Bile acid transport by basal membrane vesicles of human term placental trophoblast

GASTROENTEROLOGY 1990;99:1431-1436

Bile Acid Transport by Basal Membrane Vesicles of Human Term Placental Trophoblast JOSE J. G. MARIN, MARIA A. SERRANO, MOHAMAD Y. EL-MIR, NELIDA ELENO, and C. A. RICHARD BOYD Department of Physiology and Pharmacology and Department of Biochemistry, University of Salamanca, Salamanca, Spain; and Department of Human Anatomy, University of Oxford, Oxford, England _

The aim of this work was to investigate the first step in the vectorial translocation of bile acids from the fetus to the mother, which is the transfer across the basal (i.e., fetal-facing) plasma membrane of the trophoblast. Thus, the uptake of [14C]taurocholate by basal plasma membrane vesicles obtained from normal human term placentas was studied. Taurocholate retention into vesicles was studied using a rapid filtration technique that was modified to reduce the taurocholate binding to the filters and to the external surface of the vesicles. Using 100 pmol/L substrate, the membrane vesicles showed a temperature-dependent, Na+-independent transport of taurocholate into an osmotically reactive intravesicular space. The initial rate of taurocholate influx in the presence of 100 mmol/L KNO, followed saturation kinetics (apparent K, for taurocholate = 670 k 128 pmol/L; V,, = 1.86 f 0.28 nmol/mg protein . 60 sat 37%). Over the 6.9-7.9 pH range neither internal nor external pH nor inward nor outward proton gradients affected the uptake of taurocholate. When the electrical potential difference across the basal membrane was manipulated by external anion replacement (Cl-, SCN-, SO,‘-, or NO,-) or by valinomycininduced K+-diffusion potential (vesicle inside negative), taurocholate uptake was not significantly modified. Taurocholate uptake was c&inhibited in the presence of 1 mmol/L glycocholate, 0.5 mmol/L 4,4’-diisothiocyanostilbene-2,9’-disulfonate and 0.5 mmol/L sulfobromophthalein. However, 1 mmol/L probenecid or 0.5 mmol/L p-aminohippurate had no effect. Moreover, preloading the vesicles with 100 mmol/L HCO,- (but not with 100 mmol/L Cl- or 50 mmol/L SO,‘-) induced a significant enhancement in the initial rate of taurocholate uptake. In summary, these findings provide strong evidence for the pres-

ence of an electroneutral transport system for taurocholate in the basal plasma membrane of human chorionic trophoblast. They also suggest that this is likely to be an anion-exchange system.

I

n the adult, bile acids represent the greatest mass of organic anions processed by the liver each day. It has been shown that serum concentrations of bile acids such as cholic acid, chenodeoxycholic acid, and lithocholic acid are somewhat higher in umbilical than in maternal blood (1). This state of hypercholanemia seems to reflect an imbalance between the relative maturity of the fetal liver (as far as its capacities to synthesize and conjugate bile acids are concerned) and the minimal excretion of these compounds into bile in the fetus (2,3). Because bile acids are potentially toxic when accumulated in the liver or blood, the transfer of these molecules across the placenta may be an important route for bile acid excretion by the fetus (4).

The placenta is thought to have excretory and nutritional functions that postnatally are performed by the liver. Experimental evidence reviewed by Watkins (4) shows that there is indeed a transfer of bile acid across this organ. Although bile acids are probably able to cross the trophoblast in both directions

Abbreviations used in this paper: bPPM, basal (fetally facing) placenta plasma membrane: BSP, sulfobromophthalein; DIDS, 4,4’diisothiocyanostilbene-2,2’-disulfonate; FCCP, carboryl cyanide 4+rlfluoromethoxy) phenylhydrazone; GCA, glycocholate; K,, diffusional constant; mPPM, microvillous (maternally facing) placenta plasma membrane: PAH, p-aminohippuric acid; V,,, maximal velocity of transport. 0 1990 by the American Gastroenterological Association

