自微乳化系统对细胞紧密连接蛋白的影响

目的从分子细胞水平考察正、负电荷自微乳化系统对细胞紧密连接蛋白复合体的影响。方法建立了模拟小肠上皮细胞结构的Caco-2细胞模型;测定对跨膜电阻和细胞间转运物质甘露醇转运评价制剂对细胞完整性的影响。采用免疫荧光法评价两种自微乳化系统不同稀释倍数对细胞紧密连接蛋白ZO-1和细胞骨架肌动蛋白(actin)的影响。结果负电荷自微乳化系统在不同稀释倍数对跨膜电阻都无显著性影响。正电荷处方在考察的3种稀释倍数显著性降低了跨膜电阻(P<0.05)。细胞单分子层经正电荷自微乳制剂处理2 h，再经过48 h培养，较高稀释倍数跨膜电阻能够完全恢复；50倍稀释的处方不能完全恢复(81.3%)。两种制剂在不同稀释倍数都能显著提高甘露醇的渗透系数(2.9～64.6倍)(P<0.05)，作用与自微乳稀释倍数具有相关性。免疫荧光结果表明，经制剂处理后，影响了actin和ZO-1细胞分布，呈现不连续性。正电荷处方由于静电吸引，可能对细胞膜产生压力，比负电荷处方更能影响actin的分布。对细胞紧密连接影响具有制剂稀释倍数的依赖性。结论自微乳化系统能够提高甘露醇的细胞间转运。通过影响actin和ZO-1的细胞膜分布的作用机制打开细胞紧密连接。     

Disruption of Epithelial Tight Junctions by Yeast Enhances the Paracellular Delivery of a Model Protein

Purpose The aim of this study was to investigate the effect of heat-killed yeast cells on the integrity of epithelial tight junctions in vitro.Methods Changes in barrier potential of Caco-2 cell monolayers were assessed by transepithelial electrical resistance (TEER) measurements and by an increasing permeability to a marker protein, horse–radish peroxidase (HRP). Visualisation of tight junction disruption was carried out directly through electron microscopy and indirectly through fluorescence confocal microscopy and immunoblotting of the tight junction-associated proteins zonula occludens ZO-1, occludin and actin.Results Yeast cells opened tight junctions in a reversible dose- and time-dependent manner, as shown by a decrease in TEER and an increase in HRP permeability. These changes to barrier potential were shown not to be due to cytotoxic effects but due to modulation of the tight junctions. ZO-1, actin and occludin proteins were demonstrated to be involved in yeast-induced tight junction opening through the use of confocal microscopy and western blotting. Electron microscopy confirmed a direct opening of tight junctions after application of yeast.Conclusion Yeast modulated epithelial tight junctions in a reversible manner by contraction of the actin cytoskeleton and shift of ZO-1 and occludin tight junction proteins from the membrane to cytoskeletal areas of the cell.     

Modulation of the Tight Junctions of the Caco-2 Cell Monolayers by H2-antagonists

Purpose. The tight junctions in the intestinal epithelium represent highly specialized intercellular junctions. Ranitidine, an H₂-antagonist, causes a tightening of the tight junctions. Hence, we have investigated the effect of ranitidine and other H₂-antagonists on the function of the intestinal tight junctions. Methods. Effect of the H₂-antagonists on the tight junctions has been investigated using the transepithelial electrical resistance (TEER) and the transport of mannitol across the Caco-2 cell monolayers. Results. Four different H₂-antagonists caused an increase in the TEER across the Caco-2 cell monolayers, accompanied by a decrease in the permeability for mannitol. The effect was concentration-dependent and saturable. Ranitidine and famotidine, caused a decrease in their own transport rate across the Caco-2 cells. Ranitidine competitively inhibited the increase in TEER caused by famotidine, whereas compounds which represent molecular fragments of ranitidine had no effect. The relative potency of the four H₂-antagonists in causing an increase in the TEER correlated inversely with the oral bioavailability of these compounds in humans. Conclusions. We hypothesize that the H₂-antagonists exert their effect on the tight junctions of Caco-2 cells by modulation of interactions among proteins associated with the tight junctional complex.     

