Transport of Antihypertensive Peptide RVPSL, Ovotransferrin 328–332

Sep 3, 2015 - When transport from the apical side to the basolateral side was investigated, the apparent permeability coefficient (Papp) was (6.97 ± ...
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Transport of Antihypertensive Peptide RVPSL, Ovotransferrin 328− 332, in Human Intestinal Caco‑2 Cell Monolayers Long Ding, Liying Wang, Yan Zhang, and Jingbo Liu* Laboratory of Nutrition and Functional Food, Jilin University, Changchun 130062, People’s Republic of China ABSTRACT: The objective of this study was to investigate the transepithelial transport of RVPSL (Arg-Val-Pro-Ser-Leu), an egg-white-derived peptide with angiotensin I-converting enzyme (ACE) inhibitory and antihypertensive activity, in human intestinal Caco-2 cell monolayers. Results revealed that RVPSL could be passively transported across Caco-2 cell monolayers. However, during the process of transport, 36.31% ± 1.22% of the initial RVPSL added to the apical side was degraded, but this degradation decreased to 23.49% ± 0.68% when the Caco-2 cell monolayers were preincubated with diprotin A (P < 0.001), suggesting that RVPSL had a low resistance to various brush border membrane peptidases. When transport from the apical side to the basolateral side was investigated, the apparent permeability coefficient (Papp) was (6.97 ± 1.11) × 10−6 cm/s. The transport route of RVPSL appears to be the paracellular pathway via tight junctions, as only cytochalasin D, a disruptor of tight junctions (TJs), significantly increased the transport rate (P < 0.001). In addition, the relationship between the structure of RVPSL and transport across Caco-2 cell monolayers was studied by mutation of RVPSL. It was found that N-terminal Pro residues were more beneficial for transport of pentapeptides across Caco-2 cell monolayers than Arg and Val. Furthermore, RVPSL could be more easily transported as smaller peptides, especially in the form of dipeptides and tripeptides. KEYWORDS: egg white, antihypertensive peptide, transport, Caco-2 cell monolayer, brush border membrane peptidase

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INTRODUCTION Cardiovascular disease (CVD) is a significant public health problem worldwide and will become the leading cause of death and disability by 2020. As a major type of CVD, hypertension often occurs with other diseases, such as stroke, atherosclerosis, diabetes and myocardial infarction.1 It has been demonstrated that diet and lifestyle are two major risk factors for hypertension.1 Therefore, in addition to synthetic antihypertensive drugs, many food components also have the potential to lower blood pressure with limited adverse effects. Among these components, food-derived bioactive peptides with angiotensin I-converting enzyme (ACE) inhibitory activity have attracted the most attention in the past decade.2 ACE-inhibitory peptides are generally produced from food proteins such as milk casein or egg white proteins after gastrointestinal digestion, fermentation, or hydrolysis by proteolytic enzymes.3 Several in vivo studies have demonstrated that many ACE-inhibitory peptides can reduce blood pressure in spontaneously hypertensive rats (SHR) after either intravenous or oral administration.4−6 However, there have been discrepancies between in vitro ACE inhibitory and in vivo antihypertensive activity, suggesting that it is very important to evaluate absorption and bioavailability of food-derived antihypertensive peptides through the human intestine after oral administration.3 The intestinal epithelium is composed of a large number of welldifferentiated and polarized epithelial cells with microvilli structures on the apical surface. Human colon carcinoma cell line Caco-2 cells can spontaneously differentiate into monolayers with tight junctions (TJs) and exhibit many properties like intestinal epithelium, which can simulate the structure and functions of human intestinal epithelium in vitro. As a consequence, Caco-2 cell monolayers have been used to study the absorption of nutrients and drugs in vitro.7 In © 2015 American Chemical Society

addition, because Caco-2 is a widely used model, a good correlation between passive transport of drugs across Caco-2 cell monolayers and absorption in vivo has been demonstrated.8 To exert antihypertensive effects in vivo, food-derived bioactive peptides must be absorbed intact into the blood circulation and then reach target organs in an active form and in a sufficient quantity after oral administration.3 However, absorption is still a challenge for many functional factors and drugs. In general, the absorption of peptides mainly occurs through the epithelium of the small intestine after extensive gastrointestinal digestion. This implies the importance of resistance to brush border membrane peptidases for successful absorption through the intestine.9 For instance, the egg-whitederived antihypertensive peptides FRADHPFL and YAEERYPIL are susceptible to brush border membrane peptidases and are cleaved into shorter fragments prior to their absorption. However, some of their fragments can be transported intact across Caco-2 cell monolayers.10 Many studies on transport through the Caco-2 cell monolayer system have indicated that the permeability of bioactive peptides depends mainly on their molecular size, hydrophobicity, and even molecular surface properties.11,12 Many dipeptides and tripeptides have been shown to be successfully transported intact through the intestinal epithelium by H+-dependent peptide transporter 1 (PepT1).13,14 However, oligopeptides, especially those larger than three amino acid residues, are not usually substrates for PepT1. Alternatively, the paracellular pathway modulated by TJs has been shown to be involved in transepithelial absorption Received: Revised: Accepted: Published: 8143

