Ammonia transport across the skin of adult rainbow trout (Oncorhynchus mykiss) exposed to high environmental ammonia (HEA)

Recent molecular evidence points towards a capacity for ammonia transport across the skin of adult rainbow trout. A series of in vivo and in vitro experiments were conducted to understand the role of cutaneous ammonia excretion (J (amm)) under control conditions and after 12-h pre-exposure to high environmental ammonia (HEA; 2 mmol/l NH4HCO3). Divided chamber experiments with bladder-catheterized, rectally ligated fish under light anesthesia were performed to separate cutaneous J (amm) from branchial, renal, and intestinal J (amm). Under control conditions, cutaneous J (amm) accounted for 4.5 % of total J (amm) in vivo. In fish pre-exposed to HEA, plasma total ammonia concentration increased 20-fold to approximately 1,000 mu mol/l, branchial J (amm) increased 1.5- to 2.7-fold, and urinary J (amm) increased about 7-fold. Urinary J (amm) still accounted for less than 2 % of total J (amm). Cutaneous J (amm) increased 4-fold yet amounted to only 5.7 % of total J (amm) in these fish. Genes (Rhcg1, Rhcg2, Rhbg, NHE-2, v-type H+-ATPase) known to be involved in ammonia excretion at the gills of trout were all expressed at the mRNA level in the skin, but their expression did not increase with HEA pre-exposure. In vitro analyses using [C-14] methylamine (MA), an ammonia analog which is transported by Rh proteins, demonstrated that MA permeability in isolated skin sections was higher in HEA pre-exposed fish than in control fish. The addition of basolateral ammonia (1,000 mu mol/l) to this system abolished this increase in permeability, suggesting ammonia competition with MA for Rh-mediated transport across the skin of HEA pre-exposed trout; this did not occur in skin sections from control trout. Moreover, in vitro J (amm) by the skin of fish which had been pre-exposed to HEA was also higher than in control fish in the absence of basolateral ammonia, pointing towards a possible cutaneous ammonia loading in response to HEA. In vitro MA permeability was reduced upon the addition of amiloride (10(-4) mol/l), but not phenamil (10(-5) mol/l) suggesting a role for a Na/H-exchanger (NHE) in cutaneous ammonia transport, as has been previously described in the skin of larval fish. Overall, it appears that under control conditions and in response to HEA pre-exposure, the skin makes only a very minor contribution to total J (amm), but the observed increases in cutaneous J (amm) in vivo and in cutaneous J (amm) and MA permeability in vitro demonstrate the capacity for ammonia transport in the skin of adult trout. It remains unclear if this capacity may become significant under certain environmental challenges or if it is merely a remnant of cutaneous transport capacity from early life stages in these fish.