Transepithelial Transport Characteristics Research Articles

Transepithelial transport characteristics of Hydroxysafflor yellow A across cellular monolayers and the effects of the influx and efflux transporters

Hydroxysafflor yellow A (HSYA), the major biologically active constituent distributed in the florets of Carthamus tinctorius L. (safflower), is concerned with potential health-promoting functions against cardiovascular diseases. However, little information on clarifying the intestinal absorption characteristics of HSYA has been reported, which has limited its widespread application in the medical and healthcare fields. The intention of this research was to systematically characterize the transepithelial transport of HSYA using in vitro cell monolayer models (Caco-2, mucus-producing Caco-2/HT29-MTX-E12, MDCKⅡ and P-gp overexpressed MDCKⅡ-MDR1 cell monolayers). The apparent permeability coefficient (Papp) from the apical to the basolateral side of HSYA is approximately (4.30 ± 0.22) × 10⁻⁸ cm/s, suggesting that HSYA is poorly absorbed across the intestinal epithelium. The transport and uptake of HSYA depends on time, concentration, pH and temperature, and the Papp values increase with ethylene diamine tetraacetic acid pretreatment, indicating that the transport mechanisms of HSYA include passive diffusion (transcellular and paracellular pathways) and carrier-mediated transport. Meanwhile, transporter inhibitors were added in transport and uptake experiments, the results showed that carrier-mediated efflux and influx transport of HSYA is relevant to P-glycoprotein and H⁺ correlative transporters (e.g., peptide transporter 1, monocarboxylic acid transporter). In addition, the mucus layer acts as a barrier to HSYA absorption, as evidenced by a decrease in Papp (22–47%) and cellular uptake (18%) in the coculture model compared to the Caco-2 cell model. Detailed knowledge on the transepithelial transport of HSYA would provide insights to improve the pharmacological activities of HSYA and expand its application in food and healthcare fields.

Novel Casein-Derived Peptide-Zinc Chelate: Zinc Chelation and Transepithelial Transport Characteristics

Casein-derived peptides are recognized as promising candidates for improving zinc bioavailability through the form of a peptide-zinc chelate. In the present work, a novel 11-residue peptide TEDELQDKIHP identified from casein hydrolysate in our previous study was synthesized to investigate the zinc chelation characteristics. Meanwhile, the digestion stability and transepithelial transport of TEDELQDKIHP-Zn were also investigated. The obtained results indicated that the carboxyl groups (from Asp and Glu), amino groups (from Lys and His), pyrrole nitrogen group of Pro, and imidazole nitrogen group of His were responsible for zinc chelation. The complexation with zinc resulted in a more ordered structure of TEDELQDKIHP-Zn. In terms of digestion stability, the chelate of TEDELQDKIHP-Zn could remain stable to a large extent after gastric (78.54 ± 0.14%) and intestinal digestion (70.18 ± 0.17%). Moreover, TEDELQDKIHP-Zn was proven to be a well-absorbed biological particle with a Papp value higher than 1 × 10-6 cm/s, and it could be transported across the intestine epithelium through transcytosis. TEDELQDKIHP-Zn exhibited more bioavailable effects on zinc absorption and ALP activity than inorganic zinc sulfate.

Biological analysis of constituents in Spatholobi Caulis by UFLC-MS/MS: Enhanced quantification and application to permeability properties study in Caco-2 cell monolayer model

