Biochimica et Biophysica Acta, 1035 (1990) 97-103
97
Elsevier BBAGEN 23340
Transport of bile acids in a human intestinal epithelial cell line, Caco-2 I s m a e l J. H i d a l g o a n d R o n a l d T . B o r c h a r d t Department of Pharmaceutical Chemistry, Unioersity of Kansas, Lawrence, KS (U.S.A.)
(Received 10 November 1989) (Revised manuscript received9 March 1990)
Key words: Caco-2; Bile acid; Taurocholicacid; Epithelial transport The transport of tanrocholic acid (TA) across Caco-2 cell monolayers was dependent on time in culture and reached a plateau after 28 days, at which time the apical (AP)-to-basolateral (131,) transport was 10-times greater than BL-to-AP transport. The amounts of TA inside the cells following application of 10 nM [I4C]TA to tile AP or BL side of the monolayers (30 min) were approximately equal (54.4 + 2.7 and 64.6 ± 2.8 f m o l / m g protein, respectively). AP-to-BL transport of TA was saturable and temperature-dependent. Vm~, and K m for transport were 13.7 pmol / mg protein per min and 49.7 p M , respectively. The transport of TA had an activation energy of 13.2 kcal- mo1-1, required Na + and glucose. AP-to-BL transport of |14C]TA was inhibited by the co-administration (on the AP side) of either unlabeled TA or deoxycholate, but it was not reduced by the presence of unlabeled TA on the BL side.
Introduction Bile acids are produced in the liver and secreted into the small intestine where by virtue of their surfactant activity they play a major role in the intestinal absorption of lipids. After exerting their action, bile acids are passively or actively transported across the intestinal mucosa and recycled back into the liver [1]. This enterohepatic recirculation ensures minimal loss of bile acids into the feces and maximizes utilization along the entire length of the small intestine. The characteristics of the ileal bile-acid carrier have been investigated in guinea-pig [2-4], rat [5,6] and human [7,8] intestine. These studies have provided valuable information on the ileal bile-acid carrier. It is clearly established that bile acids are taken up by ileal brush border through a carrier-mediated, Na+-depen dent process. Structure-activity studies have suggested that the shape of the steroid nucleus, as well as the negative charge in the side chain of the bile acid, are
Abbreviations: pAH, p-aminohippuric acid; TA, tauroeholic acid; AP, apical; BL, basolateral; DMEM, Dulbecco's Modified Eagle's medium; FBS, fetal bovine serum; NEAA, non-essentialamino acids; HBSS, Hanks' balanced salt solution; 2-DG, 2-deoxyglucose;TEER, transepithelial electrical resistance; KRB, Krebs-Ringer buffer; DC, dcoxycholicacid. Correspondence: R.T. Borchardt, Department of Pharmaceutical Chemistry, University of Kansas, Lawrence,KS 66045, U.S.A.
essential for an efficient coupling with the carrier system [1,2]. Furthermore, optimal interaction between Na + and the bile acid are believed to be a requirement for transport [2]. The N a ÷ gradient needed for the uphill transport of bile acids is maintained by Na+/K+-ATPase, which is located in the basolateral membrane of the enterocyte. Previously, we presented evidence that the human colon carcinoma cell line, Caco-2, when grown on a microporous membrane, form a polarized monolayer of enterocytic cells which can be useful in the study of intestinal epithelial transport of drugs, peptides, proteins and macromolecules [10,11]. In this report, we show that Caco-2 cells possess a bile-acid carrier system which may be useful in the investigation of the various processes underlying the intestinal uptake and the vectoriai transport of bile acids at the cellular level. Portions of this work were presented at the Annual Meeting of the American Association of Pharmaceutical Scientists, 2 November 1988, and published in abstract form in Ref. 24. Materials and Methods Materials
The Caco-2 cell line was obtained from American Type Culture Collection (Rockville, MD) and used between passages 59 and 70. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and non-
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98 essential amino-acids (NEAA) were obtained from Hazleton Research (Lenexa, KS). Transwell T M clusters, PVP free, 24.5 mm in diameter (4.71 cm 2 surface area) and 3.0 # m pore size were purchased from Costar (Bedford, MA). Rat tail collagen (Type I) was from Collaborative Research (Lexington, MA). Penicillin, streptomycin and fungizone were obtained as a mixture from Gibco Laboratories (Grand Island). Lucifer yellow C H was from Molecular Probes (Eugene, OR); [14C]Taurocholic acid ([14C]TA, 56 m C i / m m o l ) from Amersham (Arlington Heights, IL); Hanks' balanced salt solution (HBSS), ouabain, N - 2 - h y d r o x y e t h y l p i p e r a z i n e - N ' - 2 - e t h a n e sulfonate (Hepes), sodium azide and 2-deoxyglucose (2-DG) from Sigma (St. Louis, MO). All other chemicals were reagent grade.
Cell culture Caco-2 cells were plated at a density of 63000 cells/cm 2 on Transwell T M polycarbonate membranes (3.0 # m pore size) which had been coated with collagen. The culture medium, consisted of DMEM, 10% FBS, 1% NEAA, 100 U / m l penicillin and 100/xg/ml streptomycin. The culture medium was replaced (1.5 ml inside and 2.6 ml outside) daily. Cells were maintained at 37°C in an atmosphere of 5% CO 2 and 90% relative humidity. All cells used in this study were between passages 59 and 70. Uptake studies The uptake of [I'IC]TA was investigated in Caco-2 cells and grown on Transwell T M for 30 days. Cells were washed twice with serum-free medium and then incubated with HBSS that contained 25 mM glucose and 10 p m o l / m l [14C]TA on the AP or BL side at 37°C. After 30 min, monolayers were washed three times with ice-cold HBSS, harvested in 1 ml 1% Triton-X that contained 0.3 M N a O H and allowed to dissolve for 30 min at 37°C. Transport studies Monolayer integrity. Prior to the transport experiments the integrity of Caco-2 cell monolayers was assessed by measuring transepithelial electrical resistances (TEER) using an EVOM epithelial voltohmmeter, World Precision Instruments (New Haven, CT). [14C]TA solutions also contained lucifer yellow (0.1-0.5 m g / m l ) to further monitor potential monolayer leakage. Sampling. The culture medium of Transwell T M grown Caco-2 monolayers was replaced with HBSS containing 25 m M glucose and 10 m M Hepes buffer (pH 7.35) (transport medium). Subsequently, cell monolayers were equilibrated at the temperature of the corresponding experiment for 30 rain before undertaking the transport studies. In AP-to-BL transport studies all wells in sixwell clusters received 2.5 ml of transport medium which
had been equilibrated at the appropriate temperature. Inserts were positioned in the first well with the outer surface of the inserts (BL side) immersed in the transport medium. [14C]TA was added to the AP side and sampling was performed by transferring the insert to subsequent wells in the cluster that contained fresh transport medium. In BL-to-AP transport studies, inserts containing 1.5 ml of transport medium were placed in 35-mm wells that contained 2.5 ml of [14C]TA solutions. In this way only the BL cell surface was exposed to the radioactive bile acid. Sampling was performed by withdrawing carefully the 1.5 ml in the insert (AP side) and replacing it with an equal volume of fresh transport medium. Measurements. The amount of TA transported at each interval was determined in a Beckman LS-5801 Liquid Scintillation Counter. LY was measured in a SLM4800 Aminco-Bowman spectrophotofluorometer. Total protein was measured by using the method of Lowry et al. [12]. Effect of time in culture on transport. Caco-2 cells, seeded at the usual density, were cultured for different lengths of time. On selected days, T E E R values were measured, and 70000 d p m / m l [14C]TA were applied to either the AP or BL side of at least four monolayers which were incubated at 37°C for 60 rain. The amounts of TA transported at 5, 10, 15, 30, 45 and 60 min were added to obtain cumulative transport.
