Brefeldin a inhibits the transcytotic vesicular transport of horseradish peroxidase in intrahepatic bile ductules isolated from rat liver

Authors

  • Antonio Benedetti,

    Corresponding author
    1. Istituto di Patologia Sperimentale and Clinica di Gastroenterologia, University of Ancona, School of Medicine, Ancona, Italy
    • Istituto di Patologia Sperimentale, C.P. 538, University of Ancona, 60100 Ancona, Italy
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  • Luca Marucci,

    1. Istituto di Patologia Sperimentale and Clinica di Gastroenterologia, University of Ancona, School of Medicine, Ancona, Italy
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  • Cristina Bassotti,

    1. Istituto di Patologia Sperimentale and Clinica di Gastroenterologia, University of Ancona, School of Medicine, Ancona, Italy
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  • Carlo Guidarelli,

    1. Istituto di Patologia Sperimentale and Clinica di Gastroenterologia, University of Ancona, School of Medicine, Ancona, Italy
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  • Anne Marie Jezequel

    1. Istituto di Patologia Sperimentale and Clinica di Gastroenterologia, University of Ancona, School of Medicine, Ancona, Italy
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Abstract

The fungal metabolite Brefeldin A (BFA) has become a valuable tool to address mechanisms of membrane transport in eukaryotic cells. The aim of the study was to investigate the action of BFA on the endocytic and transcytotic pathways in the biliary epithelium. Intrahepatic bile ductules were isolated from rat liver by collagenase digestion and mechanical separation of biliary tree from parenchymal tissue. Tissue remnants were first incubated in L-15 culture medium in absence or presence of BFA (10 or 20 μmol/L) or a BFA-inactive analog (B-36, 10 or 20 μmol/L) for 20 minutes at 37°C. They were then exposed to horseradish peroxidase (HRP) (10 mg/mL) for 3 minutes at 37°C and finally prepared for electron microscopy immediately (time 0) or after further 5,10, 15, 20, 60, or 120 minutes' incubation in HRP-free medium with or without BFA. In control cells, HRP was predominantly found in regularly shaped, spherical vesicles. In the presence of BFA but not of its analog, HRP was retained in a prominent tubular juxtanuclear network. Part of this network was labeled for thiamine pyrophosphatase (TPP), a Golgi enzyme marker. A morphometric analysis of HRP-containing structures was performed to quantify the intracellular distribution of HRP. In presence of BFA, the volume density (VD = % area) of HRP-containing structures in the basolateral region was not significantly different with respect to control cells at 0 (1.08 ± 0.11 vs. 1.32 ± 0.11) or 5 minutes, respectively (1.33 ± 0.19 vs. 1.40 ± 0.13). On the contrary, VD of HRP-containing structures in the apical region at 15 minutes decreased from 1.95 ± 0.19 in control cells to 1.12 ± 0.20 (P < .02) in BFA-treated cells. Most striking were the changes in VD of HRP-containing elements in the intermediate (juxtanuclear) area, showing a value of 0.45 ± 0.22 in control cells against 2.59 ± 0.50 in BFA-treated cells at 15 minutes (n = 10) (P < .01). Finally, taurocholic acid (TCA, 50 μmol/L) significantly increased the VD of HRP-labeled structures in the apical region only in the absence of BFA (2.22 ± 0.20, n = 5 vs. 1.14 ± 0.32 in BFA-treated samples at 5 minutes P < .02). These data suggest that in rat biliary epithelial cells, BFA does not interfere with fluid-phase endocytosis but inhibits the transcytotic vesicular pathway, as shown by the retention of HRP in the Golgi juxtanuclear area. These properties make BFA of interest for the study of intracellular mechanisms involved in bile ductular secretion. (HEPATOLOGY 1995; 22:194–201.)

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