Modulation of tight junctions does not predict oral absorption of hydrophilic compounds: Use of caco-2 and calu-3 cells

Permeability estimates using Caco-2 cells do not accurately predict the absorption of hydrophilic drugs that are primarily absorbed via the paracellular pathway. The objective of this study was to investigate whether modulation of tight junctions would help differentiation of paracellularly absorbed compounds. Tight junctions in Caco-2 cell monolayers were manipulated using calcium depletion approaches to decrease the transepithelial electrical resistance (TEER) of the monolayers, and permeability of hydrophilic compounds were measured under these conditions. Permeability of these compounds were also measured in Calu-3 cells, which have tighter junctions than Caco-2 cells. Calcium depletion loosened the tight junctions of Caco-2 cells to varying levels as measured by the decrease in TEER values of the monolayers. While the absolute permeability of all the model compounds increased as the tight junctions were loosened, the ratios of their permeability relative to mannitol permeability were similar. The permeability of these compounds in the tighter Calu-3 cells were also found to be similar to each other. Altering the tight junctions of Caco-2 cells to obtain leakier cell monolayers, or using a cell line with tighter junctions like Calu-3 cells, did not improve differentiation between well absorbed and poorly absorbed hydrophilic drugs. Mere manipulation of the tight junctions to increase or decrease transepithelial electrical resistance does not appear to be a viable approach to predict human absorption for hydrophilic compounds that are primarily absorbed via the paracellular pathway.     

Effect of Chitosan on Epithelial Cell Tight Junctions

PURPOSE: Chitosan has been proposed as a novel excipient for transepithelial drug-delivery systems. Chitosan is thought to disrupt intercellular tight junctions, thus increasing the permeability of an epithelium. The effect of chitosan on tight junction complex was investigated at the molecular level. METHODS: Changes in barrier properties of Caco-2 cell monolayers, including transepithelial electrical resistance and permeability to horseradish peroxidase (HRP), were assessed in response to chitosan treatment. Changes in subcellular localization of the tight junction proteins zona occludens 1 (ZO-1) and occludin by immunofluorescence and Western blotting of cellular fractions were also assessed. RESULTS: Chitosan was found to cause a dose-dependent reduction in transepithelial electrical resistance of Caco-2 monolayers of up to 83%. A corresponding increase in horseradish peroxidase permeability of up to 18 times greater than the control was also observed across the monolayer. Immunofluorescent localization of ZO-1 revealed loss of membrane-associated ZO-1 from discrete areas. Analysis of cellular fractions revealed a dose-dependent loss of ZO-1 and occludin from the cytosolic and membrane fractions into the cytoskeletal fraction. These changes did not occur because of chitosan-mediated ATP depletion. CONCLUSIONS: Chitosan-mediated tight junction disruption is caused by a translocation of tight junction proteins from the membrane to the cytoskeleton.     

Dodecylphosphocholine-mediated enhancement of paracellular permeability and cytotoxicity in Caco-2 cell monolayers

The intestinal epithelium is a significant barrier for oral absorption of hydrophilic drugs because they cannot easily traverse the lipid bilayer of the cell membrane and their passage through the intercellular space (paracellular transport) is restricted by the tight junctions. In this report we show that dodecylphosphocholine (DPC) can improve the paracellular permeability of hydrophilic compounds across Caco-2 cell monolayers by modulating the tight junctions. The results show that the alkyl chain as well as the zwitterionic head group of DPC are required for its activity. DPC appears to act by modulating the permeability of tight junctions as evidenced by the fact that treatment of Caco-2 cell monolayers by this agent results in a decreased transepithelial electrical resistance (TEER), increased permeability of paracellular markers (e. g., mannitol) with no change in the permeability of the transcellular marker testosterone, and redistribution of the tight junction-associated protein ZO-1. The effect of DPC on Caco-2 cells (e.g., decrease in TEER) is reversible, and is not caused by gross cytotoxicity (as indicated by the MTT test) or by nonspecific disruption of the cell membrane (as indicated by only slight nuclear staining due to the nonpermeable DNA-specific dye propidium iodide). We propose in the present study a parameter, potency index, that allows comparison of various enhancers of paracellular transport in relation to their cytotoxicity. The potency index is a ratio between the IC(50) value (concentration at which 50% inhibition of control mitochondrial dehydrogenase activity occurs in the MTT test) and the EC(50) value (concentration at which TEER drops to 50% of its control (untreated) value). By this parameter, DPC is significantly safer than the commonly used absorption enhancer palmitoyl carnitine (PC), which has the potency index of approximately 1 (i.e., no separation between effective and toxic concentration).     