Characterization of the bile acid carrier system Concentration and time-dependency. Several concentrations of [~4C]TA ranging from 1 to 100 # M were applied to the AP side of the monolayers and samples removed from the BL side at 15, 30, 45, 60, 90 and 120 min. Radioactivity measurements were used to calculate the amount of TA that underwent AP-to-BL transport at each interval. Temperature-dependency. The effect of temperature on TA transport was investigated by applying 0.05/tM [14C]TA to the AP side of monolayers which were then incubated either at 37, 23 or 4°C for 60 min. Samples were taken from the BL side at the same time-intervals indicated above. Activation energy for transport. The rates of transport of TA at 23 and 3 7 ° C were utilized to obtain an Arrhenius plot and calculate E a, the activation energy for transcellular transport of this bile acid. Transport against concentration gradient. [laC]TA was applied to the AP side of two groups of monolayers. In the control group, the BL (receptor) side contained only transport medium and in the test group the BL side contained 10 # M unlabeled TA dissolved in transport medium. Inhibition studies. To determine the energy-dependence of the bile-acid carrier, the transport of TA was studied in glucose-free HBSS and in the presence of 1
99 m M sodium azide plus 50 mM 2-DG. The sodium-dependence of TA transport was examined by carrying out the experiment in Krebs-Ringer buffer (KRB, p H 7.35) (control), in KRB that contained K ÷ or choline instead of N a +, and in the presence of KRB that contained 2.5 mM ouabain. The transport of [14C]TA was carried out in the presence of 25, 125 and 250 /~M concentration of deoxycholic acid (a dihydroxylated bile acid) and TA to assess the affinity of this bile-acid carrier. Results
The development of polarity in Caco-2 cells is reflected in an increased expression of the marker enzymes sucrase-isomaltase, alkaline phosphatase and aminopeptidases [10,12]. For that reason, to determine the optimal time for maximal bile-acid transport, TA transport studies in Caco-2 cells were carried out at different times in culture. Between days 6 and 13, both AP-to-BL and BL-to-AP transport of TA showed a decrease (Fig. 1A) which was most likely due to an increased sealing capacity of the occluding junctions. From day 13 to day 28, however, there was a gradual increase in AP-to-BL transport and a slight decrease in the already low BL-to-AP transport (Fig. 1A). Since the flux of the leakage marker, LY (MW 457), was less than 0.25%/h at all times after day 13, it can be concluded that the translocation of TA across Caco-2 monolayers cultured for 13 or more days was mainly the result of transcellular transport. This
indicates that maximal expression of the bile-acid carrier takes longer than the morphological differentiation (2 weeks). Indeed, at day 28 + in culture the rate of transport in the AP-to-BL direction was more than 10-times faster than that in the opposite direction (Fig. 1B). To characterize the bile-acid carrier, only monolayers with T E E R values greater than 250 ohms. cm 2 and lucifer yellow (LY) fluxes of less than 0.25%/h were utilized. The number of monolayers that had to be excluded due to excessive leakage was less than 5%. The amounts of TA inside the cells after 30 rnin incubation with the labeled bile-acid on the AP (n = 4) and BL (n = 4) side were 54.4+ 2.7 and 6 4 . 6 + 2.8 f m o l / m g protein, respectively. Appearance of TA on the BL side following AP application exhibited an initial lag time after which transport proceeded at a faster, yet constant rate (Fig. 2). The appearance of []4C]TA on the basolateral side is not likely to be due to exchange of radioactive for non-radioactive TA (initially in the BL side), since the BL-to-AP transport of TA was minimal compared to AP-to-BL transport. Moreover, TA transport increased linearly at concentrations up to 10 /~M, but saturation of the transport system was observed above this concentration (Figs. 2B, 3). Vmax and K m values for transport were 13.7 p m o l / m g protein per min and 49.7/~M, respectively. TA was transported against a concentration gradient (10 × ) at the same rate as in the absence of such a gradient (Fig. 4). To determine the temperature-dependence of transport, 0.05 /xM [14C]TA was applied to the AP side of
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TIME (MIN) Fig. 1. Effect of time in culture on T A transport. (A) O n specified days [14C]TA (1.0 p M , 70000 d p m / m l ) was applied to the A P side (1.5 ml) ol BL side (2.5 ml). The monolayers were incubated under 5% CO 2 for 1 h at 37°C and samples were taken at 15, 30, 45 and 60 min and the cumulative a m o u n t s of T A transported both in the AP-to-BL ([]) and BL-to-AP (11) direction were determined. (B) Time-course of AP-to-BI transport at 37 ° C (D) and 4 ° C ( o ) and BL-to-AP transport at 37 ° C (m) in 30-day-old monolayers. Values are m e a n + S.D. (n = 4).
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Fig. 2. Concentration-dependency of TA transport. 30-32 day-old Caco-2 cell monolayers were administered 2 ml transport medium containing 70000 d p m / m l []4C]TA (on the AP side). Panel A shows 1 (o), 5(*) and 10 (12) /AM and panel B 25 (e), 50 (A) and 100 ( - - - - - - ) #M. After incubating for 60 rain under the same conditions described in Fig. 1, samples were withdrawn from the BL side at selected times (indicated). Values shown are mean + S.D. (n = 4).
