title: 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 (NH₃ + NH₄+) 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 NH₄+ 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 IC50 = 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.
authors.affiliation: Foundation Clinic for Cattle, University of Veterinary Medicine Hannover, 30173 Hannover, Germany.
authors.forename: Anne-Kathrin
authors.initials: AK
authors.lastname: Blaesse
authors.forename: Gunnar
authors.initials: G
authors.lastname: Broehan
authors.forename: Heiko
authors.initials: H
authors.lastname: Meyer
authors.forename: Hans
authors.initials: H
authors.lastname: Merzendorfer
authors.forename: Dirk
authors.initials: D
authors.lastname: Weihrauch
mesh_terms: D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000641:Ammonia / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport / Q000187:drug effects; D015152:Blotting, Northern; D002412:Cations; D003001:Cloning, Molecular; D041981:Gastrointestinal Tract / Q000378:metabolism*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D018402:Manduca / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D011188:Potassium / Q000378:metabolism; D017433:Protein Structure, Secondary; D011522:Protons; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism
chemical_list: D002412:Cations; D011522:Protons; D012333:RNA, Messenger; D017923:Sodium-Hydrogen Exchangers; D000641:Ammonia; D000584:Amiloride; D025262:Vacuolar Proton-Translocating ATPases; D011188:Potassium; C039614:ethylisopropylamiloride