The Transport of Lysine Across Monolayers of Human Cultured Intestinal Cells

7/27/2019 The Transport of Lysine Across Monolayers of Human Cultured Intestinal Cells

1/15

The transport of lysine across monolayers of human culturedintestinal cells (Caco-2) depends on Napositive-dependent andNapositive-independent mechanisms on different plasma membranedomainsFerruzza, Simonetta;Ranaldi, Giulia;Di Girolamo, Mario;Sambuy, Yula.TheJournal of Nutrition 125.10 (Oct 1995): 2577-85.

Aktifkan sorotan temuan untuk peramban bicara

Abstrak (ringkasan)TerjemahkanAbstrak

To characterize the mechanisms involved in the intestinal absorption of the essential amino acid L-

lysine from the diet, the transepithelial transport of L-lysine was studied in monolayers of cultured

human intestinal cells (Caco-2) grown and differentiated on microporous membrane supports. L-lysine

was transported mainly in the apical (AP) to basolateral (BL) direction and the BL to AP transport wasapproximately one order of magnitude lower at all concentrations tested. Non-linear regression

analysis of the transport in the AP to BL and the BL to AP direction identified, in both cases, single

saturable components with similar Km but different Vmax and a nonsaturable diffusional component.

The AP to BL L-lysine transport was highly energy- and sodium-dependent and was unaffected by an

unfavorable concentration gradient. Selective replacement of sodium ions in the AP or the BL

compartment and determination of both AP to BL transport and the intracellular soluble lysine pool

showed that uptake occurs via a sodium-independent mechanism, not significantly influenced by

membrane potential, whereas efflux is a sodium-dependent process. Competition experiments showed

that L-lysine uptake is highly stereospecific and is shared by cationic and large neutral amino acids.

This study demonstrates the presence of a sodium-dependent mechanism of lysine efflux across the

BL membrane of intestinal cells, which may be essential for lysine transport into the blood circulation.

Overall, these results support the use of the Caco-2 cell model for studies of intestinal nutrient

transport.

Teks Lengkap

TerjemahkanTeks lengkap

The cationic amino acid L-lysine belongs to the class of the nutritionally essential, or indispensable,

amino acids (Young 1994). As such, its transport mechanisms across the human intestinal mucosa are

of particular interest to nutritionists. Unfortunately, with the only exception of a study conducted on

brush border vesicles isolated from fetal intestine (Malo 1991), studies on human tissue are lacking

because of difficulties in obtaining material for experiments. The intestinal transport of cationic amino

acids has been investigated in various animal models, employing different experimental approaches

and tissue preparations; these studies have identified both sodium-dependent and sodium-
http://search.proquest.com/indexinglinkhandler/sng/au/Ferruzza,+Simonetta/$N?accountid=62694http://search.proquest.com/indexinglinkhandler/sng/au/Ferruzza,+Simonetta/$N?accountid=62694http://search.proquest.com/indexinglinkhandler/sng/au/Ranaldi,+Giulia/$N?accountid=62694http://search.proquest.com/indexinglinkhandler/sng/au/Ranaldi,+Giulia/$N?accountid=62694http://search.proquest.com/indexinglinkhandler/sng/au/Di+Girolamo,+Mario/$N?accountid=62694http://search.proquest.com/indexinglinkhandler/sng/au/Di+Girolamo,+Mario/$N?accountid=62694http://search.proquest.com/indexinglinkhandler/sng/au/Di+Girolamo,+Mario/$N?accountid=62694http://search.proquest.com/indexinglinkhandler/sng/au/Sambuy,+Yula/$N?accountid=62694http://search.proquest.com/indexinglinkhandler/sng/au/Sambuy,+Yula/$N?accountid=62694http://search.proquest.com/indexinglinkhandler/sng/au/Sambuy,+Yula/$N?accountid=62694http://search.proquest.com/pubidlinkhandler/sng/pubtitle/The+Journal+of+Nutrition/$N/34400/DocView/197427547/fulltext/$B/1?accountid=62694http://search.proquest.com/pubidlinkhandler/sng/pubtitle/The+Journal+of+Nutrition/$N/34400/DocView/197427547/fulltext/$B/1?accountid=62694http://search.proquest.com/pubidlinkhandler/sng/pubtitle/The+Journal+of+Nutrition/$N/34400/DocView/197427547/fulltext/$B/1?accountid=62694http://search.proquest.com/pubidlinkhandler/sng/pubtitle/The+Journal+of+Nutrition/$N/34400/DocView/197427547/fulltext/$B/1?accountid=62694http://search.proquest.com/pubidlinkhandler/sng/pubtitle/The+Journal+of+Nutrition/$N/34400/DocView/197427547/fulltext/$B/1?accountid=62694http://search.proquest.com/docview/197427547/fulltext?accountid=62694http://search.proquest.com/docview/197427547/fulltext?accountid=62694http://search.proquest.com/docview/197427547/fulltext?accountid=62694#centerhttp://search.proquest.com/docview/197427547/fulltext?accountid=62694#centerhttp://search.proquest.com/docview/197427547/fulltext?accountid=62694#centerhttp://search.proquest.com/docview/197427547/fulltext?accountid=62694#centerhttp://search.proquest.com/indexingvolumeissuelinkhandler/34400/The+Journal+of+Nutrition/01995Y10Y01$23Oct+1995$3b++Vol.+125+$2810$29/125/10?accountid=62694http://search.proquest.com/indexingvolumeissuelinkhandler/34400/The+Journal+of+Nutrition/01995Y10Y01$23Oct+1995$3b++Vol.+125+$2810$29/125/10?accountid=62694http://search.proquest.com/docview/197427547/fulltext?accountid=62694#centerhttp://search.proquest.com/docview/197427547/fulltext?accountid=62694#centerhttp://search.proquest.com/docview/197427547/fulltext?accountid=62694http://search.proquest.com/docview/197427547/fulltext?accountid=62694http://search.proquest.com/pubidlinkhandler/sng/pubtitle/The+Journal+of+Nutrition/$N/34400/DocView/197427547/fulltext/$B/1?accountid=62694http://search.proquest.com/pubidlinkhandler/sng/pubtitle/The+Journal+of+Nutrition/$N/34400/DocView/197427547/fulltext/$B/1?accountid=62694http://search.proquest.com/pubidlinkhandler/sng/pubtitle/The+Journal+of+Nutrition/$N/34400/DocView/197427547/fulltext/$B/1?accountid=62694http://search.proquest.com/pubidlinkhandler/sng/pubtitle/The+Journal+of+Nutrition/$N/34400/DocView/197427547/fulltext/$B/1?accountid=62694http://search.proquest.com/indexinglinkhandler/sng/au/Sambuy,+Yula/$N?accountid=62694http://search.proquest.com/indexinglinkhandler/sng/au/Di+Girolamo,+Mario/$N?accountid=62694http://search.proquest.com/indexinglinkhandler/sng/au/Ranaldi,+Giulia/$N?accountid=62694http://search.proquest.com/indexinglinkhandler/sng/au/Ferruzza,+Simonetta/$N?accountid=62694