April 12, 2015 September 1, 2015 September 3, 2015 September 3, 2015 DOI: 10.1021/acs.jafc.5b01824 J. Agric. Food Chem. 2015, 63, 8143−8150

Article

Journal of Agricultural and Food Chemistry of oligopeptides.15 For example, the antihypertensive oligopeptides VLPVP and HLPLP have been shown to be transported through Caco-2 cell monolayers mainly via the paracellular pathway.17,18 Additionally, it has been reported that the antihypertensive tripeptide VPP, derived from milk β-casein, is also transported across Caco-2 cell monolayers via the paracellular pathway rather than the PepT1-mediated pathway.18 However, the molecular mechanism behind the regulation of paracellular transport has not been clearly identified. As such, further studies are needed to assess the transepithelial transport of bioactive peptides, especially oligopeptides, through the human intestine. As a good protein source for bioactive peptides, chicken egg white, which is composed predominantly of ovalbumin, ovotransferrin, ovomucoid, globulins, and lysozyme, is widely used to prepare antihypertensive peptides.19 In our previous study, egg-white-derived peptide RVPSL (ovotransferrin 328− 332) was identified with in vitro ACE-inhibitory and in vivo antihypertensive activities, making it a prospective functional food candidate and providing an approach for the therapy of hypertension.20−22 Before studying the bioavailability and pharmacokinetics of the antihypertensive peptide RVPSL in vivo, it is necessary to evaluate its permeability through the intestinal epithelium. Therefore, the objective of this paper was to investigate the resistance to brush border membrane peptidases and the transepithelial transport of RVPSL in the human intestinal Caco-2 cell monolayers. In addition, the structure−transport relationship of RVPSL across Caco-2 cell monolayers was also studied.

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Transport Experiments. The bidirectional transepithelial transport of antihypertensive peptide RVPSL in human intestinal Caco-2 cell monolayers was conducted as previously described.23 Caco-2 cell monolayers were rinsed with HBSS twice and then incubated with HBSS for 30 min at 37 °C in 5% CO2 prior to transport experiments. Then the HBSS was removed and replaced with 0.5 mL of 5 mM RVPSL (dissolved in HBSS, pH 6.0) on the apical side and 1.5 mL of fresh HBSS on the basolateral side (pH 7.4). Alternatively, to research the efflux of RVPSL, 0.5 mL of fresh HBSS on the apical side (pH 6.0) and 1.5 mL of 5 mM RVPSL (dissolved in HBSS, pH 7.4) on the basolateral side were used. All of the plates were incubated at 37 °C in 5% CO2 for 2 h. Then the RVPSL concentrations on both the apical and basolateral sides were determined by HPLC analysis. The resistance of RVPSL to brush border membrane peptidases was studied as described previously by use of diprotin A, an inhibitor of membrane peptidase dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5), which is a major brush border membrane peptidase.24 Briefly, Caco-2 cell monolayers were preincubated with 1 mM diprotin A at 37 °C in 5% CO2 for 30 min before transport experiments. Then 0.5 mL of 5 mM RVPSL was added on the apical side. After 2 h of incubation, samples were collected both from apical and basolateral sides and detected by HPLC analysis. The transepithelial transport mechanism of RVPSL across Caco-2 cell monolayers was investigated by use of transport inhibitors as described previously.25 Namely, the Caco-2 cell monolayer was preincubated with cytochalasin D (a TJ disruptor, 0.5 μg/mL), wortmannin (a transcytosis inhibitor, 500 nM), sodium azide (an ATP synthesis inhibitor, 10 mM), or Gly-Sar (a peptide transport PepT 1 substrate, 25 mM) for 30 min, followed by addition of 0.5 mL of 5 mM RVPSL to the apical side. Cytochalasin D, wortmannin, and sodium azide were dissolved in DMSO (the final concentration of DMSO in HBSS was 0.044%), and Gly-Sar was dissolved in HBSS. As a control, DMSO was used. After 2 h of incubation, the RVPSL on the basolateral side was detected by HPLC analysis. The relationship between the structure of RVPSL and transport across Caco-2 cell monolayers was investigated by analyzing the intact transport of a number of peptides, which were re-formed and synthesized based on the structure of RVPSL. These peptides contained two parts: 10 pentapeptides derived from RVPSL by amino acid replacement with Pro residue, and six terminal fragment smaller peptides of RVPSL, including two tetrapeptides, two tripeptides, and two dipeptides. In the transport experiments, peptides (5 mM, pH 6.0) were added on the apical side. After 2 h of incubation, peptide concentrations on the basolateral side were detected by HPLC analysis. The apparent permeability coefficient (Papp, centimeters per second) was calculated as follows:

