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First published online September 15, 2004
Journal of Experimental Biology 207, 3707-3716 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01203

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Na⁺ competes with K⁺ in bumetanide-sensitive transport by Malpighian tubules of Rhodnius prolixus

Juan P. Ianowski, Robert J. Christensen and Michael J. O'Donnell^*

Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada, L8S 4K1

View larger version (18K): [in a new window]   Fig. 1. Schematic diagram of the current model for transepithelial ion transport by cells in the upper (secretory) segment of the Rhodnius Malpighian tubule. Basolateral and transepithelial potentials are indicated.

View larger version (17K): [in a new window]   Fig. 2. Time course of changes in fluid secretion rate (mean ± S.E.M.) after addition of 10–6 mol l–1 serotonin at t=0 min to Malpighian tubules bathed in control saline (N=6), K+-free saline (N=8) or Na+-free saline (N=24).

View larger version (19K): [in a new window]   Fig. 3. Effects of serotonin on apical membrane potential (Vap). The data are plotted as –Vap so that upward shifts correspond to more lumen-positive potentials. The left panels show representative recordings, and the right panels show values of –Vap (mean + S.E.M.) for each of the three phases of the response to serotonin. Addition of 10–6 mol l–1 serotonin is indicated by downward arrows. Addition of 10–5 mol l–1 bumetanide is indicated by upward arrows. Tubules were bathed in control saline (top panels; replotted from Ianowski and O'Donnell, 2001), K+-free saline (middle panels, N=6) or Na+-free saline (bottom panels, N=4). Different letters in each panel denote columns that differ significantly (P<0.05; one-way ANOVA and Tukey–Kramer multiple comparisons).

View larger version (20K): [in a new window]   Fig. 4. Effects of bumetanide on fluid secretion rates (mean ± S.E.M.) of tubules bathed in (A) control saline, (B) K+-free saline or (C) Na+-free saline. Tubules were exposed to the vehicle (; 0.1% ethanol) alone or to vehicle plus bumetanide at the following concentrations: , 10–6 mol l–1; , 0.5x10–5 mol l–1; , 10–5 mol l–1; , 10–4 mol l–1. Serotonin (10–6 mol l–1) was added at t=0 min. Fluid secreted between 0 and 15 min was discarded and the first droplet was collected at t=20 min. Arrows indicate the time of addition of bumetanide. The number of tubules per group is indicated in parentheses.

View larger version (12K): [in a new window]   Fig. 5. Dose–response curves showing % inhibition of fluid secretion rate (mean ± S.E.M., N=6–14) versus bumetanide concentration for tubules bathed in control saline () and K+-free saline (). Percentage inhibition was calculated as 1–(secretion rate of bumetanide-treated tubule/mean secretion rate of control tubules)x100. Data were calculated using the t=55 min values in Fig. 4 and were fit to the Michaelis–Menten equation using non-linear regression analysis, giving r2 values for the control and K+-free data of 0.94 and 0.98, respectively.

View larger version (17K): [in a new window]   Fig. 6. Effect of 10–4 mol l–1 hydrochlorothiazide on secretion rate (mean ± S.E.M.) of tubules bathed in control saline and K+-free saline. Tubules were exposed to the vehicle alone (filled symbols; 0.1% ethanol) or to the vehicle plus hydrochlorothiazide (open symbols). Arrows indicate the time of addition of hydrochlorothiazide. The number of tubules per group is indicated in parentheses.

View larger version (18K): [in a new window]   Fig. 7. Effects of bath K+ concentration (10 or 6 mmol l–1) on fluid secretion rate (mean ± S.E.M.) and ion flux (mean ± S.E.M.). (A) Fluid secretion rates of tubules bathed in saline containing 10 or 6 mmol l–1 K+ are shown before (filled columns) and 10 min after (open columns) addition of 10–5 mol l–1 bumetanide. (B) Na+ fluxes and K+ fluxes of tubules bathed in saline containing 10 or 6 mmol l–1 K+. Different letters in each panel denote columns that differ significantly by (A) two-way ANOVA and Tukey HSD for unequal sample sizes or (B) one-way ANOVA and Tukey–Kramer multiple comparisons. N=5–10 tubules per column.

View larger version (44K): [in a new window]   Fig. 8. Effects of variation in saline Na+ concentration and K+ concentration on fluid secretion rate (mean ± S.E.M.). The key indicates saline K+ concentration in mmol l–1. N=8–10 tubules per column.

View larger version (19K): [in a new window]   Fig. 9. Effects of variation in saline ion composition on K+ flux. (A) Effects of changes in Na+ concentration and K+ concentration on K+ flux (mean ± S.E.M.). The key indicates saline K+ concentration in mmol l–1. (B) Double-reciprocal plots of K+ flux vs bathing saline K+ concentration for salines containing the three Na+ concentrations (in mmol l–1) indicated in the key. Data fit three straight lines described by the function y=0.62x+0.066 for tubules in 137.1 mmol l–1 Na+, y=0.47x+0.071 for tubules bathed in 120 mmol l–1 Na+ and y=0.395x+0.068 for tubules bathed in 98 mmol l–1 Na+. N=8–10 tubules.

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