Constitutive in a human

expression of the taurine colon carcinoma cell line

transporter

CHINNASWAMY TIRUPPATHI, MATTHIAS BRANDSCH, YUSEI MIYAMOTO, VADIVEL GANAPATHY, AND FREDERICK H. LEIBACH Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30912-2100

substrates has been demonstrated in the intestinal brush-border membrane (2, 22-24, 26). This transport system is active, driven by three different energy human colon carcinoma cell line. Am. J. Physiol. 263 (Gassources: a transmembrane Na+ gradient, a transmemtrointest. Liver Physiol. 26): G625G631, 1992.-The human brane Cl- gradient, and a membrane potential. The incolon carcinoma cell line HT-29, when grown to confluence, was testinal p-amino acid transporter exhibits high affinity found to take up taurine and accumulate it against a concenfor taurine. A similar transporter has also been detration gradient from a NaCl-containing uptake medium. Rescribed in other tissues such as kidney, liver, brain, retplacement of NaCl with choline chloride almost totally abolinal pigment epithelium, placenta, and Ehrlich ascites ished the uptake. Taurine uptake was dependent not only on tumor cells (3, 5, 6, 11, 16, 19-21). The studies on the Na+ but also on Cl-, because other anions failed to support the uptake in the presence of Na+. The uptake process was specific intestinal transport of taurine have so far utilized either for P-amino acids such as taurine, hypotaurine, and ,&alanine. intestinal perfusion techniques or isolated brush-border Apparently, a single transport system with a Michaelis-Menten membrane vesicles and have solely concentrated on the constant of 10.6 t 0.3 PM was responsible for the uptake. Stomechanistic aspects of the transporter. Availability of ichiometric analyses revealed that the Na+:taurine coupling raan in vitro cell culture system as an experimental model tio was 2:1, whereas the Cl-:taurine coupling ratio was 1:l. to investigate the intestinal taurine transport would cerCulture of the cells in the presence of taurine caused downregulation of the uptake system. These cells were also capable of tainly expand the scope of the studies that can be done, especially with regard to the regulatory aspects of the accumulating ,&alanine against a concentration gradient in the transporter. Such an approach has been successfully empresence of NaCl. @-Alanine uptake occurred via a single transport system with an apparent Michaelis-Menten constant of 36 ployed for the studies involving taurine transport in the t 2 PM. Taurine and p-alanine exhibited mutual interaction kidney and the placenta. Established ceil lines of renal during uptake. Kinetic experiments strongly suggested that a (LLC-PK1 and MDCK) and placental (JAR) origin have common transporter was responsible for the uptake of these two been shown to be suitable models to study regulation of ,&amino acids. It is concluded that the HT-29 cells constitutaurine transport in the kidney and placental tissues, tively express the taurine transporter and that this cell line may respectively (14, 15). To identify such a cell culture be a suitable model for investigations of intestinal taurine transmodel system for the intestinal transport of taurine, we porter. investigated the characteristics of taurine transport in a sodium chloride dependence; stoichiometry; p-alanine human colon carcinoma cell line (HT-29). The results of the present study show that the HT-29 cells constituTHERE HAS BEEN INCREASING INTEREST in recent years tively express a taurine transporter whose properties are in the mechanisms involved in the intestinal absorption similar to those of the taurine transporter described in of taurine from dietary sources. The primary reason for the normal small intestine, suggesting that this cell line this interest is the uniqueness of various aspects of taucan be used in future studies to investigate the regularine metabolism in animals. Taurine is a nonprotein tory aspects of the intestinal taurine transport. amino acid that is present in millimolar concentrations in many tissues including the brain, heart, small intesMATERIALS AND METHODS tine, and retina (4, 13, 38). Taurine deficiency leads to [2-“H(n)]taurine (sp radioact 25.6 Ci/mmol), ,8-[l-14C]alaretinal degeneration and cardiac dysfunction in laboranine (sp radioact 60 mCi/mmol), ,&[3-3H(n)]alanine (sp radiotory animals as well as humans (9, 12, 27, 35). The act 120 Ci/mmol), 3-O-[ methyL-3H]methyl-r>-glucose (sp raconcentration of taurine in the brain (expressed as dioact 79 Ci/mmol), and [carbo;lcy-l*C]inulin (sp radioact 30.3 mol/g tissue) is developmentally related, being the highCi/g) were purchased from Du Pont-New England Nuclear est in the fetus and the newborn and gradually decreas- (Boston, MA). The HT-29 human colon carcinoma cell line was obtained from American Type Culture Collection (Rockville, ing with age to reach its adult values (34). In contrast, MD). Unlabeled amino acids were from Sigma (St. Louis, MO). even though taurine can be synthesized endogenously All other chemicals were of analytical grade. Dulbecco’s modifrom methionine and cysteine in the liver and in the brain, the biosynthetic capacity is the lowest in the fetus fied Eagle’s medium containing D-glucose and L-glutamine (catno. 320-1965 AJ) and fetal bovine serum were purchased and the newborn and increases with age to reach its alog from GIBCO (Grand Island, NY). adult values (17, 31). These unique aspects of taurine Cell culture. The HT-29 cells were routinely cultured in 75 metabolism suggest that the intestinal absorption of di- cm2 Corning culture flasks with Dulbecco’s modified Eagle meetary taurine is obligatory for the maintenance of the dium containing 10% fetal bovine serum. The cells were mainhigh taurine levels in developing animals. tained at 37°C and 5% C02. Confluent cultures were trypsinized The existence of a specific transport system that ex- with phosphate-buffered saline containing 0.1% trypsin and and subcultures started elusively accepts p-amino acids, including taurine, as 0.25 mM ethylenediaminetetraacetate Tiruppathi, Chinnaswamy, Matthias Brandsch, Yusei Miyamoto, Vadivel Ganapathy, and Frederick H. Leibath. Constitutive expression of the taurine transporter in a