2/15

independent transport mechanisms (Cassano et al. 1983, Munck and Munck 1992, Paterson et al.

1981, Wolfram et al. 1984). However, because wide interspecies variations occur in amino acid

transport (Hopfer 1987), a human cell model would be extremely useful to unravel the mechanisms

and the factors regulating intestinal cationic amino acid transport.

In recent years, the human intestinal cell line Caco-2 has increasingly been used as a model to

investigate the transepithelial transport of nutrients and drugs across the intestinal mucosa (Alvarez-

Hernandez et al. 1994, Chen et al. 1994, Hidalgo and Borchardt 1990a, Raffaniello et al. 1992, Ranaldi

et al. 1992, Ranaldi et al. 1994, Riley et al. 1991, Thwaites et al. 1993).

This cell line, derived from a human colon adenocarcinoma (Fogh et al. 1977), has been shown to

express in culture some differentiated traits of the small intestinal absorptive enterocyte, such as a

polarized morphology, a well-developed brush border, functional tight junctions and the expression of

enterocyte-specific enzymatic activities on the apical surface (disaccharidases and peptidases) and of

several active transport carriers (Zweibaum et al. 1991).

In the present study we investigated the transepithelial transport of L-lysine across monolayers of

differentiated Caco-2 cells grown on microporous membrane supports and identified and characterized

an energy-and sodium-dependent transport, operating in the apical (AP)(4) to basolateral (BL)

direction. Further analysis of this system has shown that the uptake into the cells occurs via one or

more sodium-independent carriers, while the efflux is highly sodium-dependent.

MATERIALS AND METHODS

Cell culture. The intestinal Caco-2 cell line was routinely grown, as previously described (Ranaldi et al.

1992, Ranaldi et al. 1994), in plastic tissue culture flasks (75 cm sup 2 growth area; Falcon, Becton

Dickinson, Italia, Milan, Italy) using Dulbecco modified minimum essential medium containing 25

mmol/L glucose, 3.7 g/L NaHCO3 and supplemented with 4 mmol/L L-glutamine, 10% heat-

inactivated fetal calf serum, 1% nonessential amino acids, 1 X 10 sup 5 U/L penicillin, 100 mg/L

streptomycin. At confluency (usually every 4-5 days) the cells were passaged at 1:6 split ratio, by

detaching them with 0.25% trypsin (1:250) and 10 mmol/ L EDTA in calcium-free and magnesium-

free PBS. Cells were used between passage 80 and 100. All cell culture reagents were from Flow

Laboratories International (Opera, Milan, Italy). The fluorescent dye bisbenzimide (H 33258;

Boehringer Mannheim, Milan, Italy) was routinely used to screen cells for mycoplasma contamination

(Chen 1977).


