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First published online December 14, 2007
Journal of Experimental Biology 211, 42-48 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.011882

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Differential actions of diuretic factors on the Malpighian tubules of Rhodnius prolixus

Andrew Donini^1,*, Michael J. O'Donnell² and Ian Orchard³

¹ Department of Biology, York University, 4700 Keele Street, Toronto, Ontario, Canada, M3J 1P3
² Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada, L8S 4K1
³ Department of Biology, University of Toronto at Mississauga, 3359 Mississauga Road North, Mississauga, Ontario, Canada, L5L 1C6

View larger version (18K): [in this window] [in a new window]   Fig. 1. Upper tubules were initially stimulated with 25 nmol l–1 5-hydroxytryptamine (5HT) in saline and fluid was collected at 10 min intervals for a total of 20 min. The tubules then received 1 µmol l–1 of either 5HT (filled squares) or ZooneDH (filled triangles) and fluid was collected for a further 55 min at 5 min intervals. The rate of fluid secretion (A) was calculated after measuring the diameter of each droplet, and ion-selective microelectrodes were used to measure the K+ (B), Na+ (C) and Cl– (D) concentration of each droplet of secreted fluid. ZooneDH and 5HT stimulate fluid secretion to a similar extent and both increase the Na+ concentration of the secreted fluid at the expense of K+ over time. Each point is the mean ± s.e.m. of 6–9 individual tubules.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Upper tubules were initially stimulated with 25 nmol l–1 5HT in saline and fluid was collected at 10 min intervals for a total of 30 min. The tubules were then maintained in 25 nmol l–1 5HT (filled triangles) or received 0.1 µmol l–1 of either RhoprDH31 (filled circles) or leucokinin I (filled squares) at the solid arrow, and fluid was collected for a further 40 min at 10 min intervals. The rate of fluid secretion (A) was calculated after measuring the diameter of each droplet, and ion-selective microelectrodes were used to measure the K+ (B), Na+ (C) and Cl– (D) concentration of each droplet of secreted fluid. RhoprDH31 and leucokinin I had no effect on the rate of fluid secretion or the ion composition of the secreted fluid. Each point is the mean ± s.e.m. of 7–9 individual tubules. Addition of 1 µmol l–1 5HT at the dashed arrow was used to confirm the viability of the Malpighian tubules.

View larger version (11K): [in this window] [in a new window]   Fig. 3. Transepithelial potential (TEP) of the upper, secretory segment of the Malpighian tubules. (A) Representative recording showing the effects of 1 µmol l–1 ZooneDH (arrow). Three distinct phases of the response are numbered on the trace. The dotted line indicates a potential of 0 mV, when both microelectrodes were placed in the bathing droplet. (B) The mean ± s.e.m. (N=5) TEP prior to the addition of ZooneDH (Rest) and during the three phases of the response. (C) Representative recording showing the effects of 0.1 µmol l–1 RhoprDH31 (solid arrow) and 1 µmol l–1 5HT (dashed arrow). The three phases of the TEP response to 5HT are numbered. (D) The mean ± s.e.m. (N=5) TEP prior to the addition of RhoprDH31 (Rest), 10 min after the addition of RhoprDH31 (DH31), and during the three phases of the 5HT response. Asterisks denote a significant difference from the resting TEP (ANOVA, followed by Tukey–Kramer).

View larger version (26K): [in this window] [in a new window]   Fig. 4. Whole Malpighian tubules were removed from animals and a modified Ramsay secretion assay employing two separate bathing droplets for the upper and lower segments of the tubules was used to collect secreted fluid. Ion-selective microelectrodes were used to measure the K+ and Cl– concentration of the secreted fluid. Upper tubules were stimulated with 1 µmol l–1 5HT and the lower tubules received 1 µmol l–1 ZooneDH (A), 0.1 µmol l–1 RhoprDH31 (B), 0.1 µmol l–1 leucokinin I (C,D), all three peptides at once (E) or 1 mmol l–1 8-bromo-cyclic AMP (F; solid arrow in A–F). In addition, the lower tubules subsequently received 1 µmol l–1 5HT (dashed arrow in A–F) as a positive control. A sample of the fluid from the upper tubule was taken at the end of the experiment by cutting the tubule at the junction of the upper and lower tubule (UMT). Values are means ± s.e.m. for A: N=4; B: N=5; C and D: N=6–11; E: N=7; F: N=6.