Confluent Caco-2 Monolayers Research Articles

Effects of Bovine Colostrum with or without Egg on In Vitro Bacterial-Induced Intestinal Damage with Relevance for SIBO and Infectious Diarrhea.

Small intestinal bacterial overgrowth (SIBO) occurs commonly, is difficult to treat, and frequently recurs. Bovine colostrum (BC) and chicken eggs contain immunoglobulins and other components that possess antimicrobial, immunoregulatory, and growth factor activities; however, it is not known if they have the ability to reduce injury caused by the presence of bacteria associated with SIBO (Streptococcus, Escherichia coli, Staphylococcus, Bacteroides, Klebsiella, Enterococcus, and Proteus) and infectious diarrhea (enteropathogenic Escherichia coli, Salmonella). We examined the effects of BC, egg, or the combination, on bacterial growth and bacteria-induced changes in transepithelial electrical resistance (TEER) and bacterial translocation across confluent Caco-2 monolayers. BC, egg, or the combination did not affect bacterial growth. Adding bacteria to monolayers reduced TEER and (with minor variations among species) increased bacterial translocation, increased monolayer apoptosis (increased caspase-3 and Baxα, reduced Bcl2), increased intercellular adhesion molecule 1 (ICAM-1), and reduced cell adhesion molecules zonulin1 (ZO1) and claudin-1. BC, egg, or the combination reduced these effects (all p < 0.01) and caused additional increases in vascular endothelial growth factor (VEGF) and Heat Shock Protein 70 (Hsp70) expression. We conclude that BC ± egg strengthens mucosal integrity against a battery of bacteria relevant for SIBO and for infectious diarrhea. Oral BC ± egg may have clinical value for these conditions, especially SIBO where eradication of precipitating organisms may be difficult to achieve.

Method development for the quantitative determination of captopril from Caco-2 cell monolayers by using LC-MS/MS

Aim. Caco-2 cells are a human colon epithelial cancer cell line used as a model of human intestinal absorption of drugs and other compounds. Although compounds were used in the original Caco-2 cells monolayer assays, compounds have been replaced in most laboratories by the use of liquid chromatography-mass spectrometry (LC-MS) and LC-tandem mass spectrometry (LC-MS/MS). Mass spectrometry not only eliminates the need for compounds, but permits the simultaneous measurement of multiple compounds. The measurement of multiple compounds per assay reduces the number of incubations that need to be carried out, thereby increasing the throughput of the experiments. Furthermore, LC-MS and LC-MS-MS add another dimension to Caco-2 assays by facilitating the investigation of the metabolism of compounds by Caco-2 cells. A simple, rapid LC-MS/MS method has been developed for determination of captopril from confluent Caco-2 monolayers and from aqueous solution. Materials and methods. Chromatography was achieved on Discovery C18, 50 × 2.1 mm, 5 μm column. Samples were chromatographed in a gradient mode (eluent A (acetonitrile – water – formic acid, 5 : 95 : 0.1 v/v), eluent B (acetonitrile – formic acid, 100 : 0.1 v/v)). The initial content of the eluent B is 0%, which increases linearly by 1.0 min to 100% and to 1.01 min returns to the initial 0%. The mobile phase was delivered at a flow rate of 0.4 mL/min into the mass spectrometer ESI chamber. The sample volume was 5 μl. Results. Under these conditions, captopril was eluted at 1.42 min. A linear response function was established at 2 – 200 ng/mL. The regression equation for the analysis was y =0.0187x+0.000248 with coefficient of correction (r2) = 0.9993. According to the Caco-2 test results, captopril showed low permeability. It should be noted that the recovery value is 103.20%. The within-run coefficients of variation ranged between 0.321% and 0.541%. The within-run percentages of nominal concentrations ranged between 99.13% and 101.12%. The between-run coefficients of variation ranged between 0.314% and 0.663%. The between-run percentages of nominal concentrations ranged between 99.17% and 101.03%.The assay values on both the occasions (intra- and inter-day) were found to be within the accepted limits. Conclusion. From results of analysis, it can be concluded that developed method is simple and rapid for determination of captopril from confluent Caco-2 monolayers and from aqueous solution. Acquired results demonstrate that proposed strategy can be effortlessly and advantageously applied for examination of captopril from Caco-2 cell monolayers.

