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Transepithelial Transport of Oligopeptides in the Human Intestinal Cell, Caco-2
Peptides, Vol. 18, No. 5, pp. 681–687, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/97 $17.00 / .00
PII S0196-9781(97)00002-8
Transepithelial Transport of Oligopeptides in the Human Intestinal Cell, Caco-2 MAKOTO SHIMIZU, 1 MAKI TSUNOGAI AND SOICHI ARAI Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113, Japan Received 9 September 1996; Accepted 24 January 1997 SHIMIZU, M., M. TSUNOGAI AND S. ARAI. Transepithelial transport of oligopeptides in the human intestinal cell, Caco-2. PEPTIDES 18(5) 681–687, 1997.—The transepithelial transport of oligopeptides (of more than 4 residues) was studied by using human intestinal Caco-2 cell monolyers. The susceptibility to the brush-border peptidases was observed to be one of the primary factors which decide the transport rate. The apical-to-basolateral transport mechanism was investigated by using bradykinin and GGYR which were resistant to cellular peptidases. The intracellular pathway, probably the adsorptive transcytosis, was suggested to be involved in the transport of bradykinin and its analogues, the transport rate being particularly dependent on the hydrophobic properties of the peptides. On the other hand, the tetrapeptide such as GGYR was suggested to be transported mainly via the paracellular pathway. q 1997 Elsevier Science Inc. Bradykinin
Caco-2
Intestinal epithelial cell
Oligopeptide
IN the past two decades, oligopeptides with such diverse physiological functions as opiatic (5,29), hypertensive (2), immunomodulatory (34) and anti-coagulative (12) have been discovered in the hydrolysates of several food proteins. These oligopeptides are thought to be produced in the digestive tract by limited hydrolysis of dietary proteins and are expected to express their physiological functions in the body. Some of the peptides have already been reported to be effective in vivo after oral administration; modulating the endocrine systems in dogs (42,43) and lowering the blood pressure of spontaneous hypertensive rats (27,33,46) are examples. These findings suggest that some of the physiologically active oligopeptides can be absorbed by the body across the intestinal epithelium, while retaining their intact structure and activity. The results of intensive studies, using electrophysiological methods, during the 1970s and 1980s have suggested the existence of a peptide transport system in the intestinal epithelium, by which peptides would be actively transported through the apical membrane under an H / gradient (14,24). However, the electrophysiological measurements strongly suggested that this transport system carried only di- or tripeptides (10). Very recently, the peptide transporter protein (PepT1) has been cloned from the rabbit intestine (11). The results of an experiment using xenopus oocyte transfected with PepT1 cRNA confirmed the specificity of this transporter for di- and tripeptides. Oligopeptides with more than 4 residues are hardly, if at all, recognized by this transport system. The absorption of such larger oligopeptides must therefore be performed by another mechanism. It is possible that these peptides may be absorbed via the transcytotic route which is known to be used for the transport of macromolecules such as
Transcytosis
Paracellular transport
proteins (20). Burton and his coworkers (6,8,28) suggested the intracellular passive transport to be another mechanism for the peptide absorption. The contribution of the paracellular route to peptide absorption (1,9,36), may also be possible. However, the role of these pathways in intestinal oligopeptide absorption has not yet been fully understood, the mechanism for oligopeptide transport across the intestinal epithelium being still obscure. Caco-2 cells derived from a human intestinal adenocarcinoma have recently been found to provide a useful cell culture model of the small intestinal epithelium. The Caco-2 cell line exhibits spontaneous enterocyte-like differentiation under standard culture conditions, showing morphological polarity and expressing brush-border hydrolases (22). In the past decade, transportermediated transport of nutrients such as hexoses (40), amino acids (23,35) and di- and tripeptides (41) has been investigated by using this cell line. The polarized transcytotic activity in Caco-2 cells has also been reported (19,21). The formation of a tight junction (22) and paracellular transport regulated by tight junctions have been observed by using this cell line (4,16–18). Caco-2 is, therefore, thought to provide a good model for investigating the mechanism for intestinal oligopeptide absorption at a cellular level. For example, the permeability characteristics of a series of peptides have been investigated by Burton et al. (1,6– 9,28) using this cell line. They found that the hydrogen-bonding capacity was the major contributing factor for the transport of peptides, by using chemically modified peptides of less than 4 amino acid residues. In the present study, the mechanism for peptide transport through the intestinal epithelium was investigated by using the
1 Requests for reprints should be addressed to M. Shimizu, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan, E-mail: [email protected]
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Caco-2 monolayer cultured on a permeable filter. Oligopeptides with 4–9 amino acid residues (native peptides without chemical modification) were used as model peptides, and the structural factors of peptides which influence the absorption rate are discussed. METHOD
Materials The human colon adenocarcinoma cell line, Caco-2, was obtained from American Type Culture Collection (Rockville, MD, USA). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Nissui Pharmacies (Tokyo, Japan). Fetal calf serum (FCS), glutamine, non-essential amino acids (NEAA), penicillin-streptomycin (10000 units/ml and 10 mg/ml in 0.9% sodium chloride, respectively), phosphate-buffered saline (PBS) and Hank’s balanced salt solutions (HBSS) were all purchased from Gibco (Gaithersburg, MD, USA). Collagen type I was purchased from Nitta Zelatin (Osaka, Japan). Plastic dishes and plates were from Corning (Corning, NY, USA), and Millicell-HA with 0.45 mm cellulose membranes of 12 or 30 mm in diameter was purchased from Millipore (Molsheim, France). Lucifer yellow CH lithium salt (LY) was from Molecular Probes (Eugine, OR, USA). Fluorescein isothiocyanate (FITC)-labeled dextran with an average molecular weight of 40000 (DX-40K), bradykinin and the bradykinin B1-receptor antagonist, Des-Arg 9-[Leu 8 ]bradykinin, and a tetrapeptide, PFGK, were all from Sigma (St. Louis, MO, USA). Papain inhibitor GGYR, b-Casomorphin-5, the bradykinin B2-receptor antagonist, D-Arg-[Hyp 3 , Thi 5,8 , DPhe 7 ]-bradykinin, and Diprotin A were purchased from Peptide Institute (Osaka, Japan). Ovokinin and the bradykinin analogs were synthesized by Anaspec (USA) and purified by using a reverse-phase YMC Dispo SPE C18 column (YMC, Japan) or by gel filtration in a PD-10 Sephadex G-25M column (Pharmacia, Upsala, Sweden) before use. All other chemicals were of reagent grade. Cell Culture Caco-2 cells were cultured in DMEM supplemented with 10% FCS, 1% NEAA, 2% glutamine, 100 U/ml of penicillin and 100 mg/ml of streptomycin, together with an appropriate amount of sodium bicarbonate. They were incubated at 377C in a humidified atmosphere of 5% CO2 in air. The monolayer became confluent 3 to 4 days after seeding at 7 1 10 5 cells per 60-mm dish, and the cells were passaged at a split ratio of 4 to 8 by trypsinizing with 0.1% trypsin and 0.02% EDTA in PBS. All the cells used in this study were between passages 38 and 70. Cells for the transport studies were grown in a Millicell-HA insert with a membrane coated with type I collagen. The cells were seeded at a density of 2 1 10 5 cells/ml of the medium, and the medium was changed every 1 or 2 days. Collagen coating was performed as described previously (16). The integrity of the cell layer was evaluated by measuring the transepithelial electrical resistance (TEER) with Millicell-ERS equipment (Millipore). The monolayer cells cultured for 14–15 days and with the TEER values of higher than 200 ohm cm2 were used for the transport experiment. Transport Studies The cell monolayers grown in the Millicell insert (30 mm in diameter) were gently rinsed with HBSS, 2 ml of HBSS then being added to the apical side of the Millicell insert, and the insert placed in a well containing 2 ml of HBSS. After incubating for 30 min at a constant temperature of 377C, 200 ml of HBSS in
