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First published online February 27, 2009
Journal of Experimental Biology 212, 878-892 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.021899

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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

T. K. N. Tsui¹, C. Y. C. Hung¹, C. M. Nawata¹, J. M. Wilson², P. A. Wright³ and C. M. Wood^1,*

¹ Department of Biology, McMaster University, Hamilton, Ontario, Canada L8S 4K1
² Ecofisiologia CIMAR, 4550-123 Porto, Portugal
³ Department of Integrative Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1

^* Author for correspondence (e-mail: woodcm{at}mcmaster.ca)

Accepted 11 December 2008

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 (K_m=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, 10 µmol 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.

Key words: Rhesus glycoproteins, Oncorhynchus mykiss, gills, ammonia transport, sodium uptake, cortisol, H⁺-ATPase, carbonic anhydrase, transepithelial potential

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