Adsorptive-mediated endocytosis of a basic peptide in
enterocyte-like Caco-2 cells
Yoshimichi
Sai1,
Masahiro
Kajita1,
Ikumi
Tamai1,
Jun
Wakama2,
Tateaki
Wakamiya2, and
Akira
Tsuji1
1 Department of Pharmaceutics,
Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa
920-0934; and 2 Department of Chemistry,
Faculty of Science and Technology, Kinki University, Higashi-osaka,
Osaka 577-8502, Japan
 |
ABSTRACT |
The internalization of a basic
peptide, 001-C8
[H-MeTyr-Arg-MeArg-D-Leu-NH(CH2)8NH2],
into enterocyte-like Caco-2 cells was evaluated. Internalization of
125I-labeled 001-C8
(125I-001-C8) increased time
dependently and reached steady state at 60 min. The steady-state
internalization of 125I-001-C8
(7.24 ± 0.41 µl/mg protein) was temperature and concentration dependent and was significantly decreased by dansylcadaverine (500 µM), protamine (1 mM),
poly-L-lysine (1 mM), E-2078 (1 mM), and ebiratide (1 mM), whereas
poly-L-glutamic acid (1 mM),
tyrosine (1 mM), and glycylglycine (25 mM) were not
inhibitory. Predigestion of acid mucopolysaccharides by heparinase I,
heparitinase, and chondroitinase ABC also decreased the
internalization. The maximal internalization, the half-saturation
constant, and the nonsaturable internalization of
125I-001-C8 were 1.13 ± 0.23 pmol/mg protein, 0.47 ± 0.43 µM, and 3.13 ± 0.19 µl/mg
protein, respectively. Confocal microscopy also indicated the
internalization of fluorescence-derived 001-C8
[001-C8-4-nitrobenz-2-oxa-1,3-diazole (001-C8-NBD)].
Granular staining seen within the cell, excluding nuclei, indicated the
sequestration of 001-C8-NBD within endocytotic vesicles.
Dansylcadaverine and protamine strongly decreased the granular
distribution of 001-C8-NBD within the cell. These results demonstrate
that 001-C8 is taken up by Caco-2 cells via adsorptive-mediated endocytosis.
intestine; transport; macromolecular uptake; absorptive-mediated
drug delivery
 |
INTRODUCTION |
SPECIFIC TRANSPORT mechanisms have been evolved in the
intestine to facilitate the uptake of various nutrients, including small peptides and macromolecules. For proteins and peptides, four
types of uptake mechanisms have been recognized. They include 1) highly specific,
receptor-mediated endocytosis (RME),
2) adsorptive-mediated endocytosis
(AME) after binding of molecules to cell surface anionic sites,
3) nonspecific fluid-phase
endocytosis of substances dissolved in the extracellular fluid, and
4) carrier-mediated transport for
small peptides (3-6, 12, 14, 16-18, 23, 25, 27). RME
mechanisms in the intestine have been shown in the transport of various
immunoglobulins and growth factors (see Ref. 18 for review).
Fluid-phase endocytosis has also been reported with horseradish peroxidase as a model protein in Caco-2 cells (4). As for AME in the
intestine, however, very little information is available. Although a
dynorphin-like analgesic peptide, E-2078 (23), an ACTH analog,
ebiratide (19), and various polycationic proteins, such as
-endorphin-cationized albumin complex (6), histone (14), and avidin
(12), are transported via AME into the brain capillaries, it remains
unknown whether such a process also occurs in intestinal epithelial
cells.
In our previous study, we prepared a novel peptide, 001-C8
[H-MeTyr-Arg-MeArg-D-Leu-NH2(CH2)8NH2],
and its derivatives with different numbers of basic and neutral amino
acids and with various carboxy-terminal structures to clarify the
structural specificity of AME of brain capillary endothelial cells
(21). 001-C8 consists of a partial amino acid sequence of E-2078 and
the carboxy-terminal structure of ebiratide, with two arginine residues
and an octanediamine residue, and was confirmed to be efficiently taken
up by brain capillary endothelial cells via AME (21).
