Ammonia uptake in Manduca sexta midgut is mediated by an amiloride sensitive cation/proton exchanger: Transport studies and mRNA expression analysis of NHE7, 9, NHE8, and V-ATPase (subunit D)

Abstract

In this study participation of cation/proton exchangers (NHE) in ammonia uptake in the medial midgut of Manduca sexta larvae was investigated employing a modified Ussing chamber. There was a mean inward net ammonia (NH 3 + NH 4 +) flux of 194 ± 17 nmol cm −2 h −1 across the isolated epithelium under conditions of 0.1 mmol L −1 ammonia on both sides of the tissue and a 100-fold inwardly directed P NH3-gradient (pH 8.5 luminal side, pH 6.5 hemolymphal side). Employing a 100-fold NH 4 + gradient amiloride applied to the luminal side inhibited the influx in a dose-dependent manner, with a maximal inhibition of 75% at 20 mmol L −1 and an estimated IC 50 = 2 mmol L −1. Amiloride also caused a dose-dependent but smaller decrease in the short-circuit current ( I sc ). No inhibition by apical or basal applied amiloride was noticed on cellular metabolic ammonia release, of which ca. 1/3 and 2/3 was secreted towards the apical and basal side, respectively. Using molecular methods full and partial sequence information of two putative cation/proton exchangers (MsNHE8, MsNHE7, 9) were obtained, both containing the characteristic amiloride binding motif. An mRNA expression analysis revealed ubiquitous expression patterns for both proteins, with similar expression levels for NHE8 in all tissues investigated and lower mRNA abundances for MsNHE7, 9 in the midgut sections of the caterpillar. In contrast, in this tissue high expression levels of the V-ATPase (D subunit) were detected, likely the sole pump responsible for energizing goblet cell K + excretion, but also involved in columnar cell ammonia uptake.