Bile acid-dependent vesicular transport of lysosomal enzymes into bile in the rat

GASTROENTEROLOGY

1993; 105:889-900

Bile Acid-Dependent Vesicular Transport of Lysosomal Enzymes Into Bile in the Rat GENE

D. LESAGE,

Diwsion of Gastroenterology

WILLIE

E. ROBERTSON,

and Department

of Biochemistry,

and

MARY

A. BAUMGART

Scott and White Clinic and Texas A&M Uwersity

Health Science Center,

Temple, Texas

Background: Bile acids may stimulate the movement of hepatocyte vesicies and enhance their fusion with the biliary canalicuius. The present study examined the effects of various biie acids on the exocytosis of the contents of hepatocyte iysosomes into the biliary canalicuius. Methods: The effects of various bile acids on hepatocyte iysosome movement and on exocytosis of the contents of hepatocyte lysosomes into the biliary canaiicuius were determined from the distribution of fluorescein isothiocyanate-dextran-labeied lysosomes in hepatocyte coupiets and by quantitating biiiary lysosomal enzyme output in rats. Results: Hydrophobic as weli as hydrophiiic and nonmicellar biie acids were found to stimulate to a simiiar degree the output of lysosomal enzymes into biie, indicating that biie acid-induced change of canaiicular or lysosomal membrane fluidity is not responsible for enhanced exocytosis. The taurochoiate-dependent increase in lysosomal enzyme excretion was compietely blocked by either microtubuie or microfiiament inhibition, suggesting that these subcelluiar structures are involved in biie acid-dependent vesicuiar transport. Fluorescent microscopy studies showed that taurocholate causes a microtubule-dependent translocation of lysosomes towards the canaiiculus in hepatocyte coupiets, which occurred at the same time as increased output of iysosomai enzymes into bile. Conclusions: The results suggest that biie acids modulate vesicie traffic towards the canaliculus by a mechanlsm unrelated to biie acid interaction with the vesicle membrane.

T

he hepatocyte vacuolar apparatus consisting of endocytic vesicles, transcytotic vesicles, Golgi

complexes, and lysosomes extends from the sinusoidal membrane to the bile canaliculus.’ This constellation of vesicles (and associated microtubules and microfilaments that modulate their movement) may represent a pathway for the transcellular transport of proteins,2’3 lipids,4,5 and organic anions’ to bile. Multiple pathways within vacuolar apparatus exist. Substances may be taken up into vesicles from the circulation by receptor-mediated endocytosis’ or fluid phase endocytosis’

and potentially vesicle

from the hepatocyte

membrane

transporter.”

cytoplasm

Transcytotic

by a

vesicles

carrying substances destined for biliary excretion interact and exchange contents mic reticulum,

may

with Golgi, endoplas-

and lysosomes before their fusion with

the biliary canaliculus.3,‘0 It has been proposed that bile acids modulate movement of intracellular vesicles towards the canaliculus.” High bile acid flux through liver cells is associated with increased size and number of pericanalicular vesicles,12 enhanced transcytosis of fluid phase markers,”

conversion

of vesicles

to tubular forms,13

and increased output of lysosomal enzymes into bile.14 Recent studies suggest that bile acid stimulation ary excretion

of phospholipids

occur by bile acid interaction vesicles Previous

of bili-

and cholesterol

may

with lipid-containing

that fuse with the canalicular

membrane.4”5

studies have not addressed how bile acids

may alter vesicular transport. Potential mechanisms for modulation of vesicular transport include altering microtubule or microfilament function,4 altering vesicie membrane fluidity, l6 increasing vesicle luminal osmolality,” or changing concentration.”

vesicle

membrane

calcium

The release of lysosomal enzymes into bile has been extensively studied as a model for vesicular transport directed towards the canaliculus.‘,‘* Exocytosis of the contents of hepatocyte lysosomes into biliary canaliculi is microtubuleand microfilament-dependent” and is accompanied by the parallel biliary release of exogenous substances that accumulate in lysosomes. 20,21This model is wel1 suited for studies of vesicular transport because it uses no exogenous markers that may alter hepatocyte function; also, the release of Abbreviations used in this paper: DIC, differentlal interference optics; DPH, 1,6-diphenyi-1,3,5hexatriene; FITC, fiuorescein isothiocyanate; LPM, iiver plasma membrane; TC, taurochoiate; TDHC, taurodehydrochoiate; TUDC, tauroursodeoxychoiate. 0 1993 by the American Gastroenteroiogicai Association 0016~5085/93/$3.00

890

LESAGE

lysosomal sicular

ET AL.

GASTROENTEROLOGY

enzymes

into bile occurs

transport,

in contrast

horseradish

peroxidase

paracellular

pathway

factors

governing

fusion

with

Thus,

markers

such as

may be transported

in addition

hepatocyte

by a

to transcytosis.”

lysosome

the canalicular

movement

membrane

for other

The and

may also con-

types of vesicles.20,23

the study of the effects of bile acids on the excre-

tory function sight

by ve-

to other

that

trol the same functions

exclusively

into

function

of hepatocyte

how

lysosomes

bile acids interact

of the remainder

could

with

give in-

and alter

of the hepatocyte

the

vacuolar

apparatus. The

present

mechanism

somal enzymes increased

studied somal

evaluated release

or plasma

properties lysosomal

membrane

the

of lyso-

suggest that the

traffic is not caused by an alteration membrane

fluidity

of bile acids, because

(hydrophobic,

augmented

systematically

acid-dependent

into bile. The findings

vesicular

in lysosomal phipathic

studies

of bile

hydrophilic, enzyme

and plasma

by the amal1 bile acids

or nonmicellar)

output

into bile but lyso-

membrane

fluidity

mea-

sured by fluorescente polarization was increased with hydrophobic bile acids only. The size of lysosomes determined

by morphometric

bile acids, suggesting molality. lysosomal crotubule

studies

no change

The taurocholate

(TC)-dependent

enzyme secretion or microfilament

cent microscopy, tubule-dependent the canaliculus

was not altered

in intralysosomal increase

in

was ablated by either miinhibition. Using fluores-

we showed that TC caused translocation of lysosomes in hepatocyte

by os-

couplets,

which

a microtowards had the

same tempo as its effect on lysosomal enzyme output into bile. The present studies indicate the mechanism of bile acid-dependent vesicular transport in the rat is not caused by bile acid effects on vesicle osmotic

effects in the interior

membrane

or

of the vesicle.