0016-5065/90/$3.00

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(4-61, the higher concentrations of primary bile acids in the fetus suggest that net flux is likely to be toward the mother. Bile acid conjugation and sulfation by the conceptus may thus play an important role in determining the overall rate of placental bile acid transfer (4). Using membrane vesicles, the transport of bile acids across the microvillous (maternally facing) plasma membrane (mPPM) derived from human chorionic trophoblast has been reported to take place via an electrogenically facilitated diffusion system (71. The electrical potential difference across the trophoblastic membrane probably contributes to the forces driving bile acid transfer as an anion from the cell into maternal blood. This microvillous transport system is somewhat similar to that present in the canalicular membrane of rat hepatocytes (8-10). The story is not clear cut, however, because it has recently been suggested that the driving force for bile acid transfer across the brush border membrane of human placenta may be the transmembrane hydroxyl gradient rather (11); hydroxylthan the electrical potential difference bile acid exchange has also been described by Blitzer et al. (12)in the basolateral plasma membrane of the hepatocyte. Because the first step in the transport of bile acids across the trophoblast in the direction from the fetus to the mother must be the transfer of these molecules across the basal (fetally facing) membrane of the trophoblast, bile acid transport by the basal plasma membrane of human chorionic trophoblast (bPPM) was investigated.

Materials and Methods Materials %-labeled taurocholic acid and L-alanine were obtained from New England Nuclear (Madrid, Spain). Unlabeled bile acids (taurocholic and glycocholic acids), bovine albumin (fraction V), valinomycin, +I’-diisothiocyanostilbene-2,2’-disulfonate (DIDS), probenecid, sulfobromophthalein (BSP) and p-aminohippuric acid (PAH) were purchased from Sigma Chemical Co. [St. Louis, MO). Carbony1 cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) was obtained from Aldrich-Chemie (Steinheim, F.R.G.). All reagents were of analytical grade.

Plasma

Membrane

Vesicles

Normal term placentas were placed on ice immediately after delivery of the baby, and the process of preparing basal membrane vesicles was begun within 30 minutes according to the method of Kelley et al. (13). Briefly, tissue was sonicated to remove the maternally facing plasma membrane and syncytial cytoplasm. It was then washed in hypotonic medium, soaked in isotonic 10 mmol/L ethylene glycol tetraacetic acid, and resonicated to free the basal

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plasma membrane from the basal lamina. Free plasma membrane fragments were isolated by differential centrifugation. Isolated membranes were usually resuspended in a medium containing 450 mmol/L sucrose, 10 mmol/L MgCl,, 0.2 mmol/L CaCl,, and 10 mmol/L HEPES/Tris (pH 7.4). This buffer system will hereafter be referred to as “standard loading medium.” High partial purification of bPPM as well as low values for contamination of mPPM similar to that reported by Kelley et al. (13) was confirmed by the presence of basal and apical syncytiotrophoblastic membrane markers, i.e., dihydroalprenolol binding (14) and alkaline phosphatase activity (EC 3.1.3.1) (15). The degrees of enrichment of the markers were 18.5- and 3.73-fold, respectively. To analyze the contamination with other cellular membranes, L(+ j-tartratesensitive acid phosphatase [EC 3.1.3.2) (lS), glucose-6phosphatase (EC 3.1.3.9) (17). and succinic dehydrogenase (EC 1.3.%X1] (18) activities were measured. The degrees of enrichment of these markers were less than threefold. Enzymes were assayed after overnight storage at 4°C. The vesicles were stored at -70°C before use. Frozen membrane preparations were first thawed and then vesiculated by six passages through a 25-gauge needle. They were later diluted to the desired protein concentration (approximately 5 mg/mL) using standard loading medium. In some experiments, the vesicles were preloaded with different media. In these cases, the vesicles (500 PL) were diluted with a large volume (50 mL) of the appropriate loading medium. After incubation at 25°C for 120 minutes they were centrifuged (100,000 x g for 30 minutes at 4”C), recovered from the pellet, and passed through a 25-gauge needle six times. Protein concentrations were determined by the method of Lowry et al. as modified by Markwell et al. [19) with bovine serum albumin as a standard.