Caco-2, a human colon carcinoma cell line, forms a highly polarized
membrane when grown to confluence on plastic dishes and exhibits
structural and functional differentiation patterns characteristic of
mature enterocytes (15). Caco-2 cells have been used as an in vitro
model in various studies, including characterization of the
intracellular sorting or cytosis of proteins (1, 4, 9), because of
their close morphological and functional similarity to intestinal
epithelium. We have also used this system in studies of the
carrier-mediated transport mechanism (11, 22, 26).
In this report, we demonstrate the AME of a basic peptide, 001-C8, in
enterocyte-like Caco-2 cells by means of inhibition, saturation
kinetic, and confocal-microscopic observation studies using radio- or
fluorescence-labeled ligands.
 |
MATERIALS AND METHODS |
Materials.
[3H]polyethylene
glycol 900 ([3H]PEG
900) (74-370 MBq/g) and
Na125I (629 GBq/mg) were
purchased from New England Nuclear (Boston, MA).
Tetramethylrhodamine-dextran was obtained from Molecular Probes
(Eugene, OR). FCS and rat tail collagen (type I) were purchased from
GIBCO (Grand Island, NY) and Collaborative Research (Bedford, MA),
respectively. Salmon roe protamine sulfate was purchased from Wako Pure
Chemical Industries (Osaka, Japan). Dansylcadaverine, heparin,
heparinase I, and neuraminidase were purchased from Sigma Chemical (St. Louis, MO). Heparitinase, chondroitinase ABC, and hyaluronidase were purchased from Seikagaku (Tokyo, Japan). E-2078, a
dynorphin-like analgesic peptide
[H-MeTyr-Gly-Gly-Phe-Leu-Arg-MeArg-D-Leu-NHC2H5], was kindly supplied by Eisai (Tokyo, Japan). The Caco-2 cells were
obtained from American Type Culture Collection (Manassas, VA).
All cells used in this study were between passages
33and 59. All other
chemicals used were commercial products of reagent grade.
Peptide synthesis and labeling.
001-C8 used in the present study was synthesized in this laboratory, as
described previously (21), and was coupled to
4-nitrobenz-2-oxa-1,3-diazole (NBD) as a fluorescence label for direct
visualization. Radioiodination of 001-C8 was performed with
Na125I and chloramine-T as
described previously (21).
125I-001-C8 obtained had a
specific activity of ~11.1 TBq/g and a chemical purity of >95%.
Assay for internalization.
The cultivation of Caco-2 cells was performed as described previously
(26). A typical uptake experiment was as follows. Monolayers of Caco-2
cells grown for 7 days after confluency on four-well multidishes (NUNC,
Raskilde, Denmark) coated with collagen were washed three times with
Hanks' balanced salt solution (HBSS) (pH 6.0) (in mM: 0.952 CaCl2, 5.36 KCl, 0.441 KH2PO4,
0.812 MgSO4, 136.7 NaCl, 0.385 K2HPO4,
25 D-glucose, and 10 MES; the
osmolarity was 315 mosmol/kg). The uptake experiment was initiated by
adding 250 µl of incubation solution (HBSS, pH 6.0) containing
125I-001-C8 (37 kBq) or
[3H]PEG 900 (37 kBq)
to cells. At designated times after incubation, cells were washed three
times with 1 ml of the ice-cold incubation solution to terminate
uptake. An acid-wash technique (24) was then used to distinguish
surface-bound and internalized
125I-001-C8. The acid treatment
removes 125I-001-C8 bound to the
cell surface. After the uptake procedure, cells were incubated for 10 min with 1 ml of ice-cold acetate-barbital buffer (28 mM
CH3COONa, 120 mM NaCl, 20 mM
barbital-HCl, pH 3.0) at 4°C. The buffer was then removed, and
cells were washed four more times with 1 ml of acetate-barbital buffer.
