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Intracellular ion activities in Malpighian tubule cells of Rhodnius prolixus: evaluation of Na⁺-K⁺-2Cl^- cotransport across the basolateral membrane

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 (13K): [in a new window]   Fig. 1. Examples of recordings of basolateral membrane potential (Vbl) and intracellular activity of (A) Na+, (B) K+ and (C) Cl- in serotonin-stimulated tubules. Vbl and ion activity were measured simultaneously using double-barrelled ion-selective microelectrodes. In this and subsequent figures, impalement is indicated by the downward-pointing arrows and the removal of the electrode from the cell is indicated by upward-pointing arrows.

View larger version (24K): [in a new window]   Fig. 2. Schematic diagram showing net electrochemical potentials (µ/F) for three cation-Cl- cotransporters in serotonin-stimulated tubules. Corresponding values for unstimulated tubules are given in parentheses. (A) Na+-K+-2Cl-, (B) Na+-Cl-, (C) K+-Cl-.

View larger version (11K): [in a new window]   Fig. 3. Effects of (A) Na+-free or (B) K+-free saline on intracellular Cl- activity in serotonin-stimulated tubules. Tubules were exposed to Na+-free or K+-free saline for the period indicated by the horizontal bar.

View larger version (12K): [in a new window]   Fig. 4. Effects of bumetanide on intracellular Cl- activity and on the effect of (A) Na+-free or (B) K+-free saline on intracellular Cl- activity in serotonin-stimulated tubule. Tubules were exposed to Na+-free or K+-free saline for the period indicated by the horizontal black bar. The horizontal grey bar indicates the time of exposure to 10-5 mol l-1 bumetanide.

View larger version (16K): [in a new window]   Fig. 5. Effects of 10-5moll-1 bumetanide on (A) fluid secretion rate, (B) K+ flux and (C) Na+ flux in serotonin-stimulated tubules. At t=10 min, bumetanide was added to the Malpighian tubules in the experimental group (open circles) and vehicle (0.1% ethanol) was added to the control group (filled circles). Data are expressed as means ± S.E.M. Asterisks indicate significant differences (P<0.05) between the control and experimental groups; N=7 for the experimental group and N=10-12 for the controls.

View larger version (13K): [in a new window]   Fig. 6. Effects of 10-4moll-1 ouabain on (A) fluid secretion rate and (B) K+ flux in serotonin-stimulated tubules. At t=10 min, ouabain was added to the Malpighian tubules in the experimental group (open circles) and an equal volume of saline was added to the control group (filled circles). Data are expressed as means ± S.E.M. The experimental and control tubules did not differ significantly; N=10 experimental tubules, N=17 for control fluid secretion and N=11 for control K+ flux.

View larger version (13K): [in a new window]   Fig. 7. Effects of 6 mmoll-1 Ba2+ on fluid secretion in serotonin-stimulated tubules. At t=20 min, Ba2+ was added to the Malpighian tubules in the experimental group (open circles) and an equal volume of saline was added to the control group (filled circles). Data are expressed as means ± S.E.M. The experimental and control tubules did not differ significantly; N=11 for the experimental tubules and N=10 for the controls.

View larger version (4K): [in a new window]   Fig. 8. Representative recording showing the effect of 6 mmoll-1 Ba2+ on basolateral membrane potential in a serotonin-stimulated tubule.