MATERIALS AND METHODS

Chemicals. Caco-2 cell lines were purchased from American Type Culture Collection. The antihypertensive peptide RVPSL and other peptides with purity over 95% were synthesized by solid-phase procedures using Fmoc-protected amino acid synthetic methods by ChinaPeptide Co., Ltd. (Shanghai, China). Diprotin A and glycylsarcosine (Gly-Sar) were purchased from ChinaPeptide Co., Ltd. (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, penicillin−streptomycin, and Hanks’ balanced salt solution (HBSS) were purchased from Gibco BRL Life Technology (Carlsbad, CA). Nonessential amino acid solution, trypsin, Lglutamine, dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA), collagen type I, cytochalasin D, wortmannin, and sodium azide were all purchased from Sigma−Aldrich (St. Louis, MO). Acetonitrile (ACN), trifluoroacetic acid (TFA), and methanol were purchased from Fisher Scientific (Waltham, MA). All other reagents were analytical-grade. Cell Culture. The Caco-2 cells were grown in DMEM containing 10% fetal bovine serum, 1% nonessential amino acid solution, 100 units/mL penicillin, 100 μg/mL streptomycin, and 4 mM L-glutamine at 37 °C in an atmosphere of 5% CO2 and 90% relative humidity. Stock cultures were grown in 75 cm2 tissue culture flasks and were split at 80−90% confluency with 0.25% trypsin and 0.02% EDTA solution. The cells from passage numbers 30−40 were used and seeded on collagen type I-coated permeable polycarbonate inserts (12 mm diameter, 0.4 μm pore size, 1.12 cm2 grown surface area; Costar, Corning, NY) in 12 transwell plates at a density of 1 × 105 cells/insert. The culture medium (0.5 mL in the apical side and 1.5 mL in the basolateral side) was replaced every 2 days for the first week and then daily until they were used for experiments 21 days after seeding. The integrity of the cell monolayers was evaluated by measuring transepithelial electrical resistance (TEER) values with an EVOM epithelial volt/ohmmeter (Millicell-ERS, Millipore, Billerica, MA). Only cell monolayers with TEER values higher than 300 Ω·cm2 were used for transport studies.

Papp =

dC 1 1 V dt A C 0

where dC/dt is the change of RVPSL concentration in the receiving chamber (micromoles per liter per minute); A is the area of membrane (square centimeters), C0 iss the initial peptide concentration in donor chamber (millimoles per liter), and V is the volume of the receiving chamber. Reverse-Phase High-Performance Liquid Chromatography. The peptide was quantified on a Shimadzu HPLC system equipped with LC solution software, a LC-20AD binary gradient pump, a SIL20AC autosampler, a CTO-20AC column oven, and SPD-20AV UV detector (Shimadzu, Kyoto, Japan). The analytical column was a C18 reverse-phase column (Symmetry C18 column, 250 × 4.6 mm, particle size 5 μm; Waters, Milford, MA). The mobile phases were solvent A (0.1% TFA in water) and solvent B (0.1% TFA in ACN). Samples were eluted with a gradient of solvent B from 10% to 55% in 15 min and held at 55% for 5 min at a flow rate of 0.5 mL/min. The injection volume was 20 μL, and the absorbance detector wavelength was set at 220 nm. The peptide concentration was calculated by a linear regression analysis of the peak area by the method of Lei et al.16 A calibration curve was constructed over a RVPSL concentration range 8144

