effect of brefeldin a on transcytotic vesicular pathway and bile secretion: a study on the isolated...

Effect of Brefeldin A on Transcytotic Vesicular Pathway and Bile Secretion: A Study on the Isolated Perfused Rat Liver

and Isolated Rat Hepatocyte Couplets

DOMENICO ALVARO,~ ANTONIO BENEDETTI,' ALESSANDRO GIGLIOZZI,' ADRIANO BINI,~ SONIA FURFARO,' CRISTINA BASSOTTI,2 TIZIANA LA ROSA,^ ANNE MARIE JEZEQUEL,3 AND LIVIO CAPOCACCIA'

This study investigated the effect of Brefeldin A (BFA) on the transcytotic vesicular pathway labeled with horseradish peroxidase (HRP) in both isolated rat hepatocyte couplets (IRHC) and the isolated perfused rat liver (IPRL). To evaluate the role of the transcytotic vesicular pathway on bile secretion, the effect of BFA on bile secretion in the P R L was then investigated. In the basolateral area of IRHC, BFA showed no effect on the density and percentage of area of HRP-labeled vesicles. However, HRP-labeled vesicles tended to accumulate in the juxtanuclear area of BFA-treated hepatocytes (P < .001 vs. controls). In the pericanalicular area, on the other hand, HRP-labeled vesicles were depleted compared with controls (P < .001). In keeping with these findings, although the early peak remained unchanged, BFA inhibited as much as 50%0 of the late peak of HRP excretion in bile, after a pulse load of HRP in the IPRL. Bile flow and the biliary secretion of bile salts (BS) and phospholipids were not modified by BFA in isolated livers perfused without BS in the perfusate or with 1 pmoY min taurocholate (TCA). In BFA-treated livers, peak bile flow and BS output decreased by 20% (P < .05 vs. controls) only when a 5 pmol TCA bolus was administered. In conclusion, this study demonstrates that BFA inhibits the transcytotic vesicular pathway in the liver. How- ever, BFA has no significant effect on bile secretion either in basal conditions or during perfusion with physiological amounts of BS. BFA slightly decreases bile flow and BS output only after an overload of BS, providing

Abbreviations: BS, bile salt; TCA, taurocholate; HRP, horseradish peroxidase; IPRL, isolated perfused rat liver; BFA, Brefeldin A; p-COP, 0-clatharin coat protein; MDCK, Madin-Darby canine kidney; IRHC, isolated rat hepatocyte couplets; bw, body weight; HEPES, N-[Z-hydroxyethyl]piperazine-N'-12- ethanesulfonic acid]; VD, volume density; TPPase, thiamine pyrophosphatase; TPP, thiamine pyrophosphate; KRB, Krebs-Ringer bicarbonate; LDH, lactate dehydrogenase; GOT, glutamic-oxaloacetic transaminase; DMSO, dimethyl sulfoxide.

From the 'I1 Department of Gastroenterology, University of Rome "La Sa- pienza" Rome; 'Department of Gastroenterology, University of Ancona, An- cona; and 'Institute of Experimental Pathology, University of Ancona, Ancona, Italy.

Received March 21, 1994; accepted August 10, 1994. Address reprint requests to: Domenico Alvaro, MD, Via Valsolda 45/i, 00141

Copyright 0 1995 by the American Association for the Study of Liver Rome, Italy.

Diseases. n 2 ~ o - ~ i 3 ~ ~ ~ 5 ~ z i o z - o o ~ ~ ~ 3 . o o ~ o

evidence against the physiological relevance of the transcytotic vesicular pathway in the process of bile formation. (HEPATOLOGY 1995;21:450-459.)

The transport of bile salts (BS) from blood to bile represents the driving force in the process of bile formation. The uptake of BS by hepatocyte basolateral membrane and their transport across canalicular membrane have been extensively investigated.' Much less is known of the mechanisms underlying the intracellular transport of BS.1-3

BS may cross the hepatocyte by a diffusion process of free or protein-bound moiety, a hypothesis supported by the rapid appearance of BS in bile after basolateral uptake. However, evidence suggests that BS intracellular transport may involve a vesicular pathway, which necessitates intact cytoskeleton components. '-' The microtubule inhibitor colchicine, for example, inhibits bile secretion stimulated by a taurocholate (TCA) load, although it has no effect on either basal bile secretion or the transport of tracer doses of TCA.4-6 Bile secretion is also inhibited by the microfilament binding toxin, phalloidin.6 Furthermore, morphological studies have indicated that the endoplasmic reticulum and the Golgi complex play a role in BS intracellular transport. BS analogues have been localized in smooth endoplasmic reticulum and Golgi s a c ~ u l e s . ~ ~ ~ BS antibodies bind to the saccules of the trans-Golgi complex during liver perfusion with the correspondent BS9 and a carrier- mediated BS transport has been described in micro- somes and in the Golgi apparatus.l0,''

In light of the above, it has been suggested that a cytosolic pathway carries BS to canalicular membranes under basal conditions and that during transport of BS load, such as that occurring in the postprandial phase, a vesicular pathway may be The relevance of the vesicular pathway in the intracellular transport of BS, and more specifically that involving the trans- Golgi complex, has been recently questioned because TCA and biliary lipid secretion are not influenced by monensin, a Na' ionophore that induces a disorganiza- tion of the Golgi complex, inhibits vesicle formation and protein secretion from the trans-Golgi, as well as

450

HEPATOLOGY Vol. 21, NO. 2, 1995 ALVARO ET AL 451

inhibits horseradish peroxidase (HRP) excretion in bile in the isolated perfused rat liver (IPRL).12

In most cells, Brefeldin A (BFA; Sigma Chemical, St. Louis, MO), a fungal isoprenoid antibiotic, causes a retrieval of the Golgi complex to the endoplasmic retic- ulum13-15 through a selective inhibition of the binding of a peripheral 110-kd protein (P-clathrin coat protein [P-COPI) to Golgi membranes, thus blocking the anterograde transport mediated by these vesicles. 13,15 In normal kidney cells, BFA induces the trans-Golgi net- work to fuse with early endosomes. This effect would appear not just to be limited to the Golgi apparatus because the traffic between endosomes and lysosomes is also involved.16 Regarding the process of transcytosis, in Madin-Darby canine kidney (MDCK) cells BFA inhibits polymeric immunoglobulin A receptor- mediated transcyt~sis, '~ whereas, transcytosis of ri- cin,17 HRP,17 and transferrin receptor'' is stimulated. In isolated hepatocytes, BFA selectively blocks the transport of secretory proteins from the endoplasmic reticulum to the Golgi complex, without any effect on endocytosis or on protein degradation.lg Because of these properties, BFA has become a valuable tool in the study of membrane transport in the secretory pathway because the mechanism of the drug appears different from other substances, such as microtubule or microfilament inhibitors, monensin, or weak bases.