Mechanism of action of ZOT-derived peptide AT-1002, a tight junction regulator and absorption enhancer

Tight junctions (TJs) are intercellular structures that control paracellular permeability and epithelial polarity. It is now accepted that TJs are highly dynamic structures that are regulated in response to exogenous and endogenous stimuli. Here, we provide details on the mechanism of action of AT-1002, the active domain of Vibrio cholerae's second toxin, zonula occludens toxin (ZOT). AT-1002, a hexamer peptide, caused the redistribution of ZO-1 away from cell junctions as seen by fluorescence microscopy. AT-1002 also activated src and mitogen activated protein (MAP) kinase pathways, increased ZO-1 tyrosine phosphorylation, and rearrangement of actin filaments. Functionally, AT-1002 caused a reversible reduction in transepithelial electrical resistance (TEER) and an increase in lucifer yellow permeability in Caco-2 cell monolayers. In vivo, co-administration of salmon calcitonin with 1 mg of AT-1002 resulted in a 5.2-fold increase in AUC over the control group. Our findings provide a mechanistic explanation for AT-1002-induced tight junction disassembly, and demonstrate that AT-1002 can be used for delivery of other agents in vivo.     

Effect of Rhamnolipids on Permeability Across Caco-2 Cell Monolayers

Purpose

This report describes the effect of rhamnolipids (RLs), an amphiphilic biosurfactant produced by the bacterium Pseudomonas aeruginosa, on the integrity and permeability across Caco-2 cell monolayers.

Methods

We measured the trans-epithelial electrical resistance (TEER) and permeability of [¹⁴C]mannitol across Caco-2 cell monolayers upon incubation with 0.01–5.0% v/v RLs as a function of incubation time (30, 60, 90, and 120 min). We also studied the recovery of RL-treated Caco-2 cell monolayers upon incubation with Kaempferol, which is a natural flavonoid that promotes the assembly of the tight junctions.

Results

TEER of Caco-2 cell monolayers incubated with 0.01–5.0% v/v RLs solution dropped to 80–28% of that of untreated cells. Decline in TEER was associated with an increase in [¹⁴C]mannitol permeability as a function of RLs concentration and incubation time with Caco-2 cells. Incubation of RLs-treated Caco-2 cell monolayers with normal culture medium for 48 h did not restore barrier integrity. Whereas, incubation of a RLs-treated Caco-2 cells with culture medium containing Kaempferol for 24 h restored barrier function indicated by the higher TEER and lower [¹⁴C]mannitol permeability values.

Conclusions

These results show the ability of RLs to modulate the integrity and permeability of Caco-2 cell monolayers in a concentration- and time-dependent fashion, which suggest their potential to function as a non-toxic permeation enhancer.     

Cyclodextrins in Nasal Delivery of Low-Molecular-Weight Heparins: In Vivo and in Vitro Studies

PURPOSE: To test the hypothesis that cyclodextrins reversibly enhance nasal absorption of low-molecular-weight heparins (LMWHs) and to investigate the mechanisms by which cyclodextrins enhance LMWH absorption via the nose. METHODS: Absorption of LMWHs was studied by measuring plasma anti-factor Xa activity after nasal administration of various LMWH formulations to anesthetized rats. In vivo reversibility studies were performed to investigate if the effects of cyclodextrins are reversible and diminish with time. The absorption-enhancing mechanisms of cyclodextrins were investigated in cell culture model. The transport of enoxaparin and mannitol, changes in transepithelial electrical resistance (TEER), and distribution of tight junction protein ZO-1 were investigated. RESULTS: Formulations containing 5% dimethyl-beta-cyclodextrin (DMbetaCD) produced the highest increase in the bioavailability of LMWH preparations tested. In vivo reversibility studies with 5% DMbetaCD showed that the effect of the absorption enhancer at the site of administration diminished with time. Transport studies using 16HBE14o(-) cells demonstrated that the increase in the permeability of enoxaparin and mannitol, reduction in TEER, and the changes in the tight junction protein ZO-1 distribution produced by 5% DMbetaCD were much greater than those produced by beta-cyclodextrin (betaCD) or hydroxyl-propyl-beta-cyclodextrin (HPbetaCD). CONCLUSIONS: Of the cyclodextrins tested, DMbetaCD was the most efficacious in enhancing absorption of LMWHs both in vivo and in vitro. The study also suggests that cyclodextrins enhance nasal drug absorption by opening of cell-cell tight junctions.     

