BREFELDIN-A INHIBITS THE TRANSCYTOTIC VESICULAR TRANSPORT OF HORSERADISH-PEROXIDASE IN INTRAHEPATIC BILE DUCTULES ISOLATED FROM RAT-LIVER

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 du ctules were isolated from rat Liver by collagenase digestion and mecha nical separation of biliary tree from parenchymal tissue. Tissue remna nts were first incubated in L-15 culture medium in absence or presence of BFA (10 or 20 mu mol/L) or a BFA-inactive analog (B-36, 10 or 20 m u mol/L) for 20 minutes at 37 degrees C. They were then exposed to hor seradish peroxidase (HRP) (10 mg/mL) for 3 minutes at 37 degrees C and finally prepared for electron microscopy immediately (time 0) or afte r further 5, 10, 15, 20, 60, or 120 minutes' incubation in HRP-free me dium with or without BPA. In control cells, HRP was predominantly foun d in regularly shaped, spherical vesicles. In the presence of BFA but not of its analog, HRP was retained in a prominent tubular juxtanuclea r network. Part of this network was labeled for thiamine pyrophosphata se (TPP), a Golgi enzyme marker. A morphometric analysis of HRP-contai ning structures was performed to quantify the intracellular distributi on of HRP. In presence of BFA, the volume density CVD = % 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 regio n 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 change s in VD of HRP-containing elements in the intermediate (juxtanuclear) area, show ing 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). Finall y, taurocholic acid (TCA, 50 mu mol/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 inhibit s 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 s ecretion.