The present study aimed to investigate the role of the transcytotic vesicular pathway in the process of bile formation. We thus evaluated the effect of BFA on the following: (1) the transcytotic vesicular pathway, labeled by the fluid phase marker HRP, in both isolated rat hepatocyte couplets (IRHC) and in IPRL; and (2) the basal- and TCA-stimulated bile secretion in the IPRL. It emerged from this study that BFA inhibits the transcytotic vesicular pathway, labeled by HRP, with no significant effects on bile flow and BS secretion, providing evidence against a role of vesicular transcytosis in the process of bile formation.

MATERIALS AND METHODS

Male Sprague-Dawley rats, CD strain (Charles River, It- aly) weighing 200 to 270 g, received humane care. They were maintained on a GLP diet in pellets (Nossan, Italy) and were given water ad Libitum. The animals were housed in plastic cages, with a wire mesh bottom providing isolation for the hygienic bedding, and were exposed to a cycle of automati- cally controlled light. Study protocols were in compliance with our institution's guidelines.

Purified HRP (type 11), BFA, sodium TCA, and bovine serum albumin (fraction V) were obtained from Sigma Chemi- cal (St. Louis, MO). I4C-TCA (specific activity 51 mCi/mmol) was purchased from New England Nuclear (Boston, MA). All other chemicals were of the highest quality commercially available.

Hepatocyte Couplet Isolation and Culture

The isolation of hepatocyte couplets from the rat liver was performed as previously described." After anesthesia (rat body weight [bw] 200 to 250 g) with sodium thiopental (Far-

motal, Farmitalia Carlo Erba, Nerviano, Milan, Italy; 100 mgkg bw, intraperitoneal), the portal vein was cannulated with a 16-gauge cannula and perfused in. situ for 10 minutes with buffer A (NaC1 142.0 mmol/L; KC1 6.7 mmoVL; glucose 5.6 mmol/L; and N-[2-hydroxyethyl1piperazine-Nf-[2-ethanesulfonic acid [HEPES] 10 mmoVL; pH 7.45) a t 37°C. The papilliform lobe was removed and the vena cava superior was cannulated. The liver was then perfused with Ca++, Mg++ containing L-15 culture medium supplemented with collagen- ase A (360 U L ) , HEPES (25 mmol/L), and bovine serum albumin (1 g L ) (Buffer B), for 10 minutes at 37°C. When the capsule began to detach, the perfusion was stopped and the liver was immersed in ice cold modified L-15 culture medium.*' The cells were released by gentle combing into 50 mL of L-15 culture medium on a Petri dish, and were then filtered and pelletted twice, as previously described.'l

The initial cell viability determined by trypan blue exclu- sion varied considerably, but was usually >80%. After a par- tial purification, obtained as previously described," the viability ranged from 87% to 92%.

Aliquots of 1.5 mL of the cell pellet were resuspended in 30 mL of L-15 culture medium, and were supplemented with 10% fetal calf serum, pH 7.40. The suspension was then lay- ered into plastic Petri dishes (10 mudish), containing glass coverslips (3 x 0.5 cm), precoated with the extracellular bio- matrix Matrigel (Collaborative Research Inc., Bedford, MA), and diluted 1:l with L-15 HEPES medium. Hepatocytes were incubated at 37°C for 2 to 6 hours, during which time they formed a subconfluent monolayer on the glass coverslips with a density of approximately lo9 cells/mm2.

Morphological Studies

After 3 to 4 hours in L-15 culture medium, hepatocytes were incubated in L-15 medium containing HRP (10 mg/mL) at 37°C for 3 minutes, washed twice with L-15 medium a t 37"C, and fixed immediately (time 0; n = 5 ) or after incubation in HRP-free L-15 for a further 5 (time 5; n = 51, 10 (time 10; n = 5), 15 (time 15; n = 51, 20 (time 20; n = 51, or 60 minutes (time 60; n = 5 ) at 37°C.

In five paired experiments for each time interval, BFA was added to the culture medium (10 pmol/L) 20 minutes before HRP incubation and was then present throughout the experiment.

Demonstration of HRP Reaction Products by Electron Microscopic Cytochemistry

After incubation, the cells were fixed with 2.5% glutaralde- hyde-0.8% paraformaldehyde in 0.1 mol/L cacodylate buffer (pH 7.4) for 30 minutes a t 4"C, washed, and incubated with 0.4 mg/mL 3,3'-diaminobenzidine tetrahydrochloride (DAB, Sigma) and 0.01% Hz02 in 0.05 Tris-HC1 buffer (pH 7.6). After rinsing in buffer, they were postfixed with 1% buffered OsOI and embedded in EpodAraldite (Fluka Chemica, Mi- lan, Italy).23

Demonstration of TPPase Reaction Sites

The activity of thiamine pyrophosphatase (TPPase), an en- zyme preferentially localized in the cisternae of the Golgi complex,24 was demonstrated with cesium chloride according to Angermuller and Fahimi,25 on isolated hepatocytes incubated in HRP-free medium with or without BFA. At various time intervals, the samples were fixed as above. After rinsing in cacodylate buffer, the cells were placed in reaction medium

452 ALVAFiO ETAL HEPATOLOGY February 1995

(thiamine pyrophosphate [TPP] 2 mmol/L, MnC12 5 mmol/L, cesium chloride 4 mmol/L, and sucrose 5% in 0.08% m o m Tris maleate buffer, pH 7.2) 2 x 30 minutes at 37°C with gentle shaking. After rinsing in cacodylate buffer, postfixa- tion in buffered osmium tetroxide and inclusion in EpodAral- dite was performed, as indicated above.

