Hepatic handling of bile salts and protein in the rat during intrahepatic cholestasis

GASTROENTEROLOGY

1983;84:978-86

Hepatic Handling of Bile Salts and Protein in the Rat During Intrahepatic Cholestasis MARK A. GOLDSMITH,

SANDRA

HULING, and ALBERT L. JONES

Cell Biology and Aging Section, Veterans Administration Department of Medicine, Anatomy and the Liver Center, California; and Biology Department, Princeton University,

17 a-Ethynyl estradiol-induced cholestasis was used to study the relationship of protein to bile salt transport in liver. The biliary secretion of horseradish peroxidase was unaltered in treated animals despite a 56% reduction in bile flow. Cytochemistry confirmed that e&radio1 caused no alteration in the handling of tracer. In a second study, the peak biliary secretion of [14C]taurocholate was reduced animals. The kinetics of lZ51by -46% in treated cholylglycylhistamine, a bile salt derivative, were identical to those of taurocholate in control and cholestatic animals. Taurocholate cmd cholylglycylhistamine secretion were markedly reduced in control animals during competition with unlabeled microscopic taurocholate. Q uan t’1t a t ive electron reautoradiography with ‘251-choJylglycyJhistamine vealed a high concentration ofgrains over the endoplasmic reticulum and the GoJgi complex including associated lysosomes and vesicles. These data demonstrate that estradiol markedly inhibits bile salt transport, but not vesicular transport of horseradish peroxidase. Furthermore, estradiol may alter the movement of bile salts through these organelles. Hepatocytes play an important role in the transfer of recirculating bile salts, as well as certain proteins, from portal blood to bile, a role that requires internalization, intracellular transport, and biliary secretion events. Although the uptake step for both proteins and bile salts has been well-characterized (lI l), the details of the intracellular transport event for

Received May 28, 1982. Accepted October 29, 1982. Address requests for reprints to Albert L. Jones, M.D., Cell Biology and Aging Section (151E), Veterans Administration Medical Center, 4150 Clement Street, San Francisco, California 94121. This work was supported by Veterans Administration and National Institutes of Health Grants AM25878 and AM26743. The authors thank Ms. Jill Mooney and Mr. Edward Kim for their assistance during this project. 0 1983 by the American Gastroenterological Association 0016.5085/83!050978-09$03.00

Medical Center, San Francisco, University of California, San Francisco, Princeton, New Jersey

bile salts are less clear. Some evidence suggests that the subcellular distribution of bile salts may be determined by the passive partitioning of the bile salt molecules between organelle membranes and The possibility that bile salts are the cytosol (12,13). translocated by membrane-bound vesicles (la), a mechanism known to be involved in protein transport (1,2), is supported by the observed increase in the number of pericanalicular vesicles during bile salt-induced choleresis (14,15). Several lines of evidence also indicate a role for the Golgi complex in processing bile salts (15,16-18). The availability of cholylglycylhistamine (CGH), a neutral bile salt derivative, has made possible the direct study of events that may be related to the intracellular transport of bile salts (Figure 1). Unlike other bile salts, CGH is poorly soluble in water and the presence of an imidazole ring permits crosslinking to cellular proteins during fixation with glutaraldehyde (19). Cholylglycylhistamine labeled with iodine 125 has been used to demonstrate a lobular concentration gradient for bile salts (19). Moreover, CGH has been shown to have comparable plasma clearance and secretion kinetics to those of physiologic bile salts (20,21). However, the CGH internalization mechanism may be simple diffusion, in contrast to the sodium-coupled energy-dependent uptake of some other bile salts (22). The studies reported here were designed to identify the relationship between protein and bile salt transport, and to elucidate the mechanism of intrahepatic cholestasis. For these studies we used three tracers: horseradish peroxidase (HRP), a plant glycoprotein whose cytochemical reaction product can be visualized in the electron microscope; taurocholate (TC); and cholylglycylhistamine. Horseradish peroxidase, for which no receptors are found in hepatocytes, was used as a nonspecific tool for studying the movement of vesicles across the cell. 17 cu-Ethynyl estradiol (EE) was selected as the cholestatic agent because it has been implicated both in reducing the