0193-1857/92

$2.00 Copyright 0 1992 the American Physiological Society

G625

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G626

TRANSPORT

OF P-AMINO

from released cells. For the uptake experiments, the cells were seeded in 35mm disposable Petri dishes (Falcon) at a density of -2 x lo6 cells/dish and allowed to grow to confluence (3 days). The cells received 2 ml of fresh culture medium 24 h after subculturing and were used in uptake measurements on the third day. Uptake measurement. Uptake of taurine or P-alanine in confluent cultures of HT-29 cells was measured at room temperature (21-22”C), as described earlier (7, 15). The culture medium was removed from the dish, and the cells were washed twice with the uptake medium before initiation of uptake measurement. The composition of the uptake medium was (in mM) 25 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES)/tris(hydroxymethyl)methane (Tris) (pH 7.5), 140 NaCl, 5.4 KCl, 1.8 CaC12, 0.8 MgSO*, and 5 D-&CoSe. After the wash, the uptake medium containing radiolabeled substrate (1 ml) was added to the dish. After incubation for a desired time, the medium was removed, and the cells were quickly washed four times with the uptake medium, after which 1 ml of 0.3 N NaOH was added to each dish. After 0.5-l h, the contents from the dish were transferred to a scintillation vial, and the radioactivity of the contents were determined by liquid scintillation spectrometry. In the experiments determining the effects of Na+, choline chloride was substituted for NaCl in the uptake medium. Where the effects of Cl- and other anions were investigated, the uptake medium was modified by replacing KCl, MgS04, and CaCl, with respective gluconate salts. Protein determination. For each experiment, protein was assayed using duplicate dishes that were cultured under conditions identical to those dishes employed in uptake measurements. After culture medium was removed, 1 ml of deionized water was added to each dish. The dishes were frozen and thawed twice after which the dish contents were suspended with the use of a l-ml syringe and 25-gauge needle before protein estimation. Protein was determined by the method of Lowry et al. (18). Determination of intracellular water space. Equilibrium (90min incubation) distribution of radiolabeled 3-O-methyl-D-glucase was used to determine the intracellular water space, and the correction for the extracellular water space was made by measuring the inulin space under identical experimental conditions. Statistics. Each experimental point was determined with duplicate or triplicate dishes. Unless indicated otherwise, each experiment was repeated two or three times with the use of separately seeded generations of the cells. Results are expressed as means & SE from these replicate determinations. Kinetic analyses were done with the computer package Statgraphics (STSC, Rockville, MD).

