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

Abstract

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 1(-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 A, 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. (c) 2006 Elsevier Ltd. All rights reserved.