3/15

For transport experiments the cells were seeded on polycarbonate filter cell culture chamber inserts

(Transwell, 24-mm diameter, 4.7-cm sup 2 area, 0.45-mu m pore diameter; Costar Europe,

Badhoevedorp, The Netherlands,) at a density of 2 X 10 sup 6 cells/filter; the high seeding density

allows confluency to be reached within 48 h. Caco-2 cells were left to differentiate for 13-21 days after

confluency; the medium was regularly changed three times a week.

Measurements of transepithelial electrical resistance (TEER). The development of functional tight

junctions during the differentiation of Caco-2 cells was monitored by determining TEER of filter-grown

cell monolayers on different days after seeding. The TEER was measured using the Millicell ERS

apparatus (Millipore, Bedford, MA), according to the manufacturer's instructions. Briefly, the

resistance system was calibrated, and electrical resistance of the monolayers was measured by placing

one electrode on either side of the polycarbonate filter; the resistance of cell-free filters was

subtracted from that of the experimental filters to give the electrical resistance of the epithelial cell

monolayer. The TEER, expressed as Omega X cm sup 2 , was also measured before each transport

experiment to monitor the intactness of cell monolayers.

Amino acid transport experiments. Transport experiments were conducted essentially as previously

described (Ranaldi et al. 1994) but using Hank's saline containing 5 mmol/L glucose as the transport

buffer. Before transport experiments the cells were depleted of amino acids by incubation for 30 min

at 37degC in Hank's saline, after which the donor and acceptor solutions were replaced with the

appropriate solutions prewarmed at 37degC. The donor solution contained L[4,5 sup -3 H] lysine HC1

(Amersham International, Amersham, Buckinghamshire, UK; sp. act. 3.7 TBq/mmol), dissolved in

Hank's saline in the presence of varying amounts of nonlabeled L-lysine to give the required final

concentration. The specific activity of sup 3 H-L-lysine in the transport medium varied between 2

MBq/mu mol at 10 mu mol/L and 0.1 MBq/mu mol at 2 mmol/L final concentration. To avoid amino

acid backflow, the acceptor medium was changed after each time point with fresh prewarmed saline.

The radioactive amino acid ( sup 3 H-lysine) in the transport medium was analyzed by liquid

scintillation spectrometry using a Beckmann LS1801 instrument (Beckmann Instruments, Fullerton,

CA) after diluting with liquid scintillation cocktail (Ready Safe; Beckmann Instruments). The rates of

transport were calculated, after subtraction of the lag period (5 min), from the slope of the linear

portion of the amino acid appearance curve (usually between 5 and 35-60 min; see Fig. 2), with time

points taken every 10 or 15 min. (Figure 2 omitted) The pH of the transport medium was always

maintained at pH 7.4.

To determine sodium dependency of transport, NaC1 in Hank's saline was replaced with equimolar

amounts of either KC1 or choline chloride, and sodium salts were replaced with their potassium


4/15


5/15

means (Kleinbaum and Kupper 1978), using the Statview 512+ computer program (Brainspower,

Calabasas, CA). Values in the text are means +/- SD

RESULTS

Caco-2 cells in culture undergo a process of in vitro differentiation starting at confluency and

continuing for 2-3 wk. The formation of an intact monolayer of cells coupled by functional tight

junctions was monitored by measuring the establishment of a TEER during the differentiation process.

As shown in Figure 1, the TEER increased over the first 10 d and reached a plateau that remained

relatively stable between 12 and 30 d after seeding. Although several morphological and functional

traits reach full expression by the 2nd wk of confluency, it has been shown that active transport

functions may not achieve full expression before the 3rd wk, as observed for bile acid transport

(Hidalgo and Borchardt 1990b). It is therefore essential to determine the effects of the time the cells

remain in culture, on the parameter under investigation. There was no significant difference in the rate

of transport of L-lysine across Caco-2 cells in the AP to BL and BL to AP direction between d 15 and 28

after seeding (variations observed on different days were similar to those detected in separate

experiments using cells of the same age in culture; data not shown). All further transport experiments

were conducted between d 18 and 23 after seeding.

The transport of L-lysine at 10 mu mol/L measured in the AP to BL and the BL to AP direction, after an

initial lag period over the first 5 min, was linear up to 35 min for AP to BL and 60 min for BL to AP

transport (Fig. 2); the lag period was due to the time required for transport across the membrane on

the donor side, through the cell and out of the membrane on the acceptor side (Fig. 2). At 10 mu

mol/L the BL to AP transport was very much lower than that in the AP to BL direction, which in vivo

corresponds to the absorption from the intestinal lumen to the portal circulation. The transport of 10

mu mol/L L-lysine in the AP to BL direction was 45.8 +/4.7 pmol (min X mg protein) and was not

significantly affected (P > 0.05) by the presence of increasing amounts (0.01, 0.1, 1 and 10 mmol/L)

of unlabeled L-lysine in the acceptor (BL) compartment, indicating that the transepithelial transport of

L-lysine occurs equally well against a concentration gradient.

