Functional modeling of tight junctions in intestinal cell monolayers using polyethylene glycol oligomers

C. J. Watson1,2, M. Rowland2, and G. Warhurst1

1 Gut Barrier Group, Section of Gastrointestinal Science, Clinical Division I, University of Manchester, Hope Hospital, Salford M6 8HD; and 2 School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester M13 9PL, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Despite significant advances in the characterization of tight junction (TJ) proteins, little is known about how molecular changes relate to function due primarily to the limitations of conventional paracellular probes. To address this, the paracellular pathway in Caco-2 and T84 cell lines was profiled by measuring the permeabilities of 24 polyethylene glycols (PEG) of increasing molecular radius (3.5-7.4 Å) analyzed by mass spectrometry. When combined with a paracellular sieving model, these data provided quantitative descriptors of the pathway under control conditions and after exposure to TJ modulators. PEG profiles in both cell lines conformed to a biphasic process involving a restrictive pore (radius 4.3-4.5 Å) and a nonrestrictive component responsible for permeability of larger molecules. PEG profiling revealed significant differences between the effects of EGTA and sodium caprate (C10). The restrictive component of EGTA-treated cells lost all size discrimination due to an increase in pore radius. Sodium caprate had no effect on pore radius but increased permeability via a different mechanism possibly involving increased numbers of functional pores. PEG profiling provides a useful tool for probing the functional regulation of the paracellular route.

paracellular permeability; Caco-2; T84; transepithelial resistance; tight junction modulation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE TIGHT JUNCTION IS A MACROMOLECULAR assembly of proteins that circumscribe the apical region of polarized epithelial cells. This complex divides apical from basolateral cell surface domains and, in tissues such as the intestine, forms a crucial protective barrier that allows the paracellular diffusion of ions and small solutes but excludes potentially toxic macromolecules and microorganisms. The electrical resistance of this seal can vary dramatically between tissue types (9, 40), and the permeability characteristics of a given barrier can be dynamically altered by a variety of physiological, pathological, and pharmacological stimuli (2, 32).

Over the past 30 years, our knowledge of tight junction structure has advanced dramatically. Information provided by early freeze-fracture studies describing the structural elements of the tight junction (9, 45) has evolved in recent years into a greater understanding of the molecular composition of this complex facilitated by the identification of occludin (14) and the claudin family (13, 37), both proposed components of the transmembrane seal, and proteins of the intracellular domain of the tight junction, such as the ZO proteins (ZO-1, ZO-2, ZO-3; Refs. 15, 18, 47).

Despite these advances, our understanding of how these proteins interact to regulate the barrier is limited. This in turn has impeded our understanding of the functional properties of the tight junction. Indeed, in contrast to our molecular understanding of the tight junction, our ideas concerning the nature of the paracellular barrier have progressed little since the 1970s when tight junction fibril numbers were correlated with paracellular electrical resistance (9) and the presence of fluctuating aqueous pores embedded in these fibrils was postulated to account for diffusion through the tight junction (8). Recent studies proposing that claudins are the pore-forming structures in the tight junction (44, 51) are interesting, yet progress in this area will be limited without the means to better functionally profile the paracellular pathway. Numerous questions remain unanswered. What are the functional dimensions of the paracellular pathway, and how are these altered when the tight junction is perturbed? How do macromolecules cross the tight junction? Is this achieved by simple dilatation of existing junctions, or do populations of pores with different sieving properties exist? Existing methods employed to functionally characterize tight junction permeability, such as monitoring the transepithelial flux of single, hydrophilic probes like mannitol, inulin, or dextran or simply measuring transepithelial resistance (TER), lack the necessary resolution to answer these questions. The commonly held view is that increases in paracellular permeability in perturbed or disease states results from dilatation of existing tight junction pores (21, 48). However, alternative explanations are possible including increases in pore number or the presence of subpopulations of pores accessible to large molecules (25).

To address these issues, we have developed an approach that simultaneously measures the paracellular permeability of 24 PEG oligomers separated by liquid chromatography-mass spectrometry (LC-MS) and allows detailed functional profiling and mathematical modeling of the paracellular route. This technique is used here to define the functional characteristics of the paracellular route in the intestinal cell models, T84 and Caco-2, and to investigate possible mechanistic differences in the actions of tight junction modulators.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials

[14C]-Mannitol (56mCi/mmol) was purchased from Amersham International (Buckinghamshire, UK); mannitol and polyethylene glycol 400/1000 were from BDH (Poole, UK); Hanks' balanced salt solution (HBSS) powder was from Sigma (Poole, UK); FITC-labeled 3-kDa dextran (FD3) was from Molecular Probes (Leiden, The Netherlands); EGTA and sodium caprate (C10) were from Sigma; and all tissue culture reagents were from GIBCO Life Technologies (Paisley, UK).

