Sodium-independent lysine uptake by the bewo choriocarcinoma cell line

Placenta

(1998),

19,

323-328

Sodium-independent

Lysine

Uptake

by the BeWo

Choriocarcinoma

Cell Line B. A. Waya, T. C. Furesz, The Edward Washington Paper

Mallinckrodt University

accepted

20

J. K. Schwarz,

A. J. Moeb

and C. H. Smithc

Department of Pediatrics and Department of School of Medicine, St Louis, Missouri 631 IO,

November

Pathology, USA

St Louis

Children’s

Hospital,

1997

Transport of t-lysine by a cultured placental trophoblast cell line was investigated by characterization of L-[3H]lysine uptake. In the mononuclear form of the BeWo clone b30 choriocarcinoma cell, at least two sodium-independent systems are present. Concentration dependence data were fitted by a two system model with K, values ( f se.) of 2 * 0.7 and 94 f 31 PM and V,,, values ( f s.e.) of 0.7 f 0.3 and 25 f 6.0 nM/mg DNA/min. A portion of sodium-independent uptake was inhibited by the sulphydryl modifying reagent N-ethylmaleimide (NE-M). Following NEM treatment, the data were fitted by a single system with Km= 10 zt 2 PM AND v,,,= 5.1 * 0.8 nM/mg DNA/min. In the absence of sodium, NEM-resistant uptake was sensitively inhibited by leucine whereas NEM-sensitive uptake was not inhibited by leucine. It is concluded that like placental basal membrane, the mononuclear BeWo cell possesses two sodium-independent L-lysine transport systems. The high-capacity, NEM-sensitive, leucine-insensitive system resembles the widespread system y+. The high-affinity, NEM-resistant, leucine-sensitive system resembles system b”,+. 0 1998 W. B. Saunders Company Ltd Placenta (1998), 19, 323-328

INTRODUCTION Fetal growth and development depend on the continuous transfer of amino acids from maternal to fetal blood across the syncytiotrophoblast (Smith, Moe and Ganapathy, 1992; Furesz, Smith and Moe, 1993). Both cationic and neutral amino acids are more concentrated in fetal than in maternal blood and the lysine concentration ratio is approximately 3 : 1 (Philipps et al., 1978). The movement of amino acids across plasma membranes is mediated and regulated by transport proteins that recognize, bind, and transport amino acids from the extracellular medium into the cell or vice versa. Cationic amino acid transporters have been described in the plasma membranes of many cell types (Kilberg, Stevens and Novak, 1993). The movement of cationic amino acids across the placental barrier is mediated by the transporters of the syncytiotrophoblast. Recently, at least two sodium-independent and one sodium-dependent transport systems mediating the uptake of cationic amino acids have been characterized (Van Winkle, Christensen and Campione, 1985; Van Winkle, Campione and Gorman, 1990; Furesz, Moe and Smith, 1991, 1995; Devts, Chavez and Boyd, 1992; Van Winkle, 1993; Eleno, De&, and Boyd, 1994). The principal a Current address: Instrumentation Laboratories, Orangeburg, 10962, USA. b Current address: Biology Department, Concordia University Forest, River Forest, IL 60305, USA. c To whom correspondence should be addressed. smith-ch@kids. wustl.edu 0143~004/98/040323+06

$12.00/O

NY River E-mail:

sodium-independent systems are the ubiquitous system yc and the leucine-sensitive systems b”,’ and y+L that have been described in mouse blastocyst, erythrocytes and other specific locations (Van Winkle, Christensen and Campione, 1985; Van Winkle, Campione and Gorman, 1990; Furesz, Moe and Smith, 1991, 1995; Devts, Chavez and Boyd, 1992; Devts, Angelo and Chavez. 1993; Eleno, De&s and Boyd, 1994). In the human placenta, we and others have recently described leucine-sensitive and leucine-insensitive sodium-independent cationic amino acid transport in basal and microvillous placental membranes (Furesz, Moe and Smith, 1991, 1995; Furesz and Smith, 1997). Here we characterize two sodium-independent cationic amino acid transport activities of the mononuclear, trophoblastlike BeWo choriocarcinoma cell. As in the placental membranes, both leucine-insensitive and leucine-sensitive transports are present.