The radioactivity in the cells was measured after solubilization with
250 µl of 1 M NaOH for 1 h at room temperature and represents
internalized 125I-001-C8.
Radioactivity was measured with a gamma counter (ARC-600; Aloka, Tokyo,
Japan) and a liquid scintillation counter (LSC-700; Aloka) for
125I and
3H, respectively. Protein contents
of cultured cells were determined as described by Lowry et al. (7) with
BSA as a standard. The number of experiments given in Tables 1 and 2
and Figs. 1-6 represents the number of wells of cultured cells used in
each measurement.
Confocal microscopy.
Caco-2 cells were grown on a glass coverslip equipped with a Flexiperm
chamber (Heraeus Instruments, Hanau, Germany) and cultured for 14 days
at 37°C with 95% air-5% CO2.
The coverslips used were precoated with rat tail collagen under
ultraviolet light. Cultured monolayers of Caco-2 cells were rinsed
three times with HBSS (pH 6.0), mounted on the microscope stage, and
incubated with 10 µM 001-C8-NBD dissolved in 200 µl of HBSS (pH
6.0). Tetramethylrhodamine-dextran (10 mg/ml, labeled overnight and
chased for 30 min) was used as an organelle marker. The stage was
equipped with a thermostabilizing device to keep the temperature at
37°C throughout the incubation period.
The confocal system used in this study consisted of an MRC-1000
Confocal Imaging System (Bio-Rad, Tokyo, Japan) and an Axiovert 135 inverted microscope equipped with a Neofluar oil objective (magnification ×63, numerical aperture 1.25) and epifluorescence optics (Carl Zeiss, Oberkochen, Germany). This instrument uses a pair
of microcomputer-controlled galvanometer mirrors to scan a 488- or
514-nm beam of light from a pinhole illuminated by an argon ion laser
in a raster pattern across the specimen being viewed. Images of each
optical section were taken, digitized, and stored in a frame buffer in
the microcomputer.
HPLC analysis.
Unchanged 125I-001-C8 and its
metabolites in the incubation solution and in the acid-resistant
fraction were analyzed by HPLC. Acid-washed cells were solubilized with
1 M NaOH. Each sample was evaporated to dryness under reduced pressure
at room temperature and reconstituted in the mobile phase used for HPLC
assay. The HPLC analysis conditions were as follows. The column was a
VYDAC 214TP54 (Separations Group, Hasperia, CA); the mobile phase was a
mixture of water, acetonitrile, and trifluoroacetic acid (15:85:0.1); and the flow rate was 1.0 ml/min. The eluates were collected with a
fraction collector (FRAC-100; Pharmacia, Tokyo, Japan), and the
radioactivity in each fraction (0.5 ml) was measured.
Data analysis.
Total and acid-resistant bindings were expressed as the cell-to-medium
ratio as follows
|
(1)
|
|
(2)
|
where
125I-R and
125I-S are the radioactivities of
125I in the acid-resistant and
acid-soluble fractions, respectively, and
125I-M is the radioactivity of
125I in the incubation medium.
The apparent acid-resistant binding of
[3H]PEG 900 was
estimated in the same manner.
 |
RESULTS |
Time course of internalization of
125I-001-C8 into cultured monolayers of
Caco-2 cells.
The surface binding and internalization of a cationic peptide,
125I-001-C8
[H-125I-MeTyr-Arg-MeArg-D-Leu-NH(CH2)8NH2],
at the apical surface of cultured monolayers of Caco-2 cells were
assessed by its total binding and acid-resistant binding and were
compared with the surface binding and internalization of the
extracellular space and a fluid-phase endocytosis marker,
[3H]PEG 900 (Fig.