Colonic Cell Lines

Caco-2 cells, obtained from the American Type Culture Collection (Rockville, MD), were maintained in Dulbecco's modified Eagle's medium (DMEM) containing high glucose (4.5 g/l), 10% fetal calf serum, 1% nonessential amino acids, 2 mM glutamine, and 50 IU/ml penicillin/50 µg/ml streptomycin. These cells (passages 98-120) were cultured in 75-cm2 T-flasks (Costar, High Wycombe, UK) at 37°C in a 5% CO2, constant humidity environment with medium replaced three times a week. Monolayers were subcultured when they reached ~80% confluency at a split ratio of 1:10 using 0.05% trypsin/0.02% EDTA. T84 cells (passages 100-120) were cultured in a similar fashion to Caco-2 cells except that they were maintained in a 1:1 mixture of DMEM/F-12 nutrient mix supplemented with 14 mM NaHCO3 and 50 IU penicillin/50 µg/ml streptomycin in 25-cm2 flasks (Costar).

For transport, experiments cells were seeded on Snapwell polycarbonate cell culture inserts with a mean pore size of 0.4 µm (Costar) at 2-3 × 105 cells/cm2 and were grown for at least 21 days before experiment.

Development of TER was monitored using an Evometer (World Precision Instruments) fitted with chopstick electrodes.

Transport Studies

Epithelial cell layers were removed from growth medium and washed twice in prewarmed HBSS (supplemented with 20 mM HEPES). Monolayers were subsequently placed in a modified Ussing chamber with a 1.0-cm2 diffusion window and allowed to equilibrate for 30 min with 5 ml HBSS in both chambers. Chambers were maintained at 37°C and under constant gassing with O2. Spontaneous tissue potential differences and TER, measured as deflection in potential differences caused by a 100-µA current pulse, were monitored throughout the experiment. After the equilibration period, paracellular markers were added at a final concentration of 2 mg/ml, 100 µM, and 10 µM for PEG 400/1000, unlabeled mannitol, and FD3, respectively, to the donor chamber. In the mannitol studies, the donor solution also contained 0.2 µCi/ml of [14C]mannitol. A 100-µl sample was removed from the donor chamber to determine the initial concentration. Samples (1 ml) from the receiving chamber were taken every 30 min and replaced with fresh buffer. Mannitol samples were mixed with 4 ml of Ultima Gold scintillation cocktail (Packard) and analyzed by liquid scintillation counting. Aliquots (200 µl) of FD3 receiver samples were placed in a 96-well plate and read using a Perkin Elmer LS50b fluorimeter at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. PEG samples were stored at -20°C until analysed by LC-MS.

Permeability values were expressed as apparent permeability (Papp) in centimeter per second, obtained according to the equation
P<SUB>app</SUB><IT>=</IT><FR><NU>dQ<IT>/</IT>d<IT>t</IT></NU><DE><IT>A·</IT>C</DE></FR> (1)
where dQ/dt is the rate at which the compound appears in the receiver compartment, A is the surface area of the tissue or monolayer, and C is the initial concentration of compound in the donor compartment.

LC-MS Analysis of PEG Oligomers

Sample preparation. Internal standard (dimethoxytetraethylene glycol) was added to 500 µl of each sample before application to HLB extraction cartridges (Waters Oasis, MA) previously conditioned with 4 × 1 ml methanol and equilibrated using 1 ml of water. The cartridge was then washed with 1 ml of 5% methanol before PEG elution with 1 ml of methanol. After drying down under a stream of nitrogen at 40°C, the concentrate was reconstituted in 500 µl of mobile phase and a 50-µl aliquot was injected into the LC system with mass spectrometric detection. The chromatographic run time for each sample was 40 min. Blank buffer was also spiked with known amounts of both PEG 400 and 1000 to provide a calibration curve covering the range of 0.05 to 10.0 µg/ml. These samples were processed and analyzed alongside study samples. Blank samples were also prepared to account for PEG oligomers derived from external sources (e.g., plasticware).

LC analysis. A Hewlett-Packard 1090 automated system with a Spherisorb C8 guard and analytical column (250 × 4.6 mm, Phenomenex, Macclesfield, UK) operating at ambient temperature were used. The mobile phase (flow rate 1 ml/min, split to give 0.2 ml/min to the mass spectrometer) was set to give a gradient elution by varying the proportions of methanol (A) to water (B) (both containing 0.1% formic acid) as follows: 0 min, 20% A:80% B; 1-8 min, 30% A:70% B; 10-19 min, 40% A:60% B; 21-28 min, 50% A:50% A; 31-40 min, 60% A:40% B. The composition was returned to the initial conditions for 5 min before the next injection.

Mass spectrometer. The detector (Micromass platform, Manchester, UK), operating in electrospray positive ion mode, was set to: source temperature, 160°C; capillary voltage, 3.71 kV; HV lens voltage, 0.6 kV. The cone voltage was ramped from 35 V at the lowest mass scanned to 90 V at the highest mass. Masses 245 (internal standard), 261-745 (representing the sodium adducts of PEG 400), and 789-1273 (representing the sodium adducts of PEG 1000) were scanned. These ionisation conditions allowed for each oligomer to generate a single ion corresponding to the sodium adduct with no fragmentation.