We also compared the transport in the AP to BL and BL to AP directions at 4degC at different L-lysine

concentrations, ranging from 10 mu mol/L to 2 mmol/L (Fig. 3). (Figure 3 omitted) The transport at

4degC was comparable in both directions and proportional to concentration. Computer fitting of the

data to the equation:

(1) v = Kd(AA)


6/15

representative of a diffusional component, where v is the velocity of transport, [AA] is the amino acid

concentration and Kd is the diffusion constant for a nonsaturable component, gave apparent Kd 0.013

+/- 0.0005 and 0.014 +/0.0003 pmol/(min X mg protein X mu mol X L sup -1 ) for AP to BL and BL to

AP transport, respectively. The transport at 4degC was much lower than the AP to BL and the BL to AP

transports at 37degC; at the lower concentrations (10-500 mu mol/L), it represented a negligible

fraction of the transport in both directions and at 2 mmol/L it was 3% of the AP to BL and 8% of the

BL to AP transport at 37degC.

The L-lysine transport was investigated at different concentrations (10 mu mol/L-2 mmol/L) in the AP

to BL and BL to AP direction; Figure 4 shows the rates of transport at all L-lysine concentrations.

(Figure 4 omitted) Kinetic analysis of the transport in both directions was performed by fitting the data

by nonlinear regression analysis to the following equation, including a saturable component and a

nonsaturable diffusion component:

(Equation 2 omitted)

where v is the velocity of transport, [AA] is the amino acid concentration, Vmax is the maximal

velocity of transport, Km is the amino acid concentration at which the velocity is half maximal and Kd

is the diffusion constant. For the AP to BL transport the calculated values of the apparent kinetic

parameters were Km 203 +/50 mu mol/L, Vmax 413 +/- 57 pmol/(min X mg protein) and Kd 0.27 +/-

0.029 pmol/(min X mg protein X mu mol X L sup -1 ). For the BL to AP transport the calculated values

of the apparent parameters were Km 255 +/- 201 mu mol/L, Vmax 71 +/- 34 pmol/(min X mg

protein) and Kd 0.14 +/- 0.015 pmol/(min X mg protein X mu mol X L sup -1 ]. These apparent

kinetic parameters result from the sum of distinct processes, including entry into the cell, diffusion and

compartmentalization in the cytoplasm and efflux from the cell.

When the transport medium was supplemented with 1 mmol/L NaN sub 3 , a metabolic inhibitor, and

with 50 mmol/L 2-deoxy-glucose to block glucose uptake and utilization, the AP to BL transport of L-

lysine was 39% of control (Table 1). (Table 1 omitted) Substitution of sodium in the transport medium

by KC1 or choline chloride also decreased the AP to BL transport dramatically: the transport in the

absence of sodium ions was approximately 20% of control (Table 1). A similar inhibition of transport,

although less pronounced (52% of control), was obtained by the addition of 1 mmol/L ouabain to the

sodium-containing transport medium (Table 1).

To analyze the characteristics of the sodium-independent component, contributing to the total AP to

BL transport, the rates of transport of L-lysine in the concentration range from 10 mu mol/L to 500

mu mol/L were measured in the presence of choline ions substituting for sodium ions (Figure 5).


7/15

(Figure 5 omitted) Kinetic analysis of the sodium-independent AP to BL transport by nonlinear curve

fitting to Equation 2 gave these apparent parameters: Km 2.6 +/3.9 mu mol/L, Vmax 9 +/- 2 pmol/

(min X mg protein) and Kd O.16 +/- 0.006 pmol/(min X mg protein X mu mol X L sup -1 ]. This

indicates that in the absence of sodium there was a saturable component with higher affinity but lower

capacity than that observed in the presence of sodium (Fig. 4), although most of the transport could

be accounted for by a nonsaturable diffusional component.

To determine the specificity of the AP to BL transport of L-lysine, experiments were undertaken in the

presence of a 100-fold excess of nonlabeled amino acids in the donor compartment. As shown in Table

2 the strongest inhibition was observed with the cationic amino acids L-arginine, L-ornithine and L-

lysine (9-14% of control); conversely, the lysine stereoisomer D-lysine did not inhibit transport

significantly. A strong inhibition was observed also with the large neutral amino acids L-phenylalanine,

L-leucine and L-methionine (17-27% of control). A weaker inhibiting effect was observed with L-

tryptophan, L-threonine and L-valine (76-86% of control). The acidic amino acid L-aspartate, the

amino acid L-proline and the small neutral amino acid glycine did not compete significantly to the

transport of L-lysine. To further characterize the inhibition by L-arginine, L-ornithine, L-phenylalanine

and L-leucine, experiments were performed in the presence of different concentrations of the

competitors. As shown in Figure 6, the amino acid exerting the strongest competition at the lowest

concentration used (10-fold excess) was L-arginine, whereas the least effective competitor was L-

phenylalanine. (Figure 6 omitted) When the competitor was added at 500-fold excess, all four amino

acids produced comparable and not significantly different inhibiting effects (Fig. 6).

