TY - JOUR
T1 - Uptake and subcellular localization of tritiated dihydro-microcystin-LR in rat liver
AU - Hooser, Stephen B.
AU - Kuhlenschmidt, Mark S.
AU - Dahlem, Andrew M.
AU - Beasley, Val R.
AU - Carmichael, Wayne W.
AU - Haschek, Wanda M.
N1 - Funding Information:
Aabwwlcdganent-These studier were supported in part by the United States Army Medical Research aad Development contract number DAMD 17-85-C-5241 . The viewr, opinions, and/or 5ndin8s contained in this report are shore of the authors and rhonld not be construed ss an of&tial Departmont of the Army position, policy, or dodsion unless so designated by other documentation.
Copyright:
Copyright 2014 Elsevier B.V., All rights reserved.
PY - 1991
Y1 - 1991
N2 - S. B. Hooser, M. S. Kuhlenschmidt, A. M. Dahlem, V. R. Beasley, W. W. Carmichael and W. M. Haschek. Uptake and subcellular localization of tritiated dihydro-microcystin-LR in rat liver. Toxicon 29, 589-601, 1991.-Microcystin-LR (MC-LR), a cyclic heptapeptide hepatotoxin (mol. wt = 994) produced by the blue-green alga (cyanobacterium), Microcystis aeruginosa, was reduced with tritium labeled sodium borohydride, converted to [3H]-dihydro-microcystin-LR ([3H]-2HMC-LR), and purified to > 99% purity by C-18 reverse-phase high-performance liquid chromatography. The uptake and subcellular distribution of [3H]-2HMC-LR were determined in suspensions of hepatocytes at 0° and 37°C, or following rifampicin pretreatment, and in perfused rat liver. The remaining cells were homogenized and subfractionated using sucrose gradient centrifugation. Suspensions of 7.5 × 106 hepatocytes also were incubated with 10 μg/ml of toxin, solubilized in Triton X-100, and ultracentrifuged to pellet the detergent insoluble fraction (containing actin). Isolated rat livers were perfused with media containing [3H]-2HMC-LR and the uptake of radiolabel was determined. Sequential biopsy samples were collected for histologic examination. The remaining liver was homogenized and subcellular fractions prepared. Uptake of radiolabel was rapid in both cell suspension at 37°C and perfused liver; however, uptake in cell suspensions was reduced by about 50% at 0°C and by rifampicin (50 μg/ml) pretreatment. Hepatocyte necrosis was observed in isolated perfused livers 45 min after initiation of perfusion with [3H]-2HMC-LR. In both hepatocyte suspensions and perfused livers 65 to 77% of the radiolabel was in the cytosolic fraction. In the hepatocyte suspensions, 13 to 18% of the radiolabel was present in the plasma membrane/nuclear fraction with lesser amounts in the other fractions. Trichloroacetic acid treatment of cytosolic fractions indicated that in hepatocyte suspensions, 50-60% of the radiolabel was bound to cytosolic protein. Studies using the perfused liver confirmed that the majority of the radiolabeled MCLR (78-88%) was bound to cytosolic protein. These data suggest that the uptake of [3H]-2HMC-LR occurs primarily by an energydependent transport process involving the rifampicin-sensitive hepatic bile acid carrier and that once inside the hepatocyte, the toxin binds to a cytosolic protein(s).
AB - S. B. Hooser, M. S. Kuhlenschmidt, A. M. Dahlem, V. R. Beasley, W. W. Carmichael and W. M. Haschek. Uptake and subcellular localization of tritiated dihydro-microcystin-LR in rat liver. Toxicon 29, 589-601, 1991.-Microcystin-LR (MC-LR), a cyclic heptapeptide hepatotoxin (mol. wt = 994) produced by the blue-green alga (cyanobacterium), Microcystis aeruginosa, was reduced with tritium labeled sodium borohydride, converted to [3H]-dihydro-microcystin-LR ([3H]-2HMC-LR), and purified to > 99% purity by C-18 reverse-phase high-performance liquid chromatography. The uptake and subcellular distribution of [3H]-2HMC-LR were determined in suspensions of hepatocytes at 0° and 37°C, or following rifampicin pretreatment, and in perfused rat liver. The remaining cells were homogenized and subfractionated using sucrose gradient centrifugation. Suspensions of 7.5 × 106 hepatocytes also were incubated with 10 μg/ml of toxin, solubilized in Triton X-100, and ultracentrifuged to pellet the detergent insoluble fraction (containing actin). Isolated rat livers were perfused with media containing [3H]-2HMC-LR and the uptake of radiolabel was determined. Sequential biopsy samples were collected for histologic examination. The remaining liver was homogenized and subcellular fractions prepared. Uptake of radiolabel was rapid in both cell suspension at 37°C and perfused liver; however, uptake in cell suspensions was reduced by about 50% at 0°C and by rifampicin (50 μg/ml) pretreatment. Hepatocyte necrosis was observed in isolated perfused livers 45 min after initiation of perfusion with [3H]-2HMC-LR. In both hepatocyte suspensions and perfused livers 65 to 77% of the radiolabel was in the cytosolic fraction. In the hepatocyte suspensions, 13 to 18% of the radiolabel was present in the plasma membrane/nuclear fraction with lesser amounts in the other fractions. Trichloroacetic acid treatment of cytosolic fractions indicated that in hepatocyte suspensions, 50-60% of the radiolabel was bound to cytosolic protein. Studies using the perfused liver confirmed that the majority of the radiolabeled MCLR (78-88%) was bound to cytosolic protein. These data suggest that the uptake of [3H]-2HMC-LR occurs primarily by an energydependent transport process involving the rifampicin-sensitive hepatic bile acid carrier and that once inside the hepatocyte, the toxin binds to a cytosolic protein(s).
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U2 - 10.1016/0041-0101(91)90053-T
DO - 10.1016/0041-0101(91)90053-T
M3 - Article
C2 - 1926162
AN - SCOPUS:0025772091
SN - 0041-0101
VL - 29
SP - 589
EP - 601
JO - Toxicon
JF - Toxicon
IS - 6
ER -