Transport

Measurement

Transport of [Yltaurocholate was measured by a rapid-filtration technique (20). In brief, uptake experiments were initiated by adding 80 rL of incubation buffer containing [%]taurocholate and unlabeled bile acid to 20 PL of basal membrane suspension (4-5 pg protein/pL). The compositions and conditions of different incubation and loading buffers are indicated in the figure legends. The time of incubation was terminated by the addition of 4 mL of ice-cold stop solution (250 mmol/L KCI, 25 mmol/L MgSO,, and 10 mmol/L HEPES/Tris, pH 7.4) and immediate filtration through 0.65-pm Millipore cellulose-nitrate filters (Millipore, Afora, Madrid, Spain). The filter was washed once again with the same stop solution and then three additional times with a similar stop solution that also contained 0.1 mmol/L of unlabeled taurocholate. This procedure was selected on the basis of preliminary studies, which revealed that [%]taurocholate retention by the filters (blank) was reduced from 40% to 10% of the total retained radioactivity. This maneuver slightly reduced the amount of [‘*C]taurocholate retained by the vesicles. When additional washes with this solution were tested [up to 51, no further reduction in [‘%]taurocholate retention by the filters alone or plus vesicles was achieved. Radioactivity on the filters was measured

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TAUROCHOLATE

1990

in a liquid scintillation counter (LS-1800-Beckman, Beckman Instruments Espaiia S.A., Madrid, Spain] using the Ready Safe Scintillation Cocktail, also from Beckman, as scintillant. Uptake values were corrected for a blank determined in each experiment by addition of ice-cold stop solution to the incubation buffer before adding the vesicles.

Statistical

400

-

200

-

TRANSPORT

ACROSS

THE PLACENTA

1433

Analysis

Unless otherwise indicated, all incubations were performed in duplicate or triplicate, and all observations were confirmed with two or more separate membrane preparations. Values are given as means + SE. The Bonferroni method of multiple-range testing was used for calculating the statistical significance of differences. Single comparisons of two means were performed using Student’s t test. Regression lines were calculated by the least-squares method. For the kinetic analysis, total uptake rates were fitted to an equation comprising the sum of saturable and diffusional components. The estimations were made by nonlinear regression analysis on a logarithmic scale as described by Van Melle and Robinson (21). Statistical analysis was performed using a Macintosh SE Computer (Apple Computer, Inc., Cupertino, CA).

Results Uptake of Taurocholate Vesicles

o

60

180

300

420540-

2,400

Time of incubation

(s)

Figure 1. Temperature-dependent uptake of taurocholate by basal membrane vesicles of human trophoblast. Loading buffer contained 450 mmol/L sucrose, IO mmol/L MgCl,, 0.2 mmol/L CaCl,, and 10 mmol/L HEPEWTris, pH 7.4. Incubation medium contained 100 pmol/L taurocholate, 250 mmol/L sucrose, 100 mmol/L KCI, 10 mmol/L MgCl,, 0.2 mmol/L CaCl,, and 10 mmol/L HEPEW Tris, pH 7.4. Uptakes were performed by incubating the vesicles at 3% (n = 8) or at WC (n = 5) for the time Indicated. Values are means + SE.

When these experiments were carried out by adding the same small volume (10 pL) of distilled water instead of Triton X-100, no reduction in taurocholate uptake was observed [data not shown).

by Basal Membrane

The time course of the uptake of taurocholate by bPPM vesicles was observed to be temperature dependent. At an incubation temperature of 37”C, the uptake of taurocholate was fast for the first few minutes, and the equilibrium value was reached after about 10 minutes of incubation. At this temperature, the rate of taurocholate uptake was linear up to approximately 1 minute of incubation (Figure l), which was therefore selected as the incubation time for the kinetic studies. At 3”C, taurocholate uptake was significantly slower, and equilibrium was still not reached by 40 minutes of incubation (Figure 1). In another set of experiments, taurocholate uptake was determined after 10 minutes of incubation in the presence of different gradients of osmolarity across the membrane. The results are shown in Figure 2. The amount of taurocholate retained by the vesicles was highly and significantly dependent on the magnitude of the osmotic gradient. The figure shows that in experiments with no osmotic gradient osmotically insensitive taurocholate retention by the vesicles (presumably binding) was approximately 35% of the total. Taurocholate uptake by vesicles incubated for 10 minutes in the absence of a gradient of osmolarity was dramatically reduced when the detergent Triton X-100 (Sigma) was added at a final concentration of 0.5% (vol/vol] and incubated for an additional 10 minutes.