Major chemical constituents in medicinal materials are often used as the marker compounds of traditional Chinese medicine (TCM) for treating various diseases. For spatholobi caulis (SPC), it contains a variety of flavones, phenolic acid esters, and lignans which exert many pharmacological effects. However, the absorption and permeability properties of these constituents of SPC are still unclear and require further investigation. Different types and major compounds of SPC were chosen as representative constituents to study their absorption and transepithelial transport characteristics in the human intestinal epithelium-like Caco-2 cell monolayer model. 35 constituents of SPC were evaluated by using ultra fast liquid chromatography combined with electrospray ionization triple quadrupole tandem mass spectrometry (UFLC-MS/MS) method, acetonitrile and water containing with 0.5 mM ammonium acetate were used as mobile phase, these analytes with good linear relationships (R2 was within 0.9967−0.9998), precision (CV values were less than 10.23 %, LLOQ was less than 13.69 %), accuracy (Mean of inter- and intra-day were within 85.02 %−111.61 % and 85.50−112.97 %, respectively) and stability (The mean was within 85.07 %−113.93 %), among which 16 analytes showed good permeability, 5 analytes were considered to be poorly permeable compounds, and the other 14 analytes were assigned for the moderately absorbed compounds in Caco-2 cell monolayer model. The further results showed that the absorption mechanism of 7 well absorbed compounds, 8-O-methylretusin (1), genistein (7), spasuberol B (16), naringenin (18), isoliquiritigenin (19), 4-hydroxy-3-methoxy cinnamic acid methyl ester (23) and (+)-epipinoresinol (31) in SPC was mainly passive diffusion, their bidirectional transport rate was correlated with the concentration and transport time. The chemical structures of these compounds could affect the permeability properties on the cell monolayer. This study demonstrated the utility of Caco-2 cell monolayer model for evaluating the absorption properties and initial mechanisms of compounds in SPC in vitro, and provided important basis for predicting oral bioavailability of SPC compounds.

Transepithelial Transport Characteristics of the Cholesterol- Lowing Soybean Peptide, WGAPSL, in Caco-2 Cell Monolayers.

Recent studies have shown that soybean protein and its peptides have cholesterol-lowering activities. However, it is not clear whether these peptides could overcome physiological barriers, such as phase II metabolism in gastrointestinal tract and poor permeability, to reach the blood stream in its intact form. Therefore, the transepithelial transport characteristics of soybean peptide Trp-Gly-Ala-Pro-Ser-Leu (WGAPSL) with cholesterol- lowering activity were investigated in Caco-2 cells. In this study; the transepithelial absorption of WGAPSL was studied using human intestinal Caco-2 cell monolayers. The results showed that WGAPSL had good stability (83.9% ±1.98%) after simulated gastric and intestinal digestion. During the apical (AP) side to basolateral (BL) side transport, WGAPSL was absorbed intact through Caco-2 cell monolayers with apparent permeability coefficient (Papp) values of 4.4 × 10−8 to 1.2 × 10−8 cm/s. Cytochalasin D loosened the tight junctions of Caco-2 cell monolayers and significantly (p < 0.05) improved the transport process. Sodium azide, wortmannin, and Gly-Pro had minimal effects on transport, demonstrating that the major transport route of WGAPVL was paracellular via tight junctions. Finally, LC-MS analysis showed that Gly-Ala-Pro (GAP) was the important part for the intact absorption of WGAPVL and Trp (W) was the most unstable amino acid residue.

The transepithelial transport mechanism of polybrominated diphenyl ethers in human intestine determined using a Caco-2 cell monolayer

Oral ingestion plays an important role in human exposure to polybrominated diphenyl ethers (PBDEs). The uptake of PBDEs primarily occurs in the small intestine. The aim of the present study is to investigate the transepithelial transport characteristics and mechanisms of PBDEs in the small intestine using a Caco-2 cell monolayer model. The apparent permeability coefficients of PBDEs indicated that tri- to hepta-BDEs were poorly absorbed compounds. A linear increase in transepithelial transport was observed with various concentrations of PBDEs, which suggested that passive diffusion dominated their transport at the concentration range tested. In addition, the pseudo-first-order kinetics equation can be applied to the transepithelial transport of PBDEs. The rate-determining step in transepithelial transport of PBDEs was trans-cell transport including the trans-pore process. The significantly lower transepithelial transport rates at low temperature for bidirectional transepithelial transport suggested that an energy-dependent transport mechanism was involved. The efflux transporters (P-glycoprotein, multidrug resistance-associated protein, and breast cancer resistance protein) and influx transporters (organic cation transporters) participated in the transepithelial transport of PBDEs. In addition, the transepithelial transport of PBDEs was pH sensitive; however, more information is required to understand the influence of pH.