ACIDS

IN HT-29

CELLS

30 60 Incubation time

90 (min)

120

Fig. 1. Time course of taurine transport in the HT-29 colon carcinoma cell line. Uptake of taurine (final co&n 50 nM) in confluent monolayer cultures of HT-29 cell line was measured using 2 different uptake media whose composition was (in mM) 25 HEPES/Tris (pH 7.5), 5.4 KCl, 0.8 MgSO,, 1.8 CaCl,, 5 glucose and either 140 NaCl (0) or 140 choline chloride (0). Monolayers were incubated with respective medium for 15 min before initiation of uptake measurement.

measured under the experimental conditions was negligible. The intracellular water space was similar whether the uptake medium contained NaCl or choline chloride (4.66 -+ 0.08 pl/mg of protein). At a concentration of 50 nM taurine in the NaCl-containing uptake medium, the amount of taurine that accumulated inside the cells was 1.67 t 0.12 pmol/mg protein at 5 min incubation, and this value increased to 26.1 t 1.2 pmol/mg protein at 120 min. These amounts are equal to an intracellular concentration of 360 nM and 5.6 PM, respectively. Thus accumulation of taurine inside the cells against a concentration gradient was evident at an incubation period of as short as 5 min. On the other hand, there was no evidence for concentrative uptake of taurine from the choline chloRESULTS ride-containing medium. Na+ and Cl- dependence of taurine uptake. Figure 1 The experiment described in Fig. 1 shows the Na+ describes the time course of taurine uptake in HT-29 cells dependence of taurine uptake in HT-29 cells. Uptake of from an uptake medium that contained NaCl and from a taurine in many tissues including the small intestine is medium in which NaCl was replaced by choline chloride. dependent on Na+ as well as Cl-. The anion is cotransThere was very little uptake from the choline chlorideported along with taurine and Na+ during the uptake process, and a transmembrane Cl .- gradient provides encontaining medium. In contrast, taurine was avidly taken up by the cells from the NaCl-containing medium. In the ergy for the concentrative uptake catalyzed by the transpresence of NaCl, uptake was linear with time at least up porter. Therefore, to determine whether the uptake of taurine in HT-29 cells also exhibits a similar dependence to 30 min. The extracellular water space calculated from equilibon Cl-, we studied the influence of various anions on the uptake of taurine (Table 1). Uptake was rium uptake of inulin (0.41 t 0.02 pl/mg of protein) was Na+-dependent minimal from uptake media containing NaI, NaF, ~10% of the total water space determined from equilibNaNOZ, or Na gluconate in place of NaCl, being ~10% of rium uptake of 3-O-methyl-D-glucose, a nonmetabolizable substrate for the facilitative glucose transporter (5.07 the control uptake measured in the presence of NaCl. t 0.08 pl/mg protein). The amount of taurine in the NaSCN could substitute for NaCl to some extent. These results show that the transport system responsible extracellular water snace contributing to the total untake

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TRANSPORT

OF P-AMINO

ACIDS

IN HT-29

G627

CELLS

Table 1. Anion specificity of taurine transport in the HT-29 colon carcinoma cell line Taurine Inorganic

Uptake

Salt pm01 - mg protein-l

- 10 min-l

%

NaCl 100 1.88t0.13 NaI 0.17&0.01 9 0.06t0.01 3 NaF NaN03 0.14t0.01 7 Na gluconate 0.02t0.01 1 NaSCN 0.41t0.02 22 Values are means t SE from a single experiment done in triplicate. Uptake of taurine (20 nM) was measured in confluent monolayer cultures of HT-29 cell line using a IO-min incubation. Uptake medium contained (in mM) 25 HEPES/Tris (pH 7.5)) 5.4 K gluconate, 1.8 Ca gluconate, 0.8 Mg gluconate, 5 glucose, and 140 of indicated inorganic salt.

for taurine dependent.

uptake in these cells is Na+ as well as Cl-

Substrate specificity. The taurine transporter described in various tissues is specific for taurine and other p-amino acids such as hypotaurine and ,&alanine and does not accept a-amino acids as substrates. The transport system in HT-29 cells that is responsible for taurine uptake also shows a similar substrate specificity (Table 2). Hypotaurine and ,&alanine, at a concentration of 100 PM, inhibited >70% of taurine uptake measured at 20 nM. Likewise, unlabeled taurine effectively competed with radiolabeled taurine for the uptake process. On the contrary, three a-amino acids (alanine, leucine, and aminoisobutyric acid) and one y-amino acid (y-aminobutyric acid) tested in the present study failed to inhibit taurine uptake to any significant extent. Kinetics and stoichiometry. The relationship between the uptake rate and the substrate concentration for taurine uptake was studied by measuring the initial uptake rates (lo-min incubation) at varying concentrations of taurine over a range of 2.5-60 PM. The uptake rate was found to be saturable with increasing concentrations of taurine, and the plot of taurine concentration versus uptake rate was hyperbolic, apparently indicating participation of a single transport system in the uptake process (Fig. 2). This conclusion was supported by the linear plot [coefficient of determination (r2 = 0.99)] of uptake

