Meeting Abstract
53.6 Jan. 6 A study on cation/H+ exchangers in the midgut of Manduca sexta larvae: Flux experiments, tissue mRNA expression analysis and cellular localization. BLAESSE, Anne-Kathrin*; BROEHAN, Gunnar; WEIHRAUCH, Dirk; University of Osnabrueck, Germany; University of Osnabrueck, Germany; University of Osnabrueck, Germany weihrauchblues@gmx.net
The midgut epithelium of M. sexta consists mainly of two cell types: the goblet cells, responsible for K+ secretion, and the columnar cells, where nutrition and ammonia uptake takes place. Ussing experiments showed that outwardly directed K+ flux and inwardly directed NH4+ transport are inhibited to different degrees by apically applied amiloride, however, both in a dose dependent manner. Thus it is suggested that at least two different members of the NHE family play here a role in K+ secretion and NH4+ uptake, respectively. Up to date we obtained full length cDNA sequences of two insect NHE isoforms, which showed 61% and 53% identity in deduced amino acid sequence to the mammalian NHE8 and NHE6, respectively. Employing semi-quantitative triplex RT-PCR mRNA expression levels of the insect NHE8 or NHE6 were compared to the expression levels of the V-ATPase subunit D and the ribosomal protein S3 (internal standard) in a particular tissue. While the insect NHE8 showed similar relative expression levels in all tissues (ant., med., post. midgut, hindgut, Mal. tubules, tracheae, ganglia, fatbody), insect NHE6 revealed differences in the expression pattern with ant. and post. midgut exhibiting significant lower relative expression levels compared to hindgut, Mal. tubules, tracheae, ganglia and fatbody. The V-ATPase, putatively energizing cation/H+ exchangers in M. sexta, is strongly expressed in all gut sections and Mal. tubules (transport epithelia) but showed significant lower expression levels in tracheae, ganglia and fatbody. Immunohistochemistry revealed apical/subapical and cytoplasmatic localization of NHE8 in the goblet cells suggesting a role in K+ excretion but not NH4+ uptake.