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Figure 1. Representative chromatograph of RVPSL with retention time of 12.78 min and total RP-HPLC run time of 20.00 min. (a) RVPSL on the apical side at 0 min (approximately 5 mM); (b) RVPSL on the basolateral side at 120 min (approximately 187 μM) in apical to basolateral transport experiment. of 10 μM to 5 mM. The HPLC analysis condition was the same as for samples. The peak area of RVPSL was measured, and the calibration curves of peak area versus concentration and the coefficient were then analyzed. Assay of ACE-Inhibitory Activity. The ACE-inhibitory activity of peptides was determined as described previously.26 Namely, an aliquot of 30 μL of 4 mM HHL was mixed with 10 μL of sample solutions and then incubated at 37 °C for 5 min. Then, 20 μL of 0.25 unit of ACE was added and the reaction mixture was incubated at 37 °C for an additional 30 min, followed by the addition of 60 μL of 1 M HCl to stop the reaction. The whole assay was conducted in borate buffer (100 mM, pH 8.3) containing 300 mM NaCl. The same buffer was used for solution preparation of the HHL, hippuric acid, peptides, and enzyme dilutions. HPLC analysis was used to quantify the hippuric acid produced by the enzymatic hydrolysis of the substrate hippuryl-Lhistidyl-L-leucine (HHL). The analytical column was a C18 reversephase column (Symmetry C18 column, 250 × 4.6 mm, particle size 5 μm; Waters, Milford, MA). The isocratic mobile phase consisted of 25% acetonitrile in deionized water (v/v) with 0.5% TFA. The injection volume was 10 μL, and HHL and hippuric acid were detected at 228 nm when the mobile phase was controlled at 0.5 mL/min. ACE inhibitory activity, as a percentage, was calculated as follows:

ACE inhibitory activity =

peptides used in this study were all previously validated by HPLC analysis. Transepithelial Flux of RVPSL across Caco-2 Cell Monolayers. In the transepithelial flux experiment, the synthetic peptide RVPSL at a concentration of 5 mM was added to the apical side of Caco-2 cell monolayers and incubated at 37 °C in 5% CO2 for 2 h. The levels of RVPSL on both the apical and basolateral sides were then determined. As shown in Figure 2, after 2 h of incubation, the accumulated

(A − B) × 100 B

Figure 2. Effect of diprotin A on accumulated amount of RVPSL on the apical and basolateral sides of Caco-2 cell monolayers. In the presence of diprotin A, the RVPSL remaining on the apical side was significantly higher than that without diprotin A (P < 0.001). However, no significant increase in the transport of RVPSL to the basolateral side was observed (P > 0.05). All values are means ± SD (n = 3). (*) P < 0.001 as compared to RVPSL level on the apical side without diprotin A.

where A is the peak area of the reaction blank, containing the same volume of buffer solution instead of the sample; B is the peak area of the reaction in the presence of both ACE and enzymatic peptide sample; and the IC50 value is defined as the concentration of inhibitor inhibiting 50% of the ACE activity under the assayed conditions. Statistical Analysis. All data were expressed as the mean ± SD (n = 3). The difference between groups was carried out by one-way analysis of variance (ANOVA) followed by least significant difference (LSD) test with the significance level at P < 0.05 by use of SPSS 19.0 software.

concentration of RVPSL on the basolateral side reached 187.46 ± 29.84 μM, and the Papp value was found to be (6.97 ± 1.11) × 10−6 cm/s, suggesting that the antihypertensive peptide RVPSL could be absorbed intact through the Caco-2 cell monolayers. However, only 2622.20 ± 61.22 μM RVPSL remained on the apical side, indicating that 47.56% ± 1.22% of the initial RVPSL added to the apical side had disappeared. Except for the portion that had been transported, there was 36.31% ± 1.22% of the initial RVPSL that may have been hydrolyzed to shorter fragments or free amino acids by brush border membrane peptidases. However, no other fragments in addition to RVPSL were detected on the basolateral side and in Caco-2 cells under the HPLC conditions used in the present study. To verify the low resistance of RVPSL to brush border membrane peptidases, the Caco-2 cell monolayer was preincubated with diprotin A, a protease inhibitor, prior to the transport experiment. As a result, the activity of DPPIV, the

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RESULTS Validation of RVPSL by RP-HPLC Analysis. As shown in Figure 1, the retention time for RVPSL was 12.78 min out of a total running time of 20.00 min. On both the apical side (Figure 1 a) and basolateral side (Figure 1 b), no matrixspecific interfering peaks were observed. To quantify the RVPSL in the sample, a calibration curve was established over a concentration range of 10 μM to 5 mM. Linear regression analysis of the peak area and concentration of RVPSL revealed a typical equation y = 446.75x + 31821 with a mean correlation coefficient r2 = 0.9994, indicating good linearity in this calibration curve and that the equation could be used to determine the concentration of RVPSL in this work. The other 8145