either the apical or basolateral side of the cell monolayer was replaced by the same volume of a peptide solution. After further incubating for an appropriate time, aliquots of the apical and basolateral solutions were taken, and the peptide concentration in each was determined by high-performance liquid chromatography (HPLC). Transport of the leakage markers, LY and DX40K, was performed in the same manner, except that determination was carried out by measuring the fluorescence intensity. Since the transport activity of the cells varied considerably among different series of experiments, probably because of the differing passage of cells or differing lots of the culture medium, the transport rate (flux) of the sample peptides was usually standardized by comparing with that of bradykinin coexisting in the sample solution. Treatment of Cells with Inhibitors After washing and preincubating the cell monolayer with HBSS, inhibitor solutions were added to give final concentrations of 0.1–1 mM (Diprotin A), 25 mM (sodium azide), 0.5 mM (2,4-dinitrophenol), 0.1–1 mM (colchicine) or 0.5 mg/ml (cytochalasin D), the monolayers then being incubated for 30 min. Transport experiments were carried out in the presence of the same concentrations of the inhibitors. High-Performance Liquid Chromatography (HPLC) A Shimadzu LC-6A liquid chromatograph (Kyoto, Japan) equipped with a C18 column (4.6 1 150 mm; ODS-H-1151, Senshu-Kagaku, Tokyo, Japan) and a Shimadzu SPD-6AV ultraviolet spectrophotometer at a wavelength of 210 nm were used. Peptides were applied to the column that had been equilibrated with 0.1% (v/v) trifluoroacetic acid and were eluted with a linear gradient of acetonitrile at a flow rate of 1.0 ml/min. The peptides were determined by measuring the peak areas. Fluorescence Measurement Fluorescence was measured by a FP777 fluorescence spectrophotometer (Jasco; Tokyo, Japan). The excitation and emission wavelengths for determining LY were 430 nm and 540 nm, respectively, and those for DX-40K were 287 nm and 518 nm. Statistical Analyses All results are expressed as the mean { SE. Student’s t-test (paired or unpaired) was used to compare means and ranges. RESULTS
Transport Rate Comparison for Different Oligopeptides The apical-to-basolateral flux across the Caco-2 monolayer was measured by using five peptides with different size and structure, namely bradykinin (RPPGFSPFR), b-casomorphin (YPFPG), ovokinin (FRADHPFL), PFGK and GGYR, and the results are shown in Fig. 1. Although the concentration of each peptide added to the apical solution was constant (400 mg/ml), the concentration of the intact peptides in the basolateral solution after a 30-min incubation was different. The transport rate was greatest for GGYR and smallest for b-casomorphin. The TEER value of the monolayer was not significantly changed by incubating with these peptides under the present experimental conditions. The concentration of each peptide in the apical solution after 60 min of incubation with the Caco-2 cell monolayer grown in a plastic dish are shown in Fig. 2. The concentration of bradykinin in the apical solution remained unchanged, while those of
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TRANSEPITHELIAL TRANSPORT OF PEPTIDES
FIG. 1. Apical-to-basolateral flux of various peptides across the Caco-2 monolayer. Bradykinin was present with one of the other peptides in an apical solution and the flux of each peptide is expressed as relative to the bradykinin value (means { SE). The number of epithelial cell layers used for the experiments was 6 (for b-casomorphin), 2 (ovokinin), 8 (PFGK) or 12 (GGYR). The paired t-test was used to evaluate the difference in fluxes between bradykinin and the other peptides. *Significantly different from the bradykinin flux (p õ 0.05); ** p õ 0.01.
ovokinin and b-casomorphin were decreased to a great extent, suggesting that these peptides were hydrolyzed quickly by the brush border peptidases. Since b-casomorphin was most rapidly cleaved at the peptide bond between P and F, and Caco-2 expressed dipeptidyl aminopeptidase IV (DPP-IV) as the major peptidase (manuscript in preparation), the contribution of DPPIV to the hydrolysis of b-casomorphin was suggested. The flux of b-casomorphin was increased by treating the cell layer with 1 mM Diprotin A, an inhibitor of dipeptidyl peptidase IV (Fig. 3). The differing degree of flux among the peptides (Fig. 1) would have been, at least partly, due to the different susceptibility of the peptides to cellular peptidases such as DPP-IV.
683
FIG. 2. Concentrations of intact peptides in the apical solutions after 60 min of incubation with the Caco-2 monolayer. The initial peptide concentration was 400 mg/ml, and results are presented as means { SE (3 epithelial layers).
route (17,22), was not significantly different between AP-BL and BL-AP (Fig. 5A), suggesting that the transport of bradykinin was not a simple diffusion process via the paracellular route. Effect of inhibitors on the transport of bradykinin. The APBL flux of bradykinin was decreased in the presence of such metabolic inhibitors as sodium azide and 2,4-dinitrophenol (Fig. 6), indicating that the transport incorporated an energy-dependent process. The flux was also partially inhibited by colchicine, an inhibitor of intracellular vesicle transport through perturbation of the microtubule structure (Fig. 6). The paracellular flux of
Transepithelial Transport of Bradykinin Effect of time and concentration on the transport rate of bradykinin. Bradykinin, which is resistant to hydrolysis by cellular peptidases (Fig. 2), was used as a model peptide to reveal the mechanism for oligopeptide transport through the intestinal epithelial cell layer. The transport rate was observed to be constant at least for 60 min (data not shown); therefore, incubation was carried out for 60 min in the subsequent experiments. The transport of bradykinin was concentration-dependent, but a saturable process was not apparent when the peptide concentration in the apical solution was less than 1.2 mg/ml (Fig. 4). Considering the sensitivity of HPLC for detecting peptides transported to the basolateral solution, 400 mg/ml was employed as the apical peptide concentration in this study unless otherwise stated. Apical-to-basolateral and basolateral-to-apical transport of bradykinin. The degrees of apical-to-basolateral (AP-BL) and basolateral-to-apical (BL-AP) flux of bradykinin were compared. As shown in Fig. 5A, the AP-BL flux was much higher than the BL-AP flux. In contrast, the flux of fluorescence reagent LY, which has been used as a leakage marker for the paracellular
FIG. 3. Effect of Diprotin A on the apical-to-basolateral flux of b-casomorphin. Diprotin A was added to both the apical and basal solutions, and the cell monolayer was incubated for 30 min before measuring the flux. The flux is expressed as the relative value to that in the control cell monolayer (without Diprotin A). The results are means { SE (4 epithelial layers for the control and 0.1 mM Diprotin A, and 3 epithelial layers for 1 mM Diprotin A). *Significantly different from the control value (p õ 0.005).
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FIG. 4. Concentration dependence of the apical-to-basolateral flux of bradykinin. Bradykinin was added to the apical solution, which was then incubated for 60 min. The results are expressed as means { SE (3 epithelial layers).
small molecular substances is known to be increased by treating the cell layer with cytochalasin D, an agent which opens tight junction by altering the cytoskeletal structure (30). The cytochalasin D treatment did not affect the flux of bradykinin, while that of Lucifer Yellow was increased (Fig. 7). These results suggest that the transcytotic process, not the paracellular process is mainly involved in the AP-BL transport of bradykinin. Transcytotic processes for bradykinin. A high-molecularweight dextran conjugated with a fluorescence reagent has been found to be useful for estimating fluid-phase transcytosis in the intestinal epithelial cell layer (22). The degrees of flux of DX-
FIG. 5. Comparative directional transport between bradykinin and Lucifer Yellow (A), Dextran-40K (B) and GGYR (C). The initial concentration of each peptide and dextran was 400 mg/ml, whereas that of Lucifer Yellow was 100 mg/ml. The basolateral-to-apical flux is expressed as relative to the apical-to-basolateral flux, the results being expressed as means { SE (3–7 epithelial layers for A, 6–8 layers for B, and 9–10 layers for C). *Significantly different from the apical-to-basolateral flux, p õ 0.005; **p õ 0.001.
FIG. 6. Effect of inhibitors on the apical-to-basolateral flux of bradykinin. Sodium azide (25 mM), 2,4-dinitrophenol (0.5 mM) or cholchicine (1 mM) was added to both the apical and basal solutions, which were then incubated for 30 min before measuring the flux. The results are expressed as means { SE (3 epithelial layers). *Significantly different from the control value (without an inhibitor), p õ 0.01; **p õ 0.001.
40K were therefore measured to evaluate the fluid-phase transcytotic activity of the Caco-2 cell monolayer. The AP-BL flux of DX-40K was higher than the BL-AP flux (Fig. 5B), suggesting that the fluid-phase transcytosis in the Caco-2 monolayer was more active in the AP-BL process than in the BL-AP process. The AP-BL flux of bradykinin was, however, much higher than that for DX-40K (p õ 0.01), although the degrees of BL-AP flux of these two compounds were not significantly different (Table 1). These results suggest that the AP-BL transport of bradykinin was performed not only by simple fluid-phase transcytosis, but also by some other mechanisms. Relationship between the transport rate and structure of peptides. In order to reveal the relationship between the flux via transcytosis and the peptide structure, some peptides having an analogous
FIG. 7. Effect of cytochalasin D on the apical-to-basolateral flux of bradykinin and Lucifer Yellow. Cytochalasin D (0.5 mg/ml) was added to both the apical and basal solutions, which were then incubated for 30 min before measuring the flux. TEER was decreased to 68% of the initial value (280 ohm cm2 ) after 30-min incubation. The results are expressed as means { SE (3 epithelial layers). *Significantly different from the control value (without cytochalasin D) p õ 0.01.
