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First published online January 12, 2004
Journal of Experimental Biology 207, 571-578 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.00495

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Electrogenic proton-regulated oxalate/chloride exchange by lobster hepatopancreatic brush-border membrane vesicles

George A. Gerencser^1,*, Frank Robbins², Jianliang Zhang² and Gregory A. Ahearn³

¹ Department of Physiology and Functional Genomics, College of Medicine, University of Florida, Gainesville, FL 32610, USA
² Department of Medicine, College of Medicine, University of Florida, Gainesville, FL 32610, USA
³ Department of Biology, College of Arts & Sciences, University of North Florida, Jacksonville, FL 32224, USA

View larger version (18K): [in a new window]   Fig. 1. Time course of 0.1 mmol l–1 [14C]oxalate uptake by hepatopancreatic brush-border membrane vesicles (BBMV). Vesicles contained 100 mmol l–1 TMA-gluconate and 50 mmol l–1 K-gluconate (, ) or 100 mmol l–1 TMA-gluconate, 25 mmol l–1 K-gluconate and 25 mmol l–1 KHCO3 (•). Incubation media contained 100 mmol l–1 TMA-gluconate and 50 mmol l–1 K-gluconate (, •) or 100 mmol l–1 Na-gluconate and 50 mmol l–1 K-gluconate (). All media contained 40 mmol l–1 Hepes-Tris and 50 µmol l–1 valinomycin at pH 7.0.

View larger version (11K): [in a new window]   Fig. 2. Effect of the chloride channel inhibitor 9-carboxyanthracene (9-AC) on [14C]oxalate uptake into hepatopancreatic BBMV. Vesicular and incubation media contained 100 mmol l–1 TMA-gluconate, 100 mmol l–1 K-gluconate and 50 µmol l–1 valinomycin, with (test) or without (control) 50 µmol l–1 9-AC.

View larger version (14K): [in a new window]   Fig. 3. Time course of 0.1 mmol l–1 [14C]oxalate uptake by hepatopancreatic BBMV. Vesicles were preloaded with 100 mmol l–1 TMA-gluconate, 25 mmol l–1 K-gluconate, 25 mmol l–1 KCl and 50 µmol l–1 valinomycin (), 150 mmol l–1 TMA-gluconate and 50 mmol l–1 µmol l–1 CCCP () or 100 mmol l–1 TMA-gluconate, 50 mmol l–1 K-gluconate and 50 µmol l–1 valinomycin (•). Incubation media contained 100 mmol l–1 TMA-gluconate and 50 mmol l–1 K-gluconate (, •) or 100 mmol l–1 K-gluconate and 50 mmol l–1 TMA-gluconate (). All media contained 40 mmol l–1 Hepes-Tris at pH 7.0.

View larger version (19K): [in a new window]   Fig. 4. Effect of membrane potential on the uptake of 0.1 mmol l–1 [14C]oxalate uptake into hepatopancreatic BBMV. Vesicles were preloaded with 100 mmol l–1 TMA-gluconate and 100 mmol l–1 TMA-Cl () or 100 mmol l–1 K-gluconate and 100 mmol l–1 TMACl (, •). Incubation media contained 100 mmol l–1 K-gluconate and 100 mmol l–1 TMA-gluconate (, ) or 200 mmol l–1 TMA-gluconate (•). All media contained 40 mmol l–1 Hepes-Tris and 50 µmol l–1 valinomycin at pH 7.0.

View larger version (15K): [in a new window]   Fig. 5. Effect of internal organic anions on 0.1 mmol l–1 [14C]oxalate uptake into hepatopancreatic BBMV. Vesicles were loaded with 100 mmol l–1 TMA-gluconate, 100 mmol l–1 K-gluconate and 5 mmol l–1 of the indicated anion (substituted iso-osmotically for TMA). Incubation media contained 100 mmol l–1 TMA-gluconate, 100 mmol l–1 K-gluconate and 0.1 mmol l–1 [14C]oxalate. All media contained 40 mmol l–1 Hepes-Tris and 50 µmol l–1 valinomycin at pH 7.0. Control anion, gluconate.