MATERIALS

AND

METHODS

Materials L-[3H]lysine (specific activity 87.4 Ci/mmol) was obtained from NEN-DuPont (Boston, MA, USA). Opti-Fluor was from Packard Instruments (Downers Grove, IL, USA). Chemically defined fetal bovine serum (FBS) was obtained from Hyclone Laboratories (Logan, UT, USA). Essential amino acids were obtained from Hazelton Biologics (Lenexa, KS, USA). 0

1998 W. B. Saunders

Company

Ltd

Placenta

324

Minimal essential medium and vitamins were from GIBCO (Grand Island, NY, USA). Gentamicin and nonessential amino acids were from the Washington University Tissue Culture Support Center. Other chemicals were obtained from Sigma (St Louis, MO, USA) or Aldrich (Milwaukee, WI, USA). Culture

measurements

L-[3H]lysine uptake by b30 cells was measured by the cluster tray technique (Gazzola et al., 1981; Kilberg, Vida and Barber, 1983; Furesz, Smith and Moe, 1993). Media was removed and cells incubated for 1 h in Earle’s balanced salt solution to reduce intracellular amino acid pools. For inhibition studies, cells were incubated for 10 min at 37°C with Earle’s balanced salt solution containing 2.5 mM N-ethylmaleimide (NEM) and the reaction stopped with 10 mM P-mercaptoethanol. Uptake measurements were begun by replacing the Earle’s solution with a substrate mixture in Kreb’s solution containing 102 mM choline chloride, 24 mM choline bicarbonate, 10 rnM choline phosphate, or the corresponding sodium salts, 5 mM glucose, 0.75 mM CaCl,, and 1 mM MgSO,, pH 7.3. L-[3H]lysine and non-radioactive lysine or other amino acids were incorporated into the incubation buffer in the final concentrations indicated below. Incubations were stopped by removing the incubation media and washing with four separate 2-ml aliquots of ice-cold phosphate-buffered saline, pH 7.4. Cells were disrupted by addition of 0.5 ml of 0.2 N NaOH-0.2 per cent sodium dodecyl sulphate to each well containing radioactivity, followed by shaking for 30 min at 37°C. An aliquot (0.3 ml) was removed from each well and added to vials containing 10 ml Opti-Fluor for counting in a liquid scintillation counter (Pharmacia LKB Nuclear, Gaithersburg, MD, USA). L-[3H]lysine uptake was expressed per unit of DNA determined in parallel wells (Kapuscinski and Skozyias, 1977). Kinetic

Vol. 19

125 1

conditions

The b30 clone was derived from the original parent BeWo line by limiting dilution (Wice et al., 1990). Cells were maintained in 25-cm2 flasks cultured in MEMB media (for composition see Moe et al., 1991) containing 10 per cent FBS, gentamicin (0.5 mg/ml) and Penicillin (100 units/ml)/streptomycin (100 ug/ml). For transport studies cells were removed with 0.05-0.02 per cent trypsin-EDTA solution, seeded into Costar (Cambridge, MA, USA) 24-well culture dishes, and incubated at 37°C in a 5 per cent CO,-95 per cent air and 100 per cent humidity atmosphere. For uniformity, cells were seeded at the same density, and experiments were performed at a constant time after plating. Uptake

(1998),

and

statistical

analyses

The Henri-Michaelis-Menten equation describing the relationship of saturable activity to concentration with an added

Time (min) Figure 1. Time course for uptake of Lj3H]lysine (3.6 x 10 s mM). Incubation medium contained 102 mM choline chloride, 24 mM choline bicarbonate, 10mM choline phosphate, 5 m.w glucose, 0.75 ItIM C&l,, and 1 Ml\/I MgSO,, pH 7.3). Data are the means f s.e. of six experiments determined in quadruplicate.

