamine synthesis, and there was no accumulation of glutamine. This is a unique observation different from those reported for other fishes in the literature. An initial switch to partial amino acid catabolism led to the accumulation of alanine and was probably associated with a decreased rate of ammonia production. Aerial exposure led to decreases in rates of ammonia and urea excretion, as well as the accumulation of tissue ammonia. As the internal ammonia levels increased, M. anguillicaudatus was able to excrete some ammonia in the gaseous form (NH(3)). The percentage of ammonia excreted as NH(3) increased with time of exposure and with increasing temperature. It appears that air-breathing through the gut is involved, with the anterior portion of the digestive tract playing a central role: it became significantly more alkaline in fish exposed to air or to environmental ammonia. The skin, which also became more alkaline during air exposure, may also be involved in ammonia volatilization in air-exposed fish. This represents the first report of a fish using volatilization of NH(3) as part of a defence against ammonia toxicity. It can be concluded that the main strategy adopted by M. anguillicaudatus confronted with ammonia loading or air exposure is to tolerate high ammonia levels in the tissues. During periods of elevated tissue ammonia levels, some ammonia is lost by volatilization via air-breathing using the gut. In addition, some ammonia may be lost across the skin during air exposure.