HPLC MS/MS method development for the quantitative determination of verapamil hydrochloride from Caco-2 cell monolayers

Aim. An understanding of the role that transporters, in particular P-glycoprotein (P-gp), can play in the absorption, distribution, metabolism and excretion (ADME) of candidate drugs, and an assessment of how these processes might impact on toxicity and the potential for drug-drug interactions in the clinic, is required to support drug development and registration. It is therefore necessary to validate preclinical assays for the in vitro evaluation of candidate drugs as substrates or inhibitors of human P-gp. 2. A simple, rapid HPLC MS/MS method was developed for determination of verapamil hydrochloride from confluent Caco-2 monolayers and from aqueous solution. Materials and methods. Chromatography was achieved on Discovery C18, 50 × 2.1 mm, 5 μm column. Samples were chromatographed in a gradient mode (eluent A (acetonitrile – water – formic acid, 5 : 95 : 0.1 v/v), eluent B (acetonitrile – formic acid, 100 : 0.1 v/v)). The initial content of the eluent B is 0%, which increases linearly by 1.0 min to 100% and to 1.01 min returns to the initial 0%. The mobile phase was delivered at a flow rate of 0.4 mL/min into the mass spectrometer ESI chamber. The sample volume was 5 μl. Results. Under these conditions, verapamil hydrochloride was eluted at 1.08 min. A linear response function was established at 1 – 100 ng/mL. The regression equation for the analysis was Y = 0.0162x + 0.00391 with coefficient of correction (R2) = 0.9992. According to the Caco-2 test results, verapamil showed low permeability. It should be noted that the recovery value for verapamil hydrochloride is 102.69%. The within-run coefficients of variation ranged between 0.336% and 0.617% for verapamil. The within-run percentages of nominal concentrations ranged between 98.82% and 100.62% for verapamil. The between-run coefficients of variation ranged between 0.334% and 0.612% for verapamil. The between-run percentages of nominal concentrations ranged between 98.97% and 101.76% for verapamil. The assay values on both the occasions (intra- and inter-day) were found to be within the accepted limits. Conclusion. From results of analysis, it can be concluded that developed method is simple and rapid for determination of verapamil hydrochloride from confluent Caco-2 monolayers and from aqueous solution. Acquired results demonstrate that proposed strategy can be effortlessly and advantageously applied for examination of verapamil hydrochloride from Caco-2 cell monolayers.

HPLC-MS/MS method development for the quantitative determination of nifedipine for Caco-2 permeability assay

Aim. Poorly water-soluble drugs such as nifedipineoffer challenging problems in drug formulation as poor solubility is generally associated with poor dissolution characteristics and thus with poor oral bioavailability (BCS class II drugs). Methods of quantitative determination of nifedipine by methods of spectrophotometry and chromatography are described in the scientific literature. However, methods are not developedfor examination of nifedipine from Caco-2 cell monolayers. Caco-2 cell monolayers have been extensively used for years as a tool to test permeability, assess the oral absorption potential and study the absorption mechanism of compounds. Therefore, the aim of this study was to develop and validate an efficient HPLC MS/MS method for determination of nifedipine from Caco-2 cell monolayers. Materials and methods. Chromatography was achieved on DiscoveryC18, 50 × 2.1 mm, 5 μm column. Samples were chromatographed in a gradient mode (eluent A (acetonitrile – water – formic acid, 5 : 95 : 0.1 v/v), eluent B (acetonitrile – formic acid, 100 : 0.1 v/v)). The initial content of the eluent B is 0%, which increases linearly by 1.0 min to 100% and to 1.01 min returns to the initial 0%. The mobile phase was delivered at a flow rate of 0.4 mL/min into the mass spectrometer ESI chamber. The sample volume was 5 μl. Results. Under these conditions, nifedipine was eluted at 1.83 min. A linear response function was established at 1 – 100 ng/mL. The regression equation for the analysis was Y = 0.0323x-0.00121 with coefficient of correction (R2) = 0.9987. According to the Caco-2 test results, nifedipine showed high permeability. The within-run coefficients of variation ranged between 0.331% and 0.619% for nifedipine. The within-run percentages of nominal concentrations ranged between 98.80% and 100.63% for nifedipine. The between-run coefficients of variation ranged between 0.332% and 0.615% for nifedipine. The between-run percentages of nominal concentrations ranged between 98.98% and 101.71% for nifedipine. The assay values on both the occasions (intra- and inter-day) were found to be within the accepted limits. Conclusion. From results of analysis, it can be concluded that developed method is simple and rapid for determination of nifedipine from confluent Caco-2 monolayers and from aqueous solution. Acquired results demonstrate that proposed strategy can be effortlessly and advantageously applied for examination of nifedipine from Caco-2 cell monolayers.

A HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY METHOD DEVELOPMENT FOR THE QUANTITATIVE DETERMINATION OF ENALAPRIL MALEATE FROM CACO-2 CELL MONOLAYERS

Objective: A simple, rapid high-performance liquid chromatography-mass spectrometry (HPLC MS/MS) method was developed for the determination of enalapril maleate from confluent Caco-2 monolayers and aqueous solution.Methods: Chromatography was achieved on Discovery C18, 50×2.1 mm, 5 μm column. Samples were chromatographed in a gradient mode (eluent A [acetonitrile-water-formic acid, 5: 95:0.1 v/v] and eluent B [acetonitrile-formic acid, 100:0.1 v/v]). The initial content of the eluent B is 0%, which increases linearly by 1.0 min to 100% and 1.01 min returns to the initial 0%. The mobile phase was delivered at a flow rate of 0.4 ml/min into the mass spectrometer ESI chamber. The sample volume was 5 μl.Results: Under these conditions, enalapril maleate was eluted at 1.52 min. According to the Caco-2 test results, enalapril showed low permeability. It should be noted that the recovery value for enalapril is 100.62 %. Low in vitro permeability data for enalapril are in good agreement with the literature data.Conclusion: From results of the analysis, it can be concluded that developed method is simple and rapid for determination of enalapril maleate from confluent Caco-2 monolayers and aqueous solution. Acquired results demonstrate that proposed strategy can be effortlessly and advantageously applied for examination of enalapril from Caco-2 cell monolayers.

QUANTITATIVE DETERMINATION OF AMLODIPINE FROM CACO-2 CELL MONOLAYERS BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY–MASS SPECTROMETRY/MASS SPECTROMETRY

Objective: A simple, rapid high-performance liquid chromatography–mass spectrometry/mass spectrometry (MS/MS) method was developed for the determination of amlodipine from confluent Caco-2 monolayers and from aqueous solution.Methods: Chromatography was achieved on Discovery C18, 50×2.1 mm, 5 μm column. Samples were chromatographed in a gradient mode (eluent A [acetonitrile-water-formic acid, 5:95:0.1 v/v], eluent B [acetonitrile-formic acid, 100:0.1 v/v]). The initial content of the eluent B is 0%, which increases linearly by 1.0 min to 100% and to 1.01 min returns to the initial 0%. The mobile phase was delivered at a flow rate of 0.400 ml/min into the MS electrospray ionization chamber. The sample volume was 5 μl.Results: Under these conditions, amlodipine was eluted at 1.61 min. According to the Caco-2 test results, amlodipine appeared to have moderate to high permeability. Recovery value for amlodipine is 52.33%. Caco-2 permeability values for amlodipine are in agreement with BCS Class I classification for these drugs and their known high bioavailability in humans.Conclusion: A new rapid method was developed for the determination of amlodipine from confluent Caco-2 monolayers and from aqueous solution. Acquired results demonstrate that proposed strategy can be effortlessly and advantageously applied for the examination of amlodipine from Caco-2 cell monolayers.

A HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY–MASS SPECTROMETRY/MASS SPECTROMETRY METHOD DEVELOPMENT FOR THE QUANTITATIVE DETERMINATION OF BISOPROLOL FROM CACO-2 CELL MONOLAYERS

Objective: A simple, rapid high-performance liquid chromatography–mass spectrometry (MS)/MS method was developed for the determination of bisoprolol from confluent Caco-2 monolayers and from aqueous solution.Methods: Chromatography was achieved on discovery C 18, 50 mm×2.1 mm, 5 μm column. Samples were chromatographed in a gradient mode (eluent A [acetonitrile:water:formic acid, 5:95:0.1 v/v] and eluent B [acetonitrile:formic acid, 100:0.1 v/v]). The initial content of the eluent B is 0%, which increases linearly by 1.0 min to 100% and to 1.01 min returns to the initial 0%. The mobile phase was delivered at a flow rate of 0.400 ml/min into the mass spectrometer ESI chamber. The sample volume was 5 μl.Results: Under these conditions, bisoprolol was eluted at 1.49 min. According to the Caco-2 test results, bisoprolol appeared to have moderate to high permeability. It should be noted that the recovery value for bisoprolol is 97.69%. Caco-2 permeability values for bisoprolol are in agreement with BCS Class I classification for these drugs and their known high bioavailability in humans.Conclusion: From results of analysis, it can be concluded that developed method is simple and rapid for the determination of bisoprolol from confluent Caco-2 monolayers and from aqueous solution. Acquired results demonstrate that proposed strategy can be effortlessly and advantageously applied for the examination of bisoprolol from Caco-2 cell monolayers.

Enterotoxigenic Escherichia coli infection and intestinal thiamin uptake: studies with intestinal epithelial Caco-2 monolayers

Infections with enteric pathogens like enterotoxigenic Escherichia coli (ETEC) is a major health issue worldwide and while diarrhea is the major problem, prolonged, severe, and dual infections with multiple pathogens may also compromise the nutritional status of the infected individuals. There is almost nothing currently known about the effect of ETEC infection on intestinal absorptions of water-soluble vitamins including thiamin. We examined the effect of ETEC infection on intestinal uptake of the thiamin using as a model the human-derived intestinal epithelial Caco-2 cells. The results showed that infecting confluent Caco-2 monolayers with live ETEC (but not with boiled/killed ETEC or nonpathogenic E. coli) or treatment with bacterial culture supernatant led to a significant inhibition in thiamin uptake. This inhibition appears to be caused by a heat-labile and -secreted ETEC component and is mediated via activation of the epithelial adenylate cyclase system. The inhibition in thiamin uptake by ETEC was associated with a significant reduction in expression of human thiamin transporter-1 and -2 (hTHTR1 and hTHTR2) at the protein and mRNA levels as well as in the activity of the SLC19A2 and SLC19A3 promoters. Dual infection of Caco-2 cells with ETEC and EPEC (enteropathogenic E. coli) led to compounded inhibition in intestinal thiamin uptake. These results show for the first time that infection of human intestinal epithelial cells with ETEC causes a significant inhibition in intestinal thiamin uptake. This inhibition is mediated by a secreted heat-labile toxin and is associated with a decrease in the expression of intestinal thiamin transporters.

Enteropathogenic E. coli Induces Intestinal Barrier Dysfunction by Exploiting Novel N-WASP-Mediated Cytoskeletal Junction Regulatory Activities