diffusion term was fitted to the uptake data using the RS/l program (Bolt, Beranek and Newman Research Systems, 1990) on a VAX computer. The program finds the least squares solution by the Marquardt-Levenberg method of iteration. Similar equations with two saturable components were also used where indicated by Furesz, Moe and Smith (1991). Oneand two-site models were compared by using the F-test as described by Draper and Smith (1966). Inhibition data were analysed by fitting the Michaelis-Menten equation with a competitive inhibitor term (l+l/&) as the coefficient of K, in the denominator (Furesz, Moe and Smith, 1991). RESULTS Time

dependence

To establish conditions for the measurement of cationic amino acid transport in mononuclear BeWo cells, we first determined the initial rates of amino acid entry. In the absence of sodium, uptake of L-[3H]lysine (36 nM) was proportional to time during the first 2 min of incubation (Figure 1). The uptake reached equilibrium by 2 h (data not shown). For uniformity, all transport experiments were performed with 2 min incubation. When sodium was added to the incubation medium, there was a small, variable increase in uptake (data not shown). As our primary interest was in evaluating sodium-independent uptake, this small sodium-stimulated component of uptake was not investigated further. Concentration sodium-independent

dependence L-lysine

of uptake

Sodium-independent uptake of L-[3H]lysine (36 nM) by BeWo clone b30 cells was measured in the presence of increasing

Way et al.: Placental

Lysine

Transport

325

30

n -8

-7

-6

-5 Log

-4 lysine

-3

-2

-1

Figure 2. Concentration dependence of L-[3H]lysine (3.6 X 10-s ~IIM) uptake by BeWo clone b30 cells in the absence of N-ethylmaleimide (NEM). Uptake was measured in the presence of increasing concentrations of L-lysine. A model containing two saturable components was fit with Michaelis-Menten constant (K,)=2.0 * 0.7 and 94 + 31 FM and maximum velocity (V,,,) values=0.7 & 0.3 and 25 It 6.0 nM/mg DNA/min. A diffusion constant (Kn) of 49 f 8.0 pmol/nM/mg DNA/min was determined. Data are means f SEM for eight experiments performed in quadruplicate.

concentrations (3.6 X 10 -s to 60 mM) of L-lysine (Figure 2) and the Henri-Michaelis-Menten equation for one and two saturable components was fitted to the data. The goodness of fit was significantly increased by the addition of a second saturable component when compared to a one-component model by F-test. K,,, values ( & se.) were 2 * 0.7 and 94 f 3 1 PM and V,,, values ( f s.e.) were 0.7 & 0.3 and 25 f 6.0 nM/mg DNA/min, respectively. The diffusion constant (KD) was 49 f 8.0 pmOkS/nM/IIIg DNA/min. NEM

-8

-7

-6

(M)

inhibition

To define independently the two sodium-independent systems present, transport was studied in the presence and absence of the sulphydryl modifying reagent, N-ethylmaleimide (NEM). NEM has recently been shown to inhibit lysine transport by a transporter identified in human erythrocytes as system y” (De&, Angelo and Chavez, 1993), and we have observed similar effects of NEM in placental microvillous and basal membranes (Furesz, Moe and Smith, 1995, Furesz and Smith, 1997). Preliminary experiments in the BeWo cell demonstrated that incubation for 10 min with 2.5 mM NEM at 37°C gave maximum inhibition of L-lysine uptake (data not shown). NEM treatment caused partial inhibition of the uptake of L-lysine measured over a wide lysine concentration range. The residual uptake fitted well with a one-component model with a DNA/min Km of 10 ZIZ2 pM and a V,,, of 5.1 & 0.8 nM/mg (Figure 3). The addition of a second saturable component did not statistically improve the fit. The similarity of the Km value for the NEM-resistant uptake to that of the high-affinity