Materials and Methods Animals and Materials Male Sprague-Dawley rats (250-350 g) were used in al1 experiments. Complete bile fistula were created in al1 rats 24 hours before the experiments, except when indicated, to deplete endogenous bile acid pool. Rats received normal saline, 1.5 mL/h intravenously, throughout al1 experiments. TC, tauroursodeoxycholate (TUDC), colchicine, phalloidin, vinblastine, fluorescein isothiocyanate (FITC) - dextran (mol wt, 70,000), collagenase, type 1 trypsin inhibitor, and 1,6-diphenyl-1,3,5_hexatriene (DPH) were obtained from Sigma Chemical Co. (St. Louis, MO). Taurodehydrocholate (TDHC) was obtained from Calbiochem (San Diego, CA). Sephadex G25 was obtained from Pharmacia (Piscataway, NJ). Al1 other chemicals were of the highest quality commercially available.

Vol. 105,

No. 3

General Experimental Procedure In the first group of experiments, bile was collected from conscious rats in 5-minute aliquots in preweighed vials. After a 20-minute baseline period, TC was administered as a bolus 5-, 10-, 15-, and 20+mol doses at 30-minute intervals. In the second group of experiments, bile was collected in 30-minute aliquots for 2 hours as a baseline. TC and TDHC were infused intravenously to bile fistula rats at progressively higher doses of 0.2, 0.4, 0.6, and 0.8 p,molmin-’ - 100 g body wt -’ for 30 minutes at each dose. TUDC was administered at 0.8, 1.6, 2.4, and 3.2 /tmol - min-’ . 100 g body wt-‘. Additional rats received TC or TDHC infusion as above but were pretreated with a microfilament inhibitor, phalloidin (50 pg/lOO g body wt) or vehicle (dimethylformamide, diluted 1:50 in normal saline) intraperitoneally daily for 4 days or the microtubule-binding agents colchicine (2 mg/kg body wt) or vinblastine (1 mg/kg body wt) or vehicle (normal saline) 4 hours before the experiment. In the third group of experiments, rats received 10 mg of FITC-dextran (mol wt, 70,000) previously purified by column chromatography (Sephadex G25) intraperitoneally 24 hours before the experiment. Bile fistula were created, and after a 30-minute baseline bile collection, 10 l.trnol of sodium TC was administered intravenously as a bolus; then bile was collected in 4-minute aliquots for 60 minutes. Hepatocytes were recovered from rat livers perfused in situ with collagenase and trypsin inhibitor as previously described.24 Viability of hepatocytes exceeded 80% in al1 experiments. Liver cells in L15 media were allowed to adhere to glass cover slips for 4 hours. Serial fluorescent microscopy photographs of rat hepatocyte couplets in monolayer culture were obtained for the 20-minute period after the addition of L15 media with or without 20 /tmol/L sodium TC. Only hepatocyte couplets with a canalicular vacuolez were studied. Photographs of hepatocyte couplets were obtained using differential interference optics (DIC) and fluorescence. The DIC image was projected onto a white screen and the canaliculus outlined by hand tracing. The fluorescent image was projected over the trace. The total number of fluorescent vesicles in each couplet and the number in the area 4 pm from the canaliculus were counted in coded photomicrographs. In additional studies, isolated rat hepatocytes were homogenized, and then subcellular distribution of FITC-dextran was determined by isopycnic centrifugation with a linear sucrose gradient as described.25 Morphometric studies of lysosomal volume density were performed as we previously reportedz6 and originally described by Weibel et al.27 on electron microscopy of livers obtained from rats infused with TC, 0.6 l.trnol - min-’ * 100 g body wt-‘, or normal saline for 1 hour before death.

Membrane Isolation and Studies of Membrane Fluidity Rat liver plasma membrane (LPM) and lysosomal membranes were isolated by discontinuous sucrose density

BILE ACID-DEPENDENT

September 1993

gradient centrifugation2* and differential centrifugation,‘” respectively. Membrane fluidity of membrane preparations was determined by fluorescente polarization at 37°C as described.30

i :

a

Analytical Procedures

891

VESICULAR TRANSPORT

’

f

Three lysosomal glycosidases, l%glucuronidase (EC 3.2.1.31), P-galactosidase (EC 3.2.1.23), and ïV-acetyl-Bglucosaminidase (EC 2.3.1.30), were assayed fluorometrically using 4-methylumbelliferyl substrates (Koek-Light Laboratories Ltd., Colnbrook, Buckinghamshire, England) as described by Peters et al. 31 0 p timal assay conditions for liver and bile have been previously established.‘* The following subcellular

marker

bile or isopycnic diesterase

enzymes

1 (plasma

Dupourque

5-phosphate and

sol), assayed by the method

by the

were

method.36 were

Bio-Rad

measured

cein to the fluorescente were passed through high-performance (Progel

kin-Elmer fluorimeter Packard,

(pH

3B fluorometer.

3B fluorometer was analyzed Avondale,

fractions In prelim-

of free fluores-

column

Chromatography

Prod-

phase was ammonium

acetate

and effluent

flow cel1 in the Per-

(Nonvalk,

CT). Output

by HPLC

integrator

from the (Hewlett-

PA).

Al1 results are presented were compared

equality

of variances

unequal,

a modified

with

the

subsequent

bolus.

120

as mean + SE. Means of two t test after testing

an F test; if the variances

the were

t test was used.

Results Bolus Administration of Sodium TC The effects of serial boluses of TC on biliary outputs of lysosomal enzymes, bile flow, and biliary output of bile acids are shown in Figure 1. With the 5and ‘10 pmol doses of TC, there was a progressive increase in the output of al1 three lysosomal enzymes with return of output nearly to baseline levels before

Output

of al1 three

enzymes

changed in a parallel fashion (correlation coefficient > 0.93 for each enzyme vs. the other two), which has been previously centration

shown

under

of the lysosomal

basal conditions.‘*

enzyme

Con-

ïV-acetyl-B-glucos-

aminidase in bile increased from 8.3 f 1.2 mU/mL before bile acid infusion to a peak value of 16.9 + 3.1 after

the

lO+tmol

dose of TC (2.5 pmol),

with student’s

90

Figure 1. Effect of serial boluses of TC on the output of three lysosomal enzymes into bile in eight bile fistula rats. 0, N-acetyl-Pglucosaminidase; ? ,?B-glucuronidase; A, P-galactosidase; 0, bile flow; ?? , total bile acids.

mU/mL

Statistical Methods groups

60

into a

(HPLC)

an HPLC

30

Bile samples

filter and then injected

7.4) at 0.5 mL/min,

passed through

0

MINUTES

490 nm; emis-

contribution

Supelco

-20

dehydrogenase isopycnic

chromatographic

PA). Mobile

0.1 mol/L

and

was

CA).35 Bile

in bile was determined.