Transepithelial transport of rosuvastatin and effect of ursolic acid on its transport in Caco-2 monolayers

The aim of this study was to determine transepithelial transport characteristics of rosuvastatin and effect of ursolic acid (P-gp potential inhibitor) and ko143 (ABC transporters selective inhibitor) on its transport in Caco-2 monolayers. A reliable Caco-2 cell monolayers model was established. The TEER value was used to inspect integrity of cell model. Apparent permeability coefficients (Papp(BL-AP) and Papp(AP-BL)) were used to analyze transepithelial transport of rosuvastatin. Uptake of rosuvastatin was time- and concentration-dependent in Caco-2 cell. The ko143 but not ursolic acid had effect on the uptake of rosuvastatin in Caco-2 cell monolayer model and affected apparent permeability coefficient and apparent permeability of rosuvastatin. Active transport and passive diffusion absorption existed in transepithelial transport of rosuvastatin in Caco-2 cell model. Ursolic acid had no effect on transport of rosuvastatin in Caco-2 cell monolayer. The result indicated that ursolic acid may not cause effect on intestinal absorption of rosuvastatin.

Determination of the enantioselectivity of six chiral aryloxy aminopropanol drugs transport across Caco-2 cell monolayers

This study aimed to determine the transepithelial transport characteristics of chiral drug enantiomers across Caco-2 cell monolayers, a model of human intestinal epithelial membrane. Six chiral aryloxy enantiomers (atenolol, sotalol, celiprolol, carvedilol, metoprolol and propafenone) were tested in bi-directional transport studies. The separation and quantitation of these enantiomers were performed by reversed-phase high-performance liquid chromatography (RP-HPLC) using 2, 3, 4, 6-tetra-O-acetyl-β-d-glucopyranosyl isothiocyanate (GITC) as a pre-column derivatizing agent. Bi-directional transport studies demonstrated that celiprolol and carvedilol exhibited significant enantioselectivity in polarized transport at the concentration range tested. The efflux ratio (ER) for (R)-(+)-celiprolol was 8.96, but it was much lower for (S)-(-)-celiprolol which is 3.42 at the concentration of 96.0μM; carvedilol had the same transport behavior as celiprolol while the difference between the ER values of two enantiomers was not as significant as celiprolol at the concentration of 5.0μM. They are 2.41 for (R)-(+)-carvedilol and 1.98 for (S)-(-)-carvedilol. But in the transport studies of racemic atenolol, sotalol, metoprolol and propafenone, no enantioselective transport were observed over the concentration range tested. Because P-glycoprotein (P-gp) is highly expressed in Caco-2 cells, we inferred that P-gp might participate in the transport processes of celiprolol and carvedilol in chirally discriminative ways.

Absorption and transportation characteristics of scutellarin and scutellarein across Caco-2 monolayer model

To investigate the absorption and transepithelial transport characteristics of scutellarin and scutellarein in the human colonic adenocarcinoma cell (Caco-2) monolayer model. The influence factors on these two compounds' absorption were investigated, such as buffer solution, duration of culture, and inhibitors of multidrug resistance-associated protein 2 (MRP(2)), breast cancer drug resistance protein (BCRP) and P-glycoprotein (P-gp). By using Caco-2 monolayer as an intestinal epithelial cell model, the transport process was studied from apical (AP) side to basolateral (BL) side or from BL to AP. The two compounds were determined by high-performance liquid chromatography coupled with diode-array-detector detection. Transport parameters and apparent permeability coeffients (P(app)) were calculated. The P(app) values of scutellarin and scutellarein were different in two buffer solutions, respectively. In phosphate buffered saline, scutellarin had no absorption from AP to BL, while its P(app) value was 0.74×10(-6) to 1.58×10(-6) cm/s from BL to AP. The P(app) values of scutellarein were 4.33×10(-6) to 6.79×10(-6) cm/s and 1.32×10(-6) to 2.56×10(-6) cm/s from AP to BL and from BL to AP, respectively. The P(app) value gradually decreased with time. The absorption of scutellarein was better than that of scutellarin. The scutellarin absorption was improved by verapamil, MK-571 and reserpine. The scutellarein absorption was improved by verapamil whereas its excretion was improved by MK-571. Absorption of scutellarin is difficult in Caco-2 monolayer cells, which contributes to its low bioavailability. Scutellarein absorption is better than scutellarin absorption. Scutellarein transepithelial transport is passive diffusion. The inhibitor of P-gp can improve scutellarin and scutellarein transportation. The inhibitors of MRP(2) and BCRP can promote transportation of scutellarin. The inhibitor of MRP(2) can promote efflux of scutellarein. The multidrug resistance-associated protein may be the second reason for low bioavailability of scutellarin.