30-day-old monolayers which were then incubated at different temperatures. The rates of transport were 0.014 and 0.0045 pmol/mg per rain at 37 and 23°C, respectively (Fig. 5). However, at 4°C, TA was not detected
on the BL side, indicating that TA transport is entirely temperature-dependent. Using the steady-state transport rate at different temperatures, an Arrhenius plot was constructed and the activation energy ( E . ) for
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CONC (pM) Fig. 3. Saturation of TA transport in Caco-2 cells. The rates of transport at different concentrations of [14C]TA were determined from the terminal (steady-state) portion of the graphs of pmoles transported per nag vs. time. Inset: double-reciprocal plot of data. l / V = ( p m o l / m g per rain) and 1 / C = ~ t M - ] . Values represent the mean + S.D. (n = 4).
~ME (Wt0 Fig. 4. Transport of TA against a concentration gradient. 0.05 /~M []4C]TA was added on the AP side of two groups of Caco-ceU monolayers which were 30-32 days old. The receiver (BL) side of the first group (12) contained only transport medium (control) while that of the second group (It) contained 10/~M tmlabelled TA. Monolayers were incubated trader the previously described conditions and sampies were taken at the specified times. Values shown are the mean + S.D. (n = 4).
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Concentration-dependent inhibition of [HC]TA transport by unlabeled taurocholic acid (TA) and deoxycholic acid (DC) The incubation time was 60 rain. Four monolayers were used in each experiment. The transport of 0.01 /xM [z4C]TA when administered alone (control) was equal to 0.09 p m o l / m g (+0.017), and this value was defined as 100%.
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Fig. 5. Effect of temperature on TA transport across Caco-2 cell monolayers. Caco-2 cell monolayers (30-32 days old) received 2 ml of 0.05 # M [14C]TA and then were incubated at 37 ° C (O), 2 3 ° C ( I ) or 4 ° C ( o ) for I h. Values shown are the mean ± S.D. (n = 4).
transcellular transport calculated to be equal to 13.2 kcal • m o l - t The transport of TA was significantly reduced in the presence of sodium azide plus 2-DG or in the absence of glucose (Table I), implying energy-dependence. The administration of ouabain and the replacement of Na + TABLE I
Effect of experimental conditions on [HC]TA transport Caco-2 monolayers were washed with and preincubated (15 min) in either Krebs-Ringer Buffer that contained 1 m g / m l glucose (KRB, control), K R B containing 1 m M sodium azide + 50 m M 2-deoxyglucose, K R B where N a + was substituted with K +, K R B where N a + was substituted with choline, 2.5 m M ouabain dissolved in K R B that contained choline instead of N a +, glucose-free KRB, K R B containing no glucose and no N a ÷, or K R B containing pAH. To determine transport, the monolayers where incubated with 0.05 # M [Z4C]TA dissolved in the appropriate buffer for 30 min at 37°C. Whenever appropriate, the osmolarity was adjusted to approx. 310 m o s M / k g with D-mannitol. The transport rate in the control group was 0.011 p m o l / m g protein per min. Statistically significant differences were evaluated using one-way analysis of variance (ANOVA) and specific differences from the control ( * P < 0.05) were identified using a multiple range t-test [23]. Each group consisted of between three and six monolayers. Conditions
Transport (% + S.D.)
K R B (Control) Azide + 2 - D G / K R B (-)Na
+,( + )K
÷/KRB
Choline buffer Ouabain (2.5 m M ) / c h o l i n e buffer (-)Ghicose/KRB (-)Na +,(-)Glucose/KRB p A n (1 m M ) / K R B p A H (10 m M ) / K R B
100 + 14 58.5 5 : 3 . 1 47.7+ 8.9 75.3+ 9.2 55.3+ 7.5 66.9+ 9.3 28.0 + 6.2 97.1 + 8.3 73.5+ 4.9
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with K + or choline resulted in a significant decrease in transport (Table I), suggesting that the process is at least partly Na+-dependent. The transport of TA was lower when both Na ÷ and glucose were absent than in the absence of either Na ÷ or glucose alone, p A H failed to inhibit the transport of 10/~M [14C]TA when co-administered at 1 mM concentration but resulted in a 27% ( P < 0.05) inhibition at 10 m M concentration (Table I). Relatively low concentrations (25 v M ) of both unlabeled TA and deoxycholic acid reduced the transport of [14C]TA by almost one-half ( P < 0.01) (Table II). Increasing the concentration of the inhibitors had a moderate effect on [zaC]TA transport (Table II). Discussion
Recently, we presented evidence that the human colon adenocarcinoma cell line, Caco-2, can be a valuable model system for the study of intestinal epithelial transport [10]. Here we demonstrate that Caco-2 cells exhibit transcellular transport of TA and may be helpful for investigating bile-acid transport across the epithelial mucosa of the small intestine. The dependency of TA transport on time in culture is hardly surprising since we found previously that although Caco-2 cells were fully differentiated by day 15, alkaline phosphatase activity continued to increase through day 20 in culture [10,13]. Similarly, aminopeptidase and sucrase-isomaltase activities were found to increase after cell differentiation was completed [13]. Comparable amounts of TA were found in the cells following the presentation of the labeled bile-acid to the AP or BL membranes. Thus, it appears that the polarized delivery of the bile-acid is determined by either the movement across the cytoplasma or the preferential efflux across the BL membrane, and not by the initial uptake.