DOI: 10.1021/acs.jafc.5b01824 J. Agric. Food Chem. 2015, 63, 8143−8150

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Journal of Agricultural and Food Chemistry main group of brush border membrane peptidases, was restricted. As shown in Figure 2, the amount of intact RVPSL on the basolateral side in the presence and absence of diprotin A was not significantly increased (P > 0.05). However, the accumulated RVPSL on the apical side increased to 3238.04 ± 34.11 μM, which was 1.23-fold higher than in the absence of diprotin A (P < 0.001), and only 35.24% ± 0.68% of the initial RVPSL had disappeared. Consequently, except for the portion that was transported, degradation of the initial RVPSL by brush border membrane peptidases decreased to 23.49% ± 0.68% (P < 0.001). A bidirectional transport experiment was also designed to determine whether the transepithelial transport of RVPSL was passive or polarized. The permeability coefficient Papp for both directions, transport from apical side to basolateral side (APBL) and transport from basolateral side to apical side (BL-AP), was tested. As shown in Figure 3, in the absence of diprotin A,

Figure 4. Effects of various compounds on transport of RVPSL across Caco-2 cell monolayers. Gly-Sar, sodium azide, and wortmannin all had little effect on Papp of RVPSL (P > 0.05). In contrast, cytochalasin D increased the transport of RVPSL significantly (P < 0.001), suggesting that RVPSL mainly transported across Caco-2 cell monolayers via the paracellular pathway mediated by TJs. All values are means ± SD (n = 3). (*) P < 0.001 as compared to the control group.

However, RVPSL transport did not decrease significantly in the presence of wortmannin (P > 0.05), indicating that the transcytosis was not involved in RVPSL transport. In contrast, in the presence of cytochalasin D, a TJ disruptor, the permeability coefficient Papp of RVPSL across the Caco-2 cell monolayers increased by 46.03%. It was significantly higher than that of the control group (P < 0.001), suggesting that RVPSL was transported across the Caco-2 cell monolayers via the paracellular pathway mediated by TJs. Structure−Transport Relationship of RVPSL Caco-2 Cell Monolayers. To investigate the relationship between RVPSL structure and transport across Caco-2 cell monolayers, Pro was used to replace one or two of the amino acid residues of RVPSL. The Papp values of 11 pentapeptides (RVPSL, PVPSL, RPPSL, RVPPL, RVPSP, PVPPL, PVPSP, RPPPL, RPPSP, PPPSL, and RVPPP) varied from (4.13 ± 0.62) × 10−6 to (2.08 ± 2.76) × 10−5 cm/s, as shown in Figure 5. The modified pentapeptide RVPSP had the highest Papp value at (2.08 ± 2.76) × 10−5 cm/s. Interestingly, Papp values of the other 10 pentapeptides were in the order PPP-X-X > RPP-X-X > PVP-X-X > RVP-X-X (where X represents Pro/P, Leu/L, or Ser/S), suggesting that the N-terminal Pro residue was more beneficial for intact transport of the pentapeptide than Val and Arg residues. Moreover, replacement of N-terminal amino acids was most informative and a high Papp arose when the Nterminal Arg residue and Val residue were both replaced by Pro, followed by replacement of Val alone, and then replacement of Arg alone. However, no similar results or other structure−transport relationships were observed at the Cterminus. Furthermore, there was no significant statistical relationship between Papp and molecular properties such as molecular weight, log D, molecular volume, molecular surface area, or polar surface area of the 11 pentapeptides. On the other hand, transport of terminal fragments resulting from cleavage of RVPSL was detected as shown in Figure 6. Except for the tetrapeptide VPSL, whose low transport rate might due to a Val residue at the N-terminal first position, all other fragment peptides had higher Papp values than the pentapeptide RVPSL, suggesting that the antihypertensive peptide RVPSL could be absorbed more easily through the Caco-2 cell monolayers in

Figure 3. Effects of diprotin A on bidirectional transport of RVPSL across Caco-2 cell monolayers. In the presence of diprotin A, Papp for BL-AP increased significantly (P < 0.001). However, this was still slightly lower than Papp for AP-BL, although the difference was not significant (P > 0.05). All values are means ± SD (n = 3). (*) P < 0.001 as compared to Papp for AP-BL in the absence of diprotin A; (#) P < 0.001 as compared to Papp for BL-AP in the absence of diprotin A.