Table 2. Substrate specificity of the taurine in the HT-29 colon carcinoma cell line [3H] taurine Unlabeled

Amino

0.04 15 30 s, Concentration

45 of taurine

v/s

0.08

60 QJM)

Fig. 2. Kinetics of taurine transport. Uptake of taurine in confluent monolayer cultures of HT-29 cell line was measured over a concentration range of 2.5-60 PM. Uptake medium contained NaCl, and time of incubation was 10 min. For each concentration of taurine, concentration of radiolabeled taurine was maintained at 40 nM. Uptake of radiolabel measured in the presence of 500 PM unlabeled taurine was used to calculate nonmediated component of taurine transport, and this value was subtracted from total uptake to determine mediated component. Inset: Eadie-Hofstee plot (v/s versus v).

rate/taurine concentration vs. uptake rate (EadieHofstee plot). The apparent Michaelis-Menten constant (KJ for the uptake process was 10.6 t 0.3 PM, and the maximal velocity ( Vmax) was 0.94 t 0.01 nmol mg protein-l 10 min-l. To determine the Na+ and Cl- stoichiometry for the taurine uptake process in these cells, we employed the activation method as described by Turner (36). It has to be pointed out here, however, that this experimental approach does not allow us to distinguish between catalytic and energetic activation of taurine uptake by Na+ and Cl-. The initial uptake rates of taurine at a fixed concentration (30 nM) were measured at varying concentrations of Na+ (5-140 mM) but at a constant Cl- concentration (140 mM). The relationship between the uptake rate and the Na+ concentration was found to be sigmoidal, suggesting participation of more than one Na ion per l

transporter 0.05 Concentration

0.1

0.15

of Na’

UW 44 0

Uptake

Acid pm01 - mg protein-l

Control Taurine Hypotaurine ,&Alanine y-Aminobutyric Alanine Leucine cu-Aminoisobutyric

1

acid

acid

2.19kO.05 0.17t0.02 0.14&0.01 0.64kO.02 1.89t0.25 2.22kO.09 2.37kO.11 2.44t0.06

- 10 min-1

%

100 8 6 29 86 101 108 111

Values are means & SE from 2 separate experiments done in duplicate (n = 4). Uptake of radiolabeled taurine (20 nM) was measured in confluent monolayer cultures of HT-29 cell line using a lo-min incubation. Uptake medium contained NaCl. When present, the concentration of unlabeled amino acids was 100 PM.

a +; 500 r ’ 250 t

1

2

3

V

Fig. 3. Na+ kinetics of taurine transport. Uptake of taurine (final concn 30 nM) in confluent monolayer cultures of HT-29 cell line was measured with a lo-min incubation in uptake media containing different concentrations of Na+ (5-140 mM). Osmolality and concentration of Cl- (140 mM) were maintained by appropriately substituting choline chloride for NaCl. Uptake measured in the absence of Na+ was subtracted from each uptake value to determine the Na+-dependent component.

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G628

TRANSPORT

OF ,&AMINO

transport of one molecule of taurine (Fig. 3). To calculate the number of Na ions involved per transport cycle, the experimental data were analyzed according to the Hilltype equation

y-m Wa+lN ’ = KONg + [Na+lN

where v is the initial uptake rate, KO 5 is the Na+ concentration necessary for half-maximal activation, and N is the Hill coefficient (i.e., the no. of Na ions involved per transport cycle). The Hill-type plot was not linear when N = 1 (v vs. v/[Na+]) but became linear (r2 = 0.97) when N was assigned a value of 2 (v vs. v/[Na+12). Thus the Na+:taurine coupling ratio appeared to be 2:l. The approximate value for KOS5for Na+ was 60 mM. Similar experiments were made to determine the Cl-:taurine coupling ratio (Fig. 4). The initial uptake rates of taurine at a concentration of 30 nM were measured at varying concentrations of Cl- (2.5-140 mM) but at a fixed concentration of Na+ (140 mM). In contrast to the Na+ kinetics, the relationship between the uptake rate and the Cl- concentration was hyperbolic, suggesting a Cl-:taurine coupling ratio of 1:1. This conclusion was supported by the linear Hill-type plot (r2 = 0.98) when N was assigned a value of 1 (v vs. v/[Cl-I). The approximate value for KOS5for Cl- (i.e., the concentration of Cl- necessary for half-maximal activation) was 40 mM.