Melittin as an Epithelial Permeability Enhancer I: Investigation of Its Mechanism of Action in Caco-2 Monolayers

Purpose Melittin is an amphipathic antimicrobial peptide which has been shown to enhance the permeability of mannitol and reduce transepithelial electrical resistance (TER) across Caco-2 monolayers. The aim of this work was to further examine the potential of melittin as a paracellular permeability enhancer and to investigate the mechanism of interaction with tight junction proteins in Caco-2. Materials and Methods The permeability of a range of fluorescent markers of differing molecular weights across monolayers was examined and immunofluorescence and western blotting analysis of tight junction proteins were also carried out. The mechanism of TER reduction was also examined using cell signalling inhibitors. Results Apical but not basolateral addition of melittin increased the permeability of a range FITC-dextrans (4–70 kDa) across monolayers. Melittin effects were reversible and no cytotoxicity was evident in polarized Caco-2 epithelia at the concentrations used. Altered expression of ZO-1, E-cadherin and F-actin was also detected. The phospholipase A2 inhibitors, aristolochic acid and indomethacin and the cyclooxygenase inhibitor, piroxicam, partially attenuated melittin-induced TER reduction, suggesting that part of the mechanism by which melittin opens tight junctions involves prostaglandin signalling. Conclusions Apically-added melittin opens tight junctions, causing dramatic TER reductions with significant increases in flux of dextrans. These effects appear mediated in part via PLA2 and involve alterations in specific tight junction proteins.     

Rho激酶在氢气保护LPS诱导的Caco-2细胞上皮屏障功能障碍中的机制研究

目的 探讨Rho激酶（ROCK）在氢气改善离体脓毒症肠屏障功能中的作用。方法 常规培养人结肠上皮细胞Caco-2,随机分为6组（n=3）：对照组、富氢培养基组、LPS处理组、富氢培养基+LPS组、Rho激酶抑制剂组、Rho激酶抑制剂+LPS组。富氢培养基组给予0.6mmol/L富氢培养基;LPS 和Y-27632处理浓度分别为100μg/ml、25μmol/L。建立Transwell小室模型,定期检测跨上皮电阻值（TEER）,当TEER达到800Ω?cm2后给予处理,于6h、12h、24h检测TEER值,24h时检测FITC-右旋糖苷通过率。细胞接种于6孔板,融合达80%~90%后给予处理,实时聚合酶链式反应（RT-PCR）技术检测ZO-1 mRNA和ROCK mRNA表达情况;蛋白免疫印迹技术（Western Blot）检测ZO-1蛋白和ROCK蛋白表达水平。结果 与对照组比较,富氢培养基组12h、24h TEER值升高（P＜0.05）,FITC-右旋糖苷通过率、ZO-1和ROCK mRNA及蛋白表达水平无显著变化（P＞0.05）;ROCK抑制剂组各时间点TEER值升高（P＜0.05）,FITC-右旋糖苷通过率无显著变化（P＞0.05）,ZO-1 mRNA表达增加,ROCK mRNA表达减少（均P＜0.05）;LPS处理组各时间点TEER值降低,FITC-右旋糖苷通过率增高,ZO-1 mRNA和蛋白表达均下降,ROCK mRNA和蛋白表达均增加（P＜0.05）。与LPS处理组比较ROCK抑制剂+LPS组TEER值增高,FITC-右旋糖苷率降低,ZO-1 mRNA表达增加（P＜0.05）。与LPS处理组比较,富氢培养基+LPS组各时间点TEER值增高,FITC-右旋糖苷通过率降低,各时间点ZO-1蛋白表达上升,ROCK蛋白表达下降（均P＜0.05）。结论 氢气可保护脓毒症肠屏障功能,改善肠上皮屏障完整性,减少肠壁通透性,增加肠细胞间紧密连接蛋白表达,这些保护机制可能与氢气抑制LPS诱导的Rho激酶过度表达有关。     