Because the transepithelial transport of substances from the intestinal lumen to the blood circulation

involves at least three distinct components, namely, entry into the cell from the AP surface, diffusion

across the cell cytoplasm and finally efflux across the BL membrane, we attempted to dissect the

requirements of these different processes by experimentally manipulating the conditions under which

transport was measured. As shown in Table 1, the substitution of the sodium ions with potassium or

choline ions significantly inhibited the transepithelial transport of L-lysine. The uptake into the cells of

cationic amino acids can be driven by the membrane electrical potential difference (Em) because of

the positive charge of the molecules and the more negative electrical potential inside the cell (White

1985). However, because a high extracellular potassium concentration leads to depolarization of the

plasma membrane while a similar high concentration of choline ions has no effect on Em (Hodgkin and

Huxley 1952), we compared the effects of either high extracellular potassium or choline on the

transport and on the intracellular pool of L-lysine. The results reported in Figure 7A and B show that

sodium replacement in the extracellular medium induced a significant (P < 0.01) reduction in AP to BL

transepithelial L-lysine transport and a large increase in the intracellular soluble pool as compared


8/15

with control. (Figure 7A and B omitted) In the presence of high extracellular potassium the effects on

transepithelial transport and on intracellular accumulation of L-lysine were only more marked than

with high extracellular choline, indicating a limited involvement of Em on the uptake of L-lysine.

Overall, the results suggested that there was a sodium requirement for the efflux rather than for the

uptake into the cell. This was further investigated by determining L-lysine transepithelial transport and

the amino acid concentration in the intracellular soluble pool under different experimental conditions,

by selectively replacing sodium ions in the AP or BL compartments with either potassium or choline.

When sodium was absent from the BL compartment, the intracellular soluble pool increased (Fig. 7B)

and the transepithelial transport decreased (Fig. 7A) significantly (P < 0.01), compared with both

sodium-containing control and with conditions in which the sodium was absent only from the AP

compartment, thus confirming the sodium requirement for efflux from the cell BL membrane. The

amount of L-lysine incorporated into newly synthesized proteins during the 60 min of the experiment

in the presence of extracellular sodium was 44.0 +/15.5 pmol/mg protein and did not changesignificantly (P > 0.05) when differences in the ionic compositions of the extracellular medium

produced the significant changes in L-lysine transport rate and soluble pool shown in Figure 7.

DISCUSSION

Caco-2 cells grown on microporous membranes were utilized to investigate the transepithelial

transport of L-lysine. L-lysine transport activity was not affected by the time of differentiation in

culture, as previously reported for the transport of large neutral amino acids (Hidalgo and Borchardt

1990a). L-lysine transport was investigated in the two directions, AP to BL and BL to AP. The AP to BL

transport, corresponding in vivo to the absorption from the intestinal lumen to the blood circulation,

was much faster than that in the opposite direction, indicating the presence in the Caco-2 cells of one

or more specific polarized systems for the transepithelial transport of cationic amino acids. In addition,

the predominantely active nature of the transport in the AP to BL direction was suggested by the

following three observations: 1) it was unaffected by the presence of an unfavorable concentration

gradient in the acceptor compartment; 2) it exhibited a strong temperature dependence; and 3) it was

markedly reduced by energy depletion. The presence of metabolic inhibitors reduced the AP to BL

transport to 39% of control, although the real energy dependence may be higher but could not be

determined because the gate function of tight junctions is itself ATP dependent and would have been

affected using stronger energy depletion (Mandel et al. 1993).

Kinetic analysis of the AP to BL and BL to AP transport identified, in both cases, a single saturable

component and a nonsaturable diffusional component. The calculated apparent Km, indicating the

affinities of the saturable components in the two directions of transport, were similar, whereas the


9/15

transport in the AP to BL direction exhibited a much higher capacity than that in the opposite

direction. The apparent Kd calculated for the diffusional component was also similar in the two

directions of transport; this nonsaturable transport component probably represents the contribution of

the paracellular pathway to the total transport of lysine across the Caco-2 cell monolayer. Tight

junctions have in fact been shown to exhibit cation selectivity (Jackson 1987) and may allow the

transport of a small positively charged molecule such as lysine. Especially during the peak of lysine

delivery to the intestinal mucosa that occurs after a meal, this transport component may significantly

contribute to lysine absorption.