1). The values were normalized using the
initial concentration of each substrate and expressed as the
cell-to-medium ratio, calculated as described in
MATERIALS AND METHODS. Total and
acid-resistant bindings of
125I-001-C8 increased time
dependently, reaching steady-state values of over 13 and 7 µl/mg
protein, respectively, at 60 min. Both values were significantly higher
than that of acid-resistant binding of
[3H]PEG 900. In the
following experiments, the acid-resistant binding at 60 min was used to
evaluate the steady-state internalization of
125I-001-C8.

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Fig. 1.
Time courses of total and acid-resistant bindings of
125I-labeled 001-C8
(125I-001-C8) and
[3H]polyethylene
glycol 900 ([3H]PEG
900) to cultured monolayers of Caco-2 cells. Caco-2 cells were
incubated with 125I-001-C8
(circles) or [3H]PEG
900 (squares) for 5 s to 120 min at 37°C. Total and acid-resistant
bindings of each substrate were determined as described in
MATERIALS AND METHODS. Data are means ± SE of 3 or 4 experiments.
|
|
Stability of the peptide in Caco-2 cells.
The stability of 001-C8 during the internalization assay period was
assessed (Fig. 2). The radio-HPLC
chromatograms of 125I-001-C8
internalized into Caco-2 cells and remaining in the assay medium at 60 min revealed that the intact peptide accounted for 76% and 89% of the
radioactivity, respectively.

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Fig. 2.
Radio-HPLC chromatograms of
125I-001-C8 internalized
(A) and remaining
(B) after incubation of
125I-001-C8 with cultured
monolayers of Caco-2 cells.
125I-001-C8 was incubated with
cultured monolayers of Caco-2 in HBSS (pH 6.0) for 60 min at 37°C.
Unchanged 125I-001-C8 and its
metabolites in the incubation solution and in the acid-resistant
fraction were then analyzed by HPLC as described in
MATERIALS AND METHODS. An HPLC
chromatogram of authentic
125I-001-C8 is also shown
(C).
|
|
Temperature dependency and effects of an endocytosis inhibitor and
various cationic peptides on internalization of
125I-001-C8 into Caco-2 cells.
Table 1 shows the temperature dependency
and effects of an endocytosis inhibitor and several cationic peptides
on the internalization of
125I-001-C8 into Caco-2 cells. At
4°C, the internalization was reduced to 60% of the control value
obtained at 37°C. An endocytosis inhibitor, dansylcadaverine (500 µM), significantly decreased the internalization. The polycationic
peptides poly-L-lysine (1 mM)
and protamine (1 mM) inhibited the internalization. It was also reduced
by the structurally analogous compounds E-2078 (1 mM) and ebiratide (1 mM). Unlabeled 001-C8 also decreased the internalization of
125I-001-C8 at 1 mM, suggesting
the existence of a saturable mechanism. No inhibition was seen with
anionic poly-L-glutamic acid (1 mM) or the constituent amino acid, tyrosine (1 mM), which is the
iodo-labeling site of 001-C8. The dipeptide glycylglycine (25 mM) was
not inhibitory, either.
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Table 1.
Effects of temperature, an endocytosis inhibitor, and various cationic
peptides on internalization of 125I-001-C8 into Caco-2
cells
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|
Saturation of the internalization process for
125I-001-C8.
The internalization of 125I-001-C8
into Caco-2 cells was saturable (Fig. 3).
The kinetic parameters for the binding of these peptides were estimated
by nonlinear least-squares analysis (28). The maximal internalization
(Bmax), the half-saturation
constant (Kd),
and the nonsaturable internalization were 1.13 ± 0.23 pmol/mg protein, 0.47 ± 0.43 µM, and 3.13 ± 0.19 µl/mg protein,
respectively.

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Fig. 3.
Concentration dependence of internalization of
125I-001-C8 into cultured
monolayers of Caco-2 cells. Caco-2 cells were incubated with
125I-001-C8 for 60 min at 37°C
with increasing concentrations of unlabeled 001-C8 (0.1 µM to 1 mM).