Intestinal Epithelial Cells Absorb γ‐Tocotrienol Faster than α‐Tocopherol

To elucidate the transepithelial transport characteristics of lipophilic compounds, the cellular uptake of tocopherol and tocotrienol isomers were investigated in Caco2 cell monolayer models. These vitamin E isomers formed mixed micelles consisting of bile salts, lysophospholipids, free fatty acid, and 2-monoacylglycerols, then the micelles were supplied to Caco2 cells. The initial accumulation of tocotrienol isomers in Caco2 cells was larger than those of corresponding tocopherol isomers. There was little difference among the cellular accumulations of four tocopherol isomers. These findings suggested that the difference between the molecular structures of the C16 hydrocarbon chain tail in tocopherol and tocotrienol was strongly responsible for the rapid epithelial transport into the Caco2 cells membranes rather than the difference in the molecular structures of their chromanol head groups. Furthermore, the secretion of alpha-tocopherol and gamma-tocotrienol from Caco2 cells was investigated using Caco2 cells plated on a transwell. The time courses of their secretions from Caco2 cells showed that the initial secretion rate of gamma-tocotrienol was also larger than that of alpha-tocopherol. To investigate the intestinal uptake of alpha-tocopherol and gamma-tocotrienol in vivo, the mice were fed single doses of alpha-tocopherol or gamma-tocotrienol with triolein. The gamma-tocotrienol responded faster in plasma than alpha-tocopherol, although the maximal level of gamma-tocotrienol was lower than that of alpha-tocopherol. This suggested that the intestinal uptake properties of administered alpha-tocopherol and gamma-tocotrienol would characterize their plasma level transitions in mice.

Transport characteristics of rutin deca (H-) sulfonate sodium across Caco-2 cell monolayers

The aim of this study was to explore potential transport mechanisms of rutin deca (H-) sulfonate sodium (RDS) across Caco-2 cell monolayers. As an in-vitro model of human intestinal epithelial membrane, Caco-2 cells were utilized to evaluate the transepithelial transport characteristics of this hydrophilic macromolecular compound. Bi-directional transport study of RDS demonstrated that the apparent permeability (P(app)) in the secretory direction was 1.4 approximately 4.5-fold greater than the corresponding absorptive P(app) at concentrations in the range 50.0 approximately 2,000 microM. The transport of RDS was shown to be concentration, temperature and pH dependent. In the presence of ciclosporin and verapamil, potent inhibitors of P-glycoprotein (P-gp)/MRP2, the absorptive transport was enhanced and secretory efflux was diminished. RDS significantly reduced the efflux ratio of the P-gp substrate rhodamine-123 in a fashion indicative of P-gp activity suppression, while rhodamine-123 competitively inhibited the polarized transport of the compound. In conclusion, the results indicated that RDS was likely a substrate of P-gp. Several efflux transporters, including P-gp, participated in the absorption and efflux of RDS and they might play significant roles in limiting the oral absorption of the compound. These observations offered important information for the pharmacokinetics of RDS.

Transepithelial transport of putrescine across monolayers of the human intestinal epithelial cell line, Caco-2.

To study the transepithelial transport characteristics of the polyamine putrescine in human intestinal Caco-2 cell monolayers to elucidate the mechanisms of the putrescine intestinal absorption. The transepithelial transport and the cellular accumulation of putrescine was measured using Caco-2 cell monolayers grown on permeable filters. Transepithelial transport of putrescine in physiological concentrations (> 0.5 mM) from the apical to basolateral side was linear. Intracellular accumulation of putrescine was higher in confluent than in fully differentiated Caco-2 cells, but still negligible (less than 0.5%) of the overall transport across the monolayers in apical to basolateral direction.EGF enhanced putrescine accumulation in Caco-2 cells by four fold, as well as putrescine conversion to spermidine and spermine by enhancing the activity of S adenosylmethionine decarboxylase. However, EGF did not have any significant influence on putrescine flux across the Caco-2 cell monolayers. Excretion of putrescine from Caco-2 cells into the basolateral medium did not exceed 50 picomoles, while putrescine passive flux from the apical to the basolateral chamber, contributed hundreds of micromoles polyamines to the basolateral chamber. Transepithelial transport of putrescine across Caco2 cell monolayers occurs in passive diffusion, and is not influenced when epithelial cells are stimulated to proliferate by a potent mitogen such as EGF.