102 That TA is targeted to the BL side despite being taken up by both the AP and BL membranes indicates that the carrier system of the AP membrane may be different from that in the BL membrane. This interpretation is supported by previous studies where an anion exchange transport system, constituted by two polypeptides ( M r 54000 and 59000), was identified in the ileal BL membrane [14,15]. This transport system is different from the polypeptide associated with the bileacid carrier found in the brush-border membrane ( M r 99 000) [16,17]. Although a direct comparison between uptake and transcellular transport is not appropriate, because uptake is a single process and transport involves uptake, movement through the cytoplasm and efflux at the opposite membrane domain, the analysis of our transport data in light of the existing data on bile-acid uptake may prove useful. The apparent Krn for transport obtained in this study (i.e., 49.7 #M) is in the same order of magnitude as that reported recently for TA uptake by human ileal brush-border membrane vesicles (37 #M) [8]. However, the corresponding Vm values differed dramatically (13.7 vs. 6560 p m o l / m g protein per min). The similarity in K m values as well as the discrepancy between Vm values can be explained if we assume that the bile-acid carrier of Caco-2 cells is reminiscent of the human ileal carrier and that such a carrier is present in fewer numbers in Caco-2 cells. An alternative explanation may be that translocation through the cytoplasm a n d / o r subsequent efflux at the BL side may be slower than the initial uptake and this limits the overall capacity for transcellular transport. The activation energy required for TA transport across Caco-2 ceils is in agreement with carrier-mediated transport, because the E a for enzyme-catalyzed reactions and carrier-mediated transport range from 7 to 25 k c a l / m o l and the E a for simple diffusion is usually lower than 4 kcal-mo1-1 [21]. Because previous reports have shown Na+-dependent TA uptake by other preparations [2,8], it was not surprising that the N a + / K + - A T P a s e inhibitor, ouabain, reduced the transport of TA. However, the magnitude of the residual transport (53%), in the presence of ouabain and when Na + was substituted with K ÷ or choline, is higher than that reported in ileal brush-border membrane vesicles [8,22] and suggests the existence of a Na+-independent pathway in Caco-2 cells and in ileal enterocytes. The results from our study did not reveal the existence of a bile-acid cartier completely dependent on the presence of N a ÷ as that encountered in the intestine. Although this difference seems to point at a dissimilarity between the Caco-2 cells and the normal enterocyte, such an apparent discrepancy may partly be due to the fact that while our study determined the overall transcellular transport in the intact, polarized
cell, previous studies have looked at uptake in isolated membrane preparations [8,22]. However, detailed experiments dealing independently with uptake by the AP membrane and efflux through the BL membrane in the intact cell will be most useful in testing this hypothesis. To assess the specificity of the bile-acid carrier system we selected one unconjugated bile-acid, deoxycholic acid (DC) and compared its ability to inhibit the transport of [14C]TA to that of unlabeled TA. Both TA and DC possess a negative charge, which has been indicated to be important for interaction with the carrier on the side chain [1]. Furthermore, since the bulkiness of the taurine conjugate in TA should have a minimal effect on transport [!], it follows that the similar inhibitions of [14C]TA transport caused by TA and DC indicate that the bile-acid carrier did not discriminate between these two compounds. These data suggest that the structural requirements of the ileal bile-acid carrier may not be the same as those of the bile-acid carrier of Caco-2 cells. It is not known whether the interaction between DC and TA took place at membrane binding sites or, less likely, through some nonspecific depression of transport caused by DC. In addition, even if DC inhibited TA binding, this would not constitute sufficient evidence that these bile acids share the same carrier because some compounds can inhibit the transport of others without undergoing transport themselves [4]. The presence of an anion transport system on the apical membrane which would transport structurally related organic anions could explain the inhibition seen with DC. However, this possibility can be ruled out (at least in part) by the lack of effect of 1 m M pAH on the AP-to-BL transport of 10/~M [14C]TA. This absence of inhibition suggests that the bulk of the AP ~ BL transport of bile acids in Caco-2 cells most likely involves a cartier system different from an anion transporter. This is supported by data showing that the AP uptake of [14C]TA was not affected by 1 m M concentrations of pAH (not shown). The significant reduction of AP-to-BL transport of [14C]TA observed following the co-administration of 10 mM pAH may be difficult to interpret at this time. While exposure to 10 and 50 mM concentrations of pAH did not result in generalized toxicity, as judged by the Trypan blue exclusion test, we do not know whether this concentration of pAH caused some nonspecific depression of cell function which may result in lower transport of bile-acid without necessarily exhibiting the type of toxicity detectable by this test. In conclusion, this study has demonstrated the existence of a bile-acid carrier system in Caco-2 cells, whose maximal expression is achieved after 28 + days in culture. Carrier-mediated transport of bile acids has been observed in human ileal but not in jejunal brushborder membrane vesicles [8]; therefore, these results suggest that Caco-2 cells exhibit ileal characteristics. Although Caco-2 cells take up the bile-acid, TA, through
103 b o t h the A P a n d BL m e m b r a n e s , the release of T A across the A P m e m b r a n e is virtually negligible, with the vast m a j o r i t y of the bile-acid b e i n g released across the BL m e m b r a n e . Such polarized delivery of bile acids to the BL m e m b r a n e s resembles that of the n o r m a l enterocytes. T r a n s p o r t b y this carrier seems to be susceptible to the same factors c o n t r o l l i n g u p t a k e in the ileal b r u s h border. However, it m a y be p r e m a t u r e to equate the bile-acid carrier f o u n d i n Caco-2 cells to that in ileal enterocytes. T o m a k e this possible, detailed studies m u s t be carried out to d e t e r m i n e the characteristics a n d relative i m p o r t a n c e of apical u p t a k e a n d basolateral efflux in the t r a n s p o r t of bile acids across Caco-2 cell monolayers. Despite the need for a d d i t i o n a l characterization of the bile-acid carrier of Caco-2 cells, it is clear that some of the similarities described in this study together with the virtually u n l i m i t e d viability of polarized Caco-2 cell m o n o l a y e r s will m a k e this system a valuable alternative tool in bile-acid t r a n s p o r t research.
Acknowledgements This work was s u p p o r t e d b y a g r a n t from the U p j o h n C o m p a n y . W e t h a n k Drs. T o m J. R a u b a n d Norm a n F. H o for r e a d i n g the m a n u s c r i p t a n d p r o v i d i n g constructive criticism.
References 1 Lack, L. and Weiner, I.M. (1966) Am. J. Physiol. 210, 1142-1152. 2 Gallagher, K., Mauskopf, J., Walker, J.T. and Lack, L. (1976) J. Lipid Res. 17, 572-577. 3 Rouse, D.J. and Lack, L. (1980) Biochim. Biophys. Acta 599, 324-329.