Several IBD-associated polymorphisms have been associated with genes encoding subunits of the Arp2/3 complex, which promotes the assembly of actin filaments. As the immediate upstream activator of the Arp complex, NWASP is poised to integrate multiple cytoskeletal regulatory pathways required for apical junction complex (AJC) stability and plasticity, and is also the target of bacterial effector proteins that enable important enteropathogens to disrupt intestinal barrier integrity. The EPEC effector protein EspF, which has been shown to disrupt intestinal barrier, specifically activates N-WASP and sorting nexin 9 (SNX9), a protein that directly interacts with N-WASP and is involved in coordinating endocytosis and endosomal sorting. To investigate the role of N-WASP in junction regulation and response to pathogens, we generated cell lines depleted of N-WASP and SNX9, and examined dynamic AJC function and the ability of EPEC to induce barrier dysfunction. Stable N-WASP (NWKD) and SNX9 knockdown (SNX9 KD) Caco-2 cell lines were generated using lentiviral constructs expressing shRNA. >90% depletion of N-WASP and SNX9 was confirmed by Western blot. Cells expressing nonsilencing siRNA for N-WASP or SNX9 were used as controls (WT). WT and KD cells were plated on 0.4 um Transwell filters and cultured over 21 days, with serial measurements of transepithelial electrical resistance (TER). Assessment of TER and immunofluorescence localization of SNX9 and junction molecules (occludin, ZO-1, E-cadherin) was performed in confluent Caco-2 monolayers at steady state and under conditions that induce TJ disassembly (calcium depletion). Fully confluent WT and NWKD cultures were infected with EPEC strain E2348/69, and TER serially assessed during the 12 hours post-infection. N-WASP knockdown led to decreased epithelial barrier integrity, reflected by lower maximal TER (14 days after plating: NWKD 153.8 Ωcm2 vs. WT 310.8 Ωcm2, P < 0.001). During calcium-switch, NWKD cells exhibited delayed translocation of occludin at 12 and 20 hours after calcium repletion, and a delay in ZO-1 localization to the AJC at 12 hours. During AJC disassembly induced by rapid removal of calcium, NWKD monolayers exhibited delayed loss of TER, reflecting loss of junction plasticity. By immunofluorescence, we found that SNX9 localized to tight junctions and co-localized with junction proteins, such as occludin, during AJC disassembly. Notably, in contrast to N-WASP knockdown, depletion of SNX9 potentiated epithelial barrier function (7 days after plating: SNX9 KD TER 748 Ωcm2 vs. WT 347.2 Ωcm2, P = 0.03). We hypothesized that EspF utilizes N-WASP to disrupt the AJC, and predicted that wild type EPEC should be attenuated in its ability to induce junction disruption in NWKD. Compared to WT controls, NWKD Caco-2 cells were resistant to EPEC-induced barrier dysfunction: four hours after infection, TER in WT cells decreased by 61.4% vs. 28.8% in NWKD monolayers (P < 0.01 compared to WT). N-WASP, the immediate upstream activator of the IBD-associated Arp2/3 complex, regulates intestinal epithelial barrier function and response to enteric pathogens. A complex of N-WASP and SNX9 specifically regulates the disassembly of intercellular junctions and may represent a future target of strategies for mitigating intestinal barrier defects central to IBD pathogenesis.

Enteral and parenteral nutrition distinctively modulate intestinal permeability and T cell function in vitro

Nutritional support is an established element of therapy for various indications. However, its impact on the mucosal barrier function is not well understood. We investigated the influence of EN and PN on intestinal epithelial cells and peripheral blood (PBMC) and lamina propria mononuclear cells (LPMC), all of which are involved in the mucosal defense against bacterial translocation and systemic inflammation. Integrity of epithelial cells was measured as transepithelial electrical resistance (TER) of confluent Caco-2 monolayers in the presence of 1% EN, PN and a parenteral amino acid mixture (AM). To determine wound healing capacities, an established migration model with IEC-6 cells was used. Furthermore, we investigated apoptosis, cell activation, proliferation and cytokine secretion of Caco-2, HT29 and of stimulated PBMC and LPMC cultured with or without 1 and 5% EN, AM or PN. We demonstrated that EN, AM and PN promoted the integrity of the epithelial monolayer and reconstituted epithelial cell continuity TGF-beta-dependently and -independently. Interestingly, only PN induced apoptosis and decreased the mitochondrial membrane potential. The activation status of PBMC was significantly reduced by EN and AM. Specifically, EN leads to an increased apoptosis rate, inhibited cell cycle progression and increased pro-inflammatory cytokine secretion. Both EN and PN reduced the activation status and the release of pro- and anti-inflammatory cytokines. Our study provides evidence that by promoting wound healing and regulating T cell function, EN, AM, and PN potently interact with the intestinal barrier and immune system, thus justifying its use in diseases accompanied by impaired mucosal barrier function.