ACIDS IN HT-29 CELLS

Cl- (data not shown). Uptake over a @alanine concentration range of lo-200 PM was found to be saturable (Fig. 6), and the linear (r2 = 0.98) Eadie-Hofstee plot (uptake rate/b-alanine concn vs. uptake rate) apparently suggested participation of a single transport agency catalyzing the uptake of P-alanine in these cells. The apparent Kt for the process was 36 t 2 PM, and the Vmax was 1.27 t 0.03 nmol mg protein-l 10 min-l. l

Interaction between taurine and @-alanine during uptake. Earlier studies from our laboratory (22, 23) with

isolated brush-border membrane vesicle have suggested that most likely a single transport system is involved in the uptake of taurine and @-alanine in the rabbit jejunum. Likewise, the characteristics of the uptake of these two ,&amino acids in HT-29 cells were also very similar, indicating that both amino acids may be transported in

Characteristics of @alanine transport in HT-29 cells.

The data given in Table 2 show that @-alanine is a potent inhibitor of taurine uptake in HT-29 cells. Because ,&alanine is commonly employed as a substrate to investigate the characteristics of the transport of ,&amino acids in general, we studied the uptake of @-alanine in HT-29 cells. As was observed in the case of taurine, the uptake of ,&alanine was also found to be concentrative and dependent on NaCl (Fig. 5). Studies on the influence of anions on the uptake of @-alanine showed that ,&alanine uptake was also obligatorily dependent on Cl-, because anions such as I-, F-, NO; and gluconate failed to substitute for

0.05 Concentration

30 60 90 120 Incubation time Onin) Fig. 5. Time course of P-alanine transport. Uptake of P-alanine (final concn, 6 PM) in confluent monolayer cultures of HT-29 cell line was measured in the presence of NaCl (a) or in the presence of choline chloride substituting for NaCl (0).

I

0.1 of Cl’ WI)

Fig. 4. Cl- kinetics of taurine transport. In this experiment, composition of uptake medium was modified from that of regular uptake medium. Medium contained (in mM) 25 HEPES/Tris (pH 7.5), 5.4 K gluconate, 1.8 Ca gluconate, 0.8 Mg gluconate, 5 glucose, and varying combinations of NaCl plus Na gluconate. Concentration of Cl- ranged between 2.5 and 140 mM, whereas that of Na+ was maintained at 140 mM. Uptake of taurine (30 nM) was measured with a lo-min incubation. Uptake measured in the absence of Cl- was subtracted from each uptake value to determine Cl-dependent component. Inset: Hill-type plot (v versus v/[Cl-I).

50 s, Concentration

0.01 “,s

0.02

100 150 of B-alanine QJM)

0.03 200

Fig. 6. P-Alanine transport kinetics. Uptake of @alanine was measured with a lo-min incubation over a concentration range of lo-200 PM. Uptake medium contained NaCl. For each concentration of P-alanine, concentration of radiolabeled @-alanine was maintained at 10 nM. Uptake of radiolabel measured in presence of 2 mM unlabeled P-alanine was used to calculate nonmediated component of transport, and this value was subtracted from total uptake to determine mediated component. Inset: Eadie-Hofstee plot (v/s versus v).