TSK 4000 PWXL;

from column

(Richmond,

(excitation,

a 0.2+m liquid

ucts, Bellefonte, buffer,

to the hy-

in bile

the possible

Suit-

Total protein

by 3-dehydrosteroid

sion, 525 nm) in a Perkin-Elmer

of

(cyto-

corresponded

per minute.

by fluorescente

inary experiments,

dehydrogenase

in al1 assays.

assay

FITC-dextran

measured

malate

by the method

were included

1 unit of activity

of 1 l.trnol of substrate

measured acids

blanks

120

90

10.8 with

of Reeves and Fimognari.34

and substrate

For al1 enzymes, drolysis

lactate

60 MINUTES

phospho-

at pH

as substrate3’;

assayed

T1

Ol”“‘,‘,“,,““,“““‘,,“,’ -20 0 30

in liver or

alkaline

assayed

(mitochondria), and Kern33;

able enzyme

fractions:

membrane),

n-nitrophenylthymidine dehydrogenase

were measured

centrifugation

E

output

bolus.

With

of total

the

lowest

bile acids in-

creased significantly (P < 0.01) from baseline of 0.24 + 0.03 to 0.78 -+ 0.12 ltrnol* min-’ - 100 g body wt-’ without appreciable change in the outputs of lysosomal enzymes. With the highest dose of TC (20 pmol), both bile flow and lysosomal enzyme output increased but to a lesser extent than the lO-ymol dose. In an additional group of rats (n = 4), the order of dose administration of TC was reversed (highest first). Peak outputs of lysosomal enzyme after each dose were not significantly different than in rats given progressively higher doses (data not shown). Output of lysosomal enzymes after TC correlated better with bile flow (t. = 0.96) than with bile acid output (r = 0.81). Biliary output of lactate dehydrogenase (a cytosolic enzyme

892

LESAGE

marker)

ET AL.

GASTROENTEROLOGY

did not change

indicating

that

effects

significantly of TC

were not caused by hepatocyte

that received

with TC boluses,

on lysosomal injury

enzyme

dent

(data not shown).

choleresis

phalloidin

Continuous Infusion of Bile Acids

colchicine

The effects of normal

mals,

and TDHC outputs

infusion

saline

on outputs

of the plasma

alone, alkaline

than controls

phospho-

chitine-, sion

each bile acid, there was a progressive

pretreatment

put of the lysosomal

enzyme

dase and total bile acids. Similar with the other two lysosomal and P-glucuronidase alkaline

The output

terase 1 did not significantly

of alkaline change

to

output

more

enzymes

(Figures

pletely

In contrast

the biliary

3-5).

with

zyme output

were significantly

with TDHC

colof ly-

rats

after TC infu-

infused

with

vinblastine,

compared

TDHC,

or phalloi-

in bile flow or total with

controls

excretion

but com-

increase

(Figures

in bili-

3-5).

infuport

bile

of lysosomal

acid-dependent enzymes

ation of LPM or lysosomal

trans-

be caused

by alter-

fluidity,

we stud-

polarization

of the membrane

membranes.

A significant

inhibi-

could membrane

vesicular

ied the effect of bile acids on fluorescente

en-

reflected

less than in control

rats

is inversely

2

In phalloidin-, rats, the output

LPM and Lysosomal Membrane Fluidity is Increased by Hydrophobic Bile Acids

and lysosomal

bile flow

In

less

of TC

Effect of Phalloidin, Colchicine, and Vinblastine on Bile Acid-Dependent Excretion of Lysosomal Enzymes baseline

ani-

was significantly

unchanged

colchicine,

enzyme

Because

tor phalloidin,

vinblastine-treated

the TDHC-dependent

ary lysosomal

phosphodies-

with the microfilament

5). In

TC infusion.

remained

with

blocked

sion.

In rats pretreated

and

with

(Figure

excretion

during

bile acid excretion

P-galactosidase

1 increased

acid

with

in rats pretreated

din did not effect the increment

were obtained

enzymes,

enzymes,

phosphodiesterase

than with TUDC.

results

(data not shown).

the effects on lysosomal

sosomal

N-acetyl-P-glucosamini-

in rats pretreated

and vinblastine-treated

diesterase 1, total bile acid, and bile flow are shown in Figure 2. With progressively higher infusion rates of in out-

bile

No. 3

3). TC-depen-

4) or vinblastine

colchicine-,

total

(Figure

3) and absent

(Figure

enzymes,

increase

vehicle

was blunted

(Figure

phalloidin-,

TC, TUDC,

of lysosomal

membrane

phalloidin

Vol. 105,

B

probe

decrease

by a decreased related

DPH

in isolated

in polarization valve

fluorescente

for polarization,

to the fluidity

(as which

of the membrane)

f * f * & *

*

pLlmmi loog body weight

gmolei

10

min/ 1009 body weighl

/

??

??

5

30

60

90

120

30

150

60

90

??

120

??

150

minutes

mlJhr/ loog body wetght

30

68

9'0 150

150

30

60

90 minutes

120

150

Figure 2. Effect of bile acid infusion on bile flow (A); bile acid output (B); biliary output of N-acetyl-Pglucosaminidase, a lysosomal enzyme (C); and alkaline phosphatase, a membrane marker enzyme (D) in bile fistula rats. Rats received no bile acid (0), TC (O), TUDC (El), or TDHC (m) at the dose indicated. Results are mean + SEM for eight animals in each group.

September

_*.

i993

r

YlLt

Infuaion rata (pmcwmlnll TCand

TDHC I-O-

.,....

_rP.r.,_.-.l_

I

ALJIJ-Vtt-CNVtN

.,rCIA.Im

1..

~~..1...-...~~

.

I KAN3tXlK

Vt31GULAK

I

..-...

OYJ

Oog body welght)

(-O.?-tO.Ty-0.61

=-A pUmin/

lO.-

.

tlzg weig

1t

?? *

. ti

.

t

5 .,

:-P

’

30

60

90

120

min! 1w bod wig

zt

ti

do

68 minutes

minutes

3

mU/hr/ loog bod weig

Figure 3. Effect of phalloidin or vehicle (dimethyl formamlde, 1:5û dllution In normal saline Intrapentoneally daily for 4 days) on bile acid-dependent changes in bile flow (A) and biliary output of bile acids (B), N-acetyl-P-glucosaminidase (C) and alkaline phosphodiesterase I (D) in bile fistula rats. Rats received TC plus phalloidin (O), TC plus vehicle (0), TDHC plus phalloidin (m), or TDHC plus vehicle (0). D^^*,I*^ _r_ 111~all ___n L I JL,“ Cf&” ,.irL.t dl111llcl13 .-.nim-,lr in r\_“k rx~5” llD L11= , Sn* I”, GlE;lII 111GaLll group. *P < 0.05, significant differente between phalloidin and vehicle-treated groups.

was noted

with TC and TUDC

LPM and lysosomal

1t

1

fractions

1 t 30

in both

(Table

1).