4 Bundy, R., Mauskopf, J., Walker, J.T. and Lack, L. (1977) J. Lipid Res. 18, 389-395. 5 Playoust, M.R. and Isselbacher, K.J. (1964) J. Clin. Invest. 43, 467-476. 6 Holt, P.R. (1964) Am. J. Physiol. 207, 1-7. 7 Krag, E. and Phillips, S.F. (1974) J. Clin. Invest. 53, 1686-1694. 8 Barnard, J.A. and Ghishan, F.K. (1987) Gastroenterology 93, 925-933. 9 Lucke, H., Stange, G., Kinne, R. and Murer, H. (1978) Biochem. J. 174, 951-958. 10 Hidalgo, I.J., Raub, T.J. and Borchardt R.T. (1989) Gastroenterology 96, 736-749. 11 Hidalgo, I.J. and Borchardt, R.T. (1988). Pharm. Res. 5, 949 (Abstr.). 12 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 13 Pinto, M., Robine-Leon, S., Appay, M.-D., Kedinger, M., Triadou, N., Dussaulx, E., Lacroix, B., Simon-Assmann, P., Haffen, K., Fogh, J. and Zweibaum, A. (1983) Biol. Cell 47, 323-330. 14 Weinberg, S.L., Burckhardt, G. and Wilson, F.A. (1986) J. Clin. Invest. 78, 44-50. 15 Lin, M.C., Weinberg, S.L., Kramer, W., Burckhardt, G. and Wilson, F.A. (1988) J. Membr. Biol. 106, 1-11. 16 Burckhardt, G., Kramer, W., Kurz, G. and Wilson, F.A. (1983) J. Biol. Chem. 258, 3618-3622. 17 Kramer, W., Burckhardt, G., Wilson, F.A. and Kurz, G. (1983) J. Biol. Chem. 258, 3623-3627. 18 Sugiyama, Y., Yamada, T. and Kaplowitz, N. (1983) J. Biol. Chem. 258, 3602-3607. 19 Grafstrom, R., Moldeus, P., Andersson, B. and Orrenius, S. (1979) Med. Biol. 57, 287-293. 20 Pinkus, L.M., Ketley, J.N. and Jakoby, W.B. (1977) Biochem. Pharmacol. 26, 2359-2363. 21 Hofer, M. and Hoggett, J.G. (1981) Transport Across Biological Membranes, p. 96, Pitman, Boston. 22 Duffy, M., Blitzer, B.L. and Boyer, J.L. (1983) J. Clin. Invest. 72, 1470-1481. 23 Schefler, W.C. (1979) Statistics for the Biological Sciences, p. 136, Addison-Wesley, Menlo Park. 24 Hidalgo, I.J. and Borchardt, R.T. (1988) Pharm. Res. 5, 5-110 (Abstr.).
97
Elsevier BBAGEN 23340
Transport of bile acids in a human intestinal epithelial cell line, Caco-2 I s m a e l J. H i d a l g o a n d R o n a l d T . B o r c h a r d t Department of Pharmaceutical Chemistry, Unioersity of Kansas, Lawrence, KS (U.S.A.)
(Received 10 November 1989) (Revised manuscript received9 March 1990)
Key words: Caco-2; Bile acid; Taurocholicacid; Epithelial transport The transport of tanrocholic acid (TA) across Caco-2 cell monolayers was dependent on time in culture and reached a plateau after 28 days, at which time the apical (AP)-to-basolateral (131,) transport was 10-times greater than BL-to-AP transport. The amounts of TA inside the cells following application of 10 nM [I4C]TA to tile AP or BL side of the monolayers (30 min) were approximately equal (54.4 + 2.7 and 64.6 ± 2.8 f m o l / m g protein, respectively). AP-to-BL transport of TA was saturable and temperature-dependent. Vm~, and K m for transport were 13.7 pmol / mg protein per min and 49.7 p M , respectively. The transport of TA had an activation energy of 13.2 kcal- mo1-1, required Na + and glucose. AP-to-BL transport of |14C]TA was inhibited by the co-administration (on the AP side) of either unlabeled TA or deoxycholate, but it was not reduced by the presence of unlabeled TA on the BL side.
Introduction Bile acids are produced in the liver and secreted into the small intestine where by virtue of their surfactant activity they play a major role in the intestinal absorption of lipids. After exerting their action, bile acids are passively or actively transported across the intestinal mucosa and recycled back into the liver [1]. This enterohepatic recirculation ensures minimal loss of bile acids into the feces and maximizes utilization along the entire length of the small intestine. The characteristics of the ileal bile-acid carrier have been investigated in guinea-pig [2-4], rat [5,6] and human [7,8] intestine. These studies have provided valuable information on the ileal bile-acid carrier. It is clearly established that bile acids are taken up by ileal brush border through a carrier-mediated, Na+-depen dent process. Structure-activity studies have suggested that the shape of the steroid nucleus, as well as the negative charge in the side chain of the bile acid, are
Abbreviations: pAH, p-aminohippuric acid; TA, tauroeholic acid; AP, apical; BL, basolateral; DMEM, Dulbecco's Modified Eagle's medium; FBS, fetal bovine serum; NEAA, non-essentialamino acids; HBSS, Hanks' balanced salt solution; 2-DG, 2-deoxyglucose;TEER, transepithelial electrical resistance; KRB, Krebs-Ringer buffer; DC, dcoxycholicacid. Correspondence: R.T. Borchardt, Department of Pharmaceutical Chemistry, University of Kansas, Lawrence,KS 66045, U.S.A.
essential for an efficient coupling with the carrier system [1,2]. Furthermore, optimal interaction between Na + and the bile acid are believed to be a requirement for transport [2]. The N a ÷ gradient needed for the uphill transport of bile acids is maintained by Na+/K+-ATPase, which is located in the basolateral membrane of the enterocyte. Previously, we presented evidence that the human colon carcinoma cell line, Caco-2, when grown on a microporous membrane, form a polarized monolayer of enterocytic cells which can be useful in the study of intestinal epithelial transport of drugs, peptides, proteins and macromolecules [10,11]. In this report, we show that Caco-2 cells possess a bile-acid carrier system which may be useful in the investigation of the various processes underlying the intestinal uptake and the vectoriai transport of bile acids at the cellular level. Portions of this work were presented at the Annual Meeting of the American Association of Pharmaceutical Scientists, 2 November 1988, and published in abstract form in Ref. 24. Materials and Methods Materials
The Caco-2 cell line was obtained from American Type Culture Collection (Rockville, MD) and used between passages 59 and 70. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and non-
0304-4165/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
98 essential amino-acids (NEAA) were obtained from Hazleton Research (Lenexa, KS). Transwell T M clusters, PVP free, 24.5 mm in diameter (4.71 cm 2 surface area) and 3.0 # m pore size were purchased from Costar (Bedford, MA). Rat tail collagen (Type I) was from Collaborative Research (Lexington, MA). Penicillin, streptomycin and fungizone were obtained as a mixture from Gibco Laboratories (Grand Island). Lucifer yellow C H was from Molecular Probes (Eugene, OR); [14C]Taurocholic acid ([14C]TA, 56 m C i / m m o l ) from Amersham (Arlington Heights, IL); Hanks' balanced salt solution (HBSS), ouabain, N - 2 - h y d r o x y e t h y l p i p e r a z i n e - N ' - 2 - e t h a n e sulfonate (Hepes), sodium azide and 2-deoxyglucose (2-DG) from Sigma (St. Louis, MO). All other chemicals were reagent grade.