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TRANSPORT

OF ,&AMINO

G629

ACIDS IN HT-29 CELLS

these cells via a common transporter. The kinetic experiments reveal that apparently a single transport system is involved in the uptake of taurine as well as in the uptake of ,&alanine. However, the possibility of the involvement of multiple transport systems that are kinetically indistinguishable under the experimental conditions employed cannot be entirely excluded. To investigate the mutual interaction of taurine and P-alanine during uptake in more detail, we studied the effects of increasing concentrations of ,&alanine on taurine uptake (Fig. 7). The uptake of taurine, which occurs via a transport system with a Kt of 10.6 PM, was almost completely inhibited by 500 PM ,&alanine. The concentration of ,&alanine required to cause a 50% inhibition (I&) was -50 PM. The apparent inhibition constant (Ki) was calculated according to the 12 3 4 5 method of Hajjar and Curran (lo), and the value was 46 Culture time (days after seeding) t 3 PM. Similarly, we studied the effects of increasing Fig. 8. Influence of growth state of cells on expression of taurine transconcentrations of taurine on ,&alanine uptake (Fig. 7). porter. Cell culture was initiated by seeding -2 x lo6 cells/dish, and uptake of taurine (20 nM, lo-min incubation) and protein content were The uptake of P-alanine, which occurs via a transport system with a Kt of 36 PM, was completely blocked by 100 determined every day for 5 days after seeding. Cells reached confluence PM taurine. The I& was -8 PM. The Ki for the process on the 3rd day. was 8.7 t 0.6 PM. Thus it was found that the Kt value for taurine was very close to the Ki value for taurine to in- transporter in HT-29 cells. The activity at the time of confluence was significantly higher than the activity in hibit ,&alanine uptake. Likewise, the Kt value for ,&alanonconfluent cells. Culturing the cells after they reached nine was approximately the same as the Ki value for confluence resulted in a substantial decrease in the up@-alanine to inhibit taurine uptake. These kinetic constants strongly suggest that taurine and fi-alanine share a take rate. Influence of taurine in culture medium on rate of common transporter in HT-29 cells. taurine uptake. We investigated the effect of the addition Taurine uptake in nonconfluent vs. confluent cells. Figure 8 describes the influence of the growth state on the of taurine in the culture medium on the rate of taurine uptake in HT-29 cells. Approximately 2 x lo6 cells were rate of taurine uptake in HT-29 cells. In this experiment, seeded in each dish and cultured with normal culture ~2 x lo6 cells were seeded in each dish, and protein content and IO-min taurine uptake were measured in medium for 2 days. Because the medium contained 10% fetal bovine serum, there might have been a small amount these cultures every day for 5 days after seeding. The protein content of the cultures was found to increase as of taurine present under these experimental conditions. On the second day, fresh culture medium with or without the number of days in culture increased. But, the specific activity of the taurine transporter (measured as the tau- 250 PM taurine was added to the cells, and the cultures continued for one more day before taurine uptake mearine uptake in pmol . mg protein-’ 10 min) increased inisurement. The rate of taurine uptake in control cells tially up to 2-3 days and then decreased considerably. The cells were found to reach confluence on the third day. grown in the absence of taurine (except for the amount of These results show that the growth status of the cells taurine contributed by the fetal bovine serum) was 1.66 t 0.03 pmol mg protein-l 10 min-l, whereas the rate in exhibits marked influence on the activity of the taurine cells grown in the presence of taurine was 0.96 t 0.03 pmol . mg protein-l 10 min? These results suggest that culturing of the cells in the presence of taurine downregulates the taurine transporter in HT-29 cells. 1

L

I

I

I

l

l

l

DISCUSSION

0 L

[f$Zk]

:%I

ITZL3l

($1

Fig. 7. Interaction between taurine and ,8-alanine for transport process. Uptake of taurine (20 nM) or ,&alanine (3pM) in confluent monolayer cultures of HT-29 cell line was measured with a lo-min incubation in NaCl-containing uptake medium. Uptake of taurine was determined in presence of varying concentrations of P-alanine (O-500 PM). Uptake of ,8-alanine was determined in presence of varying concentrations of taurine (O-100 PM). Only the mediated component of uptake was used in calculations.

Because of marked differences in the biosynthetic capacity among different animal species, there is a wide species variation in the dietary requirement for taurine. Taurine is not an essential amino acid in the rat but is an important essential amino acid in the cat (8). In the human, taurine is a conditionally essential nutrient. The need for dietary requirement for taurine in animals to maintain whole body taurine homeostasis also exhibits age dependence. Intestinal absorption of taurine is important particularly in the newborn, because tissue concentrations of taurine are highest at this stage of development and yet the capacity of the liver to produce taurine endogenously is not fully developed. There is also evidence to suggest that taurine plays a special role in