Na+ Independence and Dose Concentration Dependence of Taurocholate Transport As shown in Figure 3, the taurocholate uptake was not different

y = 229,9601 r= 1.00;

time course of in experiments

+ 174,7482x

P
600

300

0’ 0

Triton

L I

2

I/Osmolarity

3

(osmolar

X-100 4

0.5% 5

-1)

Figure 2. Effect of medium osmolarity (n = 8, W)or the presence of 0.5% Triton X-100 in the incubation buffer (n = 4, 0) on the lo-minute uptake of taurocholate at WC. Trophoblastic basal membrane vesicles were preloaded with 250 mmol/L sucrose, 100 mmol/L KCl, 10 mmol/L MgCl,, 0.2 mmol/L CaCl,, and 10 mmol/L HEPES/Tris, pH 7.4. Incubation buffers contained 100 pmol/L taurocbolate, 100 mmol/L KCl, 10 mmol/L MgCl,, 0.2 mmol/L CaCl,, 10 mmol/L HEPES/Tris, pH 7.4, and varying concentrations of sucrose to yield the final osmolarity in the incubation media plotted on abscissa. Values are means + SE.

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8oo R Sucrose

600 r

0

Bicarbonate

0

Chloride

? ?Sulfate 6oo

100 mM out > 0 mM in + Na+, out>in -O- K+, out>in

0

600

1200

I800

Time of Incubation

2400

(s)

Figure 3. Lack of effect of sodium gradient on taurocholate uptake by human tropboblastic basal membrane vesicles. Loading medium contained 450 mmol/L sucrose, IO mmol/L MgCi,, 0.2 mmoi/L CaCi,, and 10 mmoi/L HEPES/Tris, pH 7.4. Vesicles were incubated at 37OCin an incubation buffer containing 250 mmoi/L sucrose, 100 rmoi/L taurochoiate, 10 mmoi/L MgCl,, 0.2 mmoi/L CaCi,, 10 mmoi/L HEPES/Tris, pH 7.4, and 100 mmoi/L KC1(n = 5) or in a similar buffer in which 100 mmoi/L KC1was replaced by 100 mmol/L NaCi (n = 5). Values are means r SE.

in which there was an NaCl or KC1 gradient across the membrane. These values are also similar to those obtained in experiments in which external NaCl or KC1 were replaced by sucrose (Figures 4 and 5). The initial (1 minute of incubation time) rate of taurocholate uptake as a function of substrate concentrations was determined in the presence of 100 mmol/L KNO, (Figure 6A) (NO,-, a highly permeable anion, is usually used in ion-transport studies to minimize the

20 s

90 s

Time of Incubation Figure 5. Effect of inversely directed gradients of sulfate, chloride, or bicarbonate on taurocboiate uptake by basal membrane vesicles at 37%. Vesicles were preioaded with 10 mmoi/L MgCi,, 0.2 mmoi/L CaCi,, and 10 mmoi/L HEPES/Tris, pH 7.4, plus either (a) 250 mmoi/L sucrose and 100 mmoi/L KC1 or KHCO,, (b) 300 mmoi/L sucrose and 50 mmoi/L K,SO,, or(c) 450 mmoi/L sucrose. Vesicles were incubated with a buffer containing 100 pmol/L taurochoiate, 450 mmoi/L sucrose, 10 mmol/L MgCi,, 0.2 mmoi/L CaCi,, and 10 mmoi/L HEPES/Tris, pH 7.4. Values are means + SE (n zz 6). *P < 0.05 compared with 100 mmoi/L KCi-loaded vesicles.

