An in vitro analysis of intestinal ammonia transport in fasted and fed freshwater rainbow trout: roles of NKCC, K+ channels, and Na+, K+ ATPase
We examined mechanisms of ammonia handling in the anterior, mid, and posterior intestine of unfed and fed freshwater rainbow trout (Oncorhynchus mykiss), with a focus on the Na+:K+:2Cl− co-transporter (NKCC), Na+:K +-ATPase (NKA), and K+ channels. NKCC was localized by immunohistochemistry to the mucosal (apical) surface of enterocytes, and NKCC mRNA was upregulated after feeding in the anterior and posterior segments. NH4+ was equally potent to K+ in supporting NKA activity in all intestinal sections. In vitro gut sac preparations were employed to examine mucosal ammonia flux rates (Jmamm, disappearance from the mucosal saline), serosal ammonia flux rates (Jsamm, appearance in the serosal saline), and total tissue ammonia production rates (Jtamm = Jsamm − Jmamm). Bumetanide (10−4 mol L−1), a blocker of NKCC, inhibited Jsamm in most preparations, but this was largely due to reduction of Jtamm; Jmamm was significantly inhibited only in the anterior intestine of fed animals. Ouabain (10−4 mol L−1), a blocker of NKA, generally reduced both Jmamm and Jsamm without effects on Jtamm in most preparations, though the anterior intestine was resistant after feeding. Barium (10−2 mol L−1), a blocker of K+ channels, inhibited Jmamm in most preparations, and Jsamm in some, without effects on Jtamm. These pharmacological results, together with responses to manipulations of serosal and mucosal Na+ and K+ concentrations, suggest that NKCC is not as important in ammonia absorption as previously believed. NH4+ appears to be taken up through barium-sensitive K+ channels on the mucosal surface. Mucosal NH4+ uptake via both NKCC and K+ channels is energized by basolateral NKA, which plays an additional role in scavenging NH4+ on the serosal surface to possibly minimize blood toxicity or enhance ion uptake and amino acid synthesis following feeding. Together with recent findings from other studies, we have provided an updated model to describe the current understanding of intestinal ammonia transport in teleost fish.