Cell culture Caco-2 cells were plated at a density of 63000 cells/cm 2 on Transwell T M polycarbonate membranes (3.0 # m pore size) which had been coated with collagen. The culture medium, consisted of DMEM, 10% FBS, 1% NEAA, 100 U / m l penicillin and 100/xg/ml streptomycin. The culture medium was replaced (1.5 ml inside and 2.6 ml outside) daily. Cells were maintained at 37°C in an atmosphere of 5% CO 2 and 90% relative humidity. All cells used in this study were between passages 59 and 70. Uptake studies The uptake of [I'IC]TA was investigated in Caco-2 cells and grown on Transwell T M for 30 days. Cells were washed twice with serum-free medium and then incubated with HBSS that contained 25 mM glucose and 10 p m o l / m l [14C]TA on the AP or BL side at 37°C. After 30 min, monolayers were washed three times with ice-cold HBSS, harvested in 1 ml 1% Triton-X that contained 0.3 M N a O H and allowed to dissolve for 30 min at 37°C. Transport studies Monolayer integrity. Prior to the transport experiments the integrity of Caco-2 cell monolayers was assessed by measuring transepithelial electrical resistances (TEER) using an EVOM epithelial voltohmmeter, World Precision Instruments (New Haven, CT). [14C]TA solutions also contained lucifer yellow (0.1-0.5 m g / m l ) to further monitor potential monolayer leakage. Sampling. The culture medium of Transwell T M grown Caco-2 monolayers was replaced with HBSS containing 25 m M glucose and 10 m M Hepes buffer (pH 7.35) (transport medium). Subsequently, cell monolayers were equilibrated at the temperature of the corresponding experiment for 30 rain before undertaking the transport studies. In AP-to-BL transport studies all wells in sixwell clusters received 2.5 ml of transport medium which
had been equilibrated at the appropriate temperature. Inserts were positioned in the first well with the outer surface of the inserts (BL side) immersed in the transport medium. [14C]TA was added to the AP side and sampling was performed by transferring the insert to subsequent wells in the cluster that contained fresh transport medium. In BL-to-AP transport studies, inserts containing 1.5 ml of transport medium were placed in 35-mm wells that contained 2.5 ml of [14C]TA solutions. In this way only the BL cell surface was exposed to the radioactive bile acid. Sampling was performed by withdrawing carefully the 1.5 ml in the insert (AP side) and replacing it with an equal volume of fresh transport medium. Measurements. The amount of TA transported at each interval was determined in a Beckman LS-5801 Liquid Scintillation Counter. LY was measured in a SLM4800 Aminco-Bowman spectrophotofluorometer. Total protein was measured by using the method of Lowry et al. [12]. Effect of time in culture on transport. Caco-2 cells, seeded at the usual density, were cultured for different lengths of time. On selected days, T E E R values were measured, and 70000 d p m / m l [14C]TA were applied to either the AP or BL side of at least four monolayers which were incubated at 37°C for 60 rain. The amounts of TA transported at 5, 10, 15, 30, 45 and 60 min were added to obtain cumulative transport.
Characterization of the bile acid carrier system Concentration and time-dependency. Several concentrations of [~4C]TA ranging from 1 to 100 # M were applied to the AP side of the monolayers and samples removed from the BL side at 15, 30, 45, 60, 90 and 120 min. Radioactivity measurements were used to calculate the amount of TA that underwent AP-to-BL transport at each interval. Temperature-dependency. The effect of temperature on TA transport was investigated by applying 0.05/tM [14C]TA to the AP side of monolayers which were then incubated either at 37, 23 or 4°C for 60 min. Samples were taken from the BL side at the same time-intervals indicated above. Activation energy for transport. The rates of transport of TA at 23 and 3 7 ° C were utilized to obtain an Arrhenius plot and calculate E a, the activation energy for transcellular transport of this bile acid. Transport against concentration gradient. [laC]TA was applied to the AP side of two groups of monolayers. In the control group, the BL (receptor) side contained only transport medium and in the test group the BL side contained 10 # M unlabeled TA dissolved in transport medium. Inhibition studies. To determine the energy-dependence of the bile-acid carrier, the transport of TA was studied in glucose-free HBSS and in the presence of 1
99 m M sodium azide plus 50 mM 2-DG. The sodium-dependence of TA transport was examined by carrying out the experiment in Krebs-Ringer buffer (KRB, p H 7.35) (control), in KRB that contained K ÷ or choline instead of N a +, and in the presence of KRB that contained 2.5 mM ouabain. The transport of [14C]TA was carried out in the presence of 25, 125 and 250 /~M concentration of deoxycholic acid (a dihydroxylated bile acid) and TA to assess the affinity of this bile-acid carrier. Results
The development of polarity in Caco-2 cells is reflected in an increased expression of the marker enzymes sucrase-isomaltase, alkaline phosphatase and aminopeptidases [10,12]. For that reason, to determine the optimal time for maximal bile-acid transport, TA transport studies in Caco-2 cells were carried out at different times in culture. Between days 6 and 13, both AP-to-BL and BL-to-AP transport of TA showed a decrease (Fig. 1A) which was most likely due to an increased sealing capacity of the occluding junctions. From day 13 to day 28, however, there was a gradual increase in AP-to-BL transport and a slight decrease in the already low BL-to-AP transport (Fig. 1A). Since the flux of the leakage marker, LY (MW 457), was less than 0.25%/h at all times after day 13, it can be concluded that the translocation of TA across Caco-2 monolayers cultured for 13 or more days was mainly the result of transcellular transport. This
indicates that maximal expression of the bile-acid carrier takes longer than the morphological differentiation (2 weeks). Indeed, at day 28 + in culture the rate of transport in the AP-to-BL direction was more than 10-times faster than that in the opposite direction (Fig. 1B). To characterize the bile-acid carrier, only monolayers with T E E R values greater than 250 ohms. cm 2 and lucifer yellow (LY) fluxes of less than 0.25%/h were utilized. The number of monolayers that had to be excluded due to excessive leakage was less than 5%. The amounts of TA inside the cells after 30 rnin incubation with the labeled bile-acid on the AP (n = 4) and BL (n = 4) side were 54.4+ 2.7 and 6 4 . 6 + 2.8 f m o l / m g protein, respectively. Appearance of TA on the BL side following AP application exhibited an initial lag time after which transport proceeded at a faster, yet constant rate (Fig. 2). The appearance of []4C]TA on the basolateral side is not likely to be due to exchange of radioactive for non-radioactive TA (initially in the BL side), since the BL-to-AP transport of TA was minimal compared to AP-to-BL transport. Moreover, TA transport increased linearly at concentrations up to 10 /~M, but saturation of the transport system was observed above this concentration (Figs. 2B, 3). Vmax and K m values for transport were 13.7 p m o l / m g protein per min and 49.7/~M, respectively. TA was transported against a concentration gradient (10 × ) at the same rate as in the absence of such a gradient (Fig. 4). To determine the temperature-dependence of transport, 0.05 /xM [14C]TA was applied to the AP side of
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TIME (MIN) Fig. 1. Effect of time in culture on T A transport. (A) O n specified days [14C]TA (1.0 p M , 70000 d p m / m l ) was applied to the A P side (1.5 ml) ol BL side (2.5 ml). The monolayers were incubated under 5% CO 2 for 1 h at 37°C and samples were taken at 15, 30, 45 and 60 min and the cumulative a m o u n t s of T A transported both in the AP-to-BL ([]) and BL-to-AP (11) direction were determined. (B) Time-course of AP-to-BI transport at 37 ° C (D) and 4 ° C ( o ) and BL-to-AP transport at 37 ° C (m) in 30-day-old monolayers. Values are m e a n + S.D. (n = 4).