Mav 1983

INTRACELLULAR

“C-TAUROCHOLIC

ACID

OH’

lz5 I-CHOLYLGLYCYLHISTAMINE ‘J” \/\

Figure

I. ‘This tigure shows the structural characteristiu rmholic: ;ic:id and cholylgi~c~lhistamine.

of tau-

transport maximum (Tm) for taurocholate (23), and in enhancing the receptor-mediated uptake and catabolism of low-density lipoproteins (LDL) (24). Our results suggest that estradiol has no effect on vesicular transport of horseradish peroxidase, while it significantly impairs bile salt transport. Furthermore, the processing of bile salts may require the endoplasmic reticulum and the Golgi complex. The transfer appears

of the

bile

salts

through

these

to be the site of the estradiol-induced

organelles inhibi-

tion.

Methods Animals

and

Estradiol

Treatment

300-350 g Male Sprague-Dawley rats weighing (Charles River Breeding Laboratories, Inc., Wilmington, Mass.) were maintained on a pelleted diet and water. Before surgery the animals were fasted for 16-18 h, and then were anesthetized with sodium pentobarbitol (Nembutal, Abbott Laboratories, North Chicago, Ill., 50 mg/kg). Experimental animals received daily subcutaneous injections of 17 Lu-ethynyl estradiol (Sigma Chemical Co., St. Louis, MO., 5 mgikg) dissolved in propylene glycol for 5 days to induce cholestasis. Control animals received injections of propylene glycol.

Secretion

of Horseradish

Peroxidase

Both biochemical and morphologic studies were performed as described (2) to examine the influence of estradiol treatment on the vesicular transport of HRP. For the biochemical studies, estradiol-treated and control rats received portal vein injections of purified horseradish peroxidase (Sigma Chemical Co., 10 pg/lOO g) dissolved in

TRAKSPORT

IIIJRINI:

CHOLRSTASIS

97!)

0.5 ml of 0.9% NaCl infused over a 60-s interval. Bile was collected via a common bile duct cannula [Clay-Adams PE-10 tubing) into tared test tubes at lo-min intervals for 80 min. The bile samples were reweighed and then assayed for the presence of HRP using a spectrophotometric assay that measures the changes in absorbance due to the oxidation of orthodianisidine (25). Because HRP is taken up by many tissues, a large excess is injected into the circulation. For this reason, HRP secretion is expressed per gram liver weight. To study the morphologic features of protein transport, treated and control animals received portal vein injections of HRP as described. At 15 and 30 min postinjection, the livers were perfusion-fixed through the portal vein with 2.5% glutaraldehyde-0.5% paraformaldehyde in 0.2 M sodium bicarbonate buffer. Tissue sections from fixed livers were incubated in a diaminobenzidine-hydrogen peroxide medium (26), and then were osmicated, dehydrated, and embedded using standard electron microscopy tissue preparation techniques. Thin sections were stained with lead citrate and uranyl acetate and examined in a Philips EM300 [Philips Electronic Instruments Inc., Mahwah, N.J.). Visualization of an electron-dense precipitate indicated the presence of HRP. Diaminobenzidine reaction controls were also performed at each time point by using an incubation medium from which hydrogen peroxide was omitted.

Secretion

of Cholylglycylhistumine

Cholylglycylhistamine obtained from (2. Karlaganis was iodinated (1 &ilwg) using a chloramine T method ‘Z’I-Cholylglycglhistamine previously described (20). (1.5-3.0 &i) dissolved in 0.5 ml of 0.01 M sodium phosphate buffer (pH 7.4) containing 0.1% serum albumin and 10% ethanol was injected over a 30-s interval into the portal veins of treated and untreated rats. Bile was collected via a bile duct cannula at 2-min intervals for 20 min. The bile samples were reweighed and the ‘“‘I-CGH colitent of each was determined in a gamma counter.

