Modulation of intestinal paracellular

permeability by intracellular mediators and

cytoskeleton

M. Pérez, A. Barber, and F. Ponz

Abstract: The influence of cytoskeletal inhibitors (cytochalasin E and colchicine) and intracellular mediators (cAMP, Ca2+,and protein kinase C) in the control of paracellular permeability to mannitol has been examined in rat jejunum in Ussing-typechambers. Cytoskeletal inhibitors, cytochalasin E (20 µmol⋅L–1) or colchicine (0.5 mmol⋅L–1), when present in mucosal,serosal, or in both mediums, significantly increase unidirectional mannitol fluxes. Exposure of mucosal and serosal intestinalsurface to 10 mmol⋅L–1 theophylline or 1 mmol⋅L–1 cyclic AMP analogue for raising the intracellular cAMP enhancesparacellular permeability. In Ca2+-free physiological medium passive permeability strongly increases. Alterations of cytosolicCa2+ levels induced by the Ca2+ ionophore A23187 (20 µmol⋅L–1) or by 0.3 mmol⋅L–1 TMB-8, a Ca2+ releasing inhibitor fromthe intracellular stores, enhance mannitol flux. Addition of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, whichactivates protein kinase C (PKC), also induces a large increase in the intestinal permeability to mannitol. These results provideevidence that tight junctions and consequently epithelial paracellular permeability can be physiologically controlled byintracellular mediators (Ca2+, cAMP, and PKC) probably through modulation of the cytoskeleton activity.

Key words: cytoskeleton, intestinal epithelium, intracellular mediators, paracellular permeability, tight junction.

Résumé: On a examiné le rôle des inhibiteurs cytosquelettiques (cytochalasine E et colchicine) et des médiateursintracellulaires (AMPc, Ca2+ et protéine kinase C) dans la régulation de la perméabilité paracellulaire au mannitol dans lejéjunum de rat maintenu dans des chambres de type Ussing. La présence des inhibiteurs cytochalasine E (20 µmol⋅L–1) oucolchicine (0,5 mmol⋅L–1) dans les milieux séreux ou muqueux, ou dans les deux milieux, a significativement augmenté lesflux unidirectionnels de mannitol. Par milieux, on entend le milieu situé du côté muqueux, le milieu situé du côté séreux, ou lemilieu situé à la fois du côté séreux et muqueux. L’exposition de la surface intestinale séreuse et muqueuse à 10 mmol⋅L–1 dethéophylline ou à 1 mmol⋅L–1 d’un analogue de l’AMP cyclique pour augmenter l’AMPc intracellulaire stimule laperméabilité paracellulaire. Dans un milieu physiologique sans Ca2+, la perméabilité passive augmente considérablement. Lavariation des taux de Ca2+ cytosoliques par l’emploi de l’ionophore Ca2+ A23187 (20 µmol⋅L–1) ou de TMB-8 (0,3 mmol⋅L–1),un inhibiteur de la libération de Ca2+ des réserves intracellulaires, stimule le flux de mannitol. L’addition de l’ester de phorbol,TPA, qui active la protéine kinase C (PKC), induit aussi une forte augmentation de la perméabilité intestinale au mannitol.Ces résultats indiquent que les médiateurs intracellulaires (Ca2+, AMPc et PKC) pourraient réguler les jonctions étanches et,par conséquent, la perméabilité paracellulaire épithéliale, et ce probablement en modulant l’activité du cytosquelette.

Mots clés : cytosquelette, épithélium intestinal, médiateurs intracellulaires, perméabilité paracellulaire, jonction étanche.[Traduit par la Rédaction]

Introduction

The intestinal epithelium normally absorbs the vast quantity ofwater, ions, and nutrients ingested daily, and it also helps toprevent the free mixing of lumen contents with underlyinginterstitial and vascular fluid. A crucial element in the latter“barrier function” of the intestinal epithelium is the intercellu-lar tight junctions (TJs) between adjacent cells, which selec-tively restrict the paracellular permeation of ions andnonelectrolytes. Abundant evidence now indicates that TJs arein fact dynamic structures that are continuously undergoingassembly and disassembly under physiological regulation and