Papp for AP-BL, (6.97 ± 1.11) × 10−6 cm/s, was approximately 3-fold higher than Papp for BL-AP, (2.52 ± 0.08) × 10−6 cm/s (P < 0.001). However, Papp for BL-AP increased significantly to (6.53 ± 0.08) × 10−6 cm/s in the presence of 1 mM diprotin A (P < 0.001). It was still slightly lower than Papp for AP-BL of (7.29 ± 0.81) × 10−6 cm/s in the presence of diprotin A; however, no significant difference was observed (P > 0.05). These results suggested that the transepithelial transport of RVPSL across Caco-2 cell monolayers was passive and nonpolarized. In addition, the susceptibility of peptides to brush border membrane peptidases might lead to an underestimate of Papp for BL-AP. Effects of Various Compounds on RVPSL Transport. To determine which transport pathways were involved in the transepithelial transport of RVPSL through the intestinal epithelium, Caco-2 cell monolayers were preincubated for 30 min before transport experiment with a range of inhibitors. As shown in Figure 4, in the presence of Gly-Sar, a good substrate for PepT1, the permeability coefficient Papp of RVPSL decreased slightly but not significantly (P > 0.05), suggesting that the transport of RVPSL across the Caco-2 cell monolayers was not mediated by PepT1. Sodium azide, an ATP synthesis inhibitor, also had little effect on the Papp of RVPSL (P > 0.05), indicating that transepithelial transport of RVPSL was energyindependent. In addition, the effect of transcytosis, initiated by endocytosis at the apical cell membrane, on RVPSL transport was examined by wortmannin, a transcytosis inhibitor. 8146

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Table 1. ACE-Inhibitory Activities of Synthetic Peptides

Figure 5. Effects of Pro residue replacement on transport of RVPSL across Caco-2 cell monolayers. Except for RVPSP, with the highest Papp value, Papp values of the other peptides were in the order PPP-X-X > RPP-X-X > PVP-X-X > RVP-X-X, suggesting that N-terminal Pro residues were more beneficial for intact transport of pentapeptides than Val or Arg residues. However, similar results were not observed at the C-terminus. All values are means ± SD (n = 3). For each measurement, the data marked by different letters are significantly different (P < 0.05).

no.

peptide sequence

IC50 (μM)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

RVPSL PVPSL RPPSL RVPPL RVPSP PVPPL PVPSP RPPPL RPPSP PPPSL RVPPP RVPS VPSL RVP PSL RV SL

20.00 5.51 >500 117.43 51.43 >500 152.97 47.25 357.18 >500 >500 >500 >500 152.39 >500 >500 >500

and RVPSP all exhibited high permeability through the Caco-2 cell monolayers and genuine ACE-inhibitory activity, suggesting their potential in functional food.

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DISCUSSION Food-derived antihypertensive peptides meet the desired properties of modulating physiological health and reducing hypertension through food. However, many of these peptides have limited practical applications due to their instability and low bioavailability in vivo.27 Thus, it is critical for bioactive peptides to overcome two physiological barriers: extensive enzymatic degradation in the gastrointestinal tract and poor permeability through the intestinal epithelium. In our previous work, the peptide RVPSL, identified from egg white ovotransferrin, had been shown to have ACE-inhibitory activity in vitro and antihypertensive activity in vivo in SHR. In addition, in a simulated gastrointestinal digestion experiment with pepsin and trypsin, 94.42% ± 0.41% of the initial RVPSL remained. From these positive results, in the present study, we investigated the transepithelial transport of RVPSL and its resistance to brush border membrane peptidases using Caco-2 cell monolayers. It has been demonstrated that Caco-2 cells express at least eight membrane peptidases in their apical surface, and these peptidases exhibit different activities at different growth stages. Among these peptidases, DPPIV has the highest activity, especially when Caco-2 cells are completely differentiated.28 DPPIV is a member of the prolyl oligopeptidase family of serine proteases and is highly expressed on endothelial cells, differentiated epithelial cells, and lymphocytes. It predominantly cleaves N-terminal dipeptides from proteins containing proline or alanine in the penultimate position.29 The activity of DPPIV can be specifically inhibited by diprotin A, which transitorily binds to the catalytic site of the enzyme and blocks the degradation of substrates.30 In the present study, we found that the antihypertensive peptide RVPSL was susceptible to brush border membrane peptidases. Approximately 36.31% ± 1.22% of the initial RVPSL (5 mM) added to the apical chamber was degraded, and this degradation significantly decreased to 23.49% ± 0.68% in the presence of diprotin A. On one hand, although diprotin A is a well-known inhibitor of DPPIV with an IC50 value of 1.1 μg/mL (3.22 μM)31,32 and is