Morphometric Analysis Morphometric studies were performed blindly on ultrathin

sections by two independent observers. The reliability of the morphometric analysis was evaluated as “overall agree- ment,” i.e., the proportion of pictures for which observers agreed on quantitative data. The sections were taken from three different blocks from each sample and examined under the electron microscope after staining with lead citrate. Four to six pictures of each couplet, including juxtacanalicular and peripheral cytoplasm, were taken at an initial magnification of ~ 7 , 0 0 0 and printed at a final magnification of X17,500. On each print, a 1-pm wide area beneath the peripheral or the pericanalicular membranes and an intermediate (juxtanuclear) area were defined. The volume density (VD, % area) of HRP-containing structures, with a diameter of 100 nm or more, present in these cytoplasmic areas, was measured by point-counting technique.

Multivesicular bodies were defined as organelles, 0.2 to 0.5 pm in diameter, containing clumps of electron-lucent vesicles, and occasionally showing small tubular appendages that were also positive for HRP. Finally, HRP-containing structures were classified as “tubular” when their length was more than twice their width.

IPRL System

Animal surgery, liver isolation and perfusion technique, and solution preparation were performed as previously de- s ~ r i b e d . ~ ~ , ~ ~ Briefly, under sodium pentobarbital anesthesia (rat bw, 250 to 270 g), the bile duct was cannulated with a PE-10 tubing (Clay Adams, Parsippany, NJ). The pancreatic- duodenal branch of the portal vein was ligated and the portal vein was cannulated with a 14-gauge Teflon intravenous catheter (DuPont, Bad Homburg, Germany). The liver was immediately perfused a t a constant flow rate of 25 mumin (37°C) with COz/Oz (5%/95%), oxygenated Krebs-Ringer bicarbonate (KRB) buffer containing 5.5 mmol/L of glucose, and 200 U of heparin per 100 mL. The diaphragm was opened, the suprahepatic vena cava cannulated, and the liver transferred into a heated perfusion chamber and then perfused in a sin- gle-pass system with C02/02 (5%/95%) oxygenated KRB buffer (pH 7.4), containing 5.5 mmol/L glucose at a flow rate of 40 mL/min. The perfusion buffer was continuously gassed by using an artificial lung, maintained at 37”C, and continuously filtered through a 2-pm filter placed in front of the portal cannula. The viability of the perfused liver was demonstrated by the following: (1) monitoring portal pressure (aver- age 10 cm HzO), with no significant changes during 90 minutes of perfusion; (2) measuring lactate dehydrogenase (LDH) and glutamic-oxaloacetic transaminase (GOT) release in the perfusate by routine methods, with no significant increase in the perfusate samples collected a t 10, 30, 60, and 90 minutes of perfusion; and (3) measuring O2 consumption (Schott-Seraete, Italscientifica, Geneva, Italy), stable a t 10, 30, 60, and 90 minutes of perfusion.

BFA was dissolved in dimethyl sulfoxide (DMSO) (stock solution) and then diluted (1:10,000, volume to volume) in the perfusate to a final concentration of 10 pmol/L (final DMSO

concentration in the perfusate 0.01%). The same amount of DMSO was also dissolved in the perfusate of control experiments.

HRP (25 mg) was dissolved in 2 mL of KRB and injected as a 1-minute bolus. Two- to ten-minute bile samples were collected in preweighed tubes and the amount was evaluated gravimetrically, HRP and 14C-TCA excretion in bile was cal- culated by correcting for the volume of the biliary cannula (20 pL) and the “dead space” of the biliary tree was considered as 2.3 p u g of liver.28

Analytical Methods

HRP was measured in 5-pL bile samples by determining the rate of oxidation of 4-aminoantipyrine at 510 11111.’’ Total biliary BS were measured by 3a-hydroxysteroid dehydroge- n a ~ e , ~ ’ choline containing phospholipids by the choline oxi- dase method.31 Radioactivity in bile was measured in dupli- cate by a Minaxi Tricarb 4000 Series liquid scintillation counter (Packard Instrument, Downers Grove, IL). Correc- tions for quenching were made by external calibration. The administered radioactivity was measured in quadruplicate in 10-pL aliquots of the medium in which 14C-TCA was dissolved.

Experimental Design

HRP was injected as a 1-minute bolus, close to the portal vein, 50 minutes after the beginning of BFA perfusion. BFA perfusion was then continued until the end of the experiments.

To evaluate the effect of BFA on bile secretion, three different experimental designs were performed.

Basal Bile Secretion. IPRLs perfused without BS (KRB only) were exposed to 10 pmol/L of BFA from 20 to 90 minutes after starting the experiment. The effect of BFA on bile flow, BS, and phospholipid biliary secretion was compared with controls.

TCA-Stimulated Bile Secretion. IPRLs were perfused with 10 p m o n BFA from 0 to 90 minutes. After 50 minutes of BFA exposure, livers were perfused with 1 pmoYmin TCA until the end of the experiment (TCA concentration in the perfusate = 25 pmoVL). The effect of BFA exposure to the TCA stimulated bile flow, BS, and phospholipid biliary secretion compared with controls.

Bile Secretion Stimulated by a Pulse Load of TCA. IPRLs were perfused with 10 pmol/L of BFA from 0 to 90 minutes. After 50 minutes of BFA exposure, a 1-minute bolus of 5 pmol TCA (TCA concentration in the perfusate = 125 pmol/L) was injected together with a tracing dose of I4C-TCA (0.5 pCi). Bile flow, total BS secretion, and total radioactivity secreted in bile were measured and compared with controls.

Statistical Analysis

All data were analyzed by the paired or unpaired Student’s t-test, as appropriate. A value of P < .05 was considered statistically significant.

RESULTS HRP Cytochemistry in IRHC

Figure 1 shows the results of morphometric analysis of HRP uptake and distribution, at increasing time intervals, after 3 minutes of incubation in HRP-containing medium, with or without BFA. At time 0 min-

ALVARO ET AL 453 HEPATOLOCY Vol. 21, NO. 2, 1995

A CONTROLS 4.0 1

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FIG. 1. VD of HRP-containing structures in the basolateral, intermediate (juxtanuclear), and apical areas of hepatocyte couplets 0, 5, 10, 15, 20, and 60 minutes after exposure to HRP (time 0 ) in the absence (A) or the presence (B) of BFA. BFA (10 pmoVL) was added 20 minutes before HRP incubation and then was present throughout the experiment. (A) a progressive increase in labeling of the apical region is visible, with significant differences at 10, 15, and 20 minutes with respect to the 5-minute value (':-I' < ,011. In the basolateral region, the VD progressively decreases with time either in the absence (A) or in the presence (B) of BFA. (B) When the samples are treated with BFA the maximal progressive increase in labeling with HRP is evident in the intermediate region, particularly a t 10 and 15 minutes with respect to the 5-minute value ("P < .01; ""P < ,001). Contrary to what we observed in Fig. lA, no progressive increase of HRP labeling is evident with time in the apical area (IB).