The Intestinal Epithelial Cell Modulates the Effect of Alcohol on Neutrophil Inflammatory Potential

Ethanol (EtOH) intoxication increases posttraumatic infectious complications. EtOH is thought to be immunosuppressive, but the effect of the interaction with the gut and associated microflora on host defense is unknown. We studied the ability of intestinal epithelial cells and bacteria to modulate EtOH effects on polymorphonuclear (PMN) cell function in vitro. Confluent Caco2 monolayers were exposed to 0.1% EtOH with or without Escherichia coli on the apical side of each chamber. Supernatants were collected from the basal side of the chambers and incubated with neutrophils. Controls were incubated with or without 0.1% EtOH alone. Chemotaxis, elastase, and superoxide anion release, and CD11b measurements were undertaken in these neutrophils and compared with controls. Alcohol exhibited differing effects on the inflammatory potential of neutrophils in regards to CD11b expression, elastase production, and superoxide production. Direct effects of EtOH were anti-inflammatory, whereas indirect effects of EtOH on neutrophils modulated by Caco2 cells were proinflammatory. Chemotaxis was significantly reduced when neutrophils were directly treated with alcohol. EtOH direct downregulates PMN inflammatory potential, but its impact of the intestinal epithelial cells produces a proinflammatory microenvironment for PMNs. The interaction of EtOH on the intestinal milieu may lead to neutrophil activation and increased organ injury after trauma in the intoxicated patient.

P260: Ethanol effects proinflammatory state of neutrophils in shock

Introduction: Ethanol (EtOH) intoxication plays an important role in the etiology of traumatic events and has often been described as having immunosuppressive effects. EtOH has been shown to effect intestinal barrier function in prior studies. The ability of gut derived factors on neutrophil function in this setting is unknown. This study looks at the role of ethanol in modulating proinflammatory states in the neutrophil in vitro. Methods: Confluent Caco2 monolayers were exposed to 0.1% EtOH and/or Escherichia Coli C-25 (EC) under normoxia (21%O2) or hypoxia (5%O2) followed by normoxic conditions (H/R).

Modulatory effects of quercetin on proliferation and differentiation of the human colorectal cell line Caco-2

The effect of the dietary flavonoid quercetin was investigated on proliferation and differentiation of the human colon cancer cell line Caco-2. Confluent Caco-2 monolayers exposed to quercetin showed a biphasic effect on cell proliferation and a decrease in cell differentiation (0.001< P<0.05). During differentiation Caco-2 cells formed 5 phase II metabolites, of which the amount of 4′- O-methyl-quercetin-3′- O-glucuronide correlated with the differentiation grade ( r=0.99, P<0.003). The increment of cell proliferation at low quercetin concentrations and the decrease in cell differentiation are effects opposite to what would be expected for a functional food ingredient with anti-carcinogenic potential.

Increased permeability of intestinal epithelial monolayers mediated by electroporation

This study assessed whether electroporation enhances transport across intact intestinal epithelial monolayers that mimic the intestinal epithelium. Confluent Caco-2 monolayers were exposed to electroporation pulses and then monitored over time for transepithelial transport of calcein, a small fluorescent tracer, or fluorescein-labeled bovine serum albumin, a large protein. Cumulative transport of both molecules across the monolayers increased significantly (up to 34-fold) after electroporation and depended on electroporation voltage and pulse length and on molecular size. Increased transport was accompanied by a decrease in the transepithelial electrical resistance of the monolayers. Further analysis of these results suggests that the increase in transport observed after electroporation is due, at least in part, to the killing of a small fraction of cells, which increased transport across “leaky” dead cells that remained adherent and increased transport through small, temporary holes left by dead cells that detached, but appeared to reseal within minutes by monolayer restitution. These findings could form the basis for the development of electroporation as a clinical tool to increase intestinal permeability and, thereby, increase the absorption of poorly absorbed drugs.