Secretion

of Taurocholute

A tracer dose of [“C]taurocholate [New England Nuclear, Boston, Mass.) dissolved in the same medium used for the CGH studies was injected into the portal veins into of control and treated rats, and bile was collected tared scintillation vials. After reweighing the samples, scintillation cocktail was used to prepare homogeneous solutions for liquid scintillation counting. j “CITaurocholate content was determined using a Beckman LS-350 LSC (Beckman Instrument, Inc., Fullerton, Calif.): an external standards-channels ratio method m:as used for quench calibration.

Secretion of CholJilglycqllhistamine and Taurocholate at Transport Maximum for the

To determine whether or not CGH and TC compete same intracellular pathway, a combined tracer

980

GOLDSMITH

GASTROENTEROLOGY

ET AL.

bolus of labeled CGH and TC was injected into the portal vein of four rats after saturation of the TC transport system by a continuous femoral vein infusion of 1.0 pmol/min per 100 g body wt of unlabeled TC. During experiments not reported here we found that at least 20 min of steady-state secretion of TC into bile (0.65 pmol/min per 100 g body wt) was observed beginning 20 min after the infusion began. At this time a combined tracer bolus of [14C]TC and lz51CGH was injected into the portal vein. Collected bile samples were assayed for radioactivity content by LSC using a method of simultaneous equations (27). The behavior of the TC bolus was a standard against which the CGH bolus could be compared in a saturated bile salt transport system.

Quantitative Electron Autoradiography

Microscopic

Six control and four estradiol-treated rats were used in quantitative autoradiography with lz51-CGH as described for labeled immunoglobulin A (1) and insulin (2). Rat livers were perfused in vivo at the following time points after portal vein injection of 17-35 PCi of the labeled bile salt analogue: 20 s (2 controls), 1.5 min (2 controls, 2 EE), 4.0 min (2 controls, 2 EE). For each animal, 8-10 random liver biopsy specimens were obtained immediately after perfusion fixation to assess the hepatic content of the label. The biopsy specimens were counted in the gamma counter, and the mean concentration (cpmig) in each liver was calculated. The tissue was processed for EM autoradiography, and the CGH distribution within parenchymal cells was determined quantitatively by circle grain analysis of the autoradiograms. One hundred to one hundred fifty grains were counted for each time point in tissue from each of the control and EE animals. Both the microscopy and grain quantitation were performed double-blind.

Table

Vol.

1. Bile Secretion

Nontreated

84, No.

in Estradiol-Treated Rats

5. Part

1

and

Mean bile secretion (&g liver nt min)

Study HRP Control EE CGH Control EE TC Control El?

1.71 2 0.30 0.74 2 0.18 1.95 + 0.37 0.81 2 0.27 2.02 ? 0.10 0.86 t 0.07

Values are shown for the studies that used horseradish peroxidase (HRP) (n = 4),cholylglycylhistamine (CGH) (n = 8), and taurocholate (TC) (n = 4). Basal secretion was constant in the three studies, and cholestatic animals had a markedly reduced rate of secretion (50%-60%). Mean values t 1 standard error are shown

lysosomes and some lysosomes contained reaction product (Figure 2). Horseradish peroxidase reaction product also was seen in the intercellular space, but the tight junctions appeared intact, thereby preventing diffusion of the HRP into the bile canalicular lumen. Some areas of vesiculation of the smooth endoplasmic reticulum were noticeable in the cholestatic livers, as was a more distinct microfilamentous web surrounding the bile canaliculus, thought to represent a partial depolymerization and disorganization of the network. Despite the marked cholestatic effect of the estradiol treatment, the vesicular transport system continued to operate without inhibition. Because the estradiol treatment causes a 17% increase in liver weight, the total hepatic transport capacity would appear to increase with estradiol treatment.

