title: The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules.
abstract: The Drosophila melanogaster homologue of an insect calcitonin-like diuretic hormone was identified in a BLAST search of the Drosophila genome database. The predicted 31-residue amidated peptide (D. melanogaster DH31; Drome-DH31) was synthesised and tested for activity on fruit fly Malpighian tubules. It increases tubule secretion by approximately 35 % of the response obtained with a myokinin from the housefly Musca domestica (muscakinin; Musdo-K) and has an EC50 of 4.3 nmol x l(-1). The diuretic activities of Drome-DH31 and Musdo-K were additive when tested at threshold and supra-maximal concentrations, which suggests that they target different transport processes. In support of this, Drome-DH31 increased the rate of secretion by tubules held in bathing fluid with a reduced Cl- concentration, whereas Musdo-K did so only in the presence of Drome-DH31. Stimulation with Drome-DH31 increased the lumen-positive transepithelial potential in the main secretory segment of the tubule. This was attributed to activation of an apical electrogenic proton-translocating V-ATPase in principal cells, since it was associated with hyperpolarisation of the apical membrane potential and acidification of secreted urine by 0.25 pH units. Exogenous 8-bromo-cyclic AMP and cyclic GMP increased tubule secretion to the same extent as Drome-DH31 and, when tested together with the diuretic peptide, their activities were not additive. Stimulation with Drome-DH31 resulted in a dose-dependent increase in cyclic AMP production by tubules incubated in saline containing 0.5 mmol x l(-1) 3-isobutyl-1-methylxanthine, whereas cyclic GMP production was unchanged. Taken together, the data are consistent with Drome-DH31 activating an apical membrane V-ATPase via cyclic AMP. Since the K+ concentration of the secreted urine was unchanged, it is likely that Drome-DH31 has an equal effect on K+ and Na+ entry across the basolateral membrane.
authors.affiliation: Department of Biology, Birkbeck College, Malet Street, London WC1E 7HX, UK. g.coast@bbk.ac.uk
authors.forename: G M
authors.initials: GM
authors.lastname: Coast
authors.forename: S G
authors.initials: SG
authors.lastname: Webster
authors.forename: K M
authors.initials: KM
authors.lastname: Schegg
authors.forename: S S
authors.initials: SS
authors.lastname: Tobe
authors.forename: D A
authors.initials: DA
authors.lastname: Schooley
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000201:enzymology*; D005260:Female; D006863:Hydrogen-Ion Concentration; D007301:Insect Hormones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D008297:Male; D008317:Malpighian Tubules / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D008969:Molecular Sequence Data; D009117:Muscidae; D009479:Neuropeptides / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D016415:Sequence Alignment; D014556:Urine / Q000737:chemistry; D025262:Vacuolar Proton-Translocating ATPases
chemical_list: C434606:Dh31 protein, Drosophila; D029721:Drosophila Proteins; D007301:Insect Hormones; D019476:Insect Proteins; D009479:Neuropeptides; D010726:Phosphodiesterase Inhibitors; C119583:myokinin; D003994:Bucladesine; D000242:Cyclic AMP; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; D006152:Cyclic GMP; D015056:1-Methyl-3-isobutylxanthine