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TRANSEPITHELIAL TRANSPORT OF PEPTIDES TABLE 1 APICAL-TO-BASOLATERAL AND BASOLATERAL-TO-APICAL FLUX FOR BRADYKININ, DEXTRAN-40K AND GGYR ACROSS THE CACO-2 CELL MONOLAYER
Sample
Apical-to-Basal Flux (ng/h/cm2) (n Å 4)
Basal-to-Apical Flux (ng/h/cm2) (n Å 3)
Bradykinin Dex-40K GGYR
346.5 { 67.9*† 140.2 { 23.4* 550.6 { 97.6
93.0 { 27.0†‡ 70.8 { 2.6 272.2 { 35.2‡
Bradykinin, FITC-dextran-40K or GGYR (400 mg/ml each) was mixed and added to either the apical or basolateral solution. After 60 min of incubation, each solution on the opposite side was recovered, and the concentrations of the peptides and dextran were evaluated by HPLC and fluorescence measurement, respectively. Results are expressed as means { SE. *Significantly different from each other, p õ 0.01; † p õ 0.05; ‡ p õ 0.05.
structure to that of bradykinin were synthesized and their transport was examined. As shown in Fig. 8, the degree of flux was markedly dependent on the peptide structure. The number of the positively charged amino acid (arginine) was negatively correlated with the flux (r Å 0.85). On the other hand, the hydrophobicity of the peptides, which was estimated from the retention time by reverse-phase HPLC (Table 2), was positively correlated with the flux (r Å 0.83), suggesting that such peptides as bradykinin were adsorbed to the apical cell membrane surface mainly through hydrophobic interaction, by which the peptides were efficiently internalized by the cells and transported across the cell layer.
685 border peptidases is the rate-limiting step for the transepithelial transport of oligopeptides. A similar observation concerning the transepithelial passage of intact b-casomorphins has been reported by using the rabbit ileum in vitro (32,45). Since the susceptibility of oligopeptides to peptidases makes it difficult to measure the real transport rate for an intact oligopeptide, bradykinin and GGYR, which were found to be resistant to cellular peptidases (Fig. 2), were chosen as the model peptides for further analyses. Bradykinin is a physiologically active peptide derived from blood serum kininogen, and is known to express a variety of functions such as arterial relaxation and smooth muscle contraction through binding to its specific receptors, B1 and B2 (39). The receptors are known to be present in various types of cells including smooth muscle and endothelial cells (39). The presence of bradykinin receptors in the colonic epithelial cells has been also reported (38). Although there is no report on bradykinin receptor B1 or B2 being present in the Caco-2 cell surface, the possibility that bradykinin transport across the Caco-2 cell layer was influenced by the receptors having been expressed on the Caco-2 cells cannot be ruled out. In order to eliminate such a possibility, we examined bradykinin transport in the presence of the B1 antagonist, [des-Arg 9 ]-bradykinin, or B2 antagonist, [Thi 5,8 ,D-Phe 7 ]-bradykinin. The bradykinin flux was not affected by these antagonists (data not shown). Microscopic observation of the cell layer and measurement of changes in the transepithelial short-circuit current after adding bradykinin also suggest that bradykinin did not exert any physiological effect on the Caco-2 cells used in this experiment (data not shown). Although the bradykinin concentrations used in this study were at least 10000 fold higher than the Kd values of bradykinin recep-
Epithelial Transport of the Tetrapeptide, GGYR The AP-BL flux of GGYR was 1.6 times that of bradykinin (Table 1). The transport of GGYR was not significantly inhibited by treating with 2,4-dinitrophenol and cholchicine (data not shown), suggesting that the major mechanism for GGYR transport did not involve an energy-dependent or cytoskeletal-dependent process. The BL-AP flux of GGYR was also much higher than that of bradykinin (p õ 0.05), the ratio of (AP-BL)/(BLAP) flux being lower than that of bradykinin (Fig. 5C). Increased flux of GGYR was also observed by treating the cell layer with cytochalasin D (data not shown). These results suggest the major transport mechanism to be different between bradykinin (9 residues) and GGYR (4 residues), the latter peptide being transported via the paracellular pathway to a large extent. DISCUSSION
The results of this study suggest that digestion by cellular peptidases is the primary factor affecting intact peptide flux across the epithelial cell layer. It has been demonstrated that Caco-2 expressed at least 8 aminopeptidases (25). The expression of the three major aminopeptidases, namely aminopeptidases N and P and dipeptidylaminopeptidase (DPP)-IV, was also observed in our study, DPP-IV having the highest activity among the three (data not shown). During the incubation of b-casomorphin-5 (YPFPG) with Caco-2 cells, N-terminal dipeptide YP was produced at the highest rate. This hydrolysis pattern was similar between the peptide incubated with intact cells and that with the cell lysate, suggesting that cell-surface DPP-IV was most responsible for the hydrolysis of b-casomorphin-5 (manuscript in preparation). The increased flux of b-casomorphin-5 by treating the cell layer with the DPP-IV inhibitor, Diprotin A (Fig. 3), strongly suggests that the hydrolysis of peptides by brush-
FIG. 8. Apical-to-basolateral flux of bradykinin and its analogous peptides (I–V). Bradykinin was present with each other peptide in an apical solution, the flux of peptides I–V being expressed as relative to the bradykinin flux. The results are expressed as means { SE (6–10 epithelial layers). The paired t-test was used to evaluate the difference in flux between bradykinin and each other peptide. *Significantly higher than the bradykinin flux, p õ 0.01; ** p õ 0.001.
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SHIMIZU, TSUNOGAI AND ARAI TABLE 2 RETENTION TIMES FOR BRADYKININ AND ITS ANALOGOUS PEPTIDES IN AN ODS COLUMN Peptide
Retention Time (min)
Bradykinin Peptide I Peptide II Peptide III Peptide IV Peptide V
17.55 23.15 25.90 21.00 8.80 18.45
Each peptide was applied to an ODS column equilibrated with 0.1% trifluoroacetic acid (pH 2.0) and then eluted by linearly increasing the concentration of acetonitrile. The retention time was used to evaluate the hydrophobicity of the peptides (45).