which may play a substantive role in nutrient uptake (Ballardet al. 1995; Holmes and Lobley 1989; Madara 1989; Madaraand Trier 1994). The integrity of the TJs is dependent on thepresence of extracellular Ca2+ (Gonzalez-Mariscal et al. 1990).The TJ structure and permeability appear to be regulated by theepithelial cells in response to intracellular mediators, includingCa2+, cAMP, G-proteins, protein kinase C (PKC), inositoltriphosphate, and calmodulin, since changes in these mediatorsaffect junctional permeability (Argenzio and Whipp 1983;Balda et al. 1991; Martinez-Palomo et al. 1980; Mullin andO’Brien 1986; Ojakian 1981). Pharmacological modulation ofthe cytoskeleton has also been reported to bring about altera-tions in occluding-junction structure and function (Madaraet al. 1986; Madara 1987; Meza et al. 1980). The absorption-enhancing effect of luminal glucose has been shown to beNa+ dependent and due to regulation of absorptive cell tightjunctions, suggesting that activation of the Na+–glucosecotransporter on the apical membrane (SGLT-1) triggers thisresponse (Pappenheimer 1988; Pérez et al. 1993; Turner and

Received August 21, 1996.

M. Pérez, A. Barber,1 and F. Ponz.Departamento deFisiología y Nutrición, Universidad de Navarra,31008 Pamplona, Spain.

1 Author for correspondence.

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Madara 1995). As final effectors of these regulations someproteins such as ZO-1, cinguline, and F-actin related to TJstructure have been suggested (Anderson and Van Itallie1995).

In the present work, the influence of cytoskeletal inhibitors,cytochalasin E and colchicine, and intracellular mediators,cAMP, Ca2+, and PKC, on mannitol transfer across rat jejunumwas studied in vitro.

Methods

The animals were obtained from CIFA (Universidad de Navarra),reared and kept under good laboratory practices, and handled accord-ing to the European Community rules and Canadian Council onAnimal Care guidelines. Male Wistar rats weighing 400–600 g andfasted for 24 h were anesthetized with sodium pentobarbital(60 mg⋅kg–1, s.c.). After the abdominal cavity was opened, a jejunalsegment 15 cm long was rapidly removed, washed with cold 0.9%NaCl solution, opened longitudinally along the mesenteric border,and mounted in Ussing-type chambers. The chamber opening ex-posed 0.78 cm2 of intestinal surface area. Both the chamber mucosaland serosal sides were attached to reservoirs containing 5 mL of thebuffer solution described below and were continuously gassed with95% O2 – 5% CO2. Reservoirs were jacketed with a circulating waterbath, which maintained the physiological solutions at 37°C. The buff-er solution consisted of 140 mmol⋅L–1 NaCl, 10 mmol⋅L–1 KHCO3,2.4 mmol⋅L–1 K2HPO4, 0.4 mmol⋅L–1 KH2PO4, 1.2 mmol⋅L–1 CaCl2,and 1.2 mmol⋅L–1 MgCl2⋅H2O, at pH 7.4, and both mucosal and se-rosal media contained 20 mmol⋅L–1 D-mannitol. [14C]Mannitol(0.5 µCi; 1 Ci = 37 GBq) was added only to the mucosal or the serosalside, depending on the unidirectional flux that was then measured. Ingeneral, the different agents tested were added to both mucosal andserosal sides. Control data for the experiment were derived from ad-jacent pieces of tissue treated identically except for the exposure tothe different agents. Cytochalasin E, A23187 Ca2+ ionophore, and12-O-tetradecanoylphorbol-13-acetate (TPA) were dissolved in di-methylsulfoxide (DMSO) to make stock solutions and then were di-luted with the electrolyte bathing solution. Final DMSO concentrationwas 0.2%, which showed no effect on mannitol fluxes (Madara et al.1986). Colchicine, cAMP analogues, EGTA, and 3,4,5-trimethoxy-benzoic acid 8-(diethylamino)octyl ester (TMB-8) were dissolved di-rectly in the incubation medium.

After a 20-min equilibration period, unidirectional mucosa-to-serosa (J m-s) and serosa-to-mucosa (J s-m) fluxes of mannitol werecalculated. Five 200-µL aliquots were taken from the unlabelled sidecorresponding to successive periods of 20 min, the first 20 min andthe last 100 min after equilibration. Equal samples were taken at thesame times from the labelled side to balance hydrostatic pressure.Radioactivity was measured by liquid scintillation counting in aWallac 1409 Pharmacia (EG-G Instruments, Barcelona, Spain).