Figure 6. Transport of terminal fragment peptides derived from RVPSL across Caco-2 cell monolayers. Most of the peptide fragments had higher Papp values than the pentapeptide RVPSL, suggesting that antihypertensive peptide RVPSL could be more easily absorbed across Caco-2 cell monolayers in smaller fragments, especially in the form of dipeptides and tripeptides. All values are means ± SD (n = 3). For each measurement, the data marked by different letters are significantly different (P < 0.05).

smaller fragments, especially in the form of dipeptides and tripeptides. Additionally, the ACE-inhibitory activities of the synthetic peptides are shown in Table 1. Besides the precursor peptide sequence RVPSL, which had an ACE-inhibitory IC50 value of 20.00 μM, peptides with the sequences PVPSL, RVPPL, RVPSP, PVPSP, RPPPL, RPPSP, and RVP all also exhibited ACE-inhibitory activity. Among these peptides, PVPSL showed the highest ACE-inhibitory activity with an IC50 value of 5.51 μM. RVPSP and RPPPL showed ACE-inhibitory activity with IC50 values of 51.43 and 47.25 μM, respectively. The remaining peptides all had very low ACE-inhibitory activities with IC50 values over 500 μM. However, the peptides PVPSL, RPPPL, 8147

DOI: 10.1021/acs.jafc.5b01824 J. Agric. Food Chem. 2015, 63, 8143−8150

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integrity and tightness of TJs of Caco-2 cell monolayers used in different laboratories may also have important influences on peptide transport. In addition, since Caco-2 cell monolayers have tighter TJs than human intestinal epithelial cells, the paracellular permeability of RVPSL in vivo in human intestinal epithelium is likely to be higher.42 There are three possible mechanisms for transport of oligopeptides: transcytosis, paracellular transport and PepT1mediated transport. There are two types of transcytosis, specific receptor-mediated transport and nonspecific pinocytotic transport, which are mainly responsible for the transport of some proteins and large molecular peptides,43 such as β-casein (193− 209), a 17-residue peptide, which may be absorbed intact through transcytosis via internalized vesicles.44 In contrast, PepT1 can transport small peptides, including a range of dipeptides and tripeptides, though not all.13 It is known that PepT1 is a low-affinity, high-capacity transporter and can be inhibited by its substrate glycyl dipeptide.45 The affinity of peptides for PepT1 depends on the structural characteristics and molecular properties of the peptides, such as charge, hydrophobicity, and side-chain flexibility.46 However, in the present study, the above two transport pathways were not involved in intact transport of antihypertensive peptide RVPSL across Caco-2 cell monolayers. Another transport mechanism is the paracellular pathway, which is mediated by TJs. The paracellular pathway involves the passive diffusion of watersoluble low molecular mass substances, including oligopeptides.47 Although TJs, which consist of several proteins such as zonula occludens 1 (ZO-1), occludins, and claudins, form a tight biological barrier against the permeation of exogenous macromolecules or cytotoxic substances, a great number of pores still exist in TJs.48 The pores usually have a radius no more than 15 Å and selectively allow some minerals and molecules to pass through.49 Studies have shown that the paracellular pathway is also involved in the transepithelial transport of many bioactive peptides. Again, the properties of peptides such as their molecular size, hydrophilicity, and some molecular surface properties all have important influences on their paracellular flux.15 In the present study, cytochalasin D, a disruptor of TJs, caused an approximately 1.5-fold increase of the permeability coefficient Papp of RVPSL compared to controls, suggesting that the paracellular pathway mediated by TJs might be the major transport route of RVPSL through the intestinal epithelium. This result is similar to that of other previously reported peptides, such as VLPVP,16 HLPLP,17 QIGLF,25 Gly-Ala-Hyp-Gly-Leu-Hyp-Gly-Pro,39 GGYR,24 and VPP.18 Proline is a key amino acid residue in ACE-inhibitory peptides and is usually found at the C-terminus of a large number of these peptides.1 In the present study on the relationship between RVPSL structure and transport, Pro was used to replace individual amino acids in RVPSL due to its structure, hydrophobicity, and relatively low molecular weight, which may play important roles in the transport of peptides.11,12,15 For instance, it had been previously demonstrated that dipeptides with C-terminal Pro residues but not Nterminal Pro residues exhibit high affinity for PepT1.50,51 However, it was also found that N-terminal, but not C-terminal, Pro residues had important roles on the transport of peptides across Caco-2 cell monolayers in this study. This finding has not been reported elsewhere and will need to be investigated further.