tors (39), the transport was not saturable, being increased by increasing the apical concentration. These results suggest that bradykinin transport observed in this study was not significantly affected by a receptor-mediated process, even if the receptors had been expressed on the surface of the Caco-2 cells. The results of the present study suggest that the energy-dependent transcytotic pathway is involved in the transport of such oligopeptides as bradykinin. Pappenheimer et al. (36), from the results of oral administration tests on rats, have suggested that a D-octapeptide (EASASYSA) was transported paracellularly. However, it is noteworthy that their peptide was administered with a high content of glucose, which had been reported to dilate the tight junction, inducing solvent drag through the absorptive cell junctions (31). Adson et al. (1) indicated that the paracellular diffusion rate was strongly dependent on molecular size, the paracellular flux of a tetrapeptide ( D-Phe)3-Gly being restricted to much higher extent than that of D-Phe-Gly. Hu et al. (26) reported that the permeability of GHRP-6, a hexapeptide-like drug, was not significantly increased after dilation of the tight junction by luminal glucose. Perturbation of the tight junction by cytochalasin D resulted in an increase in the Lucifer Yellow flux by increasing the pore radius, whereas the bradykinin flux was not affected by this treatment (Fig. 7). Under normal condition, transcytosis would be the most possible pathway, at least for 9-residue peptides such as bradykinin. Transcytosis is a transport system via internalized vesicles carrying specifically bound ligands (receptor-mediated transcytosis) or non-specifically adsorbed ligands (adsorptive transcytosis) or fluids (fluid-phase transcytosis) from their sites of entry to sites on the opposite side of the cell (13,44). The transport of albumin, transferrin and insulin in endothelial cells and that of IgA and transferrin in epithelial cells is carried out by the receptor-mediated process (13,44). Since receptor-mediated transcytosis of bradykinin is not likely to have occurred as already described, bradykinin transport would seem to be performed by fluid-phase or adsorptive transcytosis. Fluid-phase transcytosis is a process started by the uptake of extracellular fluid. The transport rate is therefore dependent on the concentration of the molecules in the extracellular fluid and
the rate of internalization (44). The significantly higher flux of bradykinin than that of DX-40K, a marker for fluid-phase transcytosis (Table 1), suggests that bradykinin transport was not simply carried out by fluid-phase transcytosis. Adsorptive transcytosis requires the interaction of solutes (molecules to be transported) with the cell membrane surface, by which the molecules are concentrated at the surface and efficiently internalized, although the mechanism involved in the internalization of cell membranes is still not clear. It was reported that molecules with a pI value between 4.5 and 7.0 did not bind to the plasma membrane (43). Positively charged macromolecules have high affinity toward a cell membrane whose surface possesses a net negative charge due to an abundance of carbohydrate such as sialic acid (37). Since bradykinin is a positively charged peptides with 2 arginine residues under neutral conditions, these two arginine residues are considered to have taken part in the adsorption of bradykinin to the cell surface, facilitating adsorptive transcytosis. Unexpectedly, the results of experiments using several bradykinin analogues of different arginine content (Fig. 8) demonstrate that the number of arginine residues was rather negatively correlated with the flux. Since the removal of N- and/or C-terminal arginine residues would make the molecule smaller (peptides I–III), the change in degree of flux may be attributable only to a change in molecular size. However, a recent experiment comparing RPPGFSPFL and acetyl-LPPGFSPFL has demonstrated that replacing N-terminal R with acetyl L increased the flux by the ratio of 1.78 (SE Å 0.17, n Å 4). Considering this observation, as well as the fact that the hydrophobicity of a peptide clearly showed positive correlation with the flux, the increased flux by decreasing the number of arginine residues could have been responsible for the accompanying increase in hydrophobicity. In any event, the present results suggest adsorptive transcytosis depending on the hydrophobicity of peptides to be the major transport mechanism, although it should be investigated whether the aggregation of hydrophobic peptides is also the factor that can influence the transport of these oligopeptides. Furthermore, the possibility that the chemically-modified, lesspolar bradykinin analogues were, at least partly, transported via the intracellular passive pathway (6–8,28) can not be ruled out. In contrast, tetrapeptide GGYR showed different transport properties. The paracellular mechanism for transport of this peptide was strongly suggested, although there is no information on whether the rate-limiting factor for paracellular transport is only the molecular size. The effect of amino acid sequence and of other properties of the peptide on diffusion through the cell junctions is not yet known. Three different transport routes, namely paracellular, fluidphase and adsorptive transcytosis, may participate in oligopeptide transport across the intestinal epithelium. The contribution of each route must be different among the peptides, depending on the molecular size and other structural properties such as hydrophobicity. The major pathway for bradykinin (molecular weight of 1060) would seem to be adsorptive transcytosis, whereas paracellular diffusion would play the major role in the transport of GGYR (molecular weight of 450). Complex features of intestinal oligopeptide transport, depending on the size and structure of a peptide, are thus indicated. ACKNOWLEDGEMENT
We are grateful to Mr. M. Satake for his skillful technical assistance.
REFERENCES 1. Adson, A.; Raub, T. J.; Burton, P. S.; Barsuhn, C. L.; Hilgers, A. R.; Audus, K. L.; Ho, N. F. H. Quantitative approaches to delineate paracel-
lular diffusion in cultured epithelial cell monolayers. J. Pharm. Sci. 83:1529–1536; 1994.
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TRANSEPITHELIAL TRANSPORT OF PEPTIDES 2. Ariyoshi, Y. Angiotensin-converting enzyme inhibitors derived from food proteins. Trends Food Sci. Technol. 4:139–144; 1993. 3. Baldwin, A. L.; Chien, S. Endothelial transport of anionized and cationized ferritin in rabbit thoracic aorta and vasa vasorum. Arteriosclerosis 4:372–382; 1984. 4. Boulenc, X.; Marti, E.; Joyeux, H.; Roques, C.; Berger, Y.; Fabre, G. Importance of the paracellular pathway for the transport of a new bisphosphate using the human Caco-2 monolayer model. Biochem. Pharmacol. 46:1591–1600; 1993. 5. Brantl, V.; Teschemacher, H.; Henschen, A.; Lottspeich, F. Novel opioid peptides derived from casein ( b-casomorphin). Hoppe–Seyler’s Z. Physiol. Chem. 360:1211–1216; 1979. 6. Burton, P. S.; Conradi, R. A.; Hilgers, A. R.; Ho, N. F. H.; Maggiora, L. L. The relationship between peptide structure and transport across epithelial cell monolayers. J. Control. Release 19:87–98; 1992. 7. Burton, P. S.; Conradi, R. A.; Hilgers, A. R.; Ho, N. F. H. Evidence for a polarized efflux system for peptides in the apical membrane of Caco-2 cells. Biochem. Biophys. Res. Commun. 190:760–766; 1993. 8. Conradi, R. A.; Hilgers, A. R.; Ho, N. F. H.; Burton, P. S. The influence of peptide structure on transport across Caco-2 cells. II. Peptide bond modification which results in improved permeability. Pharm. Res. 9:435–439; 1992. 9. Conradi, R. A.; Wilkinson, K. F.; Rush, B. D.; Hilgers, A. R.; Ruwart, M. J.; Burton, P. S. In vitro/in vivo models for peptide oral absorption: Comparison of Caco-2 cell permeability with rat intestinal absorption of renin inhibitory peptides. Pharm. Res. 10:1790–1792; 1993. 10. Daniel, H.; Morse, E. L.; Adibi, S. A. Determinants of substrate affinity for the oligopeptide/H / symporter in the renal brush border membrane. J. Biol. Chem. 267:9565–9573; 1992. 11. Fei, Y.-J.; Kanai, Y.; Nussberger, S.; Ganapathy, V.; Leibach, F.; Romero, M. F.; Singh, S. K.; Boron, W. F.; Hediger, M. A. Expression cloning of a mammalian proton-coupled oligopeptide transporter. Nature 368:563–566; 1994. 12. Fiat, A.-M.; Levy-Toledano, S.; Caen, J. P.; Jolles, P. Biologically active peptides of casein and lactotransferrin implicated in platelet function. J. Dairy Res. 56:351–355; 1989. 13. Gardner, M. L. Gastrointestinal absorption of intact proteins. Ann. Rev. Nutr. 8:329–350; 1988. 14. Ganapathy, V.; Leibach, F. H. Is intestinal peptide transport energized by a proton gradient? Am. J. Physiol. 249:G153–G160; 1985. 15. Hancock, W. S.; Sparrow, J. T. HPLC Analysis of Biological Compounds. New York: Marcel Dekker Inc.; 1984. 16. Hashimoto, K.; Shimizu, M. Epithelial properties of human intestinal Caco-2 cells cultured in serum-free medium. Cytotechnology 13:175– 184; 1993. 17. Hashimoto, K.; Matsunaga, N.; Shimizu, M. Effect of vegetable extracts on the transepithelial permeability of the human intestinal Caco-2 cell monolayer. Biosci. Biotech. Biochem. 58:1345–1346; 1994. 18. Hashimoto, K.; Takeda, K.; Nakayama, T.; Shimizu, M. Stabilization of tight junction of intestinal Caco-2 cell monolayer by milk whey proteins. Biosci. Biotech. Biochem. 58:1951–1952; 1995. 19. Hassan, I. F.; Parsons, I. J.; Mackay, M. W. Receptor-mediated transport of cobalamin in Caco-2 cells: Intracellular localization of transcobalamin II. In: Wilson, G.; Davis, S. S.; Illum, L.; Zweibaum, A. eds. Parmaceutical Application of Cell and Tissue Culture to Drug Transport. New York: Plenum Press; 1991:107–120. 20. Heyman, M.; Desjeux, J. F. Significance of intestinal food protein transport. J. Pediat. Gastroenterol. Nutr. 15:48–57; 1992. 21. Heyman, M.; Crain-Denoyelle, A.-M.; Nath, S. K.; Desjeux, J. F. Quantification of protein transcytosis in the human colon carcinoma cell line Caco-2. J. Cellular Physiol. 143:391–395; 1990. 22. Hidalgo, I. J.; Raub, T. J.; Borchardt, R. T. Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology 96:736–749; 1989. 23. Hidalgo, I. J.; Borchardt, R. T. Transport of a large neutral amino acid (phenylalanine) in a human intestinal epithelial cell line: Caco2. Biochim. Biophys. Acta 1028:25–30; 1990. 24. Hoshi, T. Proton-coupled transport of dipeptides across renal and intestinal brush border membranes. In: Alvarado, F.; Van Os, C. H.,