couplets

were

100

isolated

L

60

90

120

FITC-dextran

of punctate

fluorescente

from

with a cana-

microscopy photomimultiple discrete areas

in the pericanalicular

120

150

of 10

minutes

at

after addition

of vesicles

hours,

couplet

90

minutes

TC

microscopy

a hepatocyte

0, 10, and 20 minutes

60

after adding

the addition preincubated

showed

30

150

(Figure 6B, C, and D, respectively). The distribution fluorescent vesicles appears closer to the canaliculus

livers from rats administered 10 mg FITC-dextran intraperitoneally 24 hours before experiments. Light liculus (Figure 6A). Fluorescent crographs (Figure 6B) showed

l

of TC compared

with images

0 and 20 minutes. Morphometric analysis of photomicrographs showed a significant increase in the number

Microscopy hepatocyte

. t

Kt

minutes

Demonstration of Bile Acid-Dependent \,^^:^..l^” Tl^...___“ & L., u-l..^“^^^^_L vtmcumr I rarisporr uy riuurebwrii Rat

mUlhr/ 1OOg bod weig

L

but not TDHC

membrane

3

D

200

area of

in the pericanalicular

area 10 minutes

after

of TC (Table 2). In cultured hepatocytes with colchicine (100 pmol/L) for 2

the distribution

of fluorescent

vesicles

was not

altered by TC. The total number of fluorescent vesicles was the same in control and TC- and colchicinetreated cells (74 k 8.9, 70 -t 9.0, and 68 k 7.3, respec-

hepatocytes, suggesting sequestration of FITC-dextran in the vacuolar apparatus. Observations of fluorescent vesicles over 10-20 seconds showed vesicles to move about in a small, restricted area within hepatocytes

tively), and vesicle number was not significantly different in the 0-, 5-, 10-, and 20-minute observation periods.

(salatory

showed FITC-dextran to have the same distribution pattern as the lysosomal enzyme B-galactosidase (Figure 7). An intravenous bolus of 10 pmol/L TC was administered to bile fistula rats which had received 10

motion).

In hepatocytes

preincubated

with

colchicine (100 l-4.mol/‘L) for 2 hours, no salatory motion was observed. NO vectorial movement of vesicles could be discerned by continuous visual inspection over time. Serial photomicrographs of couplets over 20 minutes were made after addition of media with and without 20 pmol/L of TC. Representative fluorescent photomicrograms show the subcellular distribution of

Isopycnic

centrifugation

of

isolated

hepatocytes

mg of FITC-dextran 24 hours before experiments. The output of P-galactosidase, total bile acids, and fluorescence increased in a coordinate fashion 6-8 minutes after a bolus of TC (Figure 8). HPLC analysis of bile showed that the increase in fluorescente observed after

,.,.a

.

-m.m.-

LE3ALlz

UY

r-

Cl

.a

C1A5 I KUtN

I tKULUbY

,..^Cllr..CrlA.

AL.

^A.,

.,

.

*

^_

_*

Vol. IUS,

NO. 3

Infusionrate (~oleiminilOOg body weight) TC and TDHC 1 - 0 - l-0.2-l-0.4-1_0.6-1-0.8-1

Pm;;’

pL/min/ 1w

1oog bod weigt;t

bod weigi?t

I

I

30

60

90 120 minutes

30

150

, LJO 120

60

I 150

minutes

Figure .e-- - 4:

weight

Effect of colchicine

wt) or vehicle hours before

100

changes

1-c

(normal

(D:2 me/100 ..w ---

saline)

experiments

in bile flow (A), biliary output of bile acids

phosphodiesterase . 3’0

ceived

.

6b

90

li0

150

3’0

6ti

TC to be FITC-dextran

rather

than

free fluorescein

To examine and consequently

li0

Size Is Not Altered if change

by Bile Acids

of lysosomal

osmolality

size cause the bile acid-dependent

of lysosomal

150

contents

into bile, electron

TC plus colchicine

*P < 0.05,

mi-

colchicine

of vesicle

with

The

second

the plasma

membrane, possibly being guided into position plasma membrane-associated microfìlament work.37 The third plasma

croscopic morphometric studies of hepatocyte lysosome-like structures were performed on six livers with and without TC infusion. There was no alteration of

between

groups.

of transport.37

apposition

alkaline

(O), TC plus vehicle (0),

significant differente

and direction

is close

and

(W), or TDHC plus vehlcle (0).

and normal saline-treated

movement event

(data not shown).

Lysosomal

$0 minutes

(C),

I (D) in bile fistula rats. Rats re-

TDHC plus colchicine

minutes

exocytosis

??

k

favor

membrane. membrane

4

on blle acid-dependent

(B), N-acetyl-P-glucosaminidase

l

G!hodv _---,

intrapentoneally

event is fusion Factors fusion

of the vesicle

by a netand the

that have been proposed

include

Ca2+ interaction

membranes” or Ca2+ regulation of lipid kinase second messengers, 38*39increased

to with

and protein vesicle size

rats (0.60 ? and control

caused by osmosis,” fusion proteins,40T41 and specialized membrane domains with increased membrane fluidity.42

livers, respectively). The largest and smallest diameters of lysosomes were similar (0.40 -t 0.04 and 0.31 i 0.04

The major findings of the present studies relate to the effects of bile acids on movement of hepatocyte

vs. 0.42 $r 0.05 and 0.32 i 0.04 Pm) in TC-treated control livers, respectively.

vesicles and their fusion with the biliary canaliculus. We evaluated two potential mechanisms for bile aciddependent vesicular transport of lysosomal enzymes

lysosomal volume density 0.08 vs. 0.57% f 0.07%

in TC-treated in TC-treated

and

nierrmecinn -.--1ww.-..

The process that precedes the secretion of hepatocyte vesicle contents into the biliary canaliculus likely follows the same sequence of events that is common to al1 eukaryotic cells before exocytosis.” The first event is translocation of a vesicle from the center of cel1 to a position near the plasma membrane. Because transport vesicles are associated with microtubules, these structures probably provide energy for

into bile, i.e., alteration of hepatocyte plasma membrane or lysosomal membrane fluidity and bile acidinduced change in the lysosomal osmolality and therefore size. Al1 bile acids studied, whether hydrophobic or hydrophilic, micellar or nonmicellar, stimulated the release of lysosomal contents into bile. Because only hydrophobic bile acids increased fluidity of isolated plasma and lysosomal membranes, bile acid-related changes in membrane fluidity is not the mecha-

September

BILE ACID-DEPENDENT

1993

VESICULAR

TRANSPORT

895

Inlusion rate (~moleiminir Oog weight) TC and TOHC I- o- p+p4~-0.6-_l-0.8-)

15

P

pL/min/ lO-1oog bod .1:t velg

&. ??

5 --

30

60

??