A novel, rapid and sensitive heterotypic cell adhesion assay for CD2–CD58 interaction, and its application for testing inhibitory peptides

The immunoglobulin CD2 is a cell adhesion molecule that mediates T-cell activation by binding to its receptor CD58 on antigen-presenting cells (APCs). Modulation or inhibition of this interaction has been shown to be therapeutically useful. E-rosetting assay is usually applied in the study of the modulation of CD2–CD58 interaction. In this study, we demonstrated a novel, rapid and sensitive heterotypic cell adhesion assay for CD2–CD58 interaction. The CD2 expression on the surface of Jurkat cells and the CD58 expression on the Caco-2 cells were confirmed by flow cytometry and ELISA studies, respectively. Then Jurkat cells were fluorescent-labeled with 2 μM of BCECF-AM for 45 min at 37 °C before adding to confluent Caco-2 monolayers cultured in 96-well culture dishes. After 30 min, non-adherent Jurkat cells were removed by washing with PBS, while the monolayer-associated Jurkat cells were lysed with 0.5 ml of 2% Triton X-100 in 0.1 M NaOH. Fluorescence (FL) was quantitated using a microplate fluorescence analyzer with BCECF's excitation maximum of 485 nm and emission maximum of 535 nm. This method was successfully applied for testing inhibitory peptides to CD2–CD58 interaction.

Quantitative determination of saquinavir from Caco-2 cell monolayers by HPLC-UV. High performance liquid chromatography.

The validation and quantitative determination of the protease inhibitor, saquinavir, from confluent Caco-2 monolayers and from aqueous solution is reported. The high performance liquid chromatographic method consisted of an Ultramex 5 C(8) reverse-phase column (250 x 4.6 mm i.d.) and a mobile phase of acetonitrile:water:triethylamine (55:44:1, v/v/v, pH 6.5). Samples were analyzed using an ultraviolet detector at 238 nm, and diltiazem hydrochloride (66 micro g/mL) was used as an internal standard. A linear response over a broad concentration range (0.4-8.0 micro g/mL, r(2) = 0.997) was obtained. The limit of detection and quantitation was set at 0.14 and 0.4 micro g/mL, respectively. Over a 4 day period, the intra-day and inter-day precision ranged from 1 to 7% with a mean of 4%, and from 1 to 2% with a mean of 1.5%, respectively. Bench-top and storage stability of saquinavir was found to be satisfactory. The permeability of saquinavir through Caco-2 monolayers was estimated using this assay.

Posttranslational Alanine Trans-Stimulation of Zwitterionic Amino Acid Transport Systems in Human Intestinal Caco-2 Cells

Background. Neutral dietary amino acids, such as alanine, are transported across the gut lumen by both Na+-dependent (System B) and Na+-independent (System L) carriers, but the nature of the acute phase of substrate-induced uptake is unknown. This study examined the effects of acute amino acid substrate exposure on the rapid modulation of apical membrane alanine transport in cultured human intestinal cells.Methods. System B and System L transport activity kinetics, as well as ATB0 mRNA levels, were measured in confluent Caco-2 monolayers treated with various metabolic agents during short-term and extended time periods.Results. Depleting the incubation medium of alanine attenuated both System B and System L uptake activities within 30 mins, with a complete return to baseline values within 3 h. Extracellular alanine added to depleted Caco-2 cells rapidly (within 5 min) increased alanine transport activities. Kinetic analysis showed that acute alanine exposure increased both Km and Vmax of each transport system, indicative of a trans-stimulation effect. Augmenting intracellular alanine levels using the cytosolic alanine aminotransferase inhibitor, aminooxyacetic acid, increased alanine uptake activity. Acute exposure to other substrates of Systems B and L also increased the uptake of alanine, while nonsubstrates did not affect alanine uptake. Cycloheximide or actinomycin did not affect substrate acute activation of System B, and the steady-state level of ATB0 mRNA was not altered by amino acid exposure.Conclusion. Increasing alanine availability to intestinal cells, by either exogenous substrate exposure or inhibition of intracellular catabolism, acutely and reversibly increases apical membrane alanine transport activity via a posttranslation trans-stimulation mechanism.