Utes and 5 minutes, in control cells exposed to HRP, the electron-dense reaction product was distributed mainly in the peripheral cytoplasm and was contained in small pinocytic vesicles, approximately 0.10 pm in diameter, in larger vesicles ranging from 0.20 to 0.25 pm, and in short tubular structures with a uniform thickness of 0.02 p8m and a length of 0.10 pm (Fig. 2). In this area, the VD of HRP-containing elements peaked at 5 minutes (1.25 ? 0.05% of cytoplasm) (Fig. 1A). In the juxtacanalicular cytoplasm, the VD of HRP- containing structures increased progressively and peaked at 20 minutes (3.23 ? 0.25%). In this area, most of the HRP was associated with tiny HRP-positive vesicles, clustering inside larger multivesicular bodies, 0.30 to 0.50 pm in diameter, and usually distributed along the inner aspect of the limiting membrane of the multivesicular bodies (Fig. 2). Few HRP-positive vesicles were observed in the intermediate area, where the peak appeared after 5 minutes (1.80 & 0.20%; Fig. 1A).

In cells pretreated with BFA, the amount and distribution of HRP-positive structures observed in the peripheral cytoplasm were not significantly different from those observed in controls. In contrast, a marked decrease of HRP-containing elements occurred up to 60 minutes in the pericanalicular cytoplasm with a VD of 0.80 F 0.20% (P < .05 vs. controls) and 0.82 i 0.38% (P < .001 vs. controls) a t 15 and 20 minutes, respectively (Fig. 1B). Especially striking in BFA-pretreated hepatocytes was the peculiar distribution of HRP-containing elements in the intermediate area. The pattern was qualitatively and quantitatively different with respect to controls. At 15 and 20 minutes, in fact, the VD of HRP-positive elements in the intermediate area was 0.58 5 0.36%1, 2.49 5 0.34%, and 1.97 7t 0.18% a t time 0, 15, and 20 minutes, respectively (P < .001 vs. con-

trols). In addition, HRP-containing elements appeared with a characteristic configuration of finely tubular, Y- shaped or horseshoe-shaped structures, 0.02 pm wide and up to 4 pm long, situated in the intermediate areas, and extending a t times a t short distances from the nuclear membrane (Fig. 3).

TPPase-Reactive Sites The reaction product was observed in limited areas of

the cytoplasm in groups of horseshoe-shaped cisternae similar to Golgi stacks, sometimes with bulbous ends (Fig. 4). The staining occasionally showed two or three reactive sites in the cell, but no reaction product was observed either in juxta-Golgi vesicles or in the endoplasmic reticulum. The pattern of reaction remained unchanged regardless of whether the cells were left in L-15 for 3 minutes o r longer (up to 60 minutes) or whether they had been exposed (Fig. 4B; 20-minute incubation with 10 pmol/L of BFA) or not to BFA (Fig. 4A).

Effect of BFA on HRP Biliary Excretion in the IPRL IPRL perfused without BS (KRB only) was exposed

to 10 pmol/L of BFA from 0 to 90 minutes (n = 5). A 25-mg HRP bolus was administered 50 minutes after the onset of BFA administration and the excretion in bile was measured in comparison with IPRL perfused without BFA (n = 5). HRP was excreted (Fig. 5) in bile in two major peaks, an earlier peak reaching 1.92 t 0.69 ng/min/g of liver a t 4 minutes after HRP injection and a later and larger peak with a maximum of 1.60 7t 0.19 ng/min/g of liver a t 20 minutes after HRP bolus administration. The first peak of HRP excretion in bile was unchanged by BFA treatment (2.0 f 0.50 ng/min/ g of liver at 4 minutes after HRP bolus). In contrast, the later peak was decreased by 50% because of BFA

454 ALVARO E T A L HEPATOLOGY February 1995

FIG. 2. The micrograph shows the aspect of a hepatocyte couplet, 15 minutes after the exposure to HRP. The dense product of reaction is present in the pericanalicular cytoplasm in numerous small vesicles, approximately 100 nm in diameter (small arrows), and in larger multivesicular bodies, approximately 300 nm in diameter (large arrows). The microvilli surrounding the lumen (L) of the canaliculus and the junctional complexes between hepatocytes (arrowhead) are well preserved. M, mitochondria; P, peroxisomes. (Original magnification ~ 1 7 , 5 0 0 ; bar = 1 pm.) (Inset) High-power view of the labeled juxtacanalicular components; in 100-nm vesicles (small arrows) as in multivesicular bodies (large arrows), the dense reaction product tends to accumulate along the inner aspect of the limiting membrane. Short (100 nm long), tubular elements (arrowhead), uniformly labeled, are also often observed, always closely apposed, occasionally connected to multivesicular bodies. (Original magnification ~30,000; bar = 0.5 pm.)

exposure (0.80 & 0.30 ng/min/g of liver a t 20 minutes after HRP injection; P < .01).

Effect of BFA on Basal Bile Secretion The effect of BFA on bile flow and BS biliary secretion

of IPRL perfused without BS (KRB only) is reported in Fig. 6. BFA (10 pmoVL; n = 7) added in the perfusate for 70 minutes (from 20 to 90 minutes) does not change bile flow nor BS secretion in bile with respect to controls (n = 7). Phospholipid biliary secretion also remained unchanged. This decreased from 1.32 ? 0.20 (10 minutes) to 0.82 ? 0.15 (90 minutes) nmol/min/g of liver in controls and from 1.28 2 0.22 (10 minutes) to 0.88 2 0.12 (90 minutes) nmol/min/g of liver in BFA-

FIG. 3. The figure shows the distribution of HRP in cells exposed to BFA, 15 minutes after treatment with HRP. Numerous tubular- shaped elements, filled with the dense reaction product, are observed throughout the cytoplasm (arrows). The tubules vary from 0.2 to 3 pm in length with a diameter of about 50 nm. No 100-nm vesicles are present in the field. The morphology of the bile canaliculus (L) is normal. Compare with Fig. 1, same magnification. N, nucleus; M, mitochondria; P, perixosomes. (Original magnification X 17,500; bar = 1 pm.)