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eds. Ion Gradient-Coupled Transport. Elsevier Science Pub. Co.: New York; 1986:183–191. Howell, S.; Kenny, A. J.; Turner, A. J. A survey of membrane peptidases in two human colonic cell lines, Caco-2 and HT-29. Biochem. J. 284:595–601; 1992. Hu, Z.; Tse, E. G. C.; Monkhouse, D. C.; Oh, C. K.; Fleisher, D. The intestinal uptake of ‘‘enzymatically-stable’’ peptide drugs in rats as influenced by D-glucose in situ. Life Sci. 54:1977–1985; 1994. Karaki, H.; Doi, K.; Sugano, S.; Uchiwa, H.; Sugai, R.; Murakami, U.; Takemoto, S. Antihypertensive effect of tryptic hydrolyzate of milk casein in spontaneously hypertensive rats. Comp. Biochem. Physiol. 96C:367–371; 1990. Kim, D.-C.; Burton, P. S.; Borchardt, R. T. A correlation between the permeability characteristics of a series of peptides using an in vitro cell culture model (Caco-2) and those using an in situ perfused rat ileum model of the intestinal mucosa. Pharm. Res. 10:1710–1714; 1993. Loukas, S.; Varoucha, D.; Zioudrou, C.; Streaty, R. A.; Klee, W. A. Opioid activities and structure of a-casein-derived exorphins. Biochemistry 22:4567–4573; 1983. Madara, J. L.; Barenberg, D.; Carlson, S. Effects of cytochalasin D on occulding junctions of intestinal absorptive cells: Further evidence that the cytoskeleton may influence paracellular permeability and junctional charge selectivity. J. Cell Biol. 102:2125–2136; 1986. Madara, J. L.; Pappenheimer, J. R. Structural basis for physiological regulation of paracellular pathways in intestinal epithelia. J. Membr. Biol. 100:149–164; 1987. Mahe, S.; Tome, D.; Dumontier, A.-M.; Desjeux, J. F. Absorption of intact b-casomorphin ( b-CM) in rabbit ileum in vitro. Reprod. Nutr. Develop. 29:725–733; 1989. Maruyama, S.; Miyoshi, S.; Kaneko, T.; Tanaka, H. Angiotensin Iconverting enzyme inhibitory activities of synthetic peptides related to the tandem repeated sequence of a maize endosperm protein. Agric. Biol. Chem. 53:1077–1081; 1989. Migliore-Samour, D.; Jolles, P. Casein, a prohormone with an immunomodulating role for the newborn? Experientia 44:188–193; 1988. Pan, M.; Malandro, A.; Stevens, B. R. Regulation of system y / arginine transport capacity in differentiating human intestinal Caco2 cells. Am. J. Physiol. 268:G578–G585; 1995. Pappenheimer, J. R.; Dahl, C. E.; Karnovsky, M. L.; Maggio, J. E. Intestinal absorption and excretion of octapeptides composed of Damino acids. Proc. Natl. Acad. Sci. USA 91:1942–1945; 1994. Persiani, S.; Shen, W. C. Increase of poly( L-lysine) uptake but not fluid phase endocytosis in neuraminidase pretreated Madin–Darby canine kidney (MDCK) cells. Life Sci. 45:2605–2610; 1989. Rangachari, P. K.; Berezin, M.; Prior, T. Effects of bradykinin on the canine proximal colon. Regul. Pept. 46:511–522; 1993. Regoli, D.; Gobeil, F.; Nguyen, Q. T.; Jukic, D.; Seoane, P. R.; Salvino, J. M.; Sawutz, D. G. Bradykinin receptor types and B2 subtypes. Life Sci. 55:735–749; 1994. Riley, S. A.; Warhurst, G.; Crowe, P. T.; Turnberg, L. A. Active hexose transport across cultured human Caco-2 cells: Characterization and influence of culture conditions. Biochim. Biophys. Acta. 1066:175–182; 1991. Saito, H.; Inui, K. Dipeptide transporters in apical and basolateral membranes of the human intestinal cell line Caco-2. Am. J. Physiol. 265:G289–G294; 1993. Schusdziarra, V.; Schick, R.; Fuente, A.; Specht, J.; Klier, M.; Brantl, V.; Pfeiffer, E. F. Effect of b-casomorphins on somatostatin release in dogs. Endocrinology 112:885–889; 1983. Schusdziarra, V.; Schick, R.; Fuente, A.; Holland, A.; Brantl, V.; Pfeiffer, E. F. Effect of b-casomorphins and analogs on insulin release in dogs. Endocrinology 112:1948–1951; 1983. Shen, W. C.; Wan, J.; Ekrami, H. Enhancement of polypeptide and protein absorption by macromolecular carriers via endocytosis and transcytosis. Adv. Drug Delivery Rev. 8:93–113; 1992. Tome, D.; Dumontier, A.-M.; Hautefeuille, M.; Desjeux, J. F. Opiate activity and transepithelial passage of intact b-casomorphins in rabbit ileum. Am. J. Physiol. 253:G737–G744; 1987. Yamamoto, N.; Akino, A.; Takano, T. Antihypertensive effect of the peptides derived from casein by an extracellular proteinase from lactobacillus helveticus CP790. J. Dairy Sci. 77:917–922; 1994.
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PII S0196-9781(97)00002-8
Transepithelial Transport of Oligopeptides in the Human Intestinal Cell, Caco-2 MAKOTO SHIMIZU, 1 MAKI TSUNOGAI AND SOICHI ARAI Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113, Japan Received 9 September 1996; Accepted 24 January 1997 SHIMIZU, M., M. TSUNOGAI AND S. ARAI. Transepithelial transport of oligopeptides in the human intestinal cell, Caco-2. PEPTIDES 18(5) 681–687, 1997.—The transepithelial transport of oligopeptides (of more than 4 residues) was studied by using human intestinal Caco-2 cell monolyers. The susceptibility to the brush-border peptidases was observed to be one of the primary factors which decide the transport rate. The apical-to-basolateral transport mechanism was investigated by using bradykinin and GGYR which were resistant to cellular peptidases. The intracellular pathway, probably the adsorptive transcytosis, was suggested to be involved in the transport of bradykinin and its analogues, the transport rate being particularly dependent on the hydrophobic properties of the peptides. On the other hand, the tetrapeptide such as GGYR was suggested to be transported mainly via the paracellular pathway. q 1997 Elsevier Science Inc. Bradykinin
Caco-2
Intestinal epithelial cell
Oligopeptide
IN the past two decades, oligopeptides with such diverse physiological functions as opiatic (5,29), hypertensive (2), immunomodulatory (34) and anti-coagulative (12) have been discovered in the hydrolysates of several food proteins. These oligopeptides are thought to be produced in the digestive tract by limited hydrolysis of dietary proteins and are expected to express their physiological functions in the body. Some of the peptides have already been reported to be effective in vivo after oral administration; modulating the endocrine systems in dogs (42,43) and lowering the blood pressure of spontaneous hypertensive rats (27,33,46) are examples. These findings suggest that some of the physiologically active oligopeptides can be absorbed by the body across the intestinal epithelium, while retaining their intact structure and activity. The results of intensive studies, using electrophysiological methods, during the 1970s and 1980s have suggested the existence of a peptide transport system in the intestinal epithelium, by which peptides would be actively transported through the apical membrane under an H / gradient (14,24). However, the electrophysiological measurements strongly suggested that this transport system carried only di- or tripeptides (10). Very recently, the peptide transporter protein (PepT1) has been cloned from the rabbit intestine (11). The results of an experiment using xenopus oocyte transfected with PepT1 cRNA confirmed the specificity of this transporter for di- and tripeptides. Oligopeptides with more than 4 residues are hardly, if at all, recognized by this transport system. The absorption of such larger oligopeptides must therefore be performed by another mechanism. It is possible that these peptides may be absorbed via the transcytotic route which is known to be used for the transport of macromolecules such as
Transcytosis
Paracellular transport