90 li0 minutes

pmolei

mini loog bod wig zt

30

150

3

nism

of bile acid-dependent

patocyte

lysosome

vesicular

size was not altered

38

transport.

were ablated

tubule

or microfilament Morphological showed that

He-

by bile acid in-

output

pathway. croscopy

mU/hr/ 1oog bod wig 1 t 100

The effects of bile acids on lysosomal

ing that these subcellular

administration

enzyme of micro-

inhibitors,

a finding

elements

are involved

suggestin this

studies using fluorescent mithe addition of physiological

doses of TC to hepatocyte

couplets

results

in microtu-

bule-dependent translocation of lysosomes to the biliary pole of the hepatocyte. Collectively, the results of

6ìJ

$0

li0

30

150

LPM

Lysosome

NOTE. Fluorescente ‘P value significantly

Added bile acid bmof/L) 0 0.25 0.5 1 2 0 0.25 0.5 1 2

the exocytosis of lysosomal liculus by a microfilament-

90 120 minutes

150

contents into biliary canaand microtubule-depen-

dent pathway. We interpret the TC-dependent redistribution of hepatocyte lysosomes shown in Figure 6 as bile aciddependent

vesicle

followed by vesicle lus. The increased somes

TC 0.340 * 0.336 f 0.334 + 0.333 f 0.329 * 0.268 + 0.267 f 0.264 + 0.26 1 f 0.259 +

60

these studies suggest that bile acids modulate the movement of lysosomes towards the biliary canaliculus and

in hepatocyte

movement

towards

movement number

away from the canalicuof pericanalicular lyso-

couplets

Table 1. Effect of Bile Saks on Lipid Fluidity in LPM and Lysosomal Membranes Determined Membrane

150

1

fusion.

by prior

$0 120 minutes

200

mUlhr/

Figure 5. Effect of vinblastine or vehicle (normal saline) intraperitoneally 4 hours before experiments on bik acid-dependent changes in bile flow (A), biliary output of bile acids (B), N-acetyl$glucosaminidase (C). and alkaline phosphodiesterase l (D) in bile fistula rats. Rats received TC plus vinblastine (0) TC plus vehicle (0), TDHC plus vinblastine (m), or TDHC plus vehicle (Cl). *P < 0.05, significant difference between vinblastine and normal salinetreated groups.

6o

0.003 0.003 0.004 0.002” 0.002” 0.002 0.003 0.003 0.003a o.oo38

beginning

by Fluorescente

the canaliculus

at 5 minutes

Polarization

TUDC

TDHC

0.338 ? 0.003 0.337 f 0.002 0.335 f 0.002 0.335 + 0.002 0.33 1 t 0.002a 0.270 f 0.002 0.270 + 0.004 0.266 z!z 0.002 0.266 + 0.003 0.264 f 0.002”

0.342 f 0.002 0.34 1 f 0.003 0.340 * 0.002 0.340 f 0.003 0.342 + 0.003 0.27 1 f 0.002 0.270 + 0.003 0.269 + 0.002 0.270 f 0.004 0.273 & 0.003

polarization was measured in membranes exposed to varying bile acid concentrations using DPH as a probe. different (P < 0.05) from values for no added bile salt. Each value represents mean f SE for three membrane

isolations.

896

LESAGE

ET AL.

GASTROENTEROLOGY

Vol. 105,

No. 3

Figure 6. (A-D) Subcellular distribubon of FITC-dextran in rat hepatocytes. Cultured hepatocyte couplets were prepared from rats grven 10 mg FITC-dextran 24 hours before death. (A) DIC microscopy photograph of a hepatocyte couplet showing canalicular vacuole (arrow) and (B-D) fluorescente microscopy photographs of the same hepatocyte couplet 0, 10, and 20 minutes. respectrvely, after adding media wrth 20 pmol/L TC. Fluorescent photographs show punctate fluorescente in the cytoplasm (open arrow), and the hand trace shows the area 4 Pm from the bile canaliculus (closed arrows). An apparent increase in the number of fluorescent vesicles within the area 4 pm from the canaliculus is noted In (C) compared with (8) and (D).

and peaking at 10 minutes after the addition of TC (Table 2) correlates with the increased biliary output of lysosomal enzymes in bile fistula rats beginning 5 minutes after TC administration (Figure 8). These findings are consistent with the concept that translocation

of vesicles to the pericanalicular area occurs in concert with exocytotic release into bile. Because the rate of release of lysosomal enzymes into bile is slow (0.25% of hepatic content per hour under basal conditions), no decrease in the total number of hepatocyte lyso-

BILE ACID-DEPENDENT

September 1993

VESICULAR TRANSPORT

897

Table 2. The Effects of TC and Colchicine on the Percentage of Fluorescent Vesicles in the Pericanalicular Area of Hepatocyte Couplets Loaded With FITC-Dextran Time after culture media change (min)

Control

0 5 10 20

22.8 24.4 25.2 23.6

t + + +

TC and Colchicine

TC

3.8 4.1 3.8 3.2

22.0 28.4 39.6 25.6

+ t+ f

2.8 4.2 4.aa 3.4

21.8? 23.6 + 24.4 + 22.8 +

3.2 3.0 3.8 4.6

NOTE.Each value represents mean t SD for 30 hepatocyte couplets. Pericanalicular area represents area within 4 pm from bile canaliculus of hepatocyte couplet. aP < 0.05, compared with controls.

somes

due to exocytotic

in our morphometric tion

of hepatocyte

after adding

studies.

Pericanalicular

lysosomes

decreased output

in biliary

bile acid infusion

of lysosomes

which

enhanced

yet not be detected

Hayakawa

et al.” excretion

(Figure

may remain

ular area after 20 minutes,

dent biliary

distribu20 minutes

increase

for the continued output, ies.

be expected

that seems inconsistent

during

population

would

TC, a finding

the persistent enzymes

discharge

with

of lysosomal 2). A smal1

in the pericanalic-

could be responsible

biliary

lysosomal

by our morphological

suggested that of horseradish

enzyme stud-

bile acid-depenperoxidase from

40

1

Figure 8. Effects of a 10.pmol/L bolus of TC (0, n = 8) or normal saline control (0, n = 6) on biliary output of P-galactosidase, a lysosomal enzyme (A), FITC-dextran (EI), and total bile acids (C).

Frequency j

jjqilactosida~

hepatocyte

vesicles

was caused

by alteration

of mem-

brane fluidity, because they found TC-stimulated but not TDHC-stimulated biliary transport of horseradish peroxidase. In contrast, our measurements of membrane fluidity and our findings of TDHC-dependent lysosomal enzyme excretion into bile suggest that I 1.05

1.10

1.15

1.20

1.25

1.30

Density Figure 7. Subcellular ciistribution of B-galactosidase, a lysosomal enzyme marker, and FITC-dextran after isopycnic centrifugation of a postnuclearfraction of rat liver on a lO%-60% sucrose gradient 1 day after the administration of 10 mg FITC-dextran. The average frequency of the components was calculated by Q/Ap CQ, in which Q represents the activity in the fraction, ZQ represents the total activity, and Ap represents the change in density from the bottom to the top of the fractlon.

changes of hepatocyte membrane mechanism for bile acid-dependent

fluidity is not the vesicular trans-

port. The different results between Hayakawa’s study” and ours is probably caused by the study of different hepatocyte vesicle populations. Enhanced vesicular transport caused by a bile acid effect on vesicle membranes unrelated to altered fluidity is unlikely because we found that TDHC stimulates lysosome transport; however, TDHC is not thought to interact with mem-

898

LESAGE

branes

ET AL.