FIG. 4. Histochemical demonstration of TPPase activity in BFA- treated (Fig. 4B; 10 pmol/L, 20-minute incubation) or in paired controls (Fig. 4A). The reaction product of enzymatic activity is exclu- sively present in the horseshoe-shaped cisternae of the Golg apparatus in the juxtanuclear area (arrows). The enzymatic activity is distributed in a similar fashion in untreated (A) and BFA-treated (B) cells. N, nucleus; M, mitochondria. (Original magnification X24,OOO; bar = 0.5 pm.)

HEPATOLOGY Vol. 21, NO. 2, 1995 ALVARO ET AL 455

perfused livers without significant differences. These findings indicate that in basal conditions BFA has no effect on bile flow and on the biliary secretion of BS and phospholipids.

Effect of BFA on TCA-Stimulated Bile Secretion The effect of BFA on TCA-stimulated bile flow and

BS biliary secretion is shown in Fig. 7. TCA perfused at a flow rate (1 pmol/min), corresponding to the portal flow of BS in a fed rat, showed a similar choleretic effect both in BFA-treated livers (n = 5) and in controls (n = 5) (Fig. 7) . Bile flow, in fact, increased from 0.90 t 0.12 to 1.34 t 0.17 pUmin/g of liver 10 minutes &r TCA administration in BFA-treated rats and from 0.84 t 0.10 to 1.39 & 0.16 pIJmin/g of liver in controls. Thereafter bile flow remained stable with no differences between BFA-treated and control IPRLs.

Nor did BS biliary secretion differ between BFA-treated livers and controls (Fig. 7). BS secretion, in fact, increased from 6.9 t 1.1 to 54.1 2 7.0 nmol/min/g of liver at 10 minutes after TCA administration in BFA-treated livers and from 7.2 5 1 to 60 t 12 nmol/min/g of liver in controls. BS biliary secretion then remained stable until the end of the experiments.

Phospholipid biliary secretion was stimulated by TCA

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FIG. 5. Effect of BFA on HRP biliary excretion in the IPRL. IPRL perfused without BS (KRB only) was exposed to 10 ImoVL of BFA from 0 to 90 minutes (filled circles; n = 5). A 25-mg HRP bolus was administered, close to the portal vein, 50 minutes after the onset of BFA administration and the excretion in bile measured in comparison with IPRL perfused without BFA (open circles; n = 5). The figure shows that the first peak of HRP excretion in bile was unchanged by BFA treatment, whereas the later peak was decreased by 50% because of BFA exposure. Data are means t SD. *P < .04; **P < .01.

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FIG. 6. Effect of BFA on basal bile secretion. In IPRL perfused without BS (KRB only), BFA (filled circles; n = 7) added in the perfusate for 70 minutes (from 20 to 90 minutes) changes neither bile flow (upper panel) nor BS biliary secretion (lower panel) with respect to controls (open circles; n = 7). Data are means 2 SD.

to the same extent in BFA-treated IPRL as in controls. Ten minutes &r TCA administration, phospholipid secretion in bile increased to 6.43 2 1.1 nmol/min/g of liver in BFA-pehsed livers and to 6.92 -+ 1.2 nmol/min/g of liver in controls and remained stable throughout the experiment (data not shown).

These findings indicate that BFA has no effect on bile secretion stimulated by physiological concentration of TCA.

Effect of BFA on Bile Flow and BS Output AfZer a Pulse Load of 5 pmol of TCA

To evaluate whether BFA could affect bile flow and BS biliary secretion after a BS overload, a 1-minute bolus of 5 ymol of TCA, together with a tracer dose of

456 ALVARO ET AL

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HEPATOLOGY February 1995

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FIG. 7. Effect of BFA on TCA-stimulated bile secretion. IPRL were perfused with 10 pmoVL of BFA from 0 to 90 minutes (filled circles; n = 5). Fifty minutes after the onset of BFA administration, IPRL was exposed to TCA (1 pmollmin), and its effect on bile flow (upper panel) and BS (lower panel) biliary secretion compared with controls (open circles; n = 5). TCA showed a similar choleretic effect in BFA-treated livers as in controls. The BS secretion in bile did not show any differences between BFA-treated and control livers. Data are means 5 SD. In some time points, SD is covered by the symbols.

14C-TCA, was administered 50 minutes after the onset of 10 yrnol/L of BFA perfusion (Fig. 8). The choleretic response to a TCA load was decreased approximately 20% by BFA. In fact, 4 minutes after the first TCA pulse, bile flow increased from 0.86 2 0.10 to 1.35 -C 0.17 in BFA-treated livers (n = 5) and from 0.79 2 0.06 to 1.61 5 0.18 in controls (n = 5; P < .05).

In keeping with the profile of bile flow, during the first 6 minutes after a 5-ymol bolus of 14C-TCA (Fig. 8), radioactivity output in bile was significantly lower (P < .05) in BFA-treated livers (n = 5) compared with controls (n = 5). The cumulative recovery of the administered radiolabel a t the end of the experiment (i.e., 40

minutes after 14C-TCA administration) did not differ between BFA-treated rats and controls (53.7 ? 6.2% vs. 54.7 ? 7.1%). However, during the first 6 minutes, the cumulative recovery of radiolabel in bile was significantly lower in BFA-treated livers (P < .04) compared with controls, whereas the radiolabel recovered from 8 to 40 minutes was significantly higher in BFA- treated livers compared with controls (P < .01). The biliary secretion of BS reached a peak of 83 t 12 nmoV midg of liver 4 minutes after TCA administration in controls and was decreased approximately 20% by BFA treatment (67 ? 10 nmollmidg of liver; P < .05). These

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FIG. 8. Effect of BFA on bile flow and BS biliary secretion stimulated by a pulse load of taurocholate. A 5-pmol bolus of TCA (injected in 1 minute), together with a tracer dose of I4C-TCA, was administered 50 minutes after the onset of 10 pmoVL of BFA perfusion (filled circles; n = 5). Peak bile flow (upper panel) and peak radioactivity (lower panel) were decreased approximately 20% by BFA treatment (n = 5) with respect to controls (open circles; n = 5). However, the total radioactivity recovered in bile was unchanged by BFA, indicat- ing a delay in BS secretion. Data are mean t SD. In some points SD is covered by the symbols. "P < .05; **P < .02.

HEPATOLOGY Vol. 21, No. 2, 1995 ALVARO ET AL 457

findings indicate a delay in the biliary excretion of BS after a BS overload.