Effects of pluronic block copolymers on drug absorption in Caco-2 cell monolayers.

The present work characterizes the effects of Pluronic copolymers on the transport of a P-gp-dependent probe, rhodamine 123 (R123) in Caco-2 cell monolayers. The accumulation and efflux studies were performed on the confluent Caco-2 monolayers using fluorescent probes with and without Pluronic copolymers. At concentrations below the critical micelle concentration single chains ("unimers") of Pluronic P85 enhanced the accumulation and inhibited the efflux of R123 in Caco-2 monolayers. The transport of the P-gp-independent probe, rhodamine 110 was not altered under these conditions. In contrast the micelles increased R123 accumulation to a much lower extent when compared to the unimers and enhanced R123 efflux in Caco-2 monolayers. Pluronic P85 unimers increase accumulation of a P-gp-dependent drug in Caco-2 monolayers through inhibition of the P-gp efflux system. The mechanism of the micelle effect is not known, however, it is very similar to the micelle effects in BBMEC. This has been previously shown to involve vesicular transport of the micelle-incorporated drug. The study suggests that Pluronic copolymers can be useful in increasing oral absorption of select drugs.

β-Carboxylic acid esterified d-Asp-Ala retains a high affinity for the oligopeptide transporter in Caco-2 monolayers

d-Asp-Ala, a metabolically stable dipeptide, possesses a relatively high affinity for the Caco-2 oligopeptide transporter ( IC 50 = 5.75 ± 0.09 mM) as demonstrated by its ability to compete with [ 14c]Gly-Sar in cellular uptake experiments. When the β-carboxylic acid of d-Asp-Ala is modified by esterification with a cyclohexyl group ( d-Asp(OcHx)-Ala) or a benzyl group ( d-Asp(OBzl)-Ala), the resulting compounds are still able to inhibit [ 14c]GlySar binding to the oligopeptide transporter, i.e., IC so values for d-Asp(OcHx)-Ala and d-Asp(OBzl)-Ala were 2.80 ± 0.11 and 2.62 ±0.35 mM, respectively. HPLC analysis shows that both d-Asp-(OcHx)-Ala and d-Asp(OBzl)-Ala are fully resistant to degradation for up to 5 h when incubated in the apical media of confluent Caco-2 monolayers. These results demonstrate that it is possible to covalently modify the side chain of one amino acid in an enzymatically stabilized dipeptide with small, aromatic molecules while enabling them to retain their affinity for the oligopeptide transporter.

Regulation of riboflavin intestinal uptake by protein kinase A: studies with Caco-2 cells.

Although the mechanism of riboflavin (RF) intestinal uptake has been the subject of many studies, virtually nothing is known about the cellular regulation of the uptake process. In the present study, we investigated the role of protein kinase A (PKA)- and C (PKC)-mediated pathways in the regulation of RF intestinal uptake using the confluent Caco-2 monolayers. Treatment of Caco-2 cells with 3-isobutyl-1-methylxanthine (IBMX), forskolin, cholera toxin, or dibutyryl adenosine 3',5'-cyclic monophosphate caused a significant inhibition in RF uptake. The inhibitory effect of IBMX was reversible and resulted from a significant decrease in the maximal velocity of the RF uptake process with no change in its apparent Michaelis constant. The IBMX-induced inhibition in RF uptake was not mediated via inhibition in the synthesis of the RF carrier protein or through inhibition in the recruitment of preexisting carrier protein into the plasma membrane. Calyculin A also inhibited RF uptake when added alone and further potentiated the inhibitory effect of IBMX when added together. Phorbol 12-myristate 13-acetate or chelerythrine, on the other hand, showed no significant effect on RF uptake. These results demonstrate for the first time that compounds that increase intracellular cAMP levels downregulate RF intestinal uptake and that this effect is mediated via a decrease in the activity of the RF uptake carrier. It is suggested that a PKA-mediated pathway(s) plays an important role in regulating RF intestinal uptake.