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

Abstract

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.