-5 Log

-4 lysine

-3

-2

-1

(M)

Figure 3. Concentration dependence of L-[3H]lysine (3.6 x 10 5 IIIM) uptake in the presence of N-ethylmaleimide (NEM). BeWo clone b30 cells were incubated with 2.5 rTIM NEM for 10 min at 37°C and the reaction stopped with 10 mM P-mercaptoethanol. Uptake was measured in the presence of increasing concentrations (3.6 x lo- 5 to 60 mM) of L-lysine. A model containing one saturable component was fitted with K,=lO f 2 ELM, V,,,=S.l + 0.8 n.&mg DNA/t&. Data are means & SEM for four experiments determined in quadruplicate.

system seen without NEM treatment indicated a suppression by NEM of the low-affinity system and provided an additional tool to define the specificity of the transporters present. Inhibition

of L-lysine

uptake

Various inhibitors were used to characterize further the two sodium-independent transport systems (Table 1). Inhibition of 1 ,UM L-lysine uptake was measured with and without NEM treatment. The cationic amino acid L-arginine sensitively inhibited both NEM-sensitive and -insensitive L-lysine transport. The anionic amino acid L-glutamate, the neutral amino acid alanine and the amino acid analog 2-(methylamino)isobutyrate (MeAIB) all failed to inhibit transport significantly under either condition. L-leucine sensitively inhibited the NEM-resistant uptake, but failed to inhibit the NEM-sensitive uptake. In order to define better the specificity of the transport systems involved, the concentration dependence of L-leucine as inhibitor of L-lysine uptake was investigated. NEM-sensitive and NEM-resistant transport of L-lysine (1 PM) was measured in the presence of a wide range of L-leucine concentrations (Table 2). L-leucine (1 FM to 5 mM) in the absence of sodium did not substantially inhibit NEM-sensitive L-lysine uptake. In contrast, NEM-resistant L-lysine uptake was sensitively inhibited by L-leucine. This inhibition was maximal at 500 pM leucine. DISCUSSION This study demonstrates the presence of at least two distinct sodium-independent L-lysine transport activities in

326

Placenta

Table

1. Inhibition

Inhibitor

Self-inhibitable NEM-sensitive (per cent)

Control Leucine Arginine Glutamate MeAlB Alanine

100 104f0 1052 93 i 9 97 zk 8 92 zk 8

of L-lysine uptake

uptake activity

Self-inhibitable NEM-resistant (per cent)

uptake activity

100 If1 11 k.6 92 * 3 94 * 7 94Xt 13

Values are means&SE. for six experiments. Inhibition of 1 pM L-lysine uptake with and without N-ethylmaleimide (NEM) treatment by 0.5 mu amino acids or 1 mM (for leucine) was measured at 2 min. NEM-resistant uptake, uptake after NEM treatment; NEMsensitive uptake, total uptake minus NEM-resistant uptake; selfinhibitable uptake, difference between uptake measured in the presence and absence of 60 mM nonradioactive L-lysine; control uptake, uptake in the presence of 1 pM L-lysine minus uptake in the presence of 0.5 mM L-lysine, was 0.27 + 0.07 and 0.18 + 0.02 nM/mg DNA/min for NEM-sensitive and NEM-resistant activity, respectively. Table

2. Inhibition

Leucine (pLIM)

Self-inhibitable NEM-sensitive (per cent)

Control 1 2 10 100 500 1000 5000

100.0 98.6 99.2 92.5 90.8 104.0 103.6 90.7

dz 1.7 f 4.4 f 9.4 zt 5.4 * 0.1 f 16.2 z!c5.8

of L-lysine uptake by leucine uptake activity

Self-inhibitable NEM-resistant (per cent)

uptake activity

100.0 102.7 i 1.8 97.1 f 3.3 75.3 z!z19.3 13.4 + 3.0 1.0 z!z1.4 14.0 + 4.9 5.0 It 17.3