Modulation of Paracellular Permeability

The changes in PEG permeation profiles after exposure of cell monolayers to the calcium chelator EGTA and the medium chain fatty acid sodium caprate (C10), both known modulators of tight junction integrity, were assessed. For the EGTA study, T84 cells grown on Snapwell inserts were mounted in modified Ussing chambers as described in Colonic Cell Lines. EGTA was added to both mucosal and serosal chambers at the desired concentration and TER, and flux measurements were carried out for up to 30 min. Sodium caprate was added to the apical compartment, basolateral samples were removed, and electrical readings were taken at desired intervals subsequent to addition of the modulator. The sodium caprate experiments were carried out using standard HBSS in the basolateral chamber and Ca2+/Mg2+-free HBSS in the apical chamber to avoid precipitation of the fatty acid. No decrease in TER stability was observed, so it was assumed that these conditions had little effect on monolayer integrity.

Mathematical Modeling of Paracellular Permeation

The hydrodynamic radii of PEG oligomers are known (42) and can be related to molecular masses (M) by the relationship r (Å) = 0.29 M0.454. The paracellular flux of PEG 400/1000 across the cell line monolayers utilized in this study yielded two-component permeation profiles (see RESULTS); component 1 (C1) is a restrictive component where PEG permeability declines with increasing molecular radius and a second component (C2) where no size restriction is apparent for this size range of markers. The sieving model employed in this study takes account of this two-component profile. C1 is viewed as having a radius rs1, with a size discrimination to PEG such that no permeation occurs through this component when the radius of the oligomer, r, equals or exceeds a critical value, rs1. The tight junctions are viewed as fluid filled cylindrical pores, and permeation through these is assumed to occur only by diffusion.

First it is necessary to evaluate permeability and the available surface area. Ps represents the permeability of an oligomer passing through tight junctions of total available surface area As. In practice the serosal surface area (AT) is typically used in permeability calculations instead of the area associated only with tight junctions

hence
P<SUB>app</SUB><IT>=</IT><FR><NU><IT>P</IT><SUB>s</SUB><IT>·A</IT><SUB>s</SUB></NU><DE><IT>A</IT><SUB>T</SUB></DE></FR> (2)
and thus
P<SUB>app</SUB><IT>A</IT><SUB>T</SUB><IT>=P</IT><SUB>s</SUB><IT>·A</IT><SUB>s</SUB> (3)
From Fick's law of diffusion, rate of permeation, dQ/dt, under the usual sink conditions prevailing, is defined generally by
<FR><NU>dQ</NU><DE>d<IT>t</IT></DE></FR><IT>=</IT><FR><NU><IT>D·A·</IT>C</NU><DE><IT>&Dgr;x</IT></DE></FR> (4)
where D is the diffusion coefficient of the compound in the tight junction of length Delta x. Given that P · A = (dQ/dt)/C, it follows that
P·A=<FR><NU>D·A</NU><DE>&Dgr;x</DE></FR> (5)
The aperture to each pore is treated as a circle of cross-sectional area pi r<UP><SUB>s</SUB><SUP>2</SUP></UP>. In addition, a sieving term is incorporated for component 1 (1 - r/rs1)2, which can be regarded as the fraction of the pore aperture that is available for diffusion (39), so that
P<SUB>app</SUB><IT>A</IT><SUB>T</SUB><IT>=</IT><IT>P</IT><SUB>s</SUB><IT>A</IT><SUB>s</SUB><IT>=</IT><FR><NU><IT>D·n</IT><SUB>s</SUB><IT>&pgr;r</IT><SUP>2</SUP><SUB><IT>s</IT>1</SUB></NU><DE><IT>&Dgr;x</IT></DE></FR><IT>·</IT><FENCE>1<IT>−</IT><FR><NU><IT>r</IT></NU><DE><IT>r</IT><SUB>s1</SUB></DE></FR></FENCE><SUP>2</SUP> (6)
where ns is the total number of tight junctions and nspi r<UP><SUB>s</SUB><SUP>2</SUP></UP> is the total surface area of tight junction. Permeation occurs across the restrictive pore so long as r < rs1; when r >=  rs1 no permeation occurs via this pathway, the molecule being too large. According to the Stokes-Einstein equation,
D=kT<IT>/</IT>6<IT>&pgr;&eegr;r</IT><SUB>h</SUB><IT>N</IT> (7)
where k is the Boltzmann constant, T the absolute temperature, eta  the viscosity of the medium, rh is the hydrodynamic (Stokes) radius of the diffusing species, and N is Avogadro's number. Assuming r = rh, which expresses radius in hydrodynamic equivalents, substituting Eq. 7 into Eq. 6, collecting terms, and incorporating the nonrestrictive component yields
(8)
Collecting all the constants within the system, theta  = kT/(AT6eta N) and introducing two further terms, alpha  and beta  
&agr;=<FR><NU>&thgr;n<SUB>s1</SUB></NU><DE><IT>&Dgr;x</IT><SUB>1</SUB></DE></FR><IT>; &bgr;=</IT><FR><NU><IT>&thgr;n</IT><SUB>s2</SUB><IT>r</IT><SUP>2</SUP><SUB>s2</SUB></NU><DE>&Dgr;x<SUB>2</SUB></DE></FR> (9)
yields on substitution Eq. 10
P<SUB>app</SUB><IT>=&agr; </IT><FR><NU><IT>r</IT><SUP>2</SUP><SUB>s1</SUB></NU><DE><IT>r</IT></DE></FR><FENCE>1<IT>−</IT><FR><NU><IT>r</IT></NU><DE><IT>r</IT><SUB>s1</SUB></DE></FR></FENCE><SUP>2</SUP><IT>+</IT><FR><NU><IT>&bgr;</IT></NU><DE><IT>r</IT></DE></FR> (10)
Applying the above model to the PEG permeability data yields three parameters; rs1 , the estimated restrictive pore radius with units of Angstroms (Å); alpha , which is directly proportional to the number of restrictive tight junction pores (and inversely proportional to junctional length, Delta x) with the units s-1; and beta , which is directly proportional to both the number and the radius of the nonrestrictive tight junction pores (units of cm2/s). The above model was fitted to permeability data using nonlinear regression analysis (WinNonlin; Scientific Consulting).