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G630

TRANSPORT

OF ,&AMINO

development (33). The importance of intestinal absorption of dietary taurine during development is underscored by the findings that taurine is a major constituent of the free amino acid pool in mammalian milk, the primary diet for the newborn (30), and that the intestinal taurine transport activity is manyfold greater in developing animals than in adults (24). Intestinal absorption of taurine and ,&amino acids in general has been investigated in many laboratories. A number of studies using isolated brush-border membrane vesicles have clearly demonstrated the presence of a ,&amino acid transporter in the small intestine (222-24, 26). In all of these studies, only the jejunal part of the small intestine was used to prepare the membrane vesicles. This jejunal P-amino acid transporter has high affinity for taurine and effectively excludes a-amino acids. In contrast, there is some eviden .ce for the presence of a second ,&amino acid transporter in the ileal part of the small intestine, which is distinct from the jejunal P-amino acid transporter (1, 25). The ileal transporter accepts a-amino acids and cationic amino acids, in addition to ,&amino acids, as substrates. On the basis of substrate specificity, it is the jejunal P-amino acid transporter that qualifies to be called the taurine transporter. It is also this transporter that exhibits characteristics that are similar to those of the taurine transporter known to be expressed in various other tissues. The present investigation was initiated with the aim to identify a cell culture model system for taurine transport in the small intestine. Such a system would be extremely useful to delineate the regulatory aspects of the taurine transport in this organ. Cultured cell lines originating from normal small intestine do not develop the typical differentiation characteristics of mature intestinal cells (28). However, colon carcinoma cell lines, particularly HT-29 and Caco-2, have been shown to exhibit entero@e-like differentiation features (32). Both ceil lines are of human origin and are being widely used as in vitro models for the study of intestinal enzymes and transport systems. The present study describes the properties of taurine transport in HT-29 cells. The results of the present study-show that confluent monolayer cultures of this HT-29 cell line actively accumulate taurine in the presence of a transmembrane NaCl gradient. The transport system responsible for taurine uptake in these cells is very specific for p-amino acids and effectively excludes a-amino acids. The system shows much higher affinity for taurine than for p-alanine. These cells also take up p-alanine very actively, and this uptake occurs exclusively via the taurine transport system. There is no evidence for the presence of any other additional transport system for ,&amino acids. Thus the transport system expressed in the HT-29 cell line closely resembles the taurine transporter present in the jejunal part of the small intestine. The ileal ,&amino acid transporter does not appear to be present in this cell line. Therefore, the HT-29 cell line should offer a convenient in vitro model system to explore various features of the intestinal taurine transporter. Cultured cells have already proved to be useful to investigate regulatory aspects of taurine transport in other organs. The kidney cell lines LLC-PK1 and MDCK ex-

ACIDS

IN HT-29

CELLS

press a taurine transporter that is subject to adaptive regulation (14) and osmoregulation (37). The taurine transporter present in the JAR human placental choriocarcinoma cell line is regulated by protein kinase C (15) and calmodulin antagonists (29) but is unaffected by adenosine 3’,5’-cyclic monophosphate (7). There is no information available on the modulation of the activity of the intestinal taurine transporter by hormones and intracellular second messengers. It should now be feasible to initiate investigations on the regulation of the intestinal taurine transporter using the HT-29 colon carcinoma cell line as a model system. The results presented in this paper already show that the activity of the taurine transporter in these cells is subject to downregulation by taurine in the culture medium. The particular clone of HT-29 cell line used in the present study grows as a monolayer on impermeable plastic support in the presence of glucose. Differentiation of these cells in culture is a growth phase-related event. These cells remain undifferentiated at confluence, but, when allowed to grow beyond the confluence stage, they polarize to form microvilli and exhibit enterocyte-like differentiation features. It is interesting that the activity of the taurine transporter is maximal at or near confluence. The activity decreases significantly after the cells reach the stationary growth phase. It is not known at present whether this phenomenon has any relevance to the observation in the normal intestine that the activity of the taurine transporter is high in neonates but decreases to adult levels as the intestine matures. Future experiments will have to be carried out to discern the relationship between the progress of the differentiation process and the change in the activity of the taurine transporter in these cells. The authors thank Marcia D. Lewis for excellent secretarial assistance. This work was supported by National Institutes of Health Grants HD-24451 and DK-28389. Address for reprint requests: F. Leibach, Dept. of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 309122100. Received

15 January

1992; accepted

in final

form

26 May

1992.

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