title: In vitro transport of an allatostatin across the foregut of Manduca sexta larvae and metabolism by the gut and hemolymph.
abstract: The degradation of synthetic cydiastatin 4 by enzymes of the foregut and hemolymph, and transport across the foregut of larvae of the tobacco hawkmoth moth, Manduca sexta, were investigated using reversed-phase high performance liquid chromatography (RP-HPLC) together with matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In the hemolymph in vitro, cydiastatin 4 had a half-life of ca. 30 min. Two degradation products were identified; cydiastatin 4(1-6), due to cleavage of the C-terminal di-peptide GL-amide, and cydiastatin 4(2-8), due to cleavage of the N-terminal A residue. This hydrolysis could be inhibited by up to 93% by 1,10-phenanthroline. Other protease inhibitors had lesser effects (<21% inhibition of degradation) including the aminopeptidase inhibitors amastatin and bestatin, and the chelator EDTA. When incubated with foregut extract in vitro, cydiastatin 4 had a half-life of 23 min, and the hydrolysis products detected were also cydiastatin 4(1-6) and cydiastatin 4(2-8). Similarly, 1-10 phenanthroline inhibited foregut enzyme degradation of cydiastatin 4 by ca. 80%, whereas amastatin, bestatin, and EDTA had very little effect (<10% inhibition). Cydiastatin 4 was transported, intact, from the lumen to the hemolymph side of foregut tissues that were mounted as flat sheets in modified Ussing chambers. This trans-epithelial flux of peptide was dose and time-dependent, but was <3% of the amount of cydiastatin 4 present in the lumen bathing saline. In contrast, no trans-epithelial transport of peptide was apparent across everted foregut sac preparations.
authors.affiliation: Central Science Laboratory, Sand Hutton, York YO41 1LZ, UK. n.audsley@csl.gov.uk
authors.forename: Neil
authors.initials: N
authors.lastname: Audsley
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Weaver
mesh_terms: D000818:Animals; D001692:Biological Transport; D002851:Chromatography, High Pressure Liquid; D004064:Digestive System / Q000378:metabolism*; D006458:Hemolymph / Q000378:metabolism*; D006868:Hydrolysis / Q000187:drug effects; D007814:Larva / Q000378:metabolism; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D018402:Manduca / Q000378:metabolism*; D009479:Neuropeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000494:pharmacology; D010618:Phenanthrolines / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D009479:Neuropeptides; D010455:Peptides; D010618:Phenanthrolines; D011480:Protease Inhibitors; C076523:allatostatin; C016977:amastatin; D007930:Leucine; C012211:ubenimex; C025205:1,10-phenanthroline