Statistical Analysis

Results are means ± SE. For statistical comparisons, Student's t-tests were performed as appropriate; P < 0.05 was considered statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Modeling of Paracellular Permeability Across Human Colonic Cell Lines Using PEG Oligomers

Initial studies examined the permeability of the 24 PEG oligomers across two established models of the intestinal epithelium, Caco-2 and T84. These cell lines, although both derived from the colon, are phenotypically different. Caco-2 exhibit an absorptive phenotype characteristic of immature small bowel enterocytes, whereas T84 are more characteristic of the chloride secretory cells of the colonic crypt (11, 20). Figure 1 shows the PEG permeation profile for both cell lines with the apparent permeability plotted against PEG hydrodynamic radius. Profiles for both lines were essentially identical showing a biphasic relationship with a first component (C1) characterized by marked decline in permeability over the four to five smallest oligomers, followed by sharp "cutoff" to a relatively constant but quantitatively minor second component (C2) composed of oligomers 458-1250 (4.68-7.39 Å). PEG cumulative flux across cell monolayers remained linear over a 2-h period over the whole range of PEG oligomers (Fig. 2). The permeation of PEG oligomers across an unseeded filter support was at least 40-fold greater than across polarized monolayers and displayed no significant size selectivity (Fig. 1C), although the free diffusion of the oligomers did decline with increasing hydrodynamic radius, consistent with the Stokes-Einstein equation (Eq. 7).


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Fig. 1.   Profiles of mucosal-to-serosal (M-S) polyethylene glycol (PEG) fluxes across Caco-2 (A), T84 (B), and an unseeded filter support (C). Cell monolayers grown on permeable supports were removed from growth medium, washed twice in prewarmed Hanks' balanced salt solution (HBSS), and mounted in modified Ussing chambers. After a 30-min equilibration period, PEG 400/1000 mix (2 mg/ml final) was added to the donor chamber. Samples (1 ml) were removed from the receiver compartment at 30-min intervals with a 100-µl sample also removed from the donor chamber to determine initial PEG oligomer concentrations. Results are means ± SE; n = 6. Papp, apparent permeability.



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Fig. 2.   Time course of M-S PEG oligomer fluxes across Caco-2 monolayers. Cells were washed twice in prewarmed HBSS and mounted in modified Ussing chambers. PEG 400/1000 mix (2 mg/ml final) was added to the donor chamber, and samples (1 ml) were removed from the receiver chamber at 30-min intervals and analyzed by liquid chromatography-mass spectrometry (LC-MS) to assess the appearance kinetics of PEG oligomers with hydrodynamic radii of 3.47 (), 3.75 (black-triangle), 4.01(black-down-triangle ), 4.25 (black-lozenge ), and 4.47 Å (). Results are means ± SE; n = 4.

The permeabilities of the smaller PEG oligomers was lower in T84 compared with Caco-2 (40% lower permeability for the smallest oligomer, PEG 238) consistent with the higher electrical resistance of T84. However, it is interesting that the difference in PEG permeability between these cell lines is relatively modest compared with the fivefold difference in their TER values (261 ± 43 and 1,315 ± 182 Omega .cm2 for Caco-2 and T84, respectively).

Application of the modified sieving model to the cell line data yields values describing C1 (rs1, the restrictive pore radius, and alpha , which is proportional to the number of restrictive tight junction pores). In addition, the model describes the contribution of C2 to PEG permeability in terms of beta , which is proportional to the number and/or radius of tight junction pores nonrestrictive for this size range of PEGs. By definition, the model cannot provide a radius value for the "unrestricted" C2 using probes of this size range. The mean values calculated for Caco-2 and T84 are shown in Table 1. No significant differences were observed in either the radius of the restrictive pore or the alpha -values calculated for the two cell lines, although alpha  did tend to be lower in T84 (5.7 ± 0.4 vs 7.5 ± 1.6) consistent with a smaller number of restrictive pores in the higher resistance line. In both lines, the value of beta , descriptor of the "unrestricted" C2, was 15- to 20-fold lower than alpha  (Table 1).

