SUMMARY 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 NH3 or ionic NH4+permeation by various mechanisms. The presence of an apical Na+/NH4+ 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 (Km=66 μmol l–1) superimposed on a large diffusive linear component. Pre-exposure to elevated ammonia (2000 μmol 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[3H]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 μmol l–1), phenamil (10 μmol l–1) and 5-(N,N-hexamethylene)amiloride (HMA, 10μmol 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 μmol 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 (∼2300 μmol l–1). We propose an apical`Na+/NH4+ exchange complex' consisting of several membrane transporters, while affirming the importance of non-ionic NH3 diffusion in ammonia excretion across freshwater fish gills.

SUMMARY The mechanism(s) of ammonia excretion in the presence of elevated external ammonia are not well understood in fish. Recent studies in other organisms have revealed a new class of ammonia transporters, Rhesus glycoprotein genes(Rh genes), which may also play a role in ammonia excretion in fish. The first objective of this study was to clone and characterize Rhgenes in a fish species with a relatively high tolerance to environmental ammonia, the mangrove killifish Kryptolebias marmoratus (formerly Rivulus marmoratus). We obtained full-length cDNAs of three Rh genes in K. marmoratus: RhBG (1736 bp), RhCG1 (1920 bp) and RhCG2 (2021 bp), which are highly homologous with other known Rh gene sequences. Hydropathy analysis revealed that all three Rh genes encode membrane proteins with 10–12 predicted transmembrane domains. RhBG, RhCG1 and RhCG2 are highly expressed in gill tissue, with RhBG also present in skin of K. marmoratus. Exposure to elevated environmental ammonia (2 mmol l–1 NH4HCO3) for 5 days resulted in a modest (+37%) increase in whole-body ammonia levels, whereas gill RhCG2 and skin RhCG1 mRNA levels were upregulated by 5.8- and 7.7-fold, respectively. RhBG mRNA levels were also increased in various tissues, with 3- to 7-fold increases in the liver and skeletal muscle. In a separate group of killifish exposed to air for 24 h, RhCG1 and RhCG2 mRNA levels were elevated by 4- to 6-fold in the skin. Thus, the multifold induction of Rh mRNA levels in excretory tissues (gills and skin) and internal tissues in response to conditions that perturb normal ammonia excretion suggests that RhBG, RhCG1 and RhCG2 may be involved in facilitating ammonia transport in this species. Furthermore, the findings support earlier studies demonstrating that the skin is an important site of ammonia excretion in K. marmoratus.

SUMMARYThe full-length cDNA sequence of a putative urea transporter (lfUT) of the facilitated diffusion UT-A type has been cloned from the African lungfish Protopterus annectens. The lfUT cDNA is 1990 bp in length and its open reading frame encodes a 409 amino acid long protein, with a calculated molecular mass of 44,723 Da. The sequence is closest to those of amphibians (~65% amino acid homology), followed by mammals and elasmobranchs (~60%), and then teleosts (~50%). lfUT was clearly expressed in gill, kidney, liver, skeletal muscle and skin. Upon re-immersion in water after 33 days of air exposure ('terrestrialization'), lungfish exhibited a massive rise in urea-N excretion which peaked at 12-30 h with rates of 2000-5000 μmol-N kg -1 h -1 (versus normal aquatic rates of <130 μmol-N kg -1 h -1) and persisted until 70 h. This appears to occur mainly through the skin. Total 'excess' urea-N excretion amounted to ~81,000-91,000 μmol-N kg -1 over 3 days. By real-time PCR, there was no difference in lfUT expression in the ventral abdominal skin between aquatic ammoniotelic controls and terrestrialized lungfish immediately after return to water (0 h), and no elevation of urea-N excretion at this time. However, skin biopsies revealed a significant 2.55-fold elevation of lfUT expression at 14 h, coincident with peak urea-N excretion. At 48 h, there was no longer any significant difference in lfUT mRNA levels from those at 0 and 14 h, or from aquatic fed controls. In accordance with earlier studies, which identified elevated urea-N excretion via the skin of P. dolloi with pharmacology typical of UT-A carriers, these results argue that transcriptional activation of a facilitated diffusion type urea transporter (lfUT) occurs in the skin during re-immersion. This serves to clear the body burden of urea-N accumulated during terrestrialization.

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