DISCUSSION Very little is known about the role of the transcytotic

vesicular pathway in the process of bile formation. This pathway has been estimated to contribute approximately 10% to basal bile flow.' A number of studies have demonstrated the importance of this pathway in the biliary excretion of secretory proteins and immunoglobulin A.1,32 A role of this pathway in the intracellular transport of organic anions, biliary lipids, and BS in BS-loaded hepatocytes has also been sugge~ted. ' .~ More recently, it has been shown that vesicular transport might well be involved in the targeting and insertion of apical membrane transport proteins, such as the C1-/ HCO, exchanger33 or the canalicular BS transporter, into the canalicular membrane.34 Characterization of this pathway has been performed mainly with morphological studies using electron-dense markers, such as HRP or immunoglobulin A, or by evaluating the biliary output of HRP as a marker of the excretory phase of the vesicular transport. Following this approach, it has been reported that the vesicular transport is regulated at the intracellular level by both CAMP^^ and is impaired in experimental c h o l e ~ t a s i s , ~ ~ and is stimulated by hydrophilic BSS.""'"~

HRP is transported in different steps, including the following: (1) a basolateral uptake by endocytosis; (2) an intracellular transport by a direct or an indirect pathway, where HRP-labeled vesicles can interact with lysosomes; and (3) a fusion of vesicles with the canalicular membrane followed by exocytosis. When HRP is pulse-loaded in the isolated liver, such as in the present study, the appearance in bile occurs in two peaks. The first early peak is thought to represent a paracellular diffusion,39 although a contribution from a fast vesicular colchicine-insensitive transcytotic transport has been recently suggested."* The second, late excretory peak is mediated by a colchicine-sensitive vesicular

In the present study, experiments were performed to evaluate the effect of BFA on the transcytotic vesicular pathway labeled by the fluid phase marker HRP. The transport of HRP was evaluated by quantitative mor- phometry in IRHC and its biliary excretion was analyzed in the IPRL. Thereafter, to investigate the role of transcytosis in bile secretion, the effect of BFA on bile flow, BS, and phospholipid biliary secretion in the IPRL was investigated. We demonstrated that in isolated hepatocyte couplets BFA blocks the anterograde transport of HRP-labeled vesicles. The uptake of HRP was not influenced by BFA, as the number and shape of HRP-labeled structures in the basolateral area were similar, regardless of the presence or absence of the drug. On the contrary, BFA significantly inhibited the anterograde progression of HRP-labeled structures, which tended to accumulate in the juxtanuclear area. In this area, the tubular retention of HRP in the inter-

transport.:35>:38,39

mediate cytoplasm and the blockade of the normal mi- gration of HRP toward the canaliculus were evident. The configuration of branched tubular structures was identical to that reported in pancreatic islet B cells treated with BFA."

Consistent with our findings on isolated hepatocyte couplets, BFA inhibited, by approximately 50%, the second excretory peak of HRP in the IPRL. Based on our findings in isolated hepatocyte couplets, an impair- ment of HRP uptake by the isolated liver may be ex- cluded. This is in keeping with previous studies showing that endocytosis is not influenced by BFA, the site of action being at an intracellular l e ~ e l . ' ~ - l ~ The lack of effect of BFA on the first early peak of HRP excretion in bile suggests that the drug does not interfere with the paracellular permeability andor the fast vesicular pathway of HRP excretion in bile. On the contrary, the inhibitory effect on the second peak of HRP excretion in bile confirms that the effect of BFA is the same in the isolated liver and in isolated hepatocytes. Our findings, recently demonstrated in bile duct fragments are in contrast with previous reports on MDCK cells, where HRP transcytosis appears to be unaffected" or stimu- lated17 by BFA. However, a variable effect of BFA in different cell types was also demonstrated concerning the morphology of the Golgi n e t ~ o r k . ~ ~ , ~ " ' ~ , ~ ~ This variability also applies to receptor-mediated transcytosis. In MDCK cells in fact, transcytosis of immunoglobulin A appears to be inhibited by BFA without modifying the Golgi ~rganization, '~ whereas transcytosis of the transferrin receptor is stimulated. '* These discrepancies might indicate the existence of multiple transcytosis pathways working in parallel, or, on the other hand, that BFA affects the sorting of receptor into transcytotic vesicles rather than the actual transcellular movement of v e ~ i c l e s . ~ ~ ~ ' ~ However, this latter hypothesis would not fit in, with the inhibitory effect on fluid phase transcytosis observed in the present study.

In spite of the clear inhibitory impact on the transcytotic vesicular pathway, both in isolated hepatocytes and in the liver, bile secretion was largely unaffected by BFA. Bile flow and the biliary secretion of BS and phospholipid were not influenced by BFA in the IPRL perfused without BS in the perfusate (basal bile secretion). Nor did BFA show any effect when bile secretion was stimulated by TCA continuously perfused at a concentration (25 pmoVL = 1 pmol/min perfusion rate) close to the physiological BS concentration in portal blood of fed rats.49 In these experimental conditions, TCA showed the same choleretic effect in BFA-treated livers and in control livers. The BS and phospholipid secretion in bile were also similar. At the same concentration, TCA has been previously shown to significantly stimulate transcytosis in the IPRL.3* Finally, the effect of BFA on bile secretion stimulated by a pulse load of 5 pmol of TCA was evaluated, This amount is higher than transport maximum in the rat44 and the corresponding concentration in the perfusate (125 pmol/L) is higher than the maximal concentration of BS in the

458 ALVARO ET AL

portal blood of fed rats (77 ~ m o l l L ) . ~ ~ TCA was injected, together with a tracer dose of 14C-TCA, to evaluate more accurately the kinetics of secretion in bile. Under these conditions of BS overload, BFA inhibits by 20% the bile flow and the peak BS output. However, the total amount of radiolabel recovered in bile remained unchanged, suggesting that during BS overload, BFA slightly delayed TCA transport through the hepatocyte. The lack of an inhibitory effect of BFA is a new argument against a significant role of vesicular intracellular transport of BS. Rather, our findings suggest a tran- cytosolic BS diffusion process, a mechanism recently supported by the evaluation of intracellular transit time of fluorescent BS analogue^.^^

Basal and TCA-stimulated phospholipid biliary secretion were not influenced by BFA. Phospholipids are secreted in native bile, in the form of vesicles coupled with BS secretion by a virtually unknown mechanism. It has been suggested that BSs, during their intracellular transport, induce the formation of vesicles targeted to the canalicular membrane by a microtubular-dependent process and then are secreted in the canalicular lumen.46 Alternatively, phospholipid vesicles might be formed by a “detergent-like” effect exerted by BS in the bile canalicular l ~ m e n . ~ ~ , ~ ~ The lack of a BFA effect on either basal or TCA-stimulated phospholipid secretion is a novel argument favoring this latter hypothesis.