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Fig. 2. Concentration-dependency of TA transport. 30-32 day-old Caco-2 cell monolayers were administered 2 ml transport medium containing 70000 d p m / m l []4C]TA (on the AP side). Panel A shows 1 (o), 5(*) and 10 (12) /AM and panel B 25 (e), 50 (A) and 100 ( - - - - - - ) #M. After incubating for 60 rain under the same conditions described in Fig. 1, samples were withdrawn from the BL side at selected times (indicated). Values shown are mean + S.D. (n = 4).
30-day-old monolayers which were then incubated at different temperatures. The rates of transport were 0.014 and 0.0045 pmol/mg per rain at 37 and 23°C, respectively (Fig. 5). However, at 4°C, TA was not detected
on the BL side, indicating that TA transport is entirely temperature-dependent. Using the steady-state transport rate at different temperatures, an Arrhenius plot was constructed and the activation energy ( E . ) for
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CONC (pM) Fig. 3. Saturation of TA transport in Caco-2 cells. The rates of transport at different concentrations of [14C]TA were determined from the terminal (steady-state) portion of the graphs of pmoles transported per nag vs. time. Inset: double-reciprocal plot of data. l / V = ( p m o l / m g per rain) and 1 / C = ~ t M - ] . Values represent the mean + S.D. (n = 4).
~ME (Wt0 Fig. 4. Transport of TA against a concentration gradient. 0.05 /~M []4C]TA was added on the AP side of two groups of Caco-ceU monolayers which were 30-32 days old. The receiver (BL) side of the first group (12) contained only transport medium (control) while that of the second group (It) contained 10/~M tmlabelled TA. Monolayers were incubated trader the previously described conditions and sampies were taken at the specified times. Values shown are the mean + S.D. (n = 4).
101 0.8
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Concentration-dependent inhibition of [HC]TA transport by unlabeled taurocholic acid (TA) and deoxycholic acid (DC) The incubation time was 60 rain. Four monolayers were used in each experiment. The transport of 0.01 /xM [z4C]TA when administered alone (control) was equal to 0.09 p m o l / m g (+0.017), and this value was defined as 100%.
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transcellular transport calculated to be equal to 13.2 kcal • m o l - t The transport of TA was significantly reduced in the presence of sodium azide plus 2-DG or in the absence of glucose (Table I), implying energy-dependence. The administration of ouabain and the replacement of Na + TABLE I
Effect of experimental conditions on [HC]TA transport Caco-2 monolayers were washed with and preincubated (15 min) in either Krebs-Ringer Buffer that contained 1 m g / m l glucose (KRB, control), K R B containing 1 m M sodium azide + 50 m M 2-deoxyglucose, K R B where N a + was substituted with K +, K R B where N a + was substituted with choline, 2.5 m M ouabain dissolved in K R B that contained choline instead of N a +, glucose-free KRB, K R B containing no glucose and no N a ÷, or K R B containing pAH. To determine transport, the monolayers where incubated with 0.05 # M [Z4C]TA dissolved in the appropriate buffer for 30 min at 37°C. Whenever appropriate, the osmolarity was adjusted to approx. 310 m o s M / k g with D-mannitol. The transport rate in the control group was 0.011 p m o l / m g protein per min. Statistically significant differences were evaluated using one-way analysis of variance (ANOVA) and specific differences from the control ( * P < 0.05) were identified using a multiple range t-test [23]. Each group consisted of between three and six monolayers. Conditions
Transport (% + S.D.)
K R B (Control) Azide + 2 - D G / K R B (-)Na
+,( + )K
÷/KRB
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100 + 14 58.5 5 : 3 . 1 47.7+ 8.9 75.3+ 9.2 55.3+ 7.5 66.9+ 9.3 28.0 + 6.2 97.1 + 8.3 73.5+ 4.9
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with K + or choline resulted in a significant decrease in transport (Table I), suggesting that the process is at least partly Na+-dependent. The transport of TA was lower when both Na ÷ and glucose were absent than in the absence of either Na ÷ or glucose alone, p A H failed to inhibit the transport of 10/~M [14C]TA when co-administered at 1 mM concentration but resulted in a 27% ( P < 0.05) inhibition at 10 m M concentration (Table I). Relatively low concentrations (25 v M ) of both unlabeled TA and deoxycholic acid reduced the transport of [14C]TA by almost one-half ( P < 0.01) (Table II). Increasing the concentration of the inhibitors had a moderate effect on [zaC]TA transport (Table II). Discussion
Recently, we presented evidence that the human colon adenocarcinoma cell line, Caco-2, can be a valuable model system for the study of intestinal epithelial transport [10]. Here we demonstrate that Caco-2 cells exhibit transcellular transport of TA and may be helpful for investigating bile-acid transport across the epithelial mucosa of the small intestine. The dependency of TA transport on time in culture is hardly surprising since we found previously that although Caco-2 cells were fully differentiated by day 15, alkaline phosphatase activity continued to increase through day 20 in culture [10,13]. Similarly, aminopeptidase and sucrase-isomaltase activities were found to increase after cell differentiation was completed [13]. Comparable amounts of TA were found in the cells following the presentation of the labeled bile-acid to the AP or BL membranes. Thus, it appears that the polarized delivery of the bile-acid is determined by either the movement across the cytoplasma or the preferential efflux across the BL membrane, and not by the initial uptake.