                              
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Table 1.   Sieving parameters derived from application of modified sieving model to the cell line data presented in Fig. 1

Comparison of PEG Oligomers with Other Paracellular Markers

Figure 3 compares the permeabilities of PEG oligomers 282, 326, and 1250 in Caco-2 with two other widely used paracellular markers, mannitol, and FD3. The hydrodynamic radius of mannitol (4.1 Å; Refs. 26, 27) is directly comparable with that of PEG 326 (4.01 Å), and both probes exhibit near identical apparent permeability values (4.2 ± 0.07 and 4.1 ± 0.06 × 10-7cm/s for M-S mannitol and PEG 326 flux, respectively). PEG 282, which is only 0.3Å smaller than mannitol, exhibits three- to fourfold greater permeability across Caco-2, confirming the extreme sensitivity of paracellular transport to hydrodynamic radius over this size range. There was no difference in the permeabilities of the larger probes PEG 1250 and FD3 even though the latter is significantly larger [17 Å (43) vs. 7.4 Å] consistent with the presence of a larger unrestricted pathway in these cells. A recent study reported asymmetric fluxes across rat colon for several paracellular markers, including mannitol and FITC-labeled 4-kDa dextran, indicative of active transport (50). However, no significant asymmetry was observed for any of the paracellular probes tested in Caco-2 (Fig. 3) or T84 (data not shown).


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Fig. 3.   Comparison of mannitol, FITC-labeled 3-kDa dextran (FD3), PEG 282, PEG 326, and PEG 1250 permeability across Caco-2 monolayers in M-S (open bars) and S-M (solid bars) directions. Caco-2 cells grown on permeable supports were mounted in Ussing chambers. After a 30-min equilibration period, 100 µM mannitol (+0.2 µCi/ml [14C]mannitol), 10 µM FD3, or 2 mg/ml PEG 400/1000 (all final concentrations) were added to the donor chamber. At 30-min intervals, samples were removed from receiver chamber and the appearance rates of probe were assessed using relevant detection methods. Results are means ± SE; n = 4. Differences between M-S and S-M flux rates were found to be not significant (P > 0.05) for all probes.

Functional Profiling of Tight Junction Modulators with PEG Oligomers

A variety of tight junction modulators have been described that act through distinct mechanisms to increase paracellular transport (2, 32). However, the precise functional changes induced by different modulators have been difficult to define due to the limitations of conventional paracellular probes. We therefore examined the effects on T84 monolayers of two agents that act via different mechanisms to modulate tight junctions. EGTA acts by chelating extracellular Ca2+ with concomitant "loosening" of the tight junction while sodium caprate (C10) acts through an intracellular mechanism involving activation of phospholipase C (29). EGTA and sodium caprate cause a dramatic, concentration-dependent decrease in TER in T84 monolayers (Fig. 4, B and C), which was quantitatively similar with both modulators although sodium caprate acted over a much longer time course than EGTA (Fig. 4A). However, analysis of PEG oligomer permeability during the period of maximum TER reduction (30 min after EGTA addition and 120 min after addition of sodium caprate) reveals markedly different effects on paracellular permeability. In the case of EGTA (Fig. 5), the permeability of all PEG oligomers increase although proportionally the increases are much greater with the larger oligomers (4-fold for 4.68 Å ; 44-fold for 7.39 Å at 2.5 mM). The result of this is to virtually abolish size selectivity across this range of probes. The profile generated by sodium caprate is, however, markedly different (Fig. 6). With the use of this modulator, although there was also a general increase in PEG permeability, this was similar across the whole range. The net effect is to produce an upward shift in the PEG profile while retaining the overall size selectivity. Application of the sieving model to these data suggests important mechanistic differences in the action of these modulators on the tight junction in T84 cells (Table 2). In the case of higher concentrations of EGTA, there is no longer evidence of a restrictive pore in that the estimated radius was far in excess of the size of even the largest PEG oligomer (7.4 Å) used in this study. At the same time, the contribution of component 2 (beta ) increases dramatically while the contribution of component 1 (alpha ) becomes insignificant. This is consistent with the major effect of EGTA being a large dilatation of existing tight junction pores or the fusing of these to form a population of larger pores. Under these circumstances of extreme tight junction perturbation, the system conforms to a single population of essentially unrestricted pores. This was confirmed by showing that the EGTA data fitted well to a simple single-component sieving model (data not shown). The changes in alpha  and beta  (Table 2) obtained from the full two-site model clearly show the EGTA-induced transition from two-component to single component. The data from sodium caprate modeling are markedly different. In this case, there is no discernible increase in pore radius but values for both alpha  and beta  increase. This suggests that, rather than pore dilatation, sodium caprate increases the number of functional pores, both small (component 1) and large (component 2), through which PEG oligomers can permeate.