The AP to BL transport of L-lysine was markedly reduced by the removal of sodium ions from the

transport medium, indicating a strong sodium dependence. In addition, kinetic analysis of the residual

L-lysine transport in the absence of sodium ions indicated the presence of a minor sodium-

independent component, characterized by a very high affinity but a low transport capacity (Fig. 5).

The characteristics of the cationic amino acid transport in the human intestine have, to the best of our

knowledge, previously been investigated only in fetal brush border vesicles, where a sodium-

independent transport of L-lysine was shown to operate (Malo 1991). Uptake studies performed on

brush border vesicles or mucosal preparations from small intestine, reported either sodium-dependent

and sodium-independent (Munck and Munck 1992, Paterson et al. 1981, Wolfram et al. 1984) or only

sodium-independent (Cassano et al. 1983) L-lysine transport systems. This discrepancy could be the

result of regulation by nutritional factors (Wolfram et al. 1984). In a recent study, the activities

responsible for L-lysine uptake in rat intestine were characterized after mRNA expression in Xenopus

laevis oocytes. Three transport activities were identified: one sodium dependent and two sodium

independent, that could be distinguished on the basis of their interactions with other amino acids

(Harvey et al. 1993); these systems had some features in common with the previously described

system B sup 0+ and b sup 0+ for cationic and neutral amino acids (Van Winkle et al. 1990), as

defined in mouse blastocysts, and with the cationic specific system y sup + described in several

tissues as well as the intestine (White 1985). However, expression of exogenous transport activities in

oocytes can characterize their function and requirements but cannot provide information on their

localization in the cell of origin. Transepithelial transport is in fact the result of at least three distinct

processes regulated by transporters located on the different plasma membrane domains: entry into

the cell, diffusion across the cytoplasm and efflux out of the cell.

At physiological pH L-lysine is positively charged and therefore its uptake from the lumen across the

AP membrane should be favored by the more negative potential difference inside the cell, whereas the

efflux across the BL membrane would have to occur against the electrochemical gradient. By


10/15

selectively removing sodium ions on either side of the cell monolayer and comparing the transport

across the cells and the intracellular soluble amino acid pool, it is possible to alter the membrane

potential and to distinguish the sodium requirement of the entry and of the efflux mechanisms. The

depolarization of the plasma membrane, produced by the replacement of sodium ions in the

extracellular medium with potassium ions, strongly inhibited the transepithelial transport of L-lysine

but did not markedly affect its uptake at the AP membrane; the amino acid accumulated in the

intracellular pool at levels not significantly different from the sodium-containing control. By

comparison, sodium replacement by choline, which does not lead to membrane depolarization,

produced a similar decrease in transepithelial transport and a large accumulation of L-lysine in the

intracellular pool. The results indicate that L-lysine entry into Caco-2 cells is not driven primarily by

the membrane potential, and it is not affected by sodium. Conversely, the efflux out of the BL

membrane is strongly inhibited by the absence of sodium in the acceptor compartment. In addition, all

experimental conditions that altered the extracellular ionic composition did not appear to affect themetabolic activity of the cells, as sown by the similar levels of sup 3 H-lysine incorporation into newly

synthesized proteins over the course of the transport experiments.

The specificity of the uptake of L-lysine was studied by competition experiments using amino acids

with different chemical characteristics, with particular attention for essential amino acids. The

transport of L-lysine was shared by the cationic amino acids L-arginine and L-ornithine, and it showed

stereospecificity for the L-isomer. The lack of competition by glycine and the marked inhibition by L-

phenylalanine, L-leucine and L-methionine indicate that the carrier(s) responsible for the uptake of L-

lysine interact with bulky neutral amino acids but not with small ones. These results are consistent

with previous studies in the intestine where it was shown that some of the systems responsible for the

transport of cationic amino acids also accept large neutral amino acids (Cassano et al. 1983, Harvey et

al. 1993, Munck and Munck 1992, Paterson et al. 1981).

In filter-grown Caco-2 cell monolayers the transepithelial transport of L-phenylalanine was partially

inhibited by L-lysine, and it was suggested that cationic and large neutral amino acids may share the

same carrier that has L-phenylalanine as the preferred substrate (Hidalgo and Borchardt 1990a). We

found a similar interaction of L-lysine and large neutral amino acids, although our results indicate that

the system highlighted by studying lysine transport has higher affinity for the cationic than for the

neutral amino acids. Our results therefore suggest that there may be one or more sodium-

independent carriers on the AP surface, able to transport cationic amino acids but also shared by large

neutral amino acids, as it was previously shown in other experimental systems (Cassano et al. 1983,

Malo 1991, Paterson et al. 1981). The fact that a sodium-dependent uptake mechanism was not

detected in our system may be related to nutritional factors; in rat intestine it was in fact shown that


11/15

the sodium-dependent uptake of L-lysine was induced by a high protein diet and may therefore not be

expressed in cell culture conditions (Wolfram et al. 1984).

