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Journal of Experimental Biology, Vol 204, Issue 10 1795-1804, Copyright © 2001 by Company of Biologists
JOURNAL ARTICLES |
GM Coast, SG Webster, KM Schegg, SS Tobe and DA Schooley
Department of Biology, Birkbeck College, Malet Street, London WC1E 7HX, UK, School of Biological Sciences, University of North Wales, Gwynedd LL57 2UW, UK, Department of Biochemistry, University of Nevada, Reno, NV 89557, USA and Department of Zoology, University of Toronto, Ontario, Canada M55 3G5. g.coast@bbk.ac.uk
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 DH(31); Drome-DH(31)) 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 EC(50) of 4.3 nmol l(-)(1). The diuretic activities of Drome-DH(31) 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-DH(31) 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-DH(31). Stimulation with Drome-DH(31) 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-DH(31) and, when tested together with the diuretic peptide, their activities were not additive. Stimulation with Drome-DH(31) resulted in a dose-dependent increase in cyclic AMP production by tubules incubated in saline containing 0.5 mmol l(-)(1) 3-isobutyl-1-methylxanthine, whereas cyclic GMP production was unchanged. Taken together, the data are consistent with Drome-DH(31) activating an apical membrane V-ATPase via cyclic AMP. Since the K(+) concentration of the secreted urine was unchanged, it is likely that Drome-DH(31) has an equal effect on K(+) and Na(+) entry across the basolateral membrane.
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