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Fig. 4.   Comparison of effects of tight junction perturbation on transepithelial electrical resistance (TER) of T84 monolayers. A: time course of TER decreases in the presence of 2 modulators. B: effects of apical and basolateral exposure (30 min) of varying doses of EGTA on TER of T84 monolayers. C: effects of apical exposure (120 min) of varying doses of sodium caprate (C10) on TER of T84 monolayers. Results are means ± SE; n = 4.



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Fig. 5.   Effect of EGTA on PEG 400/1000 permeability profile of T84. T84 cells grown on permeable supports were removed from growth medium, washed twice in prewarmed HBSS, and mounted in modified Ussing chambers. After a 30-min equilibration period, PEG 400/1000 mix (2 mg/ml final) was added to the donor chamber. Control fluxes (1 ml) were sampled (black-triangle) and then 1.5 (), 2.0 (black-lozenge ), or 2.5 mM () (final concentration) EGTA was added to both apical and basolateral chambers. One-millimeter samples were removed 30 min after EGTA addition to assess PEG oligomer permeation rates in that 30-min period by LC-MS analysis. Results are means ± SE; n = 4.



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Fig. 6.   Effect of sodium caprate (C10) on PEG 400/1000 permeability profile of T84. T84 cells grown on permeable supports were removed from growth medium, washed twice in prewarmed HBSS, and mounted in modified Ussing chambers. Ca2+/Mg2+-free HBSS was used in the apical chamber After a 30-min equilibration period 2 (black-triangle), 5 (black-down-triangle ), or 10 mM (black-lozenge ) (final concentration), sodium caprate was added to the apical compartment. After 90-min, PEG 400/1000 mix (2 mg/ml final) was added to the donor chamber. One-millimeter samples were removed 120 min after sodium caprate addition to assess PEG oligomer permeation rates between 90 and 120 min after sodium caprate addition by LC-MS analysis. Time-matched control fluxes () were also sampled. Results are means ± SE; n = 4.


                              
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Table 2.   Sieving parameters derived from application of modified sieving model to the T84 data presented in Figs. 5 and 6

The differential effects of EGTA and sodium caprate could not be explained by cytotoxicity because neither modulator stimulated the release of lactate dehydrogenase from T84 monolayers at the concentrations used in this study (data not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Recent advances are providing an ever more detailed picture of the tight junction at the molecular level (52). However, our understanding of how changes at the molecular level relate to function has lagged behind due primarily to the limitations of conventional paracellular probes. To address this, we have utilized an approach that allows functional profiling of the paracellular route using polydispersed PEG oligomers separated by LC-MS. With the use of this technique, the paracellular permeabilities of 24 PEG oligomers of increasing molecular radii (3.5-7.39 Å) can be measured simultaneously. This produced a permeation profile that can be further analyzed by a mathematical sieving model yielding quantitative descriptors of the paracellular pathway. In the present study, a PEG oligomer methodology has been used to profile paracellular permeation in two colonic epithelial cell lines, Caco-2 and T84, and to examine the functional changes elicited by tight junction modulators. A similar methodology has been used previously to identify species differences in the intestinal paracellular route in vivo (19) but to our knowledge this is the first attempt to use this approach to profile the paracellular pathway in in vitro models of intestinal epithelium.

PEG has been used extensively as a marker of intestinal permeability since the 1970s (5) although in most studies it has been treated as a single molecular weight marker and its polydispersed nature was ignored. Some investigators have questioned the suitability of PEG as a paracellular probe. For example, the intestinal permeation rate of PEG 400 is significantly higher than mono- , di-, and trisaccharide markers of comparable molecular weight (36). This, together with claims that PEG had significant lipid solubility (24), led to suggestions of a transcellular permeation route. However, Ma and et al. (31) showed PEG 400 to be strongly hydrophilic and found a previous determination of its lipophilic properties to be technically flawed. In addition, there was no evidence for significant PEG permeation into isolated brush-border membrane vesicles (23) and very similar size discrimination in intestinal epithelia was seen between PEG oligomers and a series of polar D-peptides, permeating via the paracellular route (19). The higher than expected permeation of PEG 400 compared with other markers of similar molecular weight is almost certainly due to its smaller molecular size. A recent study using monodispersed PEG oligomers showed PEG 194 permeation to be sixfold greater than mannitol in cell monolayers despite being of comparable molecular weight (3). However, the findings of the present study suggest this can be explained by the smaller hydrodynamic radius of PEG 194 (3.2 Å) compared with mannitol (4.1 Å). A relatively small difference in the radii of the probes will have profound effects on permeability particularly where they lie close to the radius of the paracellular pore.