Because the efflux from the BL membrane must occur against the electrochemical gradient, the

presence of a sodium-dependent efflux mechanism may be required for efficient transport out of the

cell. This may be particularly important in the case of L-lysine, which needs to be efficiently obtained

from the diet. An actively regulated eFflux system for cationic amino acids has been suggested as the

rate-limiting step in the transepithelial transport of these amino acids (Cheeseman 1992). The

presence of a sodium-dependent efflux mechanism in the Caco-2 cells may therefore reflect a

physiological need for the whole organism. Although its presence in normal human intestine still needs

to be demonstrated, such a mechanism would play a very important nutritional role, allowing the

eFflux and transport into the circulation of an essential amino acid, thus increasing its bioavailability

from the diet. The presence of a physiologically essential transport mechanism for cationic amino acids

on the BL membrane of intestinal cells is strongly suggested by the defects associated with the

autosomal recessive phenotype lysinuric protein intolerance (Mc Kusick number 222700). This

condition, producing severe effects on growth and development of affected children, is believed to

arise from selective impairment of cationic amino acid efflux at the BL membrane in renal and

intestinal epithelium; it is characterized by greatly reduced levels of plasma cationic amino acid

concentrations, hyperammonaemia and cationic aminoaciduria (Smith et al. 1987).

The characterization of the transepithelial transport in the Caco-2 cell line of the cationic amino acid L-

lysine and the different requirements for its uptake at the AP membrane and its efflux from the BL

membrane were the object of this paper. The results confirm that, in the absence of alternative

human models, the Caco-2 cell line provides a useful experimental system to elucidate intestinal

amino acid transport mechanisms on both AP and BL plasma membrane domains. In addition, this

cellular model can be used to study metabolic, dietary or other extracellular environmental effects on

the intestinal bioavailability of nutrients; in the case of essential nutrients, such as lysine, these

studies could contribute to the reassessment of the recommended daily allowances of essential amino

acids that were recently under discussion (Young 1994).

ACKNOWLEDGMENTS

We thank Maria Antonietta Spadoni (INN) and Khalid Islam (Lepetit Research Center) for helpful

comments and suggestions, Amleto De Amicis (INN) for expert advice on the statistical analysis of the

data and Giuseppe Crocchioni (INN) for excellent technical assistance. Alain Zweibaum, INSERM,

Villejuif, France, donated the intestinal Caco-2 cell line.


12/15

1 Supported by National Research Council of Italy, Special Project RAISA, Subproject 4, Paper 1889

and Target Project Biotechnology and Bioinstrumentation and by the Italian Ministry of Education, MPI

60%.

2 The costs of publication of this article were defrayed in part by the payment of page charges. This

article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734

solely to indicate this fact.

3 To whom correspondence and reprint requests should be addressed.

4 Abbreviations used: AP, apical; BL, basolateral; Em, membrane electrical potential difference; TCA,

trichloroacetic acid; TEER, transepithelial electrical resistance; TLC, thin layer chromatography.

LITERATURE CITED

Alvarez-Hernandez, X., Smith, M. & Glass, J. (1994) Regulation of iron uptake and transport by

transferrin in Caco-2 cells, an intestinal cell line. Biochim. Biophys. Acta 1192: 215-222.

Cassano, G., Leszczynska, B. & Murer, H. (1983) Transport of L-lysine by rat intestinal brush border

vesicles. Pflugers Archs. Ges. Physiol. 397: 114-120.

Cheeseman, C. (1992) Role of intestinal basolateral membrane in absorption of nutrients. Am. J.

Physiol. 262: R482-R488.

Chen, J., Zhu, Y. & Hu, M. (1994) Mechanisms and kinetics of uptake and efflux of L-methionine in an

intestinal epithelial model Caco-2. J. Nutr. 124: 1907-1916.

Chen, T. R. (1977) In situ detection of mycoplasma contamination in cell cultures by fluorescent

Hoechst 33258 stain. Exp. Cell. Res. 104: 255-262.

Fogh, J., Fogh, J. M. & Orfeo, T. (1977) One hundred and twenty seven cultured human tumor cell

lines producing tumors in nude mice. J. Natl. Cancer Inst. 59: 221-226.

Harvey, C. M., Muzyka, W. R., Yao, S. Y.-M., Cheeseman, C. I. & Young, J. D. (1993) Expression of rat

intestinal L-lysine transport systems in isolated oocytes of Xenopus laevis. Am. J. Physiol. 265: G99-

G106.

Hidalgo, I. J. & Borchardt, R. T. (1990a) Transport of a large neutral amino acid (phenylalanine) in a

human intestinal epithelial cell line. Biochim. Biophys. Acta 1028: 25-30.