Values are means&SE. for three experiments performed in quadruplicate. Inhibition of 1 ELM L-lysine uptake by leucine with and without N-ethylmaleimide (NEM) treatment was measured at 2 min. NEM-resistant uptake, uptake after NEM treatment; NEM-sensitive uptake; total uptake minus NEM-resistant uptake; self-inhibitable uptake, difference between uptake measured in the presence and absence of 60 mM nonradioactive L-lysine. Control uptake, uptake in the presence of 1 pM L-lysine, was 0.28 rt 0.03 nM/mg DNA/min and 0.14 + 0.01 nM/mg DNA/min for NEM-sensitive and NEMresistant activity, respectively. For NE-M-resistant activity, Ki (inhibition constant) for leucine=lO + 4 PM.

mononuclear BeWo choriocarcinoma cells. Lysine uptake was principally sodium independent although a minimal amount of sodium-dependent uptake was observed (data not shown). Using conditions (NEM inhibition) under which the transport systems could be studied independently, we were able to characterize each transporter kinetically and with respect to their interaction with other amino acids. The first transport activity was NEM sensitive, with high Km and V,,, values and was not inhibited by neutral amino acids in the absence

(1998),

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of sodium. The second activity was NEM resistant with low Km and Vmax values and was inhibited by leucine (at FM concentrations) in the absence of sodium. Three sodium-independent systems are known to mediate the transport of cationic amino acids each with different characteristics: system y+ (White, 1985; Moe, 1995), (Van Winkle, Campione and Gorman, 1988; Van Winkle, Campione and Gorman, 1990; Furesz, Moe and Smith, 1991) system b”,’ (Van Winkle, Campione and Gorman, 1988; Furesz, Moe and Smith, 1991) and system y+L (De&, Chavez and Boyd, 1992; De&s, Angelo and Chavez, 1993; Furesz, Moe and Smith, 1995; Furesz and Smith, 1997). The identification and characterization of the transport systems of a particular cell is based not simply on comparisons of Km and Y,,, values but on the total of their characteristics (Christensen, 1985; Kilberg, Stevens and Novak, 1993). Comparison of the two sodiumindependent systems with known transport systems lead us to conclude that they are similar to the ubiquitous system yt (high capacity) and system b”>+ (high affinity), found in mouse blastocysts and basal placental membranes. The kinetic parameters of the transport activities were determined under two conditions: with both systems acting together using two-site fit analysis, and for the low Km system acting alone after NEM treatment. The low Km system comprises only a small fraction of the uptake in the two-site analysis. Not surprisingly, the uncertainties of the parameters of this system determined from the two-site fit were greater than those determined under conditions in which it acted alone (CV values were approximately 30-40 per cent for two-site fit versus 15-20 per cent for the single-site fit of the low Km system). It is logical to place more credence on the values from the single site fit in the presence of NEM in which the low Km system is responsible for essentially all of the uptake. System y+ is a well-characterized, widely distributed mediator of cationic amino acid transport (White and Christensen, 1982a,b; Christensen, 1984; White, 1985; Van Winkle, Campione and Gorman, 1988). The high-capacity, low-affinity cationic amino acid transport system in the BeWo cell has many characteristics of system y+. It is sodium independent, of relatively high capacity and sensitive to NEM. Its failure to interact with neutral amino acids in the absence of sodium is similar to the yc-like system described by Furesz, Moe and Smith (1991) in placental syncytiotrophoblast basal membrane and the ubiquitous system y+ (De&, Angelo and Chavez, 1993; Kilberg, Stevens and Novak, 1993). System b”‘+ is a broad-specificity, high-affinity, NEMresistant, sodium-independent transporter of cationic amino acids and some neutral amino acids (Van Winkle, Campione and Gorman, 1988, 1990). The low Km system of the trophoblast-like BeWo cell has characteristics similar to the system b”,+ in mouse blastocysts, and placental basal membrane. It is of high affinity, sensitive to leucine in the absence of sodium, and NEM resistant. Its kinetic parameters (Km of 10 i 2 pM and V,,, of 5.1 + 0.8 nM/mg DNA/min) are very much lower than those of system yt and are similar to those of system b”J+ m mouse blastocysts and placental basal membrane

