Molecular Physiological Characterization of Ammonia Transport in Freshwater Rainbow Trout

Abstract

Ammonia excretion from the freshwater fish gill is thought to occur mainly via passive diffusion of NH3 aided by a favourable plasma-to-water ammonia gradient sustained by a pH gradient formed by an acidified gill boundary layer. Rhesus (Rh) proteins are the newest members of the ammonia transporter superfamily. In this thesis research, ten rainbow trout Rh cDNA sequences were cloned and characterized. Rhcg2 mRNA and H+-ATPase mRNA and activity levels were upregulated in the trout gill pavement cells in response to experimentally elevated plasma ammonia, concurrent with enhanced ammonia excretion. Controversially, Rh proteins are thought to transport C02. However, Rh mRNA levels in most tissues of hypercapnia-exposed trout remained stable suggesting that trout Rh proteins likely do not conduct C02. Xenopus oocytes expressing trout Rh proteins facilitated the bi-directional transport of methylamine, an ammonia analogue. Methylamine transport was inhibited by ammonia and sensitive to a pH gradient and the concentration of the protonated species. Use of the scanning ion electrode technique (SIET) indicated that trout Rh proteins have an ammonia affinity within the physiological range, which is greater than that for methylamine, and they transport ammonia more rapidly than methylamine. A model of ammonia excretion in the trout gill pavement cell is proposed wherein ammonia enters via basolateral Rhbg and exits via apical Rhcg2, binding to these channels as NH4+ but transiting as NH3. In the gill boundary layer, NH3 combines with an H+ ion released from H+-ATPase and/or Na+/H+ exchange, forming NH4+. As low-affinity, high-capacity ammonia transporters, Rh proteins in the trout gill would exploit the favourable pH gradient formed by the acidic boundary layer to facilitate rapid ammonia efflux when plasma ammonia levels are elevated. Basal plasma ammonia levels are likely maintained by simple passive NH3 diffusion with a smaller role for Rh proteins under these conditions.