The ability to simultaneously monitor permeabilities of a 24-member homologous series produced detailed and highly reproducible permeation profiles in cell monolayers, which were used as the basis for mathematical modeling of the paracellular route. We explored the inclusion of the Renkin term (39) in the model for the restrictive pore, which aims to take account of frictional resistance as molecules move through the pore. However, the validity of the Renkin correction is in doubt when the parameter r/rs1 exceeds 0.4 [as is the case for all the PEG oligomers used in this study, with even the smallest measurable oligomer PEG 238 having a radius (3.5 Å) of 75% of the estimated size of the restricted pore (4.5 Å)]. Under these conditions, the Renkin term tends to exaggerate diffusional resistance within the pore resulting, as we found, in both a poor fit of the model to the data and unsatisfactory estimates of sieving parameters. Other investigators have expressed similar concerns about including the Renkin term when modeling paracellular permeability of compounds the size of the PEG oligomers (16, 27). Therefore, this aspect was not pursued any further.

With the use of the model, two distinct permeation components could be defined in both T84 and Caco-2, a restrictive component with a sharp molecular size cut-off (component 1) and a second component that is not restrictive for these size molecules (component 2). The model allows a quantitative assessment of the contribution of these two components to paracellular transport (alpha  and beta ) as well as estimating the pore size of the restrictive component. The fact that, under control conditions, the value for alpha  is ~20-fold greater than beta  illustrates the dominance of the restrictive pathway for paracellular permeation of small molecules of radii <4.0 Å. In contrast, the nonrestrictive pathway (defined by beta ) provides the permeation route for larger molecules (>4.0 Å). A previous study demonstrated biphasic oral absorption vs. molecular size profiles of PEG oligomers in rat, dog, and humans qualitatively similar to those presented here for epithelial monolayers. However, the greater complexity of tissues makes it more difficult to draw conclusions regarding the biological basis for the biphasic profiles observed in intestinal preparations. For example, they may reflect distinct populations of tight junction pores associated with villus tip and intestinal crypt cells as suggested by Hollander (22). This theory would not explain the two-component profile observed in intestinal monolayers.

Despite intense interest in the structure and regulation of the intestinal tight junction, its functional dimensions have remained poorly defined. Madara and Dharmsathaphorn (33) suggested a pore size for T84 cells in the range 3.6-15 Å based on the ability of two probes of widely different size, mannitol and inulin, to cross T84 monolayers. Similarly, Ma et al. (30) proposed that the paracellular pore in rat colon was accessible to molecules with a radius >11 Å on the basis of significant permeation of inulin. However, a systematic determination of the dimensions of the paracellular pore radius requires the use of multiple probes spanning a defined range of molecular size. A recent study by Knipp et al. (27) addressed the problem using a group of structurally unrelated compounds of known hydrodynamic radii. With the use of this approach, the pore radius of Caco-2 was estimated to be 5.2 Å, although the fact that all of the probes used were smaller than the estimated pore size was an acknowledged limitation. The PEG methodology described here takes this approach further by using a stable chemical series with radii that span that of the restrictive paracellular pore. As a result, we would argue that the radius calculated for Caco-2 and T84 (4.5 and 4.3 Å, respectively) in this study represents a more accurate estimate of the small molecular size-restrictive pore in these systems. The in vivo PEG absorption profiles produced for rat, dog, and human (19) were not modeled but did show a significant molecular mass cut-off at ~600 Da, which corresponds to a hydrodynamic radius of ~5.3 Å. This compares well with the estimates obtained in Caco-2 and T84 described in this study. The relatively small standard errors associated with the estimates of pore radius suggest that the paracellular pathway in these cells is relatively homogeneous, particularly compared with intestinal tissues.

The similarity in sieving parameters estimated for Caco-2 and T84 is intriguing given the 10-fold difference in TER values between the two lines. However, several recent reports have noted a disparity between TER and paracellular flux, suggesting that permeation pathways for ions and larger hydrophilic solutes may be functionally dissociated (4, 17). The sieving model indicates that differences in TER in Caco-2 and T84 cannot be explained by differences in either pore radius or the number of restrictive pores as calculated by the sieving model. A correlation between strand number and paracellular resistance has been proposed (8, 9) but, to our knowledge, there have been no direct comparisons of fibrillar arrangements in Caco-2 and T84. However, the TER of high- and low-resistance MDCK cells varies by two orders of magnitude despite possessing similar arrangements of tight junction strands (46). Recent evidence suggests that differences in occludin phosphorylation between MDCK strains may mediate the observed differences in TER (54). Further studies will be needed to determine whether a similar mechanism can explain the observed differences in TER between T84 and Caco-2.