High glucose concentration in isotonic media alters Caco-2 cell permeability

Caco-2 cell permeability was evaluated in isotonic media containing high (25mM) or physiological (5.5mM) glucose concentrations. Transepithelial electrical resistance (TEER) and membrane fluidity were measured to assess glucose-induced alterations in physical barrier properties. In parallel, distribution of the actin filament (F-actin) and zonula occludens-1 (ZO-1) proteins was assessed by confocal microscopy. Transepithelial fluxes of mannitol, hydrocortisone, digoxin, and glycyl sarcosine (Gly-Sar) that permeate the intestinal mucosa by various pathways were measured to quantify the effect of glucose-induced changes on Caco-2 cell permeability. High glucose decreased maximum TEER of cell monolayers by 47%, whereas membrane fluidity at the hydrophobic core and lipid/polar head interphase was significantly increased. F-actin distribution in high glucose cells appeared more diffuse while ZO-1 was unchanged. Mannitol and hydrocortisone fluxes across Caco-2 cells cultured in high glucose increased by 65% and 24%, respectively. In addition, high glucose decreased the maximum transport capacity (Vmax) of PepT-1. P-glycoprotein activity, however, was unchanged. In conclusion, high extracellular glucose concentration in isotonic media significantly alters physical barrier properties of Caco-2 cell monolayers, which predominantly affects transepithelial transport of solutes permeating the cell barrier by paracellular and transcellular passive diffusion and facilitated transport mediated by the proton-dependent oligopeptide transporter (PepT-1).     

Transport of octreotide and evaluation of mechanism of opening the paracellular tight junctions using superporous hydrogel polymers in Caco-2 cell monolayers

The purpose of this study was to investigate the mechanism of opening of tight junctions in Caco-2 cell monolayers using superporous hydrogel (SPH) and SPH composite (SPHC) polymers as permeation enhancers for peptide drug delivery. Moreover, the transport of octreotide across Caco-2 cell monolayers was assessed by application of SPH and SPHC polymers on Caco-2 cell monolayers. In these experiments, N,N,N-trimethyl chitosan chloride with 60% quaternization (TMC60) was used as a positive control for opening of tight junctions. Transepithelial electrical resistance (TEER) studies showed that all three polymers (TMC60, SPH, and SPHC) were able to decrease TEER values to approximately 30% of the initial values, indicating the ability of these polymers to open the tight junctions. Recovery TEER studies showed that the effects of the polymers on Caco-2 cell monolayers were reversible, indicating viability of the cells after incubation with polymers. Both SPH and SPHC (compared with TMC60) were able to increase the paracellular transport of octreotide by their mechanical pressures on tight junctions. The mechanistic studies showed that junctional proteins, including actin, occludin, and claudin-1, were influenced by application of SPH and SPHC polymers to the Caco-2 cell monolayers. SPH and SPHC induced clear changes in the staining pattern of all three proteins compared with the control, indicating that the expression of these proteins in the tight junctions was increased, most likely due to the mechanical pressure of the polymers on the junctional proteins.     

乙醇对肠上皮细胞紧密连接蛋白ZO-1表达的影响

目的探讨乙醇引起肠黏膜屏障的破坏以及这种破坏机制是否与紧密连接蛋白ZO-1相关。方法培养人结肠腺癌细胞株Caco-2,分别设正常对照组:不加刺激物及干预因素;实验组:加入不同体积分数(1%、2.5%、5%、7.5%、10%)乙醇分别刺激不同时间(0～3h),四甲基偶氮唑盐(MTT)比色法检测细胞生存率;测定跨上皮电阻(TEER)和荧光黄的透过量反映肠上皮细胞单层通透性;选定体积分数5%乙醇作为实验浓度,应用蛋白印迹法检测紧密连接蛋白ZO-1表达的动态变化。结果体积分数为5%的乙醇未影响到细胞的生存率。不同浓度乙醇作用20min后,细胞单层通透性增加,60min达高峰,TEER下降,荧光黄透过增加,以体积分数5%乙醇最明显。蛋白印迹法检测体积分数5%乙醇在1h内致Caco-2细胞表达ZO-1减少。结论乙醇可引起肠上皮黏膜屏障破坏,其机制可能和紧密连接蛋白ZO-1的破坏相关。     

Permeation enhancer effect of chitosan and chitosan derivatives: Comparison of formulations as soluble polymers and nanoparticulate systems on insulin absorption in Caco-2 cells.