102 That TA is targeted to the BL side despite being taken up by both the AP and BL membranes indicates that the carrier system of the AP membrane may be different from that in the BL membrane. This interpretation is supported by previous studies where an anion exchange transport system, constituted by two polypeptides ( M r 54000 and 59000), was identified in the ileal BL membrane [14,15]. This transport system is different from the polypeptide associated with the bileacid carrier found in the brush-border membrane ( M r 99 000) [16,17]. Although a direct comparison between uptake and transcellular transport is not appropriate, because uptake is a single process and transport involves uptake, movement through the cytoplasm and efflux at the opposite membrane domain, the analysis of our transport data in light of the existing data on bile-acid uptake may prove useful. The apparent Krn for transport obtained in this study (i.e., 49.7 #M) is in the same order of magnitude as that reported recently for TA uptake by human ileal brush-border membrane vesicles (37 #M) [8]. However, the corresponding Vm values differed dramatically (13.7 vs. 6560 p m o l / m g protein per min). The similarity in K m values as well as the discrepancy between Vm values can be explained if we assume that the bile-acid carrier of Caco-2 cells is reminiscent of the human ileal carrier and that such a carrier is present in fewer numbers in Caco-2 cells. An alternative explanation may be that translocation through the cytoplasm a n d / o r subsequent efflux at the BL side may be slower than the initial uptake and this limits the overall capacity for transcellular transport. The activation energy required for TA transport across Caco-2 ceils is in agreement with carrier-mediated transport, because the E a for enzyme-catalyzed reactions and carrier-mediated transport range from 7 to 25 k c a l / m o l and the E a for simple diffusion is usually lower than 4 kcal-mo1-1 [21]. Because previous reports have shown Na+-dependent TA uptake by other preparations [2,8], it was not surprising that the N a + / K + - A T P a s e inhibitor, ouabain, reduced the transport of TA. However, the magnitude of the residual transport (53%), in the presence of ouabain and when Na + was substituted with K ÷ or choline, is higher than that reported in ileal brush-border membrane vesicles [8,22] and suggests the existence of a Na+-independent pathway in Caco-2 cells and in ileal enterocytes. The results from our study did not reveal the existence of a bile-acid cartier completely dependent on the presence of N a ÷ as that encountered in the intestine. Although this difference seems to point at a dissimilarity between the Caco-2 cells and the normal enterocyte, such an apparent discrepancy may partly be due to the fact that while our study determined the overall transcellular transport in the intact, polarized
cell, previous studies have looked at uptake in isolated membrane preparations [8,22]. However, detailed experiments dealing independently with uptake by the AP membrane and efflux through the BL membrane in the intact cell will be most useful in testing this hypothesis. To assess the specificity of the bile-acid carrier system we selected one unconjugated bile-acid, deoxycholic acid (DC) and compared its ability to inhibit the transport of [14C]TA to that of unlabeled TA. Both TA and DC possess a negative charge, which has been indicated to be important for interaction with the carrier on the side chain [1]. Furthermore, since the bulkiness of the taurine conjugate in TA should have a minimal effect on transport [!], it follows that the similar inhibitions of [14C]TA transport caused by TA and DC indicate that the bile-acid carrier did not discriminate between these two compounds. These data suggest that the structural requirements of the ileal bile-acid carrier may not be the same as those of the bile-acid carrier of Caco-2 cells. It is not known whether the interaction between DC and TA took place at membrane binding sites or, less likely, through some nonspecific depression of transport caused by DC. In addition, even if DC inhibited TA binding, this would not constitute sufficient evidence that these bile acids share the same carrier because some compounds can inhibit the transport of others without undergoing transport themselves [4]. The presence of an anion transport system on the apical membrane which would transport structurally related organic anions could explain the inhibition seen with DC. However, this possibility can be ruled out (at least in part) by the lack of effect of 1 m M pAH on the AP-to-BL transport of 10/~M [14C]TA. This absence of inhibition suggests that the bulk of the AP ~ BL transport of bile acids in Caco-2 cells most likely involves a cartier system different from an anion transporter. This is supported by data showing that the AP uptake of [14C]TA was not affected by 1 m M concentrations of pAH (not shown). The significant reduction of AP-to-BL transport of [14C]TA observed following the co-administration of 10 mM pAH may be difficult to interpret at this time. While exposure to 10 and 50 mM concentrations of pAH did not result in generalized toxicity, as judged by the Trypan blue exclusion test, we do not know whether this concentration of pAH caused some nonspecific depression of cell function which may result in lower transport of bile-acid without necessarily exhibiting the type of toxicity detectable by this test. In conclusion, this study has demonstrated the existence of a bile-acid carrier system in Caco-2 cells, whose maximal expression is achieved after 28 + days in culture. Carrier-mediated transport of bile acids has been observed in human ileal but not in jejunal brushborder membrane vesicles [8]; therefore, these results suggest that Caco-2 cells exhibit ileal characteristics. Although Caco-2 cells take up the bile-acid, TA, through
103 b o t h the A P a n d BL m e m b r a n e s , the release of T A across the A P m e m b r a n e is virtually negligible, with the vast m a j o r i t y of the bile-acid b e i n g released across the BL m e m b r a n e . Such polarized delivery of bile acids to the BL m e m b r a n e s resembles that of the n o r m a l enterocytes. T r a n s p o r t b y this carrier seems to be susceptible to the same factors c o n t r o l l i n g u p t a k e in the ileal b r u s h border. However, it m a y be p r e m a t u r e to equate the bile-acid carrier f o u n d i n Caco-2 cells to that in ileal enterocytes. T o m a k e this possible, detailed studies m u s t be carried out to d e t e r m i n e the characteristics a n d relative i m p o r t a n c e of apical u p t a k e a n d basolateral efflux in the t r a n s p o r t of bile acids across Caco-2 cell monolayers. Despite the need for a d d i t i o n a l characterization of the bile-acid carrier of Caco-2 cells, it is clear that some of the similarities described in this study together with the virtually u n l i m i t e d viability of polarized Caco-2 cell m o n o l a y e r s will m a k e this system a valuable alternative tool in bile-acid t r a n s p o r t research.
Acknowledgements This work was s u p p o r t e d b y a g r a n t from the U p j o h n C o m p a n y . W e t h a n k Drs. T o m J. R a u b a n d Norm a n F. H o for r e a d i n g the m a n u s c r i p t a n d p r o v i d i n g constructive criticism.
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