proteins (20). Burton and his coworkers (6,8,28) suggested the intracellular passive transport to be another mechanism for the peptide absorption. The contribution of the paracellular route to peptide absorption (1,9,36), may also be possible. However, the role of these pathways in intestinal oligopeptide absorption has not yet been fully understood, the mechanism for oligopeptide transport across the intestinal epithelium being still obscure. Caco-2 cells derived from a human intestinal adenocarcinoma have recently been found to provide a useful cell culture model of the small intestinal epithelium. The Caco-2 cell line exhibits spontaneous enterocyte-like differentiation under standard culture conditions, showing morphological polarity and expressing brush-border hydrolases (22). In the past decade, transportermediated transport of nutrients such as hexoses (40), amino acids (23,35) and di- and tripeptides (41) has been investigated by using this cell line. The polarized transcytotic activity in Caco-2 cells has also been reported (19,21). The formation of a tight junction (22) and paracellular transport regulated by tight junctions have been observed by using this cell line (4,16–18). Caco-2 is, therefore, thought to provide a good model for investigating the mechanism for intestinal oligopeptide absorption at a cellular level. For example, the permeability characteristics of a series of peptides have been investigated by Burton et al. (1,6– 9,28) using this cell line. They found that the hydrogen-bonding capacity was the major contributing factor for the transport of peptides, by using chemically modified peptides of less than 4 amino acid residues. In the present study, the mechanism for peptide transport through the intestinal epithelium was investigated by using the
1 Requests for reprints should be addressed to M. Shimizu, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan, E-mail: [email protected]
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Caco-2 monolayer cultured on a permeable filter. Oligopeptides with 4–9 amino acid residues (native peptides without chemical modification) were used as model peptides, and the structural factors of peptides which influence the absorption rate are discussed. METHOD
Materials The human colon adenocarcinoma cell line, Caco-2, was obtained from American Type Culture Collection (Rockville, MD, USA). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Nissui Pharmacies (Tokyo, Japan). Fetal calf serum (FCS), glutamine, non-essential amino acids (NEAA), penicillin-streptomycin (10000 units/ml and 10 mg/ml in 0.9% sodium chloride, respectively), phosphate-buffered saline (PBS) and Hank’s balanced salt solutions (HBSS) were all purchased from Gibco (Gaithersburg, MD, USA). Collagen type I was purchased from Nitta Zelatin (Osaka, Japan). Plastic dishes and plates were from Corning (Corning, NY, USA), and Millicell-HA with 0.45 mm cellulose membranes of 12 or 30 mm in diameter was purchased from Millipore (Molsheim, France). Lucifer yellow CH lithium salt (LY) was from Molecular Probes (Eugine, OR, USA). Fluorescein isothiocyanate (FITC)-labeled dextran with an average molecular weight of 40000 (DX-40K), bradykinin and the bradykinin B1-receptor antagonist, Des-Arg 9-[Leu 8 ]bradykinin, and a tetrapeptide, PFGK, were all from Sigma (St. Louis, MO, USA). Papain inhibitor GGYR, b-Casomorphin-5, the bradykinin B2-receptor antagonist, D-Arg-[Hyp 3 , Thi 5,8 , DPhe 7 ]-bradykinin, and Diprotin A were purchased from Peptide Institute (Osaka, Japan). Ovokinin and the bradykinin analogs were synthesized by Anaspec (USA) and purified by using a reverse-phase YMC Dispo SPE C18 column (YMC, Japan) or by gel filtration in a PD-10 Sephadex G-25M column (Pharmacia, Upsala, Sweden) before use. All other chemicals were of reagent grade. Cell Culture Caco-2 cells were cultured in DMEM supplemented with 10% FCS, 1% NEAA, 2% glutamine, 100 U/ml of penicillin and 100 mg/ml of streptomycin, together with an appropriate amount of sodium bicarbonate. They were incubated at 377C in a humidified atmosphere of 5% CO2 in air. The monolayer became confluent 3 to 4 days after seeding at 7 1 10 5 cells per 60-mm dish, and the cells were passaged at a split ratio of 4 to 8 by trypsinizing with 0.1% trypsin and 0.02% EDTA in PBS. All the cells used in this study were between passages 38 and 70. Cells for the transport studies were grown in a Millicell-HA insert with a membrane coated with type I collagen. The cells were seeded at a density of 2 1 10 5 cells/ml of the medium, and the medium was changed every 1 or 2 days. Collagen coating was performed as described previously (16). The integrity of the cell layer was evaluated by measuring the transepithelial electrical resistance (TEER) with Millicell-ERS equipment (Millipore). The monolayer cells cultured for 14–15 days and with the TEER values of higher than 200 ohm cm2 were used for the transport experiment. Transport Studies The cell monolayers grown in the Millicell insert (30 mm in diameter) were gently rinsed with HBSS, 2 ml of HBSS then being added to the apical side of the Millicell insert, and the insert placed in a well containing 2 ml of HBSS. After incubating for 30 min at a constant temperature of 377C, 200 ml of HBSS in
either the apical or basolateral side of the cell monolayer was replaced by the same volume of a peptide solution. After further incubating for an appropriate time, aliquots of the apical and basolateral solutions were taken, and the peptide concentration in each was determined by high-performance liquid chromatography (HPLC). Transport of the leakage markers, LY and DX40K, was performed in the same manner, except that determination was carried out by measuring the fluorescence intensity. Since the transport activity of the cells varied considerably among different series of experiments, probably because of the differing passage of cells or differing lots of the culture medium, the transport rate (flux) of the sample peptides was usually standardized by comparing with that of bradykinin coexisting in the sample solution. Treatment of Cells with Inhibitors After washing and preincubating the cell monolayer with HBSS, inhibitor solutions were added to give final concentrations of 0.1–1 mM (Diprotin A), 25 mM (sodium azide), 0.5 mM (2,4-dinitrophenol), 0.1–1 mM (colchicine) or 0.5 mg/ml (cytochalasin D), the monolayers then being incubated for 30 min. Transport experiments were carried out in the presence of the same concentrations of the inhibitors. High-Performance Liquid Chromatography (HPLC) A Shimadzu LC-6A liquid chromatograph (Kyoto, Japan) equipped with a C18 column (4.6 1 150 mm; ODS-H-1151, Senshu-Kagaku, Tokyo, Japan) and a Shimadzu SPD-6AV ultraviolet spectrophotometer at a wavelength of 210 nm were used. Peptides were applied to the column that had been equilibrated with 0.1% (v/v) trifluoroacetic acid and were eluted with a linear gradient of acetonitrile at a flow rate of 1.0 ml/min. The peptides were determined by measuring the peak areas. Fluorescence Measurement Fluorescence was measured by a FP777 fluorescence spectrophotometer (Jasco; Tokyo, Japan). The excitation and emission wavelengths for determining LY were 430 nm and 540 nm, respectively, and those for DX-40K were 287 nm and 518 nm. Statistical Analyses All results are expressed as the mean { SE. Student’s t-test (paired or unpaired) was used to compare means and ranges. RESULTS
Transport Rate Comparison for Different Oligopeptides The apical-to-basolateral flux across the Caco-2 monolayer was measured by using five peptides with different size and structure, namely bradykinin (RPPGFSPFR), b-casomorphin (YPFPG), ovokinin (FRADHPFL), PFGK and GGYR, and the results are shown in Fig. 1. Although the concentration of each peptide added to the apical solution was constant (400 mg/ml), the concentration of the intact peptides in the basolateral solution after a 30-min incubation was different. The transport rate was greatest for GGYR and smallest for b-casomorphin. The TEER value of the monolayer was not significantly changed by incubating with these peptides under the present experimental conditions. The concentration of each peptide in the apical solution after 60 min of incubation with the Caco-2 cell monolayer grown in a plastic dish are shown in Fig. 2. The concentration of bradykinin in the apical solution remained unchanged, while those of
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TRANSEPITHELIAL TRANSPORT OF PEPTIDES
FIG. 1. Apical-to-basolateral flux of various peptides across the Caco-2 monolayer. Bradykinin was present with one of the other peptides in an apical solution and the flux of each peptide is expressed as relative to the bradykinin value (means { SE). The number of epithelial cell layers used for the experiments was 6 (for b-casomorphin), 2 (ovokinin), 8 (PFGK) or 12 (GGYR). The paired t-test was used to evaluate the difference in fluxes between bradykinin and the other peptides. *Significantly different from the bradykinin flux (p õ 0.05); ** p õ 0.01.
ovokinin and b-casomorphin were decreased to a great extent, suggesting that these peptides were hydrolyzed quickly by the brush border peptidases. Since b-casomorphin was most rapidly cleaved at the peptide bond between P and F, and Caco-2 expressed dipeptidyl aminopeptidase IV (DPP-IV) as the major peptidase (manuscript in preparation), the contribution of DPPIV to the hydrolysis of b-casomorphin was suggested. The flux of b-casomorphin was increased by treating the cell layer with 1 mM Diprotin A, an inhibitor of dipeptidyl peptidase IV (Fig. 3). The differing degree of flux among the peptides (Fig. 1) would have been, at least partly, due to the different susceptibility of the peptides to cellular peptidases such as DPP-IV.
