Meeting Abstract
6.7 Jan. 4 Active ammonia uptake in the midgut of the tobacco hornworm Manduca sexta. WEIHRAUCH, Dirk; University of Osnabrueck, Osnabrueck, Germany weihrauchblues@gmx.net
In Ussing chamber experiments the mid- and hindgut of Manduca sexta larvae were tested for their ammonia transport properties. In the presence of 0.1 mM ammonia on both sides of the isolated epithelium, active transepithelial ammonia absorption (aTEPA) was observed in all midgut sections, with greatest transport rates (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 the V-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 Barium ions had no effect on aTEPA. Using molecular methods, full cDNA sequence of a Rhesus-like ammonia transporter (RhMS) was found with low mRNA expression levels in midgut tissues, but high expression levels in the hindgut, Malpighian tublules and ganglia. Due to its low expression in the midgut, it is likely that RhMS plays no major role in the transport process. However, presumable excess hemolymph ammonia is secreted in a regulatory mode into the Malpighian tubules, where high tissue ammonia concentrations (ca. 30 �mol/g fresh weight) but also high levels of RhMS mRNA were detected. Ammonia is then finally released for excretion into the hindgut where, in contrast to the midgut sections (ca. 1 mM), very high ammonia concentrations (ca. 60 mM) were measured.