Results In all of the studies with estradiol, the bile secretion of EE animals was reduced by 50%60% of the control secretion rate (Table 1). In addition, none of the tracers significantly affected the basal flow rate. Secretion

of Horseradish

Peroxidase

Biliary secretion of HRP by control animals peaked at 47 * SE 6 min after administration of the bolus, and by EE rats at 43 + 7 min. Total HRP secretion in the 80-min collection period was 2.41 5 0.26 pug/g liver weight in controls, and 2.35 + 0.70 pgig liver weight in EE rats. Electron microscopic evaluation of the perfusion-fixed livers also revealed no alteration in the handling of HRP by EE rats, as vesicles containing the cytochemical reaction product were observed endocytosing at the plasma membrane and fusing with the bile canalicular membrane. Vesicles were also seen localized near

Secretion

of Taurocholate

Taurocholate was rapidly internalized and secreted with kinetics quite different from those of HRP. As demonstrated by the curves (Figure 3), TC secretion was retarded noticeably by treatment with estradiol; peak secretion by was 39.3% ? 0.1% injected dose at 4 min, and by EE rats 21.2% +1.9% at 6 min. Both control and EE rats cleared essentially the entire injected load within 10 min. The secretion of TC not only is a more rapid event than the secretion of HRP, but the TC transport mechanism is sensitive to e&radio1 treatment.

c&t&

Secretion

of Cholylglycylhistamine

Cholylglycylhistamine was secreted with identical kinetics to those of TC [Figure 4). In control animals peak secretion was 43.6% -+ 3.7% of injected dose at 4 min, and 18.8% 2 1.6% at 6 min in

INTRACELLULAR

TRANSPORT

UIJRINC: (:HOLESTASIS

981

ly the same concentration as did the controls, wherewas the same or as at 4.0 min, the concentration higher in EE animals (Table 2). These observations confirm that the CGH was internalized but not secreted as efficiently in cholestatic rats. In both the EE and control animals the majority of all the grains in the cell were seen over the endoplasmic reticulum, the mitochondria, and the Golgi complex and its associated lysosomes and vesicles (Figure 6). The proportion of the grains over the mitochondria never significantly exceeded the expected relative volume density (19%) of the organelle, suggesting that no active concentrating mechanism is operative for bile salts in the mitochondria. The grains over the mitochondria usually were scored also as rough endoplasmic reticulum grains because of the close proximity of these organelles. Only the distribution of grains over the endoplasmic reticulum and over the Golgi and its associated lysosomes and vesicles was considered significant (Figure i’), because only these structures had grain concentrations well in excess of their expected relative volume densities within the cells (legend, FigFigure

2. Electron micrograph of a pericanalicular region of a c,holestatic liver. Vesicles containing horseradish perclxidase (HRP) (urrow,s) are seen near the bile canalic.. alar membrane prior to the exocytic. release of the !)rotein into the biliar\r space. X 18.000.

A 44.042.040.0-

cholestatic animals. The similarity of the CGH and TC secretion curves, both in normal and EE animals, suggests that the two compounds follow related intracellular pathways, both of which are, at least in part, independent of the HRP vesicular transport system.

38.038.0-

/l II II II II / \

34.032.030.0-

Secretion of CholyJgJycyJhistamine and Taurocholate at the Transport Mechanism The secretion of both TC and CGH during a steady-state infusion of unlabeled TC was reduced almost fourfold from the nonsaturation conditions shown in Figures 3 and 4 (Figure 5). Cholylglycylhistamine secretion peaked at 10.2% 2 1.7% at 4 min, and TC secretion peaked at 12.2% + 2.2% at 4 min. The apparent competition of CGH for at least part of the TC transport mechanism strongly supports the use of CGH as a marker for studying TC transport.

4.0 2.0 -

II 2

Quantitative Autoradiography ‘L51-ChoJyJgiycyJhistamine Comparison at 1.5 min showed

of the radioactivity that the EE animals

with

in the livers had essential-

=*-*_a IllllI 1111~ 4 8 8 10 12 14 18 18 20 22

Time (min) Biliary secretion of [ ‘-‘C]taurocholate Peak secretion was both delayed and reduced in the estradiol-treated (2%) (solid line) animals. Brokctl lines indicate nontreatment (*SE).

982

GOLDSMITH

ET AL

GASTROENTEROLOGY

Vol.

84, No.