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FIG. 2. Concentrations of intact peptides in the apical solutions after 60 min of incubation with the Caco-2 monolayer. The initial peptide concentration was 400 mg/ml, and results are presented as means { SE (3 epithelial layers).
route (17,22), was not significantly different between AP-BL and BL-AP (Fig. 5A), suggesting that the transport of bradykinin was not a simple diffusion process via the paracellular route. Effect of inhibitors on the transport of bradykinin. The APBL flux of bradykinin was decreased in the presence of such metabolic inhibitors as sodium azide and 2,4-dinitrophenol (Fig. 6), indicating that the transport incorporated an energy-dependent process. The flux was also partially inhibited by colchicine, an inhibitor of intracellular vesicle transport through perturbation of the microtubule structure (Fig. 6). The paracellular flux of
Transepithelial Transport of Bradykinin Effect of time and concentration on the transport rate of bradykinin. Bradykinin, which is resistant to hydrolysis by cellular peptidases (Fig. 2), was used as a model peptide to reveal the mechanism for oligopeptide transport through the intestinal epithelial cell layer. The transport rate was observed to be constant at least for 60 min (data not shown); therefore, incubation was carried out for 60 min in the subsequent experiments. The transport of bradykinin was concentration-dependent, but a saturable process was not apparent when the peptide concentration in the apical solution was less than 1.2 mg/ml (Fig. 4). Considering the sensitivity of HPLC for detecting peptides transported to the basolateral solution, 400 mg/ml was employed as the apical peptide concentration in this study unless otherwise stated. Apical-to-basolateral and basolateral-to-apical transport of bradykinin. The degrees of apical-to-basolateral (AP-BL) and basolateral-to-apical (BL-AP) flux of bradykinin were compared. As shown in Fig. 5A, the AP-BL flux was much higher than the BL-AP flux. In contrast, the flux of fluorescence reagent LY, which has been used as a leakage marker for the paracellular
FIG. 3. Effect of Diprotin A on the apical-to-basolateral flux of b-casomorphin. Diprotin A was added to both the apical and basal solutions, and the cell monolayer was incubated for 30 min before measuring the flux. The flux is expressed as the relative value to that in the control cell monolayer (without Diprotin A). The results are means { SE (4 epithelial layers for the control and 0.1 mM Diprotin A, and 3 epithelial layers for 1 mM Diprotin A). *Significantly different from the control value (p õ 0.005).
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FIG. 4. Concentration dependence of the apical-to-basolateral flux of bradykinin. Bradykinin was added to the apical solution, which was then incubated for 60 min. The results are expressed as means { SE (3 epithelial layers).
small molecular substances is known to be increased by treating the cell layer with cytochalasin D, an agent which opens tight junction by altering the cytoskeletal structure (30). The cytochalasin D treatment did not affect the flux of bradykinin, while that of Lucifer Yellow was increased (Fig. 7). These results suggest that the transcytotic process, not the paracellular process is mainly involved in the AP-BL transport of bradykinin. Transcytotic processes for bradykinin. A high-molecularweight dextran conjugated with a fluorescence reagent has been found to be useful for estimating fluid-phase transcytosis in the intestinal epithelial cell layer (22). The degrees of flux of DX-
FIG. 5. Comparative directional transport between bradykinin and Lucifer Yellow (A), Dextran-40K (B) and GGYR (C). The initial concentration of each peptide and dextran was 400 mg/ml, whereas that of Lucifer Yellow was 100 mg/ml. The basolateral-to-apical flux is expressed as relative to the apical-to-basolateral flux, the results being expressed as means { SE (3–7 epithelial layers for A, 6–8 layers for B, and 9–10 layers for C). *Significantly different from the apical-to-basolateral flux, p õ 0.005; **p õ 0.001.
FIG. 6. Effect of inhibitors on the apical-to-basolateral flux of bradykinin. Sodium azide (25 mM), 2,4-dinitrophenol (0.5 mM) or cholchicine (1 mM) was added to both the apical and basal solutions, which were then incubated for 30 min before measuring the flux. The results are expressed as means { SE (3 epithelial layers). *Significantly different from the control value (without an inhibitor), p õ 0.01; **p õ 0.001.
40K were therefore measured to evaluate the fluid-phase transcytotic activity of the Caco-2 cell monolayer. The AP-BL flux of DX-40K was higher than the BL-AP flux (Fig. 5B), suggesting that the fluid-phase transcytosis in the Caco-2 monolayer was more active in the AP-BL process than in the BL-AP process. The AP-BL flux of bradykinin was, however, much higher than that for DX-40K (p õ 0.01), although the degrees of BL-AP flux of these two compounds were not significantly different (Table 1). These results suggest that the AP-BL transport of bradykinin was performed not only by simple fluid-phase transcytosis, but also by some other mechanisms. Relationship between the transport rate and structure of peptides. In order to reveal the relationship between the flux via transcytosis and the peptide structure, some peptides having an analogous
FIG. 7. Effect of cytochalasin D on the apical-to-basolateral flux of bradykinin and Lucifer Yellow. Cytochalasin D (0.5 mg/ml) was added to both the apical and basal solutions, which were then incubated for 30 min before measuring the flux. TEER was decreased to 68% of the initial value (280 ohm cm2 ) after 30-min incubation. The results are expressed as means { SE (3 epithelial layers). *Significantly different from the control value (without cytochalasin D) p õ 0.01.
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TRANSEPITHELIAL TRANSPORT OF PEPTIDES TABLE 1 APICAL-TO-BASOLATERAL AND BASOLATERAL-TO-APICAL FLUX FOR BRADYKININ, DEXTRAN-40K AND GGYR ACROSS THE CACO-2 CELL MONOLAYER
Sample
Apical-to-Basal Flux (ng/h/cm2) (n Å 4)
Basal-to-Apical Flux (ng/h/cm2) (n Å 3)
Bradykinin Dex-40K GGYR
346.5 { 67.9*† 140.2 { 23.4* 550.6 { 97.6
93.0 { 27.0†‡ 70.8 { 2.6 272.2 { 35.2‡
Bradykinin, FITC-dextran-40K or GGYR (400 mg/ml each) was mixed and added to either the apical or basolateral solution. After 60 min of incubation, each solution on the opposite side was recovered, and the concentrations of the peptides and dextran were evaluated by HPLC and fluorescence measurement, respectively. Results are expressed as means { SE. *Significantly different from each other, p õ 0.01; † p õ 0.05; ‡ p õ 0.05.
structure to that of bradykinin were synthesized and their transport was examined. As shown in Fig. 8, the degree of flux was markedly dependent on the peptide structure. The number of the positively charged amino acid (arginine) was negatively correlated with the flux (r Å 0.85). On the other hand, the hydrophobicity of the peptides, which was estimated from the retention time by reverse-phase HPLC (Table 2), was positively correlated with the flux (r Å 0.83), suggesting that such peptides as bradykinin were adsorbed to the apical cell membrane surface mainly through hydrophobic interaction, by which the peptides were efficiently internalized by the cells and transported across the cell layer.