A number of studies have shown that BFA affects the structure and/or the function of the Golgi appara-

In 24-hour cultured hepatocytes, BFA blocks the transport of secretory proteins from the endoplasmic reticulum to the G ~ l g i . ~ ~ , ~ ~ This has been reported to occur without any major morphological al- terationlg or with a disassembly of the Golgi complex,41 a variability that seems to depend on incubation conditions. In hepatoma cells, BFA induced an abnormal distribution of TPPase-reaction product.42 We found no abnormalities in the Golgi morphology or in the distribution of TPPase reaction product in freshly isolated hepatocytes incubated with BFA. These discrepancies could be caused by a different metabolism of BFA in freshly isolated hepatocytes with respect to hepatoma cells or 24-hour cultured hepatocytes where marked phenotypic changes of drug-metabolizing enzymes are known to occur.49

In conclusion, this article demonstrates that BFA inhibits HRP-labeled transcytosis in both IRHC and IPRL, whereas bile secretion and BS transport were largely unaffected. This indicates that the transcytotic vesicular pathway plays a minor physiological role in the process of bile formation and BS transport. This does not exclude the possibility that important biliary components, such as secretory proteins, could be secreted by this pathway. The lack of a significant effect of BFA on bile secretion and BS transport is in keeping with findings obtained for monensin, l2 but differs from the effect exerted by microtubular (colchicine) or microfilament (phalloidin) inhibitors. Given the complexity of vesicular transcytosis, these apparent discrepancies

tus. 16-l9,41,42

HEPATOLOGY February 1995

may be explained by a specific effect on different pathways of a more general and to date unknown process.

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REFERENCES

Nathanson HM, Boyer JL. Mechanism and regulation of bile secretion. HEPATOLOGY 1991;14:551-566. Crawford JM, Gollan JL. Transcellular transport of organic anions in hepatocytes: still a long way to go. HEPATOLOGY

Erlinger S. Role of intracellular organelles in the hepatic transport of bile acids. Biomed Pharmachother 1990;44:409-416. Crawford JM, Berken CA, Gollan JL. Role of the hepatocyte microtubular system in the excretion of bile salts and biliary lipid: implications for intracellular vesicular transport. J Lipid Res 1988;29:144-156. Crawford JM, Crawford AR, Strahs DCJ. Microtubule-dependent transport of bile salts through hepatocytes: cholic vs. tauro- cholic acid. HEPATOLOGY 1993;18:903-911. Dubin M, Maurice M, Feldmann G, Erlinger S. Influence of colchicine and phalloidin on bile secretion and hepatic ultrastruc- ture in the rat: possible interaction between microtubules and microfilaments. Gastroenterology 1980;79:646-654. Suchy FJ, Balistreri WF, Hung J , Miller P, Garfield SA. Intracel- Iular bile acid transport in rat liver as visualized by electron microscope autoradiography using a bile acid analogue. Am J Physiol 1983;245:G681-G689. Kitamura T, Gatmaitan Z, Arias IM. Serial quantitative image analysis and confocal microscopy of hepatic uptake, intracellular distribution and biliary secretion of a fluorescent bile acid analog in rat hepatocyte couplets. HEPATOLOGY 1990;12:1358-1364. Lamri Y, Roda A, Dumont M, Feldmann G, Erlinger S. Immuno- peroxidase localization of bile salts in rat liver cells: evidence for a role of the Golgi apparatus in bile salt transport. J Clin Invest

Reuben A, Allen RM. Taurocholate transport by rat liver micro- somes [Abstract]. Gastroenterology 1988;94:A584. Simion FA, Fleisher B, Fleisher S. Two distinct mechanisms for taurocholate uptake in subcellular fractions from rat liver. J Biol Chem 1984;259:10814-10822. Reynier MO, Hashieh IA, Crotte C, Carbuccia N, Richard B, Gerolami A. Monensin action on the Gole complex in perfused rat liver: evidence against bile salt vesicular transport. Gastro- enterology 1992;102:2024-2032. Doms RW, Russ G, Yewdell JW. Brefeldin A redistributes resi- dent and itinerant Golgi proteins to the endoplasmic reticulum. J Cell Biol 1989;109:61-72. Hunziker W, Whitney JA, Mellman I. Selective inhibition of transcytosis by Brefeldin A in MDCK cells. Cell 1991;67:617- 627. Orci L, Tagaya M, Amherdt M, Perreley A, Donaldson JG, Lip- pincott-Schwartz J , Klausner RD, et al: Brefeldin A, a drug that blocks secretion, prevents the assembly of non-clathrin-coated buds on Golgi cisternae. Cell 1991;64:1183-1195. Lippincott-Schwartz J , Yuan L, Tipper C, Amherdt M, Orci L, Klausner RD. Brefeldin A’s effects on endosomes, lysosomes and the TGN suggest a general mechanism for regulating organelle structure and membrane traffic. Cell 1991;67:601-616. Prydz K, Hansen SH, Sandvig K, van Deurs B. Effects of Brefel- din A on endocytosis, transcytosis and transport to the Golgi complex in polarized MDCK cells. J Cell Biol 1992;119:259-272. Wan J , Taub ME, Shah D, Shen WC. Brefeldin A enhances receptor-mediated transcytosis of transferrin in filter-grown Madin Darby canine kidney cells. J Biol Chem 1992;267:13446-13450. Misumi Y, Misumi Y, Miki K, Takatsuki A, Tamura G, Ikehara Y. Novel blockade by Brefeldin A of intracellular transport of

1991;14:192-197.

1988;82:1173-1182.

secretory proteins in cultured rat hepatocytes. J Biol-Chem 1986;261: 11398-1 1403. Benedetti A, Strazzabosco M, Corasanti JG, Haddad P, Graf J, Boyer JL. Cl-/HCO; exchanger in isolated rat hepatocytes: role in regulation of intracellular pH. Am J Physiol 1991;261:G512- 522.