Transepithelial transport of diphenhydramine across monolayers of the human intestinal epithelial cell line Caco-2.

The transepithelial transport characteristics of the antihistamine, diphenhydramine, were studied in human intestinal Caco-2 cell monolayers to elucidate the mechanisms of its intestinal absorption. The transepithelial transport and the cellular accumulation of diphenhydramine were measured using Caco-2 cell monolayers grown in Transwell chambers. The transepithelial transport of diphenhydramine from the apical to basolateral side was saturable, and the flux and cellular accumulation of diphenhydramine were dependent on the apical extracellular pH (pH 7.4 > 6.5 > 5.5). Transport and accumulation of diphenhydramine from the apical side were inhibited by another antihistamine, chlorpheniramine, while typical substrates for the renal organic cation transport system such as tetraethylammonium, cimetidine and guanidine had no effect. The transepithelial transport and cellular accumulation of diphenhydramine from the basolateral side were also pH-dependent and inhibited by chlorpheniramine. In addition, intracellular diphenhydramine preloaded was preferentially effluxed to the apical side, suggesting the involvement of the secretory pathway in diphenhydramine transport. Furthermore, diphenhydramine uptake from both the apical and basolateral sides was stimulated by preloading monolayers with chlorpheniramine (trans-stimulation effect). Transepithelial transport of diphenhydramine across Caco-2 cells is mediated by pH-dependent, specific transport systems that exist in both the apical and basolateral membranes.

TRANSEPITHELIAL TRANSPORT CHARACTERISTICS OF DIPHENHYDRAMINE ACROSS Caco-2 CELL MONOLAYERS

Transepithelial transport characteristics of diphenhydramine, an antihistamine, were studied in cultured human intestinal Caco-2 cell monolayers. In the apical-to-basolateral transport studies, the flux and cellular accumulation of diphenhydramine were influenced by the apical extracellular pH values. The accumulation and transepithelial transport were saturable and inhibited by chlorpheniramine. In the basolateral-to-apical transport, the accumulation and transport of diphenhydramine were also inhibited by chlorpheniramine. Diphenhydramine accumulation from both sides was stimulated by preloading the cells with chlorpheniramine, indicating the existence of specific transport systems in the both apical and basolateral membranes. Furthermore diphenhydramine was preferentially transported and effluxed to the apical direction. Finally, it was suggested that this transport system recognized tertiary amine compounds having a N-dimethyl or -diethyl residue in their structures. These results suggest that novel pH-dependent transport systems contribute to the intestinal absorption and secretion of tertiary amine compounds such as diphenhydramine.

Uptake and transport of the ACE-inhibitor ceronapril (SQ 29852) by monolayers of human intestinal absorptive (Caco-2) cells in vitro

The angiotensin-converting enzyme (ACE)-inhibitor ceronapril (SQ 29852) is shown to be a substrate of the intestinal dipeptide transporter. Uptake by Caco-2 cells, grown as confluent monolayers, follows a major saturable pathway ( K m, 0.91 ± 0.11 mM; ∼90% at 1 mM) together with a minor passive component ( k J, 32.3 ± 6.6 ng (10 6 cells) −1 (20 min) −1. Uptake was inhibited by competition with dipeptides such as l-AIa- l-Pro (K i, 2.96 mM) and l-Phe-Gly ( K i, 3.84 mM) but not by cephalosporins such as cephalexin. In contrast, transport was non-saturable, flux increased linearly with concentration and data were consistent with a passive transepithelial transport mechanism. Transport profiles showed a biphasic dependence upon time with an initial flux of 0.83 ± 0.02 ng insert −1 min −1 ( k 1) and a terminal value of 1.65 ± 0.08 ng insert −1 min −1 ( (k 2 ) at 100 μM. It is concluded that the basolateral efflux is retarded so that the passive paracellular transport controls the overall transepithelial transport characteristics in the Caco-2 model. Carrier-mediated uptake into intestinal enterocytres, followed by rate-limiting basolateral efflux, may explain the extended t max in vivo following oral administration.