685 border peptidases is the rate-limiting step for the transepithelial transport of oligopeptides. A similar observation concerning the transepithelial passage of intact b-casomorphins has been reported by using the rabbit ileum in vitro (32,45). Since the susceptibility of oligopeptides to peptidases makes it difficult to measure the real transport rate for an intact oligopeptide, bradykinin and GGYR, which were found to be resistant to cellular peptidases (Fig. 2), were chosen as the model peptides for further analyses. Bradykinin is a physiologically active peptide derived from blood serum kininogen, and is known to express a variety of functions such as arterial relaxation and smooth muscle contraction through binding to its specific receptors, B1 and B2 (39). The receptors are known to be present in various types of cells including smooth muscle and endothelial cells (39). The presence of bradykinin receptors in the colonic epithelial cells has been also reported (38). Although there is no report on bradykinin receptor B1 or B2 being present in the Caco-2 cell surface, the possibility that bradykinin transport across the Caco-2 cell layer was influenced by the receptors having been expressed on the Caco-2 cells cannot be ruled out. In order to eliminate such a possibility, we examined bradykinin transport in the presence of the B1 antagonist, [des-Arg 9 ]-bradykinin, or B2 antagonist, [Thi 5,8 ,D-Phe 7 ]-bradykinin. The bradykinin flux was not affected by these antagonists (data not shown). Microscopic observation of the cell layer and measurement of changes in the transepithelial short-circuit current after adding bradykinin also suggest that bradykinin did not exert any physiological effect on the Caco-2 cells used in this experiment (data not shown). Although the bradykinin concentrations used in this study were at least 10000 fold higher than the Kd values of bradykinin recep-
Epithelial Transport of the Tetrapeptide, GGYR The AP-BL flux of GGYR was 1.6 times that of bradykinin (Table 1). The transport of GGYR was not significantly inhibited by treating with 2,4-dinitrophenol and cholchicine (data not shown), suggesting that the major mechanism for GGYR transport did not involve an energy-dependent or cytoskeletal-dependent process. The BL-AP flux of GGYR was also much higher than that of bradykinin (p õ 0.05), the ratio of (AP-BL)/(BLAP) flux being lower than that of bradykinin (Fig. 5C). Increased flux of GGYR was also observed by treating the cell layer with cytochalasin D (data not shown). These results suggest the major transport mechanism to be different between bradykinin (9 residues) and GGYR (4 residues), the latter peptide being transported via the paracellular pathway to a large extent. DISCUSSION
The results of this study suggest that digestion by cellular peptidases is the primary factor affecting intact peptide flux across the epithelial cell layer. It has been demonstrated that Caco-2 expressed at least 8 aminopeptidases (25). The expression of the three major aminopeptidases, namely aminopeptidases N and P and dipeptidylaminopeptidase (DPP)-IV, was also observed in our study, DPP-IV having the highest activity among the three (data not shown). During the incubation of b-casomorphin-5 (YPFPG) with Caco-2 cells, N-terminal dipeptide YP was produced at the highest rate. This hydrolysis pattern was similar between the peptide incubated with intact cells and that with the cell lysate, suggesting that cell-surface DPP-IV was most responsible for the hydrolysis of b-casomorphin-5 (manuscript in preparation). The increased flux of b-casomorphin-5 by treating the cell layer with the DPP-IV inhibitor, Diprotin A (Fig. 3), strongly suggests that the hydrolysis of peptides by brush-
FIG. 8. Apical-to-basolateral flux of bradykinin and its analogous peptides (I–V). Bradykinin was present with each other peptide in an apical solution, the flux of peptides I–V being expressed as relative to the bradykinin flux. The results are expressed as means { SE (6–10 epithelial layers). The paired t-test was used to evaluate the difference in flux between bradykinin and each other peptide. *Significantly higher than the bradykinin flux, p õ 0.01; ** p õ 0.001.
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SHIMIZU, TSUNOGAI AND ARAI TABLE 2 RETENTION TIMES FOR BRADYKININ AND ITS ANALOGOUS PEPTIDES IN AN ODS COLUMN Peptide
Retention Time (min)
Bradykinin Peptide I Peptide II Peptide III Peptide IV Peptide V
17.55 23.15 25.90 21.00 8.80 18.45
Each peptide was applied to an ODS column equilibrated with 0.1% trifluoroacetic acid (pH 2.0) and then eluted by linearly increasing the concentration of acetonitrile. The retention time was used to evaluate the hydrophobicity of the peptides (45).
tors (39), the transport was not saturable, being increased by increasing the apical concentration. These results suggest that bradykinin transport observed in this study was not significantly affected by a receptor-mediated process, even if the receptors had been expressed on the surface of the Caco-2 cells. The results of the present study suggest that the energy-dependent transcytotic pathway is involved in the transport of such oligopeptides as bradykinin. Pappenheimer et al. (36), from the results of oral administration tests on rats, have suggested that a D-octapeptide (EASASYSA) was transported paracellularly. However, it is noteworthy that their peptide was administered with a high content of glucose, which had been reported to dilate the tight junction, inducing solvent drag through the absorptive cell junctions (31). Adson et al. (1) indicated that the paracellular diffusion rate was strongly dependent on molecular size, the paracellular flux of a tetrapeptide ( D-Phe)3-Gly being restricted to much higher extent than that of D-Phe-Gly. Hu et al. (26) reported that the permeability of GHRP-6, a hexapeptide-like drug, was not significantly increased after dilation of the tight junction by luminal glucose. Perturbation of the tight junction by cytochalasin D resulted in an increase in the Lucifer Yellow flux by increasing the pore radius, whereas the bradykinin flux was not affected by this treatment (Fig. 7). Under normal condition, transcytosis would be the most possible pathway, at least for 9-residue peptides such as bradykinin. Transcytosis is a transport system via internalized vesicles carrying specifically bound ligands (receptor-mediated transcytosis) or non-specifically adsorbed ligands (adsorptive transcytosis) or fluids (fluid-phase transcytosis) from their sites of entry to sites on the opposite side of the cell (13,44). The transport of albumin, transferrin and insulin in endothelial cells and that of IgA and transferrin in epithelial cells is carried out by the receptor-mediated process (13,44). Since receptor-mediated transcytosis of bradykinin is not likely to have occurred as already described, bradykinin transport would seem to be performed by fluid-phase or adsorptive transcytosis. Fluid-phase transcytosis is a process started by the uptake of extracellular fluid. The transport rate is therefore dependent on the concentration of the molecules in the extracellular fluid and
the rate of internalization (44). The significantly higher flux of bradykinin than that of DX-40K, a marker for fluid-phase transcytosis (Table 1), suggests that bradykinin transport was not simply carried out by fluid-phase transcytosis. Adsorptive transcytosis requires the interaction of solutes (molecules to be transported) with the cell membrane surface, by which the molecules are concentrated at the surface and efficiently internalized, although the mechanism involved in the internalization of cell membranes is still not clear. It was reported that molecules with a pI value between 4.5 and 7.0 did not bind to the plasma membrane (43). Positively charged macromolecules have high affinity toward a cell membrane whose surface possesses a net negative charge due to an abundance of carbohydrate such as sialic acid (37). Since bradykinin is a positively charged peptides with 2 arginine residues under neutral conditions, these two arginine residues are considered to have taken part in the adsorption of bradykinin to the cell surface, facilitating adsorptive transcytosis. Unexpectedly, the results of experiments using several bradykinin analogues of different arginine content (Fig. 8) demonstrate that the number of arginine residues was rather negatively correlated with the flux. Since the removal of N- and/or C-terminal arginine residues would make the molecule smaller (peptides I–III), the change in degree of flux may be attributable only to a change in molecular size. However, a recent experiment comparing RPPGFSPFL and acetyl-LPPGFSPFL has demonstrated that replacing N-terminal R with acetyl L increased the flux by the ratio of 1.78 (SE Å 0.17, n Å 4). Considering this observation, as well as the fact that the hydrophobicity of a peptide clearly showed positive correlation with the flux, the increased flux by decreasing the number of arginine residues could have been responsible for the accompanying increase in hydrophobicity. In any event, the present results suggest adsorptive transcytosis depending on the hydrophobicity of peptides to be the major transport mechanism, although it should be investigated whether the aggregation of hydrophobic peptides is also the factor that can influence the transport of these oligopeptides. Furthermore, the possibility that the chemically-modified, lesspolar bradykinin analogues were, at least partly, transported via the intracellular passive pathway (6–8,28) can not be ruled out. In contrast, tetrapeptide GGYR showed different transport properties. The paracellular mechanism for transport of this peptide was strongly suggested, although there is no information on whether the rate-limiting factor for paracellular transport is only the molecular size. The effect of amino acid sequence and of other properties of the peptide on diffusion through the cell junctions is not yet known. Three different transport routes, namely paracellular, fluidphase and adsorptive transcytosis, may participate in oligopeptide transport across the intestinal epithelium. The contribution of each route must be different among the peptides, depending on the molecular size and other structural properties such as hydrophobicity. The major pathway for bradykinin (molecular weight of 1060) would seem to be adsorptive transcytosis, whereas paracellular diffusion would play the major role in the transport of GGYR (molecular weight of 450). Complex features of intestinal oligopeptide transport, depending on the size and structure of a peptide, are thus indicated. ACKNOWLEDGEMENT
We are grateful to Mr. M. Satake for his skillful technical assistance.
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