5, Part

1

A 44.0-

:

s

44.

o-

42.0-

42.0-

4&O-

a

40.0-

ii 5

3&O36.0-

1' I'

E

34.032.0-

1' / 1

36.03&O34.032.0ki

30.0-

g

26.0-

8

26.0-

+

24.0-

Y

22.0-

z'

20.0-

c

16.0-

0&D

16.014.012.0lO.O-

)// r

2 2

4

6

6

10

12 14

16

16 20

6

.i lb 1'2 1'4 1'6 1'6 io

TIME (min)

22

TIME (min) Figure

4

Figure

5

4. Biliarp secretion of “‘I-cholylglycylhistamine. Secretion kinetics closely followed those of taurocholate, with a delayed and reduced peak in estradiol-treated (&SE) (solid lines] animals. Broken lines indicate nontreatment (&SE].

Discussion The role of vesicular transport in the handling of proteins by liver parenchymal cells has been well established (1,2). Evidence strongly indicates that the transport system actually comprises two distinct cellular routes, one leading directly from the sinusoidal plasma membrane to the bile canaliculus, and the other leading through the lysosomes or GERL regions with some catabolic products reaching the bile canaliculus (2). Estradiol has been used previously to enhance the use of the indirect pathway by LDL (281, a phenomenon that is reported to be the result of a sharp increase in the number of LDL receptors at the plasma membrane (24,291. In our study, neither morphologic nor kinetic data suggested any alteration in the uptake, transport, and biliary secretion of HRP per unit of liver. The possibility still remains that physiologic proteins requiring specific receptors may have altered uptake or transport during estrogen therapy. The movement of vesicles across the cytoplasm may involve cytoskeletal components (30,31). An altered appearance of canalicular microfilaments in intrahepatic cholestatsis has been reported (32) and

Biliary secretion of [“C]taurocholate (solid line) and lZ”I-cholylglycylhistamine [broken line] during continuous intravenous infusion of unlabelled taurocholate at Tm. Peak secretion of both bile salts was reduced in amount, while the timing of the peak was unaltered. The two curves are superimposable through 10 min.

is supported by our own observations. Like other cholestatic agents (33), estradiol may disrupt the canalicular network, which is thought to facilitate bile flow, but this phenomenon appears to be independent of the vesicular transport system. Table

2. Concentration Livers Fixed

Time of fixation (min) 1.5

of Cholylglycylhistamine for Autoradiography

# 1 # 2

1.69 ir 0.54 1.38

+

0.23

#

1

1.58

t

0.25

2

1.25

-c 0.21

Control

#

1

1.53

f

0.39

Control

#

2

0.79

+

0.11

#

1

1.75

r

0.29

EE # 2

2.28

t

0.40

Control Control EE#

EE

Eight to 10 random

Concentration of CGH (cpmig liver wt X 10)

Source of biopsy specimens

EE

4.0

in

samples were obtained immediately after perfusion fixation. Each sample was weighed and counted in the gamma counter. At each time point all 4 animals received identical injections. No comparison is shown for 20-s point because no EE animals were used. At 1.5 min. the concentration in all animals is the same, while at 4.0 min the EE had the same or higher concentration than the controls. Mean values f 1 standard error are shown

May

INTRACELLULAR

1983

TRANSPORT

DL’RING CHOLESTASIS

autoradiogram of a portion of a liver cell from a control animal. ‘Z”I-Chol~lglycylhistamine Figur -e 6. Electron microscopic vrisible over the Golgi complex (G) and over the endoplasmic reticulum (ER) within 20 s following injection x38.000

Compounds other than proteins, such as bile salts, may also use a vesicular transport system for traversing part of the cell. That the final canalicular event may be exocytosis to release the bile salts into the bile (34) is consistent with the apparent calcium dependence of both exocytosis (35) and bile flow

grains

983

are

of the isott 3pe.