In this study four quaternized derivatives of chitosan: trimethyl chitosan (TMC), dimethylethyl chitosan (DMEC), diethylmethyl chitosan (DEMC) and triethyl chitosan (TEC) with degree of substitution of approximately 50+/-5% were synthesized and their effect on the permeability of insulin across intestinal Caco-2 monolayers was studied and compared with chitosan both in free-soluble form and in nanoparticulate systems. Transepithelial electrical resistance (TEER) studies revealed that all four chitosan derivatives in free-soluble forms were able to decrease the TEER value in the following order TMC>DMEC>DEMC=TEC>chitosan, indicating their abilities to open the tight junctions. Recovery studies on the TEER showed that the effect of the polymers on Caco-2 cell monolayer is reversible and proves the viability of cells after incubation with all polymers. A similar rank order was also observed when measuring the zeta-potentials of the various polymers in solution form. Transport studies of insulin together with the soluble polymers across Caco-2 cell layers showed the following ranking: TMC>DMEC>DEMC>TEC>chitosan which is in agreement with the strength of the cationic charge of the polymer. In comparison to the free-soluble polymers, the nanoparticles prepared by ionic gelation of the chitosan and its quaternized derivatives had much lower effect on decreasing the TEER by opening of the tight junctions. This can be explained by the reduced available amount of positive charge at the surface of the nanoparticles. In accordance with these results, the insulin loaded nanoparticles showed much less permeation across the Caco-2 cell monolayer in comparison to the free-soluble polymers. Mass balance transport studies revealed that a substantial amount of the nanoparticles has been entrapped into the Caco-2 monolayer or attached to the cell surface. It can thus be stated that while free-soluble polymers can reversibly open the tight junctions and increase the permeation of insulin, the nanoparticles had basically only a low effect on the opening of the tight junction and the paracellular transport of insulin across the Caco-2 cell monolayer. These data convincingly show that nanoparticles consisting of chitosan and its quaternary ammonium derivatives loaded with insulin are less effective in facilitating paracellular transport across Caco-2 cell monolayers than the corresponding free polymers.     

Copper treatment alters the barrier functions of human intestinal Caco-2 cells: involving tight junctions and P-glycoprotein

This study investigated the effects of copper on paracellular permeability and P-glycoprotein (P-gp) in Caco-2 cells. Apical treatment with 100-300 microM CuSO4 in Hanks' balanced salt solution (HBSS, up to 3 hours) induced a time- and concentration-dependent increase in permeability of Caco-2 cell monolayers monitored by transepithelial electrical resistance (TEER). Copper treatment also induced a concentration-dependent reduction of F-actin stain, but not of tight junctional protein ZO-1. In addition, without any adverse effects on TEER, apical treatment with 300 microM CuSO4 in complete medium (for 24 hours) could reduce basolateral-to-apical transport, and increase apical-to-basolateral transport of rhodamine-123 (Rho-123) and accumulation of Rho-123 in Caco-2 cells. Treatment with 10-100 microM CuSO4 in HBSS (up to 3 hours) also induced a time- and concentration-dependent increase in accumulation of Rho-123 in Caco-2 cells. The results indicated that copper treatment increased the paracellular permeability probably by perturbing F-actin skeleton, and inhibited P-gp, thus altering the barrier functions of Caco-2 cells.     

Evaluation of the Caco-2 Monolayer as a Model Epithelium for Iontophoretic Transport

Purpose. To assess the Caco-2 monolayer as a model for iontophoresis of drugs across a model epithelium. Methods. The apparent permeability co-efficient (Papp) of mannitol, thyrotrophin releasing hormone (TRH), dexamethasone and a range of sizes of fluorescein isothiocyanate (FITC) dextrans across Caco-2 monolayers was measured under passive and electrically stimulated conditions. Trans-epithelial electrical resistance (TEER) was determined throughout. Transmission electron micrographs (TEM) of the monolayers were taken. Confocal laser scanning microscopy (CLSM) was used to visualize the iontophoretic transport route of FITC-Dextran (MW = 20 kDa) across a Caco-2 monolayer. Results. Application of 14.3 -Eq.cm^–2 across the monolayer evoked a transient drop in TEER. The drop in TEER was accompanied by statistically significant increases in fluxes of all the agents in the mucosal to serosal direction except for FD-70. TEM of test samples exhibited tight junction dilatation, in addition to intracellular vacuolisation. The iontophoresis of FD-20 was visualised with confocal laser scanning microscopy and was localised in paracellular spaces of the monolayer. Conclusions. The fluxes of mannitol, TRH, dexamethasone, FD-4, FD-10 and FD-20 across the Caco-2 monolayer were significantly enhanced when electric field was applied. The iontophoretic effect appeared to be directly upon tight junctions     

Disruption of the F-actin cytoskeleton and monolayer barrier integrity induced by PAF and the protective effect of ITF on intestinal epithelium

