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K⁺ transport in the mesonephric collecting duct system of the toad Bufo bufo : microelectrode recordings from isolated and perfused tubules

Nadja Møbjerg^*, Erik Hviid Larsen and Ivana Novak

August Krogh Institute, Department of Zoophysiology, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen Ø, Denmark

View larger version (99K): [in a new window]   Fig. 1. Photographs of isolated and perfused tubules taken using an inverted Zeiss microscope equipped with Nomarski optics. Tubules were identified according to their position within the nephron. The cellular boundaries in collecting ducts appear sharper under the light microscope when compared to collecting tubules. (A) Isolated and perfused ramification of collecting ducts. The pipette arrangement consists, on the perfusion side (right), of a constricted holding pipette and a single-barrelled perfusion pipette. On the left, a holding pipette holds the tubule. Scale bar, 300 µm. (B) Isolated and perfused collecting duct. Individual cells are clearly recognizable. The apical surface of the intercalated cells possesses microvilli and bulges slightly into the lumen of the tubule. Scale bar, 50 µm. (C) Isolated and perfused collecting tubule. Note the perfusion pipette, which has been advanced into the lumen of tubule. Scale bar, 50 µm. (D) Cellular impalement of a perfused collecting tubule. Cells were impaled across the basal cell membrane with glass microelectrodes. The tip of the microelectrode is below resolution limits. Scale bar, 10 µm. cp, constriction pipette; el, electrode; hp, holding pipette; ic, intercalated cell; lu, lumen; pp, perfusion pipette.

View larger version (10K): [in a new window]   Fig. 2. Frequency distribution of Vbl in 120 cells from collecting tubules and collecting ducts. (A) The Vbl of collecting duct cells (N=50) fell into two groups, one being more hyperpolarized than the other. (B) Vbl of collecting tubule cells (N=70) showed a broader distribution with the mean Vbl falling inbetween that of the two collecting duct groups.

View larger version (22K): [in a new window]   Fig. 3. Effect of bath and luminal [K+] steps on Vbl. (A) Voltage trace from a collecting duct cell. The effect of changing bath [K+] from 3 to 20 mmol l-1. (B) Summary data illustrating the effect of increasing bath [K+] from 3 to 20 mmol l-1 on Vbl from collecting duct cells. (C) Summary data illustrating the effect of the bath [K+] step on Vbl from collecting tubule cells. (D) Voltage trace from a collecting duct cell. Effect of the luminal [K+] step. (E) Summary data illustrating the effect of increasing luminal [K+] from 3 to 20 mmol l-1 on Vbl from collecting duct cells. (F) Summary data illustrating the effect of the luminal [K+] step on Vbl from collecting tubule cells. C, control solution; K20,b, high [K+] in bath; K20,l, high [K+] in lumen.

View larger version (18K): [in a new window]   Fig. 4. (A) Voltage trace from a collecting tubule cell illustrating the effect of high [K+] in bath, and of 1 mmol l-1 BaCl2 in bath control solution and in high [K+] solution, on Vbl. Basally applied Ba2+ depolarized Vbl. In the presence of Ba2+ the high [K+] solution did not depolarize Vbl further. (B) Summary data showing the effect of high [K+] solution, and of 1 mmol l-1 BaCl2 in bath control solution and in high [K+] solution, on Vbl. Bab, Ba2+ in bath; C, control solution; K20,b, high [K+] in bath.

View larger version (19K): [in a new window]   Fig. 5. Effect of luminal Ba2+. (A) Effect on Vbl of luminal [K+] step (3-20 mmol l-1) and of 1 mmol l-1 BaCl2 in luminal control solution and in high [K+] solution. In this cell, Ba2+ in the control solution had no effect on Vbl. Depolarization by the high [K+] solution was abolished by Ba2+. (B) Effect of luminal perfusion with Ba2+ on Vbl in the same cell as in A in an experiment where the cell was depolarized by high bath [K+]. (C) Summary data showing the effect of a luminal [K+] step (3-20 mmol l-1) and of 1 mmol l-1 BaCl2 in luminal control solution and in high [K+] solution on Vbl. The asterisk marks the cell shown in A and B. (D) Summary of experiments showing the effect on Vbl of luminal perfusion with Ba2+ in the control solution, while depolarizing the cell with a high basal [K+] solution. The asterisk marks the cell shown in A and B. Bab, Ba2+ in bath; Bal, Ba2+ in lumen; C, control solution; K20,b, high [K+] in bath; K20,l, high [K+] in lumen.

View larger version (12K): [in a new window]   Fig. 6. (A) Voltage trace from a collecting tubule cell illustrating the effect on Vbl of 1 mmol l-1 ouabain in bath control solution. (B) Effect of 1 mmol l-1 ouabain on Vbl. (C) Effect of 10-5 mol l-1 furosemide on Vbl. Inhibitors were applied to the bath for 2-4 min. C, control solution; Fur, furosemide; Oua, ouabain.

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