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First published online September 14, 2007
Journal of Experimental Biology 210, 3484-3493 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.008300

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Heavy metal detoxification in crustacean epithelial lysosomes: role of anions in the compartmentalization process

Kenneth M. Sterling¹, Prabir K. Mandal¹, Barbara A. Roggenbeck¹, Sean E. Ahearn¹, George A. Gerencser² and Gregory A. Ahearn^1,*

¹ Department of Biology, University of North Florida, 4567 St Johns Bluff Road, S., Jacksonville, FL 32224, USA
² Department of Physiology, University of Florida, Gainesville, FL, 32610, USA

View larger version (11K): [in this window] [in a new window]   Fig. 1. (A) Effects of intravesicular polyvalent inorganic anions and mannitol on the time course of 25 µmol l–1 65Zn2+ uptake by hepatopancreatic lysosomal membrane vesicles (LMV). Vesicles were loaded with 200 mmol l–1 mannitol, 20 mmol l–1 Hepes/Tris, pH 7.0, and 25 µmol l–1 K2SO4, K3PO4 or mannitol, and were then incubated in a medium containing 200 mmol l–1 mannitol, 25 µmol l–1 65Zinc-sulfate, 0.5 mmol l–1 NTA, 0.2 mmol l–1 ATP and 20 mmol l–1 Hepes/Tris, pH 7.0. (B) Effects of intravesicular monovalent inorganic anions (Cl–1), polyvalent organic anions (oxalate2–) and mannitol at pH 7.0 on the time course of 25 µmol l–1 65Zn2+ uptake by LMV. Uptake conditions as in A except that in this instance vesicles were loaded with 25 µmol l–1 oxalic acid, NaCl or mannitol. Experiments were conducted in triplicate; values are means ± 1 s.e.m. at each time point.

View larger version (8K): [in this window] [in a new window]   Fig. 2. Effect of varying intravesicular SO42– concentration on the time course of 25 µmol l–1 65Zn2+ uptake by LMV. Vesicles were loaded with 200 mmol l–1 mannitol, 20 mmol l–1 Hepes/Tris, pH 7.0 and K2SO4 concentrations of 25, 50, 100, 250, 500 or 1000 µmol l–1, and were then incubated in a medium containing 200 mmol l–1 mannitol, 25 µmol l–1 65Zinc-sulfate, 0.5 mmol l–1 NTA, 0.2 mmol l–1 ATP, and 20 mmol l–1 Hepes/Tris, pH 7.0. Experiments were conducted in triplicate; values are means ± 1 s.e.m. at each time point.

View larger version (7K): [in this window] [in a new window]   Fig. 3. Effects of intravesicular pH (pH 7.5–5.0), extravesicular ATP (1 mmol l–1), and induced membrane potential on the influx (10 s uptake) of 25 µmol l–1 65Zn2+. A transmembrane electrical potential was induced by equilibrating the vesicles with 50 µmol l–1 valinomycin, varying the extravesicular K+ concentration [0 mmol l–1 (Ki>Ko), 100 mmol l–1 (Ki=Ko) or 200 mmol l–1 (Ki<Ko)], and maintaining a constant intravesicular K+ concentration (100 mmol l–1). Vesicles were loaded with 100 mmol l–1 K2SO4, either 20 mmol l–1 Hepes/Tris (pH 6.5–7.5) or Mes/Tris (pH 5.0–6.0), 50 µmol l–1 valinomycin, and appropriate mannitol to maintain osmolarity. Loaded vesicles were then incubated in media containing 25 µmol l–1 65Zinc-sulfate, 0, 100 or 200 mmol l–1 K2SO4 0.2 or 1.0 mmol l–1 ATP (+ATP), 0.5 mmol l–1 NTA, 20 mmol l–1 Hepes/Tris, pH 7.0, and mannitol to maintain osmolarity. The experiment was conducted in triplicate; values are means ± 1 s.e.m. at each time point.

View larger version (11K): [in this window] [in a new window]   Fig. 4. Effect of preloaded Cl– on the time course of 35SO42– and 14C-oxalate2– uptake by hepatopancreatic lysosomal membrane vesicles. (A) Vesicles were loaded with 200 mmol l–1 mannitol, 20 mmol l–1 Hepes/Tris, pH 7.0, with or without 0.5 mmol l–1 KCl, and were incubated in media containing 200 mmol l–1 mannitol, 20 mmol l–1 Hepes/Tris, pH 7.0, with or without 0.5 mmol l–1 potassium gluconate and 5 mmol l–1 K235SO4. (B) Vesicles were loaded with 200 mmol l–1 mannitol, 20 mmol l–1 Hepes/Tris, pH 7.0, with or without 0.5 mmol l–1 KCl, and were incubated in media containing 200 mmol l–1 mannitol, 20 mmol l–1 Hepes/Tris, pH 7.0, with or without 0.5 mmol l–1 potassium gluconate, and 5 mmol l–1 14C-oxalate2–. Experiments were in triplicate; values are means ± 1 s.e.m. at each time point.