development of transmembrane electrical potential differences that might exert stimulatory or inhibitory effects on taurocholate uptake). The results are given in Figure 6B as an Eadie-Hoftee plot (initial uptake rate/substrate concentrations vs. initial uptake rate). The plot is linear (r = 0.997; P < O.OOl),suggesting that the taurocholate uptake system is saturable and that it obeys Michaelis-Menten kinetics for a single transport system. The best fit for the data shown in Figure 6A was a Michaelis-Menten equation plus a diffusional term, whose K, was very low [l x lo-” ( + 2.48 x 10-13, SD) nL/60 s - mg protein]. The values for apparent Michaelis constant (K,) and maximal velocity of the transport (V,,,) were 670 (k 128, SD] pmol/L and 1.86 (kO.28, SD) nmol/mg protein - 60 s at 37”C, respectively. Effect of pH and Membrane Differences

2400

90

Time of Incubation

(s)

Figure 4. Effect of giycochoiate (GCA), DIDS, BSP, probenecid (PB), and PAH on taurocboiate uptake. Vesicles were loaded with 450 mmol/L sucrose, 10 mmoi/L MgCi,, 0.2 mmol/L CaCl,, and 10 mmoi/L HEPES/Tris, pH 7.4. Uptake experiments were performed at 37OCwith incubation buffer (100 pmol/L taurochoiate, 450 mmoi/L sucrose, 10 mmoi/L MgCi,, 0.2 mmol/L CaCi,, and 10 mmoi/L HEPEWTris, pH 7.4) either containing or lacking the above-indicated inhibitors. Values are means + SE (n 2 5). ‘P < 0.05 compared with experiments using incubation medium containing no inhibitor.

40 min

Potential

Imposed pH gradients across the basal membrane had no effect on the uptake of 50 pmol/L taurocholate. Table 1 shows the data obtained at 1 and 40 minutes of incubation time in experiments with vesicles loaded with media at either pH 6.9 or 7.9 and incubated with incubation media at a pH of either 6.9 or 7.9. When electrical potential differences across the bPPM were manipulated by anion replacement, as shown in Table 2, or by adding valinomycin to vesicles preloaded with potassium (Table 3), the uptake of taurocholate was not significantly modified.

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TALJROCHOLATE TRANSPORT ACROSS THE PLACENTA

1990

r=l.OO;

1435

PCO.05

1

0

300

Go

600

Taurocholate

900

Concentration

2

(PM)

V/s (pmollminlmg

3

protein)/pM

Figure 6. Kinetics of taurocholate uptake by basal membrane vesicles of human trophoblast. Vesicles were loaded with 250 mmol/L sucrose, 100 mmol/L KNO,, 10 mmol/L MgCl,, 0.2 mmol/L CaCl,, and 10 mmol/L HEPEWTris, pH 7.4 and incubated at 37T for 60 seconds with incubation buffers similar to the loading medium except that they contained varying concentrations of taurocholate. Values are means it SE (n = 10,P < 0.001). Apparent K, = 670 amol/L, V,, = 1.66 nmol/mg protein . 60 s, K, = 1 x 10 ‘*nL/60 s . mg protein. A. Initial rates (60 seconds] of taurocholate B. Eadie-Hoftee

uptake (V) as a function of substrate concentrations.

plot of A.

Inhibition Uptake

and Stimulation

of Taurocholate

Taurocholate uptake was not cis-inhibited in the presence of 1 mmol/L PB or 0.5 mmol/L PAH (Figure 4). However, the addition of 1 mmol/L GCA, 0.5 mmol/L DIDS, or 0.5 mmol/L BSP to the incubation medium (i.e., to reach a final concentration lo- or 5-fold that of the substrate) markedly reduced taurocholate uptake after short-term incubation (Figure 4). In these experiments, the amount of taurocholate retained after long-term incubation was also decreased, a phenomenon also observed by Inoue et al. in canalicular membrane vesicles from rat liver (22j;

they suggest that it may reflect competition between these inhibitors and taurocholate for intravesicular binding. To test whether 1 mmol/L GCA might induce the reduction of taurocholate retention by the membrane vesicles because of the disruption of the vesicles rather than the competition for intravesicular binding sites, 12 additional experiments were carried out in vesicle preparations obtained from four different placentas. Retention of [14C]L-alanine by trophoblastic bPPM vesicles after long-term incubation (40 minutes) in the absence or presence of 1 mmol/L GCA was measured. Values, expressed as means + SE, were 78.9 + 3.3 pmol/mg protein and 76.4 * 4.9 pmol/mg protein (P > 0.051, respectively.