GASTROENTEROLOGY

in membrane-based

transport

systems.43 In ad-

effects of membrane-bound idity occur

dition to modulation of vesicular transport, bile acid changes of liver membrane fluidity44 has been sug-

likely

gested as a mechanism

cium

lipid excretion,45 increase

for bile salt coupling

and a membrane

of Na+, K+-adenosine

is a mechanism

to biliary

fluidity-dependent

triphosphatase

for alteration correlation

of bile acid hydrophobicity

and the bile acid-dependent biliary excretion of a plasma membrane enzyme in the present study and in a

bile

enzymes.

needed

to examine

sicular

transport.

We showed movement

that

both

et a1.59 found

mem-

excretion

solubilization

of enzymes

from

the canalicular

brane into the biliary space. 4s-50 It is unlikely that lysosomal enzyme release into bile occurs by similar mechanism,

because

we observed

similar

increased

output

with bile acids with low as wel1 as high detergent erties. creased

For the same reason, biliary

output

it is unlikely

of lysosomal

These

hours basal

is caused

intact

investigators

are ve-

vesicle

and exocytosis

in

microtubule

or micro-

to our study,

Marinelli biliary

was not sensitive evaluated

to col-

the effects

of

before biliary lysosomal enzyme excreby colchicine,” whereas in our study and vinblastine

after administration biliary

vesicustudies

dehydrocholate-stimulated

of acid phosphatase

dehydrocholate tion is inhibited

couplet

In contrast

that

the effects of colchicine

prop-

that the in-

enzymes

chitine.

function.

further

bile acid-dependent

in the hepatocyte

to the

filament

of

role of

its role in bile acid-dependent

plasma

has been attributed

cal-

the potential

attractive;

to our findings with lysoprevious study48 contrasts somal enzymes. Bile acid-dependent excretion of enzymes

that

release

for bile acid-dependent

remains

flu-

it seems un-

studies

acid-dependent

the bile fistula rat require

membrane

Therefore,

Nevertheless,

as a mediator

lar transport

bile flo~.~~,~’ The direct

modulates

calcium

activity

after 30 minutes.55

No. 3

on membrane

on the basis of these previous

lysosomal

of bile acid-dependent

calcium

Vol. 105,

lysosomal

when enzyme

these

were studied agents

excretion.‘”

4

inhibit It is un-

by bile acid-induced hepatocyte toxicity by a hydrophobic bile acid, a conclusion that is further supported

likely that the effect of microtubule or microfilament blockade on ablating bile acid-dependent biliary ex-

by the finding of a lack of effect of bile acid infusions on biliary excretion of a cytosolic marker enzyme.

cretion

We observed

no change

in lysosome

size with

acid infusion determined by morphometric suggesting that bile acids do not modulate traffic by an osmotic ular osmolality

mechanism.

and vesicle

cytosis just before

Increased

size is observed

membrane

fusion”;

with

bile

studies, lysosomal during

exoa

smal1 population of lysosomes could swell and be missed by our morphometric studies. In previous studies, bile acids were found to have variable hepatocyte vesicle size. 11,12Cell-free fusion lysosomes and tially determine

canalicular membranes could potenif bile acids affect vesicular size before

fusion.51B52 Another factor changes

effects on studies of

that

in intracellular

shown calcium-dependent zymes from hepatocytes.53

may regulate calcium.

exocytosis

is by

We have previously

enzymes

is caused by interference

to the site where

bile acids

modulate vesicular transport, because TDHC-dependent biliary lysosomal enzyme excretion, but not TDHC

intravesic-

consequently,

of lysosomal

bile acid transport

transport

into

bile,

is affected

by colchicine,

vinblastine, and phalloidin. Although there is no evidence for a component of TDHC transport that is mithis hypothetical fraction could crotubule dependent,4 be responsible for our observation of colchicine-sensitive TDHC-dependent

vesicular

A clear understanding vesicular transport recent experimental

transport.

of the effects of bile acids on

is important because a number of findings point to the possibility

that hepatocyte vesicles and associated microtubules and microfilaments contribute to bile solute and water excretion. Studies by Lake et al. using fluid phase markers

suggest that water may be released

from hepa-

release of lysosomal enMembrane-bound Ca2+ may

tocyte vesicles into bile. 60,61We have shown a choleresis in the sucrose-loaded rat and attributed the in-

neutralize fixed membrane charge54 or alter membrane fluidity favoring membrane fusion.55 Ca2+ also regulates microfilament and microtubule function by modulating membrane binding of F-actin4’ and microtubule polymerization. 56 Bile acids have been shown to alter hepatocyte intracellular calcium stores57,58 and alter membrane calcium content.55 In contrast to the effects of bile acids on lysosomal enzyme transport, the effects of bile acids on intracellular calcium pool are dependent on hydrophobicity of the bile acid, and the

creased flow to fluid released into canaliculi from the hepatocyte vacuolar apparatus.26 Bile acids,62 proteins,3 and lipids63 are localized to the hepatocyte vacuolar apparatus and may be released into bile by exocytosis. After fusion with biliary canaliculus, hydrophilic components are released into the canaliculus from the interior of the vesicle and hydrophobic components could be removed from the vesicle membrane by the detergent effects of bile acids.45 Inhibition of microtubules and/or microfilament function have

September

been shown biliary

to decrease

elements

Likewise,

implicated potential

dysfunction

nonosmotic

of vesicular into

hepatocyte initial with

event biliary

bile.

vesicles

of bile acids,64

anions,6*66 biliary that

play a major

in a variety

dent bile formation vesicles

excretion

bile fl~w,~ findings

subcellular

tion

biliary

lipids,5,65 organic

and basal tion.

BILE ACID-DEPENDENT

1993

suggest

mechanism and

for bile acid-depen-

release

in the bile acid-lipid

stimula-

of solutes

Bile acid-stimulated containing

has been

of cholestasis.65*67 A

could be bile acid-induced traffic

these

role in bile forma-

of these elements

of models

proteins,2 that

from

movement

of

lipids may represent

the

coupling

20.

21.