Internalization was determined as described in Fig. 1 legend. Each
point is mean ± SE of 3 or 4 experiments.
|
|
Effect of cell surface deglycosylation on internalization of
125I-001-C8 into Caco-2 cells.
To investigate the chemical nature of the membrane-associated anionic
sites, the apical surface of Caco-2 cells was treated, before exposure
of the cell to 125I-001-C8, with
heparinase I, heparitinase, chondroitinase ABC, hyaluronidase, or
neuraminidase to remove heparin, heparan sulfates, chondroitin
sulfates, hyaluronic acid, or sialic acid, respectively (Table
2). The internalization of
125I-001-C8 was significantly
decreased by heparinase I, heparitinase, and chondroitinase ABC. The
effects of these enzymes in combination were approximately additive.
Digestion with hyaluronidase had little effect, whereas neuraminidase
treatment increased the internalization. We also examined the influence
of heparin, which was found to cause a significant decrease in
internalization. None of these enzymes affected internalization of
[3H]PEG 900.
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Table 2.
Effects of cell surface deglycosylation on internalization of
125I-001-C8 and [3H]PEG 900 into Caco-2 cells
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Confocal microscopic analysis of internalization of 001-C8-NBD in
Caco-2 cells.
Figure 4 shows the time-dependent
internalization into Caco-2 cells of 001-C8 labeled with the
fluorescent probe NBD (001-C8-NBD). Faint fluorescence was detected at
an incubation time of 10 min. At 60 min, significant fluorescence
appeared within the cells, excluding the nuclei. The granular staining
indicated the sequestration of 001-C8-NBD within endocytotic vesicles.
Tetramethylrhodamine-dextran was used as a marker for
lysosomal distribution and compared with the distribution of 001-C8-NBD
(Fig. 5). A part of NBD-derived fluorescence was detected in the same granule with
tetramethylrhodamine-dextran. Accordingly, a part of 001-C8-NBD is
suggested to be localized in lysosomes.

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Fig. 4.
Confocal photomicrographs showing internalization of
fluorescence-derived 001-C8 [001-C8-4-nitrobenz-2-oxa-1,3-diazole
(001-C8-NBD)] into cultured monolayers of Caco-2 cells. Caco-2
cells grown on a glass coverslip were loaded with 001-C8-NBD, mounted
on the microstage, and incubated at 37°C. Digital confocal
microscopy images were taken at 10 (A), 20 (B), and 60 (C) min. Cell image obtained by the
trans-detector is shown in D. Scale
bar, 25 µm.
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Fig. 5.
Subcellular distribution of rhodamine-dextran and internalization of
001-C8-NBD into cultured monolayers of Caco-2 cells. Caco-2 cells were
preloaded with tetramethylrhodamine-dextran (10 mg/ml) overnight and
chased for 30 min at 37°C to mark secondary lysosomes (arrowheads).
Cells were then further incubated with 001-C8-NBD. Digital fluorescence
images of tetramethylrhodamine (A)
and NBD (C) were independently taken
with the use of respective optical filter blocks, and both images were
merged electronically (B). Scale
bar, 25 µm.
|
|
Effect of an endocytosis inhibitor and a cationic protein on
internalization of 001-C8-NBD into Caco-2 cells.
We assessed the effects of an endocytosis inhibitor, dansylcadaverine,
and a cationic protein, protamine, on the internalization of 001-C8-NBD
into Caco-2 cells (Fig. 6).
Dansylcadaverine (500 µM) strongly decreased the granular
distribution of 001-C8-NBD within the cells. Protamine (1 mM) also
strongly reduced the internalization.

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Fig. 6.
Effect of an endocytosis inhibitor and a cationic protein on
internalization of 001-C8-NBD into cultured monolayers of Caco-2 cells.