View larger version (13K): [in this window] [in a new window]   Fig. 5. Influx kinetics (10 s uptake) of polyvalent inorganic (35SO42–) and organic (14C-oxalate2–) anions into Cl–-loaded (25 mmol l–1 Cl–) LMV. (A) Vesicles were loaded with 200 mmol l–1 mannitol, 25 mmol l–1 KCl, 20 mmol l–1 Hepes/Tris, pH 7.0 and were incubated in media containing 200 mmol l–1 mannitol, 20 mmol l–1 Hepes/Tris, pH 7.0, and K235SO4 concentrations from 1 to 50 mmol l–1. (B) Vesicles were loaded with 200 mmol l–1 mannitol, 25 mmol l–1 KCl, 20 mmol l–1 Hepes/Tris, pH 7.0 and incubated in media containing 200 mmol l–1 mannitol, 20 mmol l–1 Hepes/Tris, pH 7.0 and 14C-oxalic acid concentrations from 0.1 to 10 mmol l–1. Experiments were in triplicate; values are means ± 1 s.e.m. Lines drawn through the curves were computed using Sigma Plot 10.0 Software (Jandal).

View larger version (10K): [in this window] [in a new window]   Fig. 6. Effect of intravesicular pH on 36Cl– influx kinetics in hepatopancreatic LMV. In both instances extravesicular pH was held at 7.0. Vesicles were loaded with 200 mmol l–1 mannitol and 20 mmol l–1 Hepes/Tris, pH 7.0 (A) or pH 9.0 (B). Loaded vesicles were then incubated in media containing 1, 5, 10, 15, 20 or 75 mmol l–1 K36Cl–, 20 mmol l–1 Hepes/Tris, pH 7.0, and mannitol to maintain osmolarity. The experiment was repeated in triplicate; values are means ± 1 s.e.m. The sigmoidal curves were drawn through the data using Sigma Plot software (Jandal).

View larger version (19K): [in this window] [in a new window]   Fig. 7. Effect of intravesicular OH– concentration on 36Cl– influx (2.5, 5, 15, and 35 mmol l–1 Cl–; 5 s uptakes) in hepatopancreatic LMV. In all instances the extravesicular pH was held at 7.0. Vesicles were loaded with 200 mmol l–1 mannitol, and either 20 mmol l–1 Hepes/Tris or Mes/Tris, pH 6.0, 7.0, 8.0 and 9.0. Loaded vesicles were then incubated in media containing 2.5, 5, 15 and 35 mmol l–1 K36Cl–, 20 mmol l–1 Hepes/Tris, pH 7.0, and mannitol to maintain osmolarity. The experiment was repeated in triplicate; values are means ± 1 s.e.m. The hyperbolic curves were drawn through the data using Sigma Plot software (Jandal).

View larger version (14K): [in this window] [in a new window]   Fig. 8. Effect of intravesicular pH on 35SO42– influx kinetics in hepatopancreatic LMV. In all cases extravesicular pH was held at pH 7.0. Vesicles were loaded with 200 mmol l–1 mannitol, 20 mmol l–1 Hepes/Tris at pH 7.0 (A), 8.0 (B) and 9.0 (C). Loaded vesicles were then incubated in media containing 2.5, 5, 10, 25 and 50 mmol l–1 K235SO4, 20 mmol l–1 Hepes/Tris, pH 7.0, and mannitol to maintain osmolarity. The experiment was repeated in triplicate; values are means ± 1 s.e.m. The sigmoidal curves were drawn through the data using Sigma Plot software (Jandel).

View larger version (11K): [in this window] [in a new window]   Fig. 9. Effect of intravesicular OH– concentration on 35SO42– influx at 5 mmol l–1 (A) and 10 mmol l–1 SO42– (B) (5 s uptakes) in hepatopancreatic LMV. In both cases the extravesicular pH was held at 7.0. Vesicles were loaded with 200 mmol l–1 mannitol and either 20 mmol l–1 Hepes/Tris or Mes/Tris, pH 7.0, 8.0 and 9.0. Loaded vesicles were then incubated in media containing either 5 or 10 mmol l–1 K235SO4, 20 mmol l–1 Hepes/Tris, pH 7.0 and mannitol to maintain osmolarity. The experiment was repeated in triplicate; values are means ± 1 s.e.m. The hyperbolic curves drawn through the data using Sigma Plot software (Jandal).

View larger version (69K): [in this window] [in a new window]   Fig. 10. Working model of the role of polyvalent anions in hepatopancreatic lysosomal heavy metal sequestration and detoxification. Membrane-bound, ATP-dependent, V-ATPase (Protein 1) transfers protons into the vesicle interior, creating a decrease in pH, an accumulation of hydrogen ions, and an inside-positive membrane potential. The outwardly directed proton gradient and positive vesicular interior provide the driving force for the asymmetric exchange of cytosolic divalent metals for intravesicular hydrogen ions by an ATP-dependent Zn2+-ATPase, or a 3H+/1Zn2+ exchanger (Protein 2). Polyvalent cytosolic anions such as sulfate2– or phosphate3– exchange with intravesicular monovalent anions such as Cl– or OH– by a second asymmetric antiporter (Protein 3), which uses the membrane potential as a driving force for exchange. Both divalent metals and polyvalent anions increase in concentration inside vesicles at acidic pH and are retained because they cannot be accommodated on the intravesicular binding sites of the exchangers. Divalent metals and polyvalent anions form precipitates (concretions) as the V-ATPase decreases in activity and the intravesicular pH rises.