References 1. LaRusso NF. Hepatocyte vacuolar apparatus. Viewpoints Dig Dis 1986;18:9-12. 2. Kloppel TM, Brown WR, Reichen J. Mechanisms of secretion of proteins into bile: studies in the perfused rat liver. Hepatology 1986;6:587-594. 3. Renston RH, Maloney DG, Jones AL, Hradek GT, Wong KY, Goldfine ID. Bile secretory apparatus: evidente for a vesicular transport mechanism for proteins in the rat, using horseradrsh peroxrdase and [‘251]insulin. Gastroenterology 1980;78: 1373-1388. 4. 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. 5. Gregory DH, Vlahcevic ZR, Prugh MF, Swell L. Mechanrsm of secretion of biliary lipids: role of a microtubular system in hepatocellular transport of biliary lipids in the rat. Gastroenterology 1978;74:93100. 6. Crawford JM, Gollan JL. Hepatocyte cotransport of taurocholate and bilirubrn glucuronides: role of microtubules. Am J Physiol Sec 1988;255:G121-G131. 7. Brown MS, Anderson RG. Goldstein JL. Recycling receptors: the round-trip itinerary of migrant membrane proteins. Cell 1983;32:663-667. 8. Besterman JM, Airhart JA, Woodworth RC, Low RB. Exocytosis of pinocytosed fluid in cultured cells: kinetic evidente for rapid turnover and compartmentation. J Cell Biol 198 1;9 1:7 16-727. 9. Simion FA, Fleischer B, Fleischer S. Two distinct mechanisms for taurocholate uptake in subcellular fractions from rat liver. J Biol Chem 1984;250:10614-10622. 10. Pastan 1, Willingham MC. Receptor-mediated endocytosis: coated pits, receptosomes and the Golgi. In: Prentis S, ed. Trends in biochemical sciences. Amsterdam: Elsevier, 1983: 250-254. ll. 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;99:2 16-228. 12. Jones AL, Schmucker DL, Mooney JS, Ockner RK, Adler RD. Alterations in hepatic pencanalicular cytoplasm during enhanced bile secretory activity. Lab Invest 1979;40:512-517. 13. Sakisaka S, Ng OC, Boyer JL. Tubulovesicular transcytotic pathway in isolated rat hepatocyte couplets in culture: effect of colchitine and taurocholate. Gastroenterology 1988;95:793-804. 14. Marinelli RA, Luguita MG, Garey EAR. Bile salt related secrehon of acid phosphatase in rat bile. Can J Physiol Pharmacol 1985;64: 1347- 1352. 15. Reuben A, Allen RM. Intrahepatic sources of biliary-like micelles. Biochim Biophys Acts 1986;876: 1- 12.

899

16. DeLisle RC, Williams JA. Regulation of membrane fusion in secretory exocytosis. Ann Rev Physiol 1986;48:225-238. 17. Holz RW. The role of osmotic forces in exocytosis from adrenal chromaffin cells. Ann Rev Physiol 1986;48: 175- 189. 18. LaRusso NF, Fowler S. Coordinate secretion of acid hydrolases in rat bile: hepatocyte exocytosis of lysosomal protein? J Clin Invest 1979;64:948-954. 19. Sewell RB, Barham SS, Zinsmeister AR, LaRusso NF. Microtubule modulation of biliary excretion of endogenous and exogenous hepatic lysosomal constituents. Am J Physiol Sec 1984;246:G8-G 15.

that occurs

lipid excretion.

VESICULAR TRANSPORT

22.

23.

24.

25. 26.

27.

28.

29.

30.

31.

32.

33.

34. 35.

36. 37.

LaRusso NF, Kost LJ, Carter JA, Barham SS. Triton WR- 1339, a lysosomotropic compound, is excreted into bile and alters the biliary excretion of lysosomal enzymes and lipids. Hepatology 1982;2:209-2 15. LeSage GD, Kost LJ, Barham SS, LaRusso NF. Biliary excretion of iron from hepatocyte lysosomes in the rat. A major excretoty pathway in experimental iron overload. J Clin Invest 1986;77:90-97. Lowe PJ, Kan KS, Barnwell SG, Sharna RK, Coleman R. Transcytosis and paracellular movements of horseradish peroxidase across liver parenchymal tissue from blood-to-bile. Effect of anaphthylisothiocyanate and colchicine. Biochem J 1985; 229:529-537. Rahman K, Coleman R. Output of lysosomal contentsand cholesterol into bile can be stimulated by taurodehydrocholate. Biochem J 1987;245:289-292. Gautam A, Ng OC, Boyer JL. Isolated rat hepatocyte couplets in short-term culture: structural characteristics and plasma membrane reorganization. Hepatology 1987;7:216-223. Beaufay J. Methods for the isolation of lysosomes. In: Dingle JL, ed. Amsterdam: North Holland, 1972: 1-45. LeSage GD, Robertson WE, Baumgart MA. Demonstration of vesicular-dependent bile flow in the sucrose-loaded rat. Gastroenterology 1990;99:478-487. Weibel ER, Staubli W, Gnagi HR, Hess FA. Correlated morphometnc and biochemical studies on the liver cell. 1. Morphometric model, stereologic methods, and normal morphometric data for rat liver. J Cell Biol 1969;42:68-91. Song CS, Robin W, Rifkin AB, Kappas A. Plasma membranes of the rat liver. Isolation and enzymatic characterization of a fraction rich in bile canaliculi. J Cell Biol 1969;41:124--132. DeDuve CB, Pressman BC, Gianetto R, Wattiaux R. Tissue fractionation studies. 6. Intracellular distribution patterns of enzymes in rat liver tissue. Biochemistry 1955;60:604-6 17. Schachter D, Shintzky. Fluorescente polarization studies of rat intestinal microvillus membranes. J Clin Invest 1977;59:536548. Peters TJ, Muller M, DeDuve C. Lysosomes of the arterial wall. 1. Isolation and subcellular fractionation of cells from normal rabbit aorta. J Exp Med 1972; 136: 1117- 1139. Beaufay H, Amar-Costesec A. Feytmans E, Thines-Sempoux D, Wibo M, Robbi M, Berthet J. Analytical study of microsomes and isolated subcellular membranes from rat liver. 1. Biochemical methods. J Cell Biol 1974;6 1: 188-200. Dupourque D, Kern F. Cytoplasmic and mitochondrial malate dehydrogenase from beef kidney. Methods Enzymol 1969; 13:116-122. Reeves WJ. Fimognari GM. Lactic dehydrogenase: Heart [H4]. Methods Enzymol 1966;9:288-294. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 1976;72:248-254. Talalay P. Enzymic analysis of steroid hormones. Methods Biochem Anal 1960;8: 1 19- 143. Plattner M, Westphal C, Tiggemann R. Cytoskeleton-secretory vesicle interactions during the docking of secretory vesicles at

900

LESAGE ET AL.