(36), which in part is dependent on bile salt secretion. That bile salt-containing vesicles may be derived from the Golgi complex is consistent with a variety of reports linking the Golgi to bile secretory processes (37). Yet, while the overall vesicular transport of proteins from sinusoid to bile requires 40-50

60

“7yl.7 ‘\

Endoplasmic reticulum

‘, ‘0. I

/ a Figure

L-

J

1

2

3

4

TIME (min) 7. Distribution of grains in livers perfused circles represent experiment No. 1. open experiment No. 1. open circles No. 2. One tradiol-treated animals. Relative volume highest concentration of grains was over grains became significantly concentrated estradiol-treated animals. The data suggest via the endoplasmic reticulum.

b

1

1

1

2

,

3

4

TIME (min)

at 20 s. 1.5 min. and 4.0 min. (a) Control, closed with “‘I-cholylglyc~lhistarni~~e circles h’o. 2; (b) 17 n-ethynyl estradiol, closed circles indiLate ethynyl estradiol hundred to 1511 grains wwe counted at each time point for each of the control and esdensities: endoplasmic reticulum. 140/,,;Golgi-rich area and lysosomes. 4L/U(I). The the rough and smooth endoplasmic reticulum and the Golgi-rich areas (GRA). The over the GRA immediately in the control animals and only atter 4 min in the the bile salts are associated with the GKA just prior to secretion. reaching the Golgi

984

GOLDSMITH

ET AL.

min to reach peak secretion, bile salts require onetenth this time. Therefore, only the final excretory step for bile salts in the biliary pole of the cell may use vesicles, but the transport of these molecules from the plasma to this region must involve different mechanisms. It should be noted, however, that despite the localization of autoradiographic grains over vesicles in the Golgi-rich region of the liver cells, the number of grain-vesicle associations in the immediate vicinity of the bile canaliculi were few in number. Because glutaraldehyde fixation does not fix cells instantaneously, and the movement of Golgiderived vesicles to the biliary space must be extremely rapid, it is conceivable that the incoming wave of fixative does not trap the last wave of vesicles moving across the pericanalicular cytoplasm. A similar problem was encountered during the study of synaptic vesicles in neurons and was resolved by the application of a new rapid freezing fixation technique (38). In contrast to the protein transport system discussed above, which is unaffected or even enhanced, the transport of bile salts is impaired significantly by estradiol treatment. The delay in the time of peak secretion possibly is due to a longer biliary transit time resulting from the slower bile flow. Because the concentration of the CGH in the fixed livers was as high or higher in the EE animals, the estradiol does not appear to alter the uptake of the bile salt analogue. The similarity of the secretion curves for TC and CGH, under both control and cholestatic conditions, suggests that the compounds share a common transport pathway. Though the mechanisms of uptake may differ (221, the identical secretion characteristics of the two compounds in a saturated bile salt transport system demonstrate that the liver does not distinguish between the synthetic and natural bile salt during rate-determining events. Cholylglycylhistamine, then, may be useful as a marker for studying the transport pathways of physiologic bile salts. The autoradiography showed that in both control and EE animals, CGH was highly concentrated over both the rough and smooth endoplasmic reticulum (ER), and over the Golgi region of the cell. Suchy et al. (39), using a different bile salt derivative, lZ51cholylglycyltyrosine, recently demonstrated in normal rats a similar hepatic ER concentration of the compound using electron microscopic autoradiography. In our studies, while the control animals immediately showed a high concentration of grains over Golgi-rich areas, the EE animals had a delayed peak concentration over the Golgi, which may correspond to the retarded biliary appearance during the kinetics studies. Although only 2 animals were used at each time point, the qualitative trends were consis-

GASTROENTEROLOGY

Vol.

84, No.

5. Part

1

tent within each pair. We assume that the association with Golgi occurs just before secretion. The high association with ER at all time points suggests that these membranes, too, may play a role in the transport of CGH. A reasonable pathway at least for CGH, then, would be from the ER to the Golgi, and finally into the biliary space. Still to be elucidated, however, is the functional significance of the ER localization of a bile salt that is already conjugated. This latter relationship may be related to an intracellular coupling of bile salt to cholesterol and phospholipid for secretion [Carey MC, personal communication). Our observation that the cholestatic animals had inhibited transport in the region of the ER is consistent with an inhibition mechanism that relies on alterations in membrane composition, which might have a significant effect on the ability of bile salts to cross the barriers. Estradiol has been reported to lead to an increase in surface membrane cholesterol and a decrease in membrane fluidity (40,41). Such changes in intracellular membranes might affect any membrane exchange during the transfer of bile salts to the Golgi. While continuities between smooth ER and Golgi membranes have been observed (42), definitive evidence has not yet been described. If a bile salt (lo), this carrier does exist in these membranes carrier itself may be altered by the estradiol. While it is likely that the translocation of bile salts from plasma to bile involves a complex series of biochemical events, it is necessary to start with a