13/15

Hidalgo, I. J. & Borchardt, R. T. (1990b) Transport of bile acids in a human intestinal epithelial cell

line, Caco 2. Biochim. Biophys. Acta 1035: 97-103.

Hodgkin, A. L. & Huxley, A. F. (1952) Currents carried by sodium and potassium ions through the

membrane of the giant axon of Loligo. J. Physiol. 116: 449-472.

Hopfer, U. (1987) Membrane transport mechanisms for hexoses and amino acids in the small

intestine. In: Physiology of the gastrointestinal tract (Johnson, L. R., ed.), pp. 1499-1526. Raven

Press, New York, NY.

Jackson, M. J. (1987) Drug transport across gastrointestinal epithelia. In: Physiology of the

gastrointestinal tract (Johnson, L. R., ed.), pp. 1597-1621. Raven Press, New York, NY.

Kleinbaum, D. & Kupper, L. (1978) Applied regression analysis and other multivariable methods.

Duxbury Press, Boston, MA.

Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) Protein measurement with the

Folin phenol reagent. J. Biol. Chem. 193: 265-275.

Malo, C. (1991) Multiple pathways for amino acid transport in brush border membrane vesicles

isolated from human fetal small intestine. Gastroenterology 100: 1644-1652.

Mandel, L. J., Bacallao, R. & Zampighi, G. (1993) Uncoupling of the molecular "fence" and paracellular

"gate" functions in epithelial tight junctions. Nature 361: 552-555.

Marquardt, D. W. (1963) An algorithm for least squares estimation of non linear parameters. J. Soc.

Ind. Appl. Math. 11: 431-441.

Munck, L. & Munck, B. (1992) Variation in amino acid transport along the rabbit small intestine.

Mutual jejunal carriers of leucine and lysine. Biochim. Biophys. Acta 1116: 83-90.

Paterson, J. Y. F., Sepulveda, F. V. & Smith, M. W. (1981) Distinguishing transport systems having

overlapping specificities for neutral and basic amino acids in the rabbit ileum. J. Physiol. (Lond.) 319:

345-354.

Raffaniello, R., Lee, S.-Y., Teichberg, S. & Wapnir, R. (1992) Distinct mechanisms of zinc uptake at

the apical and basolateral membranes of Caco-2 cells. J. Cell. Physiol. 152: 356-361.


14/15

Ranaldi, G., Islam, K. & Sambuy, Y. (1992) Epithelial cells in culture as a model for the intestinal

transport of antimicrobial agents. Antimicrob. Agents Chemother. 36: 1374-1381.

Ranaldi, G., Islam, K. & Sambuy, Y. (1994) D-cycloserine uses an active transport mechanism in the

human intestinal cell line Caco 2. Antimicrob. Agents Chemother. 38: 1239-1245.

Riley, S. A., Warhurst, G., Crowe, P. T. & Turnberg, L. A. (1991) Active hexose transport across

cultured human Caco 2 cells: characterization and influence of culture conditions. Biochim. Biophys.

Acta 1066: 175-182.

Smith, D., Scriver, C., Tenenhouse, H. & Simell, O. (1987) Lysinuric protein intolerance mutation is

expressed in the plasma membrane of cultured skin fibroblasts. Proc. Natl. Acad. Sci. U.S.A. 84:

7711-7715.

Thwaites, D. T., Mc Ewan, G. T. A., Cook, M. J., Hirst, B. & Simmons, N. (1993) H sup + -coupled (Na

sup + -independent) proline transport in human intestinal (Caco 2) epithelial cell monolayers. FEBS

Lett. 333: 78-82.

Van Winkle, L. J., Campione, A. L. & Gorman, J. M. (1990) Inhibition of transport system b sup 0,+ in

blastocysts by inorganic and organic cations yields insight into the structure of its amino acid receptor

site. Biochim. Biophys. Acta 1025: 215-224.

White, M. F. (1985) The transport of cationic amino acids across the plasma membrane of mammalian

cells. Biochim. Biophys. Acta 822: 355-374.

Wolfram, S., Giering, H. & Scharrer, E. (1984) Na sup + -gradient dependence of basic amino acid

transport into rat intestinal brush border membrane vesicles. Comp. Biochem. Physiol. 78A: 475-480.

Young, V. R. (1994) Adult amino acid requirements: the case for a major revision in current

recommendations. J. Nutr. 124: 1517S-1523S.

Zweibaum, A., Laburthe, M., Grasset, E. & Louvard, D. (1991) Use of cultured cell lines in studies of

intestinal cell differentiation and function. In: Handbook of Physiology: The Gastrointestinal System.

(Field, M. & Frizzel, R. A., eds.), vol. 4, pp. 223-255. Am. Physiol. Soc., Bethesda, MD.

Jumlah kata: 5742

Copyright American Institute of Nutrition Oct 1995


15/15

Documents

The Transport of Lysine Across Monolayers of Human Cultured Intestinal Cells