the cel1 membrane in Paramecium

GASTROENTEROLOGY Vol. 105, No. 3

cells. J Cell Biol

55. Storch J, Schachter D, Inoue M, Wolkoff AW. Lipid flurdity of he-

1982;92:368-377. 38. Berridge MJ. Inositol triphosphate and diacylglycerol as second messengers. Biochem J 1984;220:345-360.

tetraurelia

patocyte plasma membrane subfractions and their differential regulation by calcium. Biochim Biophys Acts 1983;727:209212. Brady ST, Crothers SD, Nosal C, McClure WO. Fast axonal transport in the presence of high Ca*+: evidente that microtubules are not required. Proc Natl Acad Sci USA 1980;77:5909-5913. Combettes L, Dumont M, Berthon B, Erlinger S, Claret M. Release of calcium from the endoplasmic reticulum by bile acids in rat liver cells. J Biol Chem 1988;263:2299-2303. Combettes L, Berthon B, Doucet E, Erlinger S, Claret M. Bile acids mobilise internal Caf2 independently of external Ca+2 in rat hepatocytes. Eur J Biochem 1990; 190:6 19-623. Marinelli RA, Carnovale CE, Rodriguez Garay EA. Brliary excretion of proteins in the rat during dehydrocholate choleresis. Can J Physiol Pharmacol 1990;68: 1286- 129 1. Lake JR, Licko V, Van Dyke RW, Scharschmidt BF. Bilrary secretion of flurd-phase markers by the isolated perfused rat liver. J Clin Invest 1985;76:676-684. Scharschmidt BF, Lake JR, Renner EL, Licko V, Van Dyke RW. Fluid phase endocytosis by cultured rat hepatocytes and perfused rat liver: implications for plasma membrane turnover and vesicular trafficking of fluid phase markers. Proc Natl Acad SCI USA 1986;83:9488-9492. Lamri Y, Roda A, Dumont M, Feldmann G, Erlinger S. Immunoperoxidase localization of bile salts in rat liver cells. Evidente for a role of the Golgi apparatus in bile salt transport. J Clin Invest 1988;82: 1 173- 1182. Dawidowicz EA. Dynamics of membrane lipid metabolrsm and

39.

Rasmussen H, Barrett P. Calcium messenger system: an integrated view. Physiol Rev 1984;64:938-984. 40. Pollard HB, Creutz CE, Fowler V, Scott J, Pazoles GJ. Calcium-dependent regulation of chromaffin granule movement, membrane contact and fusion during exocytosis. Cold Spring Harb Symp Quant Biol 1982;46:8 19-834. Black MR, Glick BS, Wilcox CA, Wieland FT, Rothman JE. Purification of an N-ethylmaleimide-sensitive catalyzing vesicular transport. Proc Natl Acad Sci USA 1988;85:7852-7856. 42. Orci L, Perrelet A, Montesano R. Differentiai filipin labeling of Iumrnal membranes lining the pancreatic acinus. J Histochem Cytochem 1983;31:952-955. 43. O’Maille ER, Hofmann AF. Relatively high biliary secretory maximum for non-micelle-forming bile acid: possible srgnificance for mechanism of secretion. Q J Exp Physiol 1986;7 1:475-482.

41.

44. Scharschmidt BF, Keeffe EB, Vessey DA, Blankenship NM, Ockner RK. In vitro effect of bile salts on rat liver plasma mem1981; brane, lipid fluidity, and ATPase activity. Hepatology 1:137-145. 45. Coleman R. Biochemistry of bile secretion. Biochem J 1987;244:249-26 1. 46. Wannagat RJ, Adler RD, Ockner RK. Bile acid-induced increase in bile acad-independent flow and plasma membrane Na+,K+-ATPase activity in rat liver. J Clin Invest 1978;61:297307. 47. Keefee EB, Scharschmidt BF, Blankenship NM, Ockner RK. Studies of relationship among bile flow, liver plasma membrane Na+,K+-ATPase, and membrane microviscosity in the rat. J Clin Invest 1979;64: 1590- 1598. 48. Hatoff DE, Hardison WG. Bile acid-dependent secretron of alkaline phosphatase in rat bile. Hepatology 1982;2:433-439. 49. Barnwell SG, Lowe PJ, Coleman R. Effect of taurochenodeoxycholate or tauroursodeoxycholate upon biliary output of phospholipids and plasma-membrane enzymes, and the extent of cel1 damage, in isolated perfused rat livers. Biochem J 1983; 216:107-1 ll. 50. Scholmerich J, Becher MS, Schmidt K, Schubert R, Kremer B, Feldhaus S, Gerok W. Influence of hydroxylation and conjugation of bile salts on their membrane-damaging properbes-studies on isolated hepatocytes and lipid membrane vesicles. Hepatology 1984;4:66 1-666. 5 1. Hurtley S, Warren G. Reconstitution of an endocytic fusion event in a cell-free system: some charactenstics of fusion in vitro. Prog Clin Biol Res 1988;270:355-360. 52. Davey J. A cell-free analysis of the endocytic pathway. Biosci Rep 1987;7:299-306. 53. LeSage GD. Modulation of lysosomal enzyme release from cultured hepatocytes by a Ca”-dependent microtubule binding agent (abstr). Gastroenterology 1985;88: 1674. 54. Rand RP, Kachar B, Reese TS. Dynamic morphology of calcruminduced interactions between phosphatidylserine vesicles. Biophys J 1985;47:483-489.

56.

57.

58.

59.

60.

61.

62.

63.

turnover. Annu Rev Biochem 1987;56:43-61. Kacich RL, Renston RH, Jones AL. Effects of cytochalastn D and colchrcine on the uptake, translocation and biliary secretion of horseradish peroxidase and [%]sodium taurocholate in the rat. Gastroenterology 1983;85:385-394. 65. Barnwell SG, Lowe PJ, Coleman R. The effects of colchicine on secretion into bile of bile salts, phospholipids, cholesterol and plasma membrane enzymes: bile salts are secreted unaccompanied by phospholipids and cholesterol. Biochem J 1984; 220:723-73 1. 66. Kawahara H, Marceau N, French SW. Effect of agents which rearrange the cytoskeleton in vitro on the structure and function of hepatocytic canaliculi. Lab Invest 1989;60:692-704. 67. Dubin M, Maurice M, Feldmann G, Erlinger S. Phalloidin-induced cholestasis in the rat: relation to changes in microfilaments. Gastroenterology 1978;75:450-455. 64.

Received August 19, 1991. Accepted April 5, 1993. Address requests for reprints to: Gene D. LeSage, M.D., Department of Medicine, Scott and White Clinic, Texas A&M University Health Science Center, 2401 South 31st Street, Temple, Texas 76508. Presented to the American Association for the Study of Liver Diseases, May 13-19, 1989, Washington, D.C. Published in abstract form in Gastroenterology (1989;96:A620) and Hepatology (1987;7: 1075).