Table I. Effect of pH on Taurocholate Uptake by Placenta

Basal Plasma Membrane Vesicles Taurocholate pH in/pH out 6.9/6.9 7.9/6.9 7.9/7.9 6.9/7.9

1 min 264 314 286 279

+ + * +

15 24 13 27

uptake (pmol/mg protein)

Table 2. Effect of External Anion Replacement on Taurocholate Uptake by Placenta Basal Plasma Membrane Vesicles

40 min 589 617 581 503

r + r +

74 36 51 52

NOTE. Plasma membrane vesicles were loaded with 250 mmol/L sucrose, 100 mmol/L KNO,, 10 mmol/L MgCl,, 0.2 mmol/L CaCl,, and 10 mmol/L HEPEWTris at pH values indicated (“pH in”], sedimented, and resuspended in the buffer used for the initial loading. Uptake experiments were performed at 37’C in the presence of incubation buffers containing 50 pmol/L taurocholate, 250 mmol/L sucrose, 100 mmol/L KNO,, 10 mmol/L MgCl,. 0.2 mmol/L CaCl,, and 10 mmol/L HEPES/Tris at pH values indicated (“pH out”). Values are means + SE (n 2 6, P > 0.05). No significant difference among groups was found by the Bonferroni method of multiple-range testing.

Taurocholate Anion

20 s

ClNO, SCNso;-

161 ? 16 170 -t 18 136 + 23 165 + 15

uptake (pmol/mg 90 s 296 288 250 294

+ 49 + 39 +-63 + 28

protein) 40 min 493 433 388 438

* 43 +-41 + 106 + 46

NOTE. Plasma membrane vesicles were loaded with 450 mmol/L sucrose, 10 mmol/L MgCl,. 0.2 mmol/L CaCl,, and 10 mmol/L HEPEWTris, pH 7.4. The incubation media contained 100 rmol/L taurocholate, 10 mmol/L MgCl,. 0.2 mmol/L CaCl,, and 10 mmol/L HEPEWTris, pH 7.4 plus either (a) 250 mmol/L sucrose and 100 mmol/L KCl, KNO,, or KSCN or (b) 300 mmol/L sucrose and 50 mmol/L K,SO,. Values are means + SE (n 2 4). No significant difference among groups was found by the Bonferroni method of multiple-range testing.

1436 MARIN ET AL.

Table 3.

GASTROENTEROLOGY

Effect of Valinomycin on Taurocholate Uptake by Placenta Basal Plasma Membrane Vesicles Taurocholateuptake (pmoVmgprotein)

Control

Valinomycin

20 s

90 s

40 min

121 + 3 117 + 4

271 t 11 273 + 17

556 k 28 572 f 31

NOTE. Membrane vesicles were loaded with 250 mmol/L sucrose, 100 mmol/L KCI, 10 mmol/L MgCl,, 0.2 mmol/L CaCl,, and 10 mmol/L HEPEWTris, pH 7.4. They were preincubated for 15 minutes at 25°C in the presence or absence of valinomycin (20 pg/mg protein). Uptake studies were carried out at 37°C with incubation buffer containing 100 pmol/L taurocholate, 250 mmol/L sucrose, 100 mmol/L NaCI, 10 mmol/L MgC&, 0.2 mmol/L CaCl,, and 10 mmol/L HEPEWTris, pH 7.4. Values are means t SE (n 2 9). No significant difference between groups was found.