Caco-2 cells were preincubated with 500 µM dansylcadaverine (DNS) or
300 µM protamine (PTM) for 30 min at 37°C. Cells were then
further incubated with 001-C8-NBD. Digital confocal microscopy images
were taken at 10 min (top) and 30 min (bottom). Scale bar, 25 µm.
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 |
DISCUSSION |
The purpose of the present study was to examine AME in intestinal
epithelial cells using a cationic model peptide, 001-C8. We had shown
previously that, among various peptide derivatives examined in brain
capillary endothelial cells, 001-C8 was internalized most efficiently
(21), but it remained unknown whether such a process is also involved
in intestinal epithelial cells. The Caco-2 cell line was used as an in
vitro model because of its close morphological and functional
similarity to the intestinal epithelium (15).
The six criteria for AME provided in our study were as
follows: 1) A cationic
nature of the model peptide 001-C8, having cationic charges at
physiological pH with an estimated isoelectric point of 12.5, is
considered to be an important determinant for AME at the blood-brain
barrier. 001-C8 was basically designed to have a part of the amino acid
residues of E-2078 (23) and the carboxy-terminal structures of
ebiratide (19), both of which have been shown to cross the blood-brain
barrier via AME. 2) Arginine and
methylarginine included in 001-C8 are important
determinants of affinity to the sites of adsorption on the cell
surface, in the case of AME at the blood-brain barrier.
3) Internalization is inhibited by
various cationic compounds such as
polyl-lysine, protamine,
E-2078, and ebiratide, which can be inhibitors of AME.
4) Internalization of the peptide is
labile to enzymatic digestion of the cell surface, which removes
membrane-associated anionic polysaccharides.
5) Internalization and granular
distribution of the peptide-derived fluorescence can be confirmed by
morphological observation. 6) Kd of 001-C8 is
comparable with previously published
Kd values of
ligands for AME, which are two to three orders higher than the values
of ligands for RME. All of the results provided in the present study
are explained by AME, which is an endocytosis mechanism that is
affected by the cationic and anionic nature of peptides and certain
components on the cell surface, respectively.
125I-001-C8 was stable within
Caco-2 cells throughout the incubation period (Fig. 2). It is also
stable in intestinal tissue homogenate, giving a half-life of about 100 min in 1 mg protein/ml of rat intestinal homogenate (16). The
metabolically stable nature of
125I-001-C8 may be ascribed to the
N-methylation of Tyr and Arg, substitution with the
D-isomer of Leu, and
modification of the carboxy-terminal structure. A minor degradation
product detected in the internalized fraction was identified as free
125I on the basis of its retention
time of 5 min in HPLC assay (Fig. 2). This property of 001-C8 is
suitable for study of AME.
We evaluated the internalization of 001-C8 caused by endocytosis both
functionally and morphologically. Because of the basic nature
(isoelectric point = 12.5) of 001-C8, measurement of cell-associated total radioactivity cannot distinguish internalization by AME from
surface binding. To separate cell surface-bound (acid-sensitive) from
internalized (acid-resistant)
125I-001-C8, an acid-wash
technique was used. The results suggest that at least one-half of the
cell-associated total radioactivity was internalized (Fig. 1). To
confirm that acid-wash-resistant binding reflects endocytosis rather
than cytosolic distribution caused by diffusion or paracellular
transfer, we used confocal microscopy. The granular distributions of
the staining and the absence of staining in nuclei in optical sections
of the center of the cells strongly indicate the sequestration of
001-C8 within endocytotic vesicles (Fig. 4). Endocytosis was also
suggested by the observation that the internalization was reduced by
dansylcadaverine and at low temperatures (Table 1). The incomplete
inhibitory effect (see Table 1) may be ascribed to the incomplete
removal of cell surface-bound
125I-001-C8 by acid washing in the
present procedure, although an involvement of uptake mechanisms other
than AME cannot be exclusively ruled out. However, involvement of the
oligopeptide transporter PepT1, which is expressed at the brush border
of absorptive epithelial cells and accepts not only di- or tripeptides
but also peptide-mimetic
-lactam antibiotics and
angiotensin-converting enzyme inhibitors as substrates
(10, 17, 27), is unlikely, because glycylglycine at 25 mM did not
affect the internalization. Furthermore, since the internalization of
001-C8 (7.24 ± 0.41 µl/mg protein) was significantly
higher than that of PEG 900 (0.81 ± 0.06 µl/mg protein), which is
a measure of fluid-phase endocytosis as well as extracellular attached
water space, 001-C8 is likely to be internalized into Caco-2 cells via
an endocytosis-type mechanism(s) other than fluid-phase endocytosis.