Way et al.: Placental

Lysine

Transport

(Van Winkle, Campione and Gorman, 1988, 1990; Van Winkle et at., 1990; Furesz, Moe and Smith, 1991). Leucine inhibition demonstrated a Ki of 10 p~ and was essentially complete at 500 PM, as has been reported for system b”,+ in placental membranes (Furesz, Moe and Smith, 1995). In both its kinetic characteristics and its sensitivity to leucine, the high affinity, sodium-independent system in the BeWo cell thus resembles most closely the corresponding system of placental basal membrane and system b”,+ of mouse blastocyst (Van Winkle, Campione and Gorman, 1988, 1990; Furesz, Moe and Smith, 1991). The most recently described transporter of cationic amino acids is system yfL (Deves, Chavez and Boyd, 1992). System y+L is a sodium-independent, NEM-resistant, temperaturesensitive, high-affinity transporter (De&s, Chavez and Boyd, 1992; Furesz and Smith, 1997). System y+L and system b”,+ are functionally very similar. Their principal difference is that system b”>+ interacts with leucine with high affinity in the absence of sodium whereas system y+L requires sodium for high-affinity leucine interaction (Furesz, Moe and Smith, 1995; Moe, 1995). Recent molecular studies have reported substantial similarities of the cloned portions of these two cationic transporters (Bertran et al., 1994). We believe additional functional and molecular studies will clarify the relationship between these two high-affinity cationic amino acid transporters. With present knowledge, we have designated the high-affinity transporter of the BeWo cell as system b”‘+ on the basis of its strong interaction with leucine in the absence of sodium. The small, variable sodium-stimulation of L-[3H]lysine transport in the BeWo cell was not characterized. As suggested in studies of the mouse blastocyst and the rat placenta, it may

327

result from activity of a broader specificity transport system such as B”>+ (Van Winkle, Christensen and Campione, 1985, Malandro et al., 1994). Because human placental basal and microvillous membrane transport is sodium independent (Furesz, Moe and Smith, 1991, 1995; Moe 1995), the origin of the small sodium-stimulated component was not investigated further. The sodium-independent cationic amino acid transport systems in the mononuclear BeWo cell resemble those of placental basal membrane. The high-capacity system (system y’) is likely to function as the major transporter in both the basal membrane in placenta (Furesz, Moe and Smith, 1991) and in the BeWo cell. The high-affinity, low-capacity system (resembling system b’,+) may, as suggested for the corresponding system in kidney (Thwaites et al., 1996) and intestine (Bertran et al., 1992), serve an exchange function in transfer from trophoblast to the fetus (Busch et al., 1994; Ahmed et al., 1995). The BeWo choriocarcinoma cell line has potential as a model for the study of transport in the developing human placental syncytiotrophoblast. Like the placental trophoblast, it has the ability to differentiate from a smooth membrane, trophoblast-like cell to a microvillous membrane-like syncytium (Wice et al., 1990). In addition, previous work in this laboratory has shown that transport in the BeWo cell is similar to placental vesicles (Furesz, Moe and Smith, 1991, 1995) and that the cell is a model for placental transport of neutral amino acids (Furesz, Smith and Moe, 1993; Moe, Furesz and Smith, 1994). Studies of cationic amino acid transport in the BeWo cell as differentiation proceeds are ongoing and will establish further its validity as a model for the cellular investigation of placental transport.

ACKNOWLEDGEMENTS The authors would like to thank Michael Zahner for assistance in this study, a grant from the National Institute of Child Health and Human Development,

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and MS Barbara HD-07562.

Hartman

for her secretarial

services.

This

work was supported

by

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