Loading of vesicles with 100 mmol/L KHCO, in the absence of any additional pH gradient induced a significant increase in taurocholate uptake (Figure 5). This trans-stimulating effect of bicarbonate was not observed if the vesicles were preloaded with KC1 or K,SO, instead of KHCO,. Discussion The present paper first describes evidence for the presence of a taurocholate-transport system in membrane vesicles derived from basal plasma membranes of human chorionic trophoblast. Because of the amphipathic properties of taurocholate, it is not always easy to clearly differentiate between its binding and translocation in studies using membrane vesicles. However, the results obtained in this study strongly suggest that although binding does exist, bPPM vesicles translocate taurocholate from the incubation media to the interior of vesicles. The criteria supporting this include the following: the much lower sensitivity of labeled taurocholate retention to washing with unlabeled taurocholate by the vesicles than by the filters probably indicates that the labeled taurocholate is intravesicular, although this finding might also be indicative of different taurocholate binding affinities to the two systems. However, the fact that more than 60% of taurocholate retained by the vesicles was in an osmotically reactive space clearly indicates that a substantial quantity of the bile acid had crossed the membrane and was intravesicular; the experiments in which the vesicles were disrupted with Triton X-100 also support this assumption. The proportion of osmotically nonreactive retained taurocholate, in addition to the low amount removed by washing with unlabeled taurocholate, is consistent with the suggestion that taurocholate binds predominantly to the inside of the vesicles [as has also been reported in liver canalicular plasma membrane vesicles (ZZ)]. The best-fit model describing uptake as a function of

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taurocholate concentration at 37°C is a MichaelisMenten equation plus a negligable diffusional term. These features, which are in agreement with the low permeability of lipid bilayers to taurocholate (231, suggest the existence of a specific saturable transport system rather than merely a simple diffusion phenomenon plus binding to vesicles. The strong temperature dependence of taurocholate uptake also suggests a carrier-mediated process. Additional evidence for the presence of a transport system for taurocholate transfer across bPPM includes trans-stimulation of taurocholate uptake by bicarbonate together with cis-inhibition by glycocholate. The apparent affinity of this transport system is lower than that of the recently described transport system for taurocholate in the mPPM of human term placenta; however, the apparent V,,, is higher for the systems located in the bPPM than that of mPPM (7). Kinetically, this probably means that membrane transport at both faces of the trophoblast will not be saturated by substrate given the probable intratrophoblast bile acid concentrations. Another difference between the two systems is the insensitivity of the basal-membrane system to the transmembrane pH gradient or electrical potential. Although it is still controversial whether bile acid transport across the mPPM is driven by electrical potential (7) or by hydroxyl exchange (ll), neither of these mechanisms seems to drive the translocation of taurocholate across the basal membrane. Thus, when the bPPM vesicles were incubated in the presence of inwardly directed gradients of KCI, KNO,, KSCN, or K,SO,, the initial rate of taurocholate uptake was not modified regardless of the permeability properties of these anions. In most membranes, decreasing membrane permeation for the selected anions is SCN- > NO,- > Cl- > SO,‘-. Anions that diffuse into vesicles more rapidly are expected to induce a relatively more negative intravesicular membrane potential, and thus the initial rate of taurocholate uptake would be inhibited if the bile acid were transported as an anion. This feature has been well documented for electrogenic taurocholate transport across the canalicular plasma membrane of rat hepatocytes (8,9). Such a conclusion is also supported by experiments using valinomycin with K+-loaded bPPM vesicles to impose an outwardly directed K+ gradient. This manipulation of transmembrane potential also had no effect on the initial rate of taurocholate uptake: neither did the imposed pH gradient across the membrane have any effect on taurocholate transport by bPPM vesicles. Therefore, clear differences exist between the taurocholate transport system described in the mPPM and that described here in the bPPM. The difference confirms, incidentally, that cross-contamination of basal by brush border membranes is minimal.