A key aim of the present study was to determine whether PEG profiling could be used to investigate the mechanism of action of tight junction modulators at the functional level. Many factors have been shown to influence tight junction integrity including physiological modulators such as cytokines and growth factors (53), bacterial toxins (12), and pharmacological agents (28). Their effects are mediated through a range of intracellular and extracellular pathways but the general assumption is that they all act to dilate the tight junction pore. The data presented here suggest this may be simplistic. EGTA and sodium caprate, C10, elicited profoundly different changes in PEG profile and sieving parameters in T84 cells despite having similar effects on TER. EGTA, a classic tight junction modulator that acts by chelating extracellular calcium (10, 35, 49), abolished size discrimination due to a marked increase in the previously restrictive pore radius, reverting essentially to a single component system consisting of large pores. Under these conditions, the two component sieving model does not allow an accurate estimate of pore radius but it is clear that after EGTA treatment the radius of paracellular pores will be far >7.4 Å (the size of the largest PEG oligomer used here). The precise mechanism behind EGTA-induced reductions in barrier function remains unclear but appears to involve a substantial redistribution of E-cadherin, a Ca2+-dependent adhesion molecule within the adherens junction (10). A downstream effect of this may be the disassembly of other junctional components, including the tight junction, mediated at least in part by protein kinase C (6, 7, 49). In contrast, sodium caprate-induced increases in permeability involve phospholipase C-dependent increases in intracellular Ca2+ concentration and a resulting contraction of perijunctional actin (29, 48). The results of PEG profiling argue strongly that pore dilatation of the tight junction is not the mechanism by which sodium caprate increases paracellular permeability. Sodium caprate markedly increased PEG permeability (indicated by the increase in alpha ) but maintained size selectivity across T84 monolayers such that the restrictive pore size was unchanged compared with control monolayers. What could produce an increase in alpha  without affecting pore size? Possible explanations include an increase in the number of restrictive pores caused by rearrangement of junctional proteins. In this respect, it is interesting that Lindmark et al. (29) showed that sodium caprate induced redistribution of ZO-1 and occludin in Caco-2 cells. It has been suggested that tight junction pores fluctuate between open and closed states (8), and it is possible that modulators such as sodium caprate could be acting to increase the open probability of restrictive pores, again mediated by alterations in junctional proteins. Alternatively, it is interesting to speculate that increased permeability in the absence of changes in size selectivity could also be caused by changes in strand complexity or path length within the restrictive pore. Recent studies in HT29-cl.19A colonocytes show that tumor necrosis factor-alpha -induced increases in mannitol flux are accompanied by a decrease in both average strand number and junctional depth (41). Glucose (34)- and protein kinase C (38)-mediated increases in paracellular permeability occur with similar alterations in tight junction morphology.

The lack of effect of sodium caprate on the size of the restrictive pore raises the question of how such modulators increase the permeability of large molecules across cell monolayers. In common with other studies, we have observed that both EGTA and sodium caprate can increase the permeability of probes that are much larger than the restrictive pore. We found that the transepithelial flux of a 10-kDa fluorescent dextran (hydrodynamic radius of 23 Å) (43) increased by 1.9-fold and 21-fold across T84 monolayers in the presence of sodium caprate and EGTA, respectively. One explanation is that larger molecules cross epithelial monolayers through an alternative pathway that is characterized in these studies by beta , the descriptor of the second component of PEG flux. This component represents a small proportion of total paracellular flux in unperturbed monolayers and is increased significantly in the presence of sodium caprate, although it remains a minor component compared with the restrictive pathway (e.g., alpha  is 3-fold larger than beta  even after exposure to sodium caprate). At present we can only speculate on the nature of the second pathway. A small population of large radius pores may exist within the tight junction strands alongside the restrictive pores and their numbers may be upregulated in response to extracellular stimuli to increase the paracellular flux of larger solutes. It may reflect heterogeneity within the junction itself. Transmission electron microscopy studies show sodium caprate induced dilations in Caco-2 tight junctions similar to those observed in hamster small intestine in the presence of glucose (1, 34). Tumor necrosis factor-alpha was reported to induce discontinuities in junctional strands in colonic monolayers, which may represent pathways for increased permeability of large molecules such as horseradish peroxidase (41). The second component could alternatively reflect heterogeneity in the monolayer such that some cells form incomplete or immature junctions that are much less restrictive. The possibility that the second component represents a nonspecific leak pathway seems unlikely given that it is upregulated by sodium caprate. Clearly, more studies are needed to characterize these pathways because they do raise important questions about the routes for movement of small and large molecules across the gut and whether this can be explained by simple dilatation of a single population of homogenous pores.

In conclusion, we have described in this study a useful technique to functionally probe the paracellular pathway and have utilized this procedure to characterize this pathway in Caco-2 and T84 cell lines. We believe this methodology yields more informative data compared with the use of conventional paracellular probes and is particularly useful when investigating junctional modulation. This technique, combined with molecular and morphological approaches, should provide valuable insights into the nature of the paracellular pathway.


    ACKNOWLEDGEMENTS

We thank Dr. Richard Stephens, Norman Higgs, Sue Murby, and Brent Collins for technical expertise.


    FOOTNOTES

This work was supported by AstraZeneca.

Address for reprint requests and other correspondence: G. Warhurst, Section of Gastrointestinal Science, Clinical Sciences Bldg., Hope Hospital, Salford, UK, M6 8HD (E-mail:gwarhurs{at}fs1.ho.man.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 28 November 2000; accepted in final form 5 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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