widely used to verify the action of DPPIV, 1 mM diprotin A may be insufficient to fully inhibit the protease. On the other hand, degradation of RVPSL may also be due to the action of other brush border membrane peptidases on the apical surface. The susceptibility of peptides to the membrane peptidases depends on their structural and chemical properties.33 Some human or bovine milk β-casomorphin-derived peptides have been reported to be very susceptible to DPPIV.34,35 In general, the N-terminal dipeptide (Xaa-Pro or Xaa-Ala) was regarded as easily cleaved by DPPIV specifically.29 In addition, N-terminal bulky hydrophobic or basic amino acid residues with an obligate free amino group are good substrates for cleavage by DPPIV.36 In terms of RVPSL, as the N-terminal Arg is a basic amino acid and contains a free amino group and Val is also a small molecular weight hydrophobic amino acid, these factors may contribute to its degradation by DPPIV. Although Pro residues in the third position are resistant to DPPIV hydrolysis,36 many natural peptides containing Xaa-Xaa-Pro at the N-terminus have been shown to have high binding affinity for DPPIV without being cleaved.37 This may also have an influence on the decrease of RVPSL on the apical side, as determined by HPLC. Caco-2 serves as an in vitro model of the human intestinal epithelium, so the expression of various brush border membrane peptidases in Caco-2 cells is essential. However, it is difficult to evaluate the real transport rate when the tested peptides are susceptible to these membrane peptidases.24 It has been found that the transport rate of some bioactive peptides increased significantly when membrane peptidases were inhibited by diprotin A.34,35 In the present study, we also determined the bidirectional transepithelial flux of RVPSL in the presence and absence of diprotin A. The results showed that the Papp value for AP-BL was 2.8-fold higher than that for BL-AP in the absence of diprotin A. However, when membrane peptidases were inhibited by diprotin A, there was a significant increase in the amount of RVPSL in the apical chamber in BLAP transport, leading to a significant increase in Papp for BL-AP transport but not AP-BL. As a consequence, no significant difference in the transport rate between AP-BL and BL-AP flux was observed in the presence of diprotin A, suggesting that the permeability of RVPSL across Caco-2 cell monolayers might be nonpolarized and passive. This result was consistent with transport of RVPSL by the paracellular pathway. Bioactive peptides, especially oligopeptides, are generally thought not to be absorbed intact through the human intestinal epithelium. Previous publications that have focused on the transport of bioactive peptides through in vivo intestine models and in vitro Caco-2 cell monolayers have shown low permeability for these peptides, with permeability coefficients Papp ranging from 1 × 10−8 to 1 × 10−6 cm/s. In this work, the antihypertensive pentapeptide RVPSL had good permeability across Caco-2 cell monolayers with a Papp value of (6.97 ± 1.11) × 10−6 cm/s, which is similar to that of human βcasomorphin-7, slightly higher than those of lactoferroxin A,34 VLPVP16 and QIGLF,25 and higher than the values of many other bioactive peptides reported elsewhere, such as KVLPVP,38 HLPLP,17 Gly-Ala-Hyp-Gly-Leu-Hyp-Gly-Pro,39 bovine β-casomorphin-5 and -7, casoxin-6,35 VYIHPF,40 and αs1-casein-(f91−97).41 Moreover, RVPSL even had a higher transport rate than the well-studied tripeptides VPP18 and YPI.10 The high permeability of RVPSL across Caco-2 cell monolayers may be due to its structural characteristics and molecular properties. On the other hand, differences in the 8148

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AUTHOR INFORMATION

Corresponding Author

*Telephone +86 431 87836351; fax +86 431 87836391; e-mail [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the National Natural Science Foundation of China (31271907, 31471597), Specialized Research Fund for the Doctoral Program of Higher Education (20130061110088), and Project of National Key Technology Research and Development Program ( 2012BAD33B03).

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ABBREVIATIONS RVPSL, Arg-Val-Pro-Ser-Leu; ACE, angiotensin-converting enzyme; SHR, spontaneously hypertensive rat; CVD, cardiovascular disease; PepT1, peptide transporter 1; TJs, tight junctions; Gly-Sar, glycyl-sarcosine; RP-HPLC, reverse-phase high-performance liquid chromatography; DMEM, Dulbecco’s modified Eagle’s medium; HBSS, Hanks’ balanced salt solution; DMSO, dimethyl sulfoxide; EDTA, ethylenediaminetetraacetic acid; ACN, acetonitrile; TFA, trifluoroacetic acid; TEER, transepithelial electrical resistance; DPPIV, dipeptidyl peptidase IV; Papp, apparent permeability coefficient

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