View larger version (21K): [in a new window]   Fig. 6. Influence of internal pH on the uptake of 0.1 mmol l–1 [14C]oxalate into hepatopancreatic BBMV. Vesicles contained 100 mmol l–1 TMA-Cl, 50 mmol l–1 K-gluconate, 50 µmol l–1 valinomycin and pH adjusted to 7.0 () or 8.0 () with 40 mmol l–1 Hepes-Tris, or pH 5.0 () or 6.0 (•) with 40 mmol l–1 Mes-Tris. Incubation media contained 100 mmol l–1 TMA-gluconate, 50 mmol l–1 K-gluconate and 40 mmol l–1 Hepes-Tris at pH 7.0.

View larger version (20K): [in a new window]   Fig. 7. Influence of external pH on the uptake of 0.1 mmol l–1 [14C]oxalate into hepatopancreatic BBMV. Vesicles contained 100 mmol l–1 TMA-Cl, 50 mmol l–1 K-gluconate, 50 µmol l–1 valinomycin and 40 mmol l–1 Hepes-Tris at pH 7.0. Incubation media contained 100 mmol l–1 TMA-gluconate and 50 mmol l–1 K-gluconate, and pH was adjusted to 7.0 (•) or 8.0 () with Hepes-Tris, or pH 5.0 () or 6.0 () with Mes-Tris.

View larger version (19K): [in a new window]   Fig. 8. Effect of various bilateral pH conditions on the uptake of 0.1 mmol l–1 [14C]oxalate into hepatopancreatic BBMV. Vesicles contained 100 mmol l–1 TMA-Cl, 50 mmol l–1 K-gluconate, 50 µmol l–1 valinomycin and adjusted to pH 7.0 (•) or 8.0 () with 40 mmol l–1 Hepes-Tris, or to pH 5.0 () or 6.0 () with 40 mmol l–1 Mes-Tris. Incubation media contained 100 mmol l–1 TMA-gluconate and 50 mmol l–1 K-gluconate with pH adjusted as above.

View larger version (28K): [in a new window]   Fig. 9. Effect of inside-negative vesicular interior on (A) [14C]oxalate/Cl– exchange and (B) 36Cl–/Ox2– exchange in hepatopancreatic BBMV. (A) Vesicles were preloaded with 100 mmol l–1 KCl and 100 mmol l–1 TMA-gluconate and incubated in media containing 0.1 mmol l–1 [14C]oxalate and either 100 mmol l–1 K-gluconate and 100 mmol l–1 TMA-gluconate (open bar) or 200 mmol l–1 TMA-gluconate (hatched bar). (B) Vesicles were preloaded with 50 mmol l–1 K2SO4 and 75 mmol l–1 TMA-gluconate and incubated in media containing 10 mmol l–1 36Cl– and either 100 mmol l–1 K-gluconate and 100 mmol l–1 TMA-gluconate (open bar) or 200 mmol l–1 TMA-gluconate (hatched bar). All media contained 40 mmol l–1 Hepes-Tris at pH 7.0.

View larger version (16K): [in a new window]   Fig. 10. Effect of potential inhibitors on [14C]Ox2–/Cl– exchange in hepatopancreatic BBMV. Vesicles were preloaded with 100 mmol l–1 TMA-gluconate, 100 mmol l–1 KCl and 50 µmol l–1 valinomycin. Incubation media contained 0.1 mmol l–1 [14C]oxalate, 100 mmol l–1 TMA-gluconate, 100 mmol l–1 K-gluconate and either 1 mmol l–1 SITS, DIDS, bumetanide or furosemide. Control incubation media contained 190 mmol l–1 TMA-gluconate and 100 mmol l–1 K-gluconate. All media contained 40 mmol l–1 Hepes-Tris at pH 7.0.

View larger version (19K): [in a new window]   Fig. 11. Effect of external Ox2– concentration on the uptake of 0.1 mmol l–1 [14C]Ox2– into hepatopancreatic BBMV. Vesicles were preloaded with 100 mmol l–1 TMA-Cl, 50 mmol l–1 K-gluconate and 50 µmol l–1 valinomycin. Incubation media contained 100 mmol l–1 TMA-gluconate, 50 mmol l–1 K-gluconate and indicated oxalate concentrations. All media contained 40 mmol l–1 Hepes-Tris at pH 7.0.