To explore whether platelet-activating factor (PAF) can disrupt the intestinal epithelial barrier directly and is associated with structural alterations of the F-actin-based cytoskeleton, and to observe the protective effect of intestinal trefoil factor (ITF), we establish an intestinal epithelia barrier model using Caco-2 cells in vitro. Transepithelial electrical resistance and unidirectional flux of lucifer yellow were measured to evaluate barrier permeability; immunofluorescent staining and flow cytometry were applied to observe morphological alterations and to quantify proteins of the F-actin cytoskeleton: the tight junction marker ZO-1 and Claudin-1 were observed using immunofluorescent staining. PAF significantly increased paracellular permeability, at the same time, F-actin and tight junction proteins were disrupted. It was thought that ITF could reverse the high permeability by restoring normal F-actin, ZO-1 and Claudin-1 structures. These results collectively demonstrated that PAF plays an important role in the regulation of mucosal permeability and the effects of PAF are correlated with structural alterations of the F-actin-based cytoskeleton and of tight junctions. ITF can protect intestinal epithelium against PAF-induced disruption by restricting the rearrangement of the F-actin cytoskeleton and of tight junctions.     

New and better protocols for a short-term Caco-2 cell culture system

The aim of the present study was to develop new and better protocols for a short-term Caco-2 cell culture system for use in rapid screening of intestinal drug absorption. Caco-2 cells were cultured according to several protocols for short-term cell culture to obtain monolayers. The effects of serum (fetal bovine serum, FBS) in the culture medium and of the period of cell culture on the barrier function and transporter activities of the monolayers were examined. The barrier function was estimated both from the transepithelial electrical resistance (TEER) and the permeability of [(14)C]mannitol. Transporter activities were monitored by measuring the permeability of [(14)C]glycylsarcosine for oligopeptide transporter (PepT1) and of rhodamine 123 for P-glycoprotein (P-gp). Caco-2 monolayers obtained by 3-day culture in the BIOCOAT HTS Caco-2 Assay System, developed by Becton Dickinson Bioscience, showed much higher permeability to hydrophilic compounds, such as mannitol, compared with those obtained by the standard 21-day culture system, due to the leaky structure of cell junctions. The newly developed 3-day protocol, which includes 10% FBS in the culture medium during the first day of culture, markedly enhanced TEER and lowered mannitol permeability of the monolayers. This protocol allowed us to better determine the rank order of permeability of compounds, giving results equivalent to those in the 21-day culture system. The longer culture period gave tighter monolayers, and the maximum value of TEER was obtained with 5 days in culture. However, after 5 days in culture, the integrity of monolayers decreased gradually. The highest activities of transporters, PepT1 and P-gp, in monolayers were obtained at days 5 or 6 of culture by the new protocol with FBS-containing medium. These results indicate that by a simple modification of the short-term culture protocol, it is possible to obtain Caco-2 monolayers with better barrier properties and higher activity of transporters that are equivalent to those found in the 21-day Caco-2 culture system.     

Structure-activity relationships for enhancement of paracellular permeability by 2-alkoxy-3-alkylamidopropylphosphocholines across Caco-2 cell monolayers

The oral route is the preferred route of delivery for a large number of drug molecules. However, the intestinal epithelium presents a formidable barrier for delivery of drugs into systemic circulation. Phospholipids are among compounds that enhance the absorption of drugs across the intestinal epithelium. In this paper, we describe structure-activity relationships for phospholipid derivatives as enhancers of paracellular permeability across Caco-2 cell monolayers. In a series of 2-alkoxy-3-alkylamidopropylphosphocholine derivatives, compounds with a long chain at C-3 (R3) and short chain at C-2 (R2) were potent in causing a decrease in transepithelial electrical resistance (TEER) and an increase in mannitol transport, but also showed significant cytotoxicity. Compounds with 9-11 carbons at C-3 and 6-10 carbons at C-2 provided good separation (up to 2.7-fold) between activity and cytotoxicity. Notably, a good correlation (r2 = 0.93) was observed between EC(50) (TEER) [concentration that caused a drop in TEER to 50% of its control (untreated) value] and EC10x (mannitol) [concentration that caused 10-fold increase in mannitol transport over the control (untreated) value], confirming that a decrease in TEER is associated with enhanced permeability of the hydrophilic compounds across Caco-2 cell monolayers. Compounds with medium to long carbon chains at C-2 and C-3, and the total carbons in the alkyl chains > 20, showed poor activity and no cytotoxicity.