Figure

8. Model for the transfer of bile salts from plasma to bile. We are postulating that bile salts (closed trinnglr) are taken up by some type of facilitated transport machanism and are carried to the ER by a carrier protein (small stick like structures). These complexes are then translocated to the Go&rich region of the cell and finally to the bile canaliculus. The mechanism(s) irlxrolved in this transport process are unknown hut ma> involve? vesicles

(see text).

May

3983

simple model that is consistent with all the known facts. Such a model would have the following characteristics (Figure 8): (a] active, carrier-mediated uptake, (b) locahzation in the ER and Golgi, and (c) vesicular transport from the Golgi to the bile canaliculus. The transmembrane carrier protein may itself also play a role in the transfer of the bile salt to the ER. perhaps in a manner analogous to that described for phospholipid exchange proteins (43). M’e speculate that bile salts are then transferred to the Golgi, before secretion, possibly by a mechanism like that for newly synthesized proteins. The final transfer from Golgi to bile may be accomplished via vesicles. The alteration in membrane characteristics during estradiol treatment makes transmembrane transfer steps likely targets of the estradiol in exerting its retarding effect. Further studies of these subcellular events are necessary to confirm this hypothesis.

INTRACELLULAR

the hepatic 14.

15.

16.

17.

18.

19

20

References 1. Renston

RH. Jones AL. Christiansen WD. Hradek GT. Underdown BJ. Evidence for a vesicular transport mechanism in hepatocytes for biliary secretion of immunoglobulin A. Science 1980;208:1276-8. DG. Jones AL, Hradek GT. \rL’ong KT. 2. Renston RH. Maloney Goldfine ID. Bile secretory apparatus: evidence for a vesicular transport mechanism for proteins in the rat, using horseradish Gastroenterology 1980:78:1373peroxidase and “ii-insulin. 3

4

5.

6.

7. 8.

9.

10.

11

12

1 :i

88. Blitzer BL. Ratoosh SL. Boyer JL. Inhibitors of Nat-coupled ion transport block taurocholate uptake by isolated rat hepatocytes (abstr). Clin Res 1980:28:273A. Glasinovich J-C, Dumont M. Duval M. et al. Hepatocellular uptake of taurocholate in the dog. J Clin Invest 1975:55:41926 lLlinder E. Paumgartner G. Disparate Na--requirement of taurocholate and indocyanine green uptake by isolated hepatocyten. Experentia 1979;35:888-90. Olinger EJ, Kerckar ES. Ostrow JD. Kinetics of sodium tauroc.holate uptake by isolated rat hepatocytes (abstr). Fed Proc 1978:37:298. Reichen J. Paumgartner G. llptake of bile acids by perfused rat liver. ;\m J Physiol 1976:231:734-42. Scharschmidt BF, Stephens JE. Transport of sodium, chloride, and taurocholate by cultured rat hepatocytes. Proc Nat1 (icad Sci I!SA 1981:78:986-90. Schwarz LR. Burr R. Schwenk M, et al. IJptake of taurocholic rc.id into isolated rat liver c.ells. Eur J Biochem 1975:55:617“3. .\ccatlno L. Simon FR. Identification and characterization of bile ar id rec;eptor in isolated liver surface membranes. J Clin Invest 1976:57:496-508. Strange RC. Cramb R, Hayes JD, et al. Partial purification of two lithocholic. acid-binding proteins from rat liver 100,000 g ?upernatants. Biochem J 1977;165:425-9. Strange RC. Chapman BT. Johnston JD, et al. Partitioning of bile ac.ids into subcellular organelles and the in viva distribution of bile acids in rat liver. Biochim Biophys Acta 1979; 573:5:(5-45, Strange R(:. Nimmo IA. Prrcv-Robb IW. Studies in the rat on

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27

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TRANSPORT

subcellular

DI’RIN(;

distribution

CHOLESTASIS

and biliary

excretion

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