The internalization of 001-C8 was inhibited by cationic peptides
including protamine,
poly-L-lysine, E-2078, and
ebiratide (Table 1; Fig. 6). E-2078 and ebiratide have been reported to be transported through the blood-brain barrier via AME based on isolated brain capillary studies (19, 23) or the use of cultured brain
capillary endothelial cells (24). The
Kd value obtained by nonlinear least-squares analysis may also be considered a criterion for AME. The observed
Kd value in this
study (0.47 µM) (Fig. 3) is comparable to those for substrates
reported to be taken up via the AME mechanism in brain capillary
endothelial cells, including cationized BSA (0.8 µM) (6), histone
(15.2 µM) (14), E-2078 (4.62 µM) (23), and ebiratide (15.9 µM)
(24). In contrast, the
Kd values for RME
reported for insulin (2.3 nM) (2), transferrin (5.6 nM) (13), and
atrial natriuretic factor (0.4 nM) (20) and that for receptor binding
of epidermal growth factor (0.35 nM) (8) are several thousand times
smaller than those for AME. These data indicate that 001-C8 is taken up
into Caco-2 cells via the AME mechanism.
The internalization of 125I-001-C8
was significantly decreased by predigestion of the apical membrane of
the Caco-2 cells with heparinase I, heparitinase, or chondroitinase ABC
(Table 2). Excess heparin also decreased the internalization. These
results suggest that the anionic site(s) on the apical membrane
involved in the internalization of 001-C8 is one of a variety of acid
mucopolysaccharides, including heparin, heparan sulfate, and
chondroitin sulfate, in the endocytosis of 001-C8 by Caco-2 cells.
Apparently incomplete inhibition by enzyme treatments may be ascribed
to the incomplete digestion by enzymes and/or an involvement of
multiple anionic components as the target for AME.
Partial identification of endocytotic granules was performed by double
staining of the cells with tetramethylrhodamine-dextran as a marker for
secondary lysosomes (Fig. 5). It was revealed that a part of the NBD
fluorescence was colocalized with rhodamine-dextran, suggesting a minor
contribution of lysosomes to this pathway.
In conclusion, the present study demonstrates that a basic peptide,
001-C8, is taken up by enterocyte-like Caco-2 cells via the AME system.
Because of its stability in the cell, 001-C8 may be passed into the
systemic circulation via adsorptive-mediated transcytosis (AMT) in
vivo. Our preliminary data (unpublished observations) using
filter-grown Caco-2 monolayers and in situ vascular perfusion of the
intestine indicate the existence of such an AMT mechanism.
 |
ACKNOWLEDGEMENTS |
This research was supported in part by a Grant-in-Aid for
Scientific Research from the Ministry of Education, Science, and Culture, Japan, and by a grant from the Japan Health Sciences Foundation, Drug Innovation Project.
 |
FOOTNOTES |
Address for reprint requests: A. Tsuji, Faculty of Pharmaceutical
Sciences, Kanazawa Univ., 13-1 Takara-machi, Kanazawa 920-0934, Japan.
Received 14 July 1997; accepted in final form 23 April
1998.
 |
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