Active ammonia absorption in the midgut of the Tobacco hornworm Manduca sexta L.: Transport studies and mRNA expression analysis of a Rhesus-like ammonia transporter

In this study the mid- and hindgut of Manduca sexta larvae were tested for their ammonia transport properties using a custom-made Ussing chamber. In the presence of 0.1 mmol l−1 ammonia on both sides of the isolated epithelium, active transepithelial ammonia absorption (aTEPA) was observed in all midgut sections, with greatest transport rates (ca. 140 nmol cm−2 h−1) detected in the median midgut. The hindgut showed no aTEPA. In the median midgut inhibition of energy metabolism by azide blocked aTEPA completely, whereas inhibition of vacuolar H+-ATPase by bafilomycin A1 reduced the active transport by 50%. The imposition of a luminal-directed NH3-gradient (pH 6.5 apical, pH 8.5 basal) lowered the aTEPA by approximately 50% but did not reverse its direction. Apical addition of amiloride reduced aTEPA by 90%, suggesting a role of carrier-mediated ammonia transport across the apical membrane via a member of the NHE family. Inhibition of the microtubule network by colchicine reduced aTEPA by ca. 50%. In contrast, blocking basal K+ channels by Ba2+ had no effect on aTEPA. Using molecular methods, evidence for intestinal expression of a Rhesus-like ammonia transporter (RhMS) was found with low mRNA expression in midgut tissues, but high expression levels in the hindgut, Malpighian tublules and ganglia.