HEPATOLOGY Vol. 21, No. 2, 1995 ALVARO ET AL 459

21. Gautam A, Ng OC, JL Boyer. Isolated rat hepatocyte couplets in short-term culture: structural characteristics and plasma membrane reorganization. HEPATOLOGY 1987;7:216-223.

22. Petrabe R, Chianale J, Gumucio J J . Physiologic significance and regulation of hepatocellular heterogenicity. Gastroenterology

23. Jezequel AM, Macarri G, Rinaldesi L, Venturini C, Lorenzini I, Orlandi F. The fate of electron opaque tracers HRP and lantha- num chloride during valproic acid induced choleresis. Liver 1986;6:341-349.

24. Goldfisher S. The internal reticular apparatus of Camillo Golgi. J Histochem Cytochem 1982;30:717-733.

25. Angermuller S, Fahini H. A new cesium-based method for cyto- chemical localization of thiamine pyrophosphatase in the Golgi complex of rat hepatocytes. Histochemistry 1984;80:107-111.

26. Bruck R, Benedetti A, Strazzabosco M, Boyer JL. Intracellular alkalinization stimulates bile flow and vesicular-mediated exocytosis in IPRL. Am J Physiol 1993;28:G347-G353.

27. Alvaro D, Cho WK, Mennone A, Boyer JL. Effect of secretin on intracellular pH regulation in isolated rat bile duct epithelial cells. J Clin Invest 1993;92:1314-1325.

28. Hacki W, Paumgartner G. Determination of biliary dead space using ''C-taurocholate as a marker. Experentia 1973;29:1091- 1093.

29. Worthington Biochemical Corporation. Enzymes and related bio- chemicals. In: Worthington manual. Freehold, NJ: Worthington Biochemical Life Sciences Division, 1987:145-146.

30. Talalay P. Enzymatic analysis of steroid hormones. Methods Bio- chem Anal 1960;8:119-149.

31. Takayama M, Itoh S, Nagasaki T, Tanimizu I. A new enzymatic method for determination of serum choline-containing phospholipids. Clin Chim Acta 1977;79:93-98.

32. Hoppe CA, Connolly TP, Hubbard AL. Transcellular transport of polymeric IgA in the rat hepatocyte: biochemical and morphological characterization of the transport pathway. J Cell Biol

33. Benedetti A, Strazzabosco M, Ng OC, Boyer JL. Regulation of activity and apical targeting of the Cl-/HCO; exchanger in rat hepatocytes. Proc Natl Acad Sci U S A 1994;91:792-796.

34. Boyer JL, McGrath JW, Ng OC. Microtubule dependent targeting of transporters to the apical membrane determines the canalicular excretion of bile acids in hepatocyte couplets [Ab- stract]. HEPATOLOGY 1993;18:197A.

35. Hayakawa T, Bruck R, Oi Cheng NG, Boyer JL. DBcAMP stimulates vesicle transport and HRP excretion in isolated rat liver. Am J Physiol 1990;259:G727-G735.

36. Beuers U, Nathanson MH, Isales CM, Boyer JL. Tauroursode- oxycholic acid stimulates hepatocellular exocytosis and mobilizes

1988;95:1130-1143.

1985;101:2113-2123.

extracellular Ca" mechanisms defective in cholestasis. J Clin Invest 1993;92:2984-2993.

37. Larkin JM, Palade GE. Transcytotic vesicular carriers for polymeric IgA receptors accumulate in rat hepatocytes after bile duct ligation. J Cell Sci 1991;98:205-216.

38. Hayakawa T, Cheng 0, Ma A, Boyer JL. Taurocholate stimulates transcytotic vesicular pathways labeled by horseradish peroxidase in the isolated perfused rat liver. Gastroenterology 1990;99:216-228.

39. Lowe PJ, Kan KS, Barnwell SG, Sharma RK, Coleman R. Trans- cytosis and paracellular movements of horseradish peroxidase across liver parenchymal tissue from blood to bile. Biochem J

40. Benedetti A, Marucci L, Bassotti C, Guidarelli C, Macarri G, Jezequel AM, Orlandi F. Brefeldin A modifies the transcytotic vesicular transport of horseradish peroxidase (HRP) in isolated rat hepatocyte couplets and bile ductular cells [Abstract]. HEPA-

41. Fujiwara T, Oda K, Yokota S, Takatsuki A, Ikehara Y. Brefeldin A causes disassembly of the Golgi complex and accumulation of secretory proteins in the endoplasmic reticulum. J Biol Chem

42. Fujiwara T, Oda K, Ikehara Y. Dynamic distribution of the Golgi marker thiamine pyrophosphatase is modulated by Brefeldin A in rat hepatoma cells. Cell Struct Funct 1989;14:605-616.

43. Cronholm T, Sjovall J. Bile acids in portal blood of rats fed different diets and cholestyramine. Eur J Biochem 1967;2:375-383.

44. Kitani K, Kanai S, Ohta M, Stao Y. Differing transport maxima values for taurine-conjugated bile salts in rats and hamsters. Am J Physiol 1986;251:G852-G858.

45. LeSage GD, Schteingart CD, Hofmann AF. Effect of bile acid hydrophobicity on biliary transit time and intracellular motility: a comparison of four fluorescent bile acid analogues [Abstract]. Gastroenterology 1994; 106:A929.

46. Gregory DH, Vhlacevic ZR, Prugh MF, Swell L. Mechanism of secretion of biliary lipids: role of a microtubular system in hepatocellular transport of biliary lipids in the rat. Gastroenterology

47. Yousef IM, Bloxan DL, Phillips MJ, Fisher MM. Liver and plasma membrane lipids and the origin of biliary phospholipids. Can J Biochem 1975;53:989-997.

48. Wheeler HO, King KK. Biliary excretion of lecithin and choles- terol in the dog. J Clin Invest 1972;51:1337-1350.

49. Guillouzo A, Begue JM, Ratanasavanh D, Chesne C, Meunier B, Guguen-Guillouzo C. Drug metabolism and cytotoxicity in long-term cultured hepatocytes. In: Guillouzo A, ed. Liver cells and drugs, Les Editions Inserm. Vol 164. London-Paris: John Libbey Eurotext, 1988:235-245.

1985;229:529-537.

TOLOGY 1994;19:431.

1988;263:18545-18552.

1978;74:93-100.

effect of brefeldin a on transcytotic vesicular pathway and bile secretion: a study on the isolated...

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