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1990

Bile acid uptake by the sinusoidal membrane of hepatocytes (22,24,25), ileal brush border membrane (26-29). and the proximal convoluted tubules of the kidney (30) is known to occur via a secondary active, Na+-dependent transport system. In this study it was tested whether an inwardly directed Na+ gradient enhances taurocholate uptake by bPPM; the results clearly show that this maneuver has no effect. Therefore, evidence for Na+-taurocholate cotransport has not been found. A bicarbonate-taurocholate exchanger system has been described in intestinal basolateral membrane vesicles (31). To examine whether taurocholate transport by bPPM is similar to that described in the ileal cells from the energetic standpoint, the possible transstimulation of taurocholate transport by a number of anions was investigated. The outwardly directed bicarbonate but not the chloride or sulfate gradients significantly enhanced the initial rate of bile acid uptake: moreover, a known inhibitor of anion-exchange systems, DID& was observed to reduce taurocholate uptake by bPPM vesicles. This was also cis-inhibitable by BSP but not by PAH or PB. As far as the physiological role of this transporter is concerned, several points should be noted. In the adult, most bile acid molecules are restricted to the enterohepatic circulation, with only small amounts leaking into the peripheral circulation. However, in the fetus, the position is both quantitatively and qualitatively different. Thus, in the newborn, serum bile acid concentrations are high, decreasing progressively after birth (32). The fetal liver synthesizes and conjugates bile acids, but the enterohepatic circulation, although probably present, is much less important than during the extrauterine life. Evidence in support of this view includes the existence of the so-called “physiological cholestasis” in the neonate and the absence of efficient fetal intestinal (33) and fetal hepatic (3,34) bile acid transport and the fact that in fetal bile total bile acid concentrations are extremely low. Therefore, the main fate for fetal bile acid molecules is probably transferral to the mother via the placenta. A schematic model is proposed in Figure 7, which depicts fetal bile acid uptake from the fetal blood to be an anion-exchanger system, whereas transfer to the maternal blood occurs via a different specific transporter system located in the mPPM. Subsequently, the maternal liver would take up these molecules and excrete them into bile. Although bicarbonate has been found to transstimulate bile acid uptake by bPPM vesicles, and the existing maternofetal-directed bicarbonate gradient might be involved in bile acid transfer to the mother, the physiological role of bicarbonate as a driving force in this process as well as the anion specificity of this exchange system require further investigation, as does

TAUROCHOLATE

t

BA

TRANSPORT

A-

BA

A

I

ACROSS

THE PLACENTA

1437

BA

BILIARY

EXCRETION

BA ENTEROHEPATIC CIRCULATION

Figure 7. Schematic representation of suggested relationship between the human maternal and fetal bile acid pools including the bile acid transfer across the placental trophoblast. BA, bile acids; A, anion (bicarbonate?).

the specificity of different bile acids for the transporter. The current preliminary results suggest a higher affinity for taurocholate than for glycocholate. This may be advantageous because the ratio of glyco- to tauro-conjugated bile acids is very low in the fetus at term (32). This feature, together with the fact that more than 90% of serum bile acids are in amidated form, suggests that tauro-conjugated bile acids may play a quantitatively more important role in the transfer across the placenta. It should he noted that although the synthesis of taurine by the fetal liver is negligible, the tissue content of this amino acid is greater in the fetus than in the adult because of an active taurine transport across the placenta (35). In summary, these findings provide strong evidence for the presence of a carrier for taurocholate in the bPPM. They indicate that driving forces other than the gradient of sodium, protons, or electrical potential must underlie the transport of the bile acid across this membrane. Finally, the current results also suggest that an anion-exchanger system may be involved in the transport of taurocholate across the basal plasma membrane of the human chorionic trophoblast.

1438 MARIN ET AL.

References 1. Colombo C, Roda A, Roda E, Buscaglia M, Dell’Agnola CA,

2. 3.

4.

5.

6.

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Received June l&1989. Accepted May 2,199O. Address requests for reprints to: Jose Juan Garcia Marin, Departamento de Fisiologia y Farmacologia, Facultad de Farmacia, Campo Charro s/n, 37007Salamanca, Spain. Dr. El-Mir was the recipient of a doctoral fellowship of “Ministerio de Asuntos Exteriores,” Spain. Supported in part by the Fondo de Investigaciones Sanitarias de la Seguridad Social, Spain (90/430). Presented in part at “The Physiological Society,” November 4-5, 1988, at Leicester, England, and has appeared in abstract form [J Physiol (Land] 1989;410:38P]. The authors thank N. Skinner and R. Picken for assistance in preparing the manuscript.