Transmural unidirectional fluxes were obtained for each piece oftissue and for each of the five 20-min periods from the rate of tracerappearance on the “cold” side and the specific activity of the “hot”side (Naftalin and Curran 1974) and were expressed as millimoles persquare centimetre per 60 min. A mean flux for the whole experimentaltime was estimated as the average of those calculated for the succes-sive periods.

The significance of differences between means was assessed byStudent’s t test. Statistical analyses were performed by running theStat View program. Both mathematical and statistical calculationswere carried out on a Macintosh computer. Results are presented asmeans ± SEM.

D-Mannitol was purchased from Merck (Darmstadt, Germany).Cytochalasin E, colchicine, bromo-cAMP, dibutyryl-cAMP, theo-phylline, EGTA, ionophore A23187, TMB-8, and TPA were pur-chased from Sigma (St. Louis, Mo.). 2-D-[1-14C]mannitol(2.0 GBq⋅mmol–1) was from Du Pont (Mississauga, Ont.).

Results

Effect of cytoskeleton inhibitors on the paracellularpermeability in rat intestine

To find a possible implication of the cytoskeletal structures inthe regulation of tight junctions, the effect of cytochalasin E,which inhibits microfilament polymerization, as well as theeffect of the microtubule-disrupting agent colchicine on theparacellular permeability to mannitol was studied.

The presence of 20 µmol⋅L–1 cytochalasin E in both mu-cosal and serosal media significantly increased mucosal to se-rosal flux of mannitol; the enhancing effect was also obtainedwhen cytochalasin E was present only in either the mucosal orserosal medium (Fig. 1). Moreover, this increase in permeabil-ity occurred as early as within the 1st h after exposure to20 µmol⋅L–1 cytochalasin, and was not significantly changedfor at least 120 min of the experiment. The same results wereobtained when serosal to mucosal flux of mannitol was meas-ured (data not shown), indicating that both mannitol J m-s andJ s-m are symetrical, in control or experimental conditions (seealso Fig. 2).

Furthermore, colchicine when present at 0.5 mmol⋅L–1 inboth mucosal and serosal sides provoked a significant increasein mannitol fluxes (Fig. 2). The increase by colchicine of mu-cosal to serosal mannitol flux was also observed when the drugwas in either the mucosal or serosal side and it appeared afterthe first 40 min (Fig. 2).

Influence of intracellular mediators on passiveparacellular permeability

Effect of changes in cAMP levelsTo raise cytosolic cAMP values, the phosphodiesterase inhibi-tor theophylline and permeant, stable cyclic AMP analogueswere added to the medium. The addition of 10 mmol·L–1

Fig. 1. Effect of cytochalasin E on mucosal to serosal mannitol fluxacross rat jejunum in vitro. After a 20-min equilibration period,mucosal to serosal (J m-s) flux (µmol⋅cm–2⋅60 min–1) was measuredin the absence (control) and presence of 20 µmol⋅L–1 cytochalasin Ein mucosal (m), serosal (s), or both mediums (m+s). Results are themean flux (±SEM) for the whole experimental time as indicated inMethods. Values in parentheses, number of data. **p < 0.01;***p < 0.001.

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theophylline or 1 mmol⋅L–1 dibutyryl-cAMP resulted in an in-crease in mannitol flux (Fig. 3). When bromo-cAMP at thelower concentration of 0.1 mmol⋅L–1 was added, mannitol per-meability was not modified (data not shown). As in previousexperiments where cytoskeleton inhibitors were used, the en-hancing effect appeared early without appreciable changethroughout time.

Influence of extra- or intra-cellular changes of calciumconcentrations

The dependence of paracellular permeability on extracellularCa2+ was tested by exposing the jejunum surface to Ca2+-freephysiological solution, prepared by removing this cation and

adding 5 mmol⋅L–1 EGTA. The mucosal to serosal mannitolflux was dramatically increased in Ca2+-free medium com-pared with control conditions (Fig. 4); the effect was observedfrom as early as the first 40 min after mucosal and serosal Ca2+

chelation and was enhanced with time of exposure to theCa2+-free medium.

The influence of cytosolic Ca2+ on mannitol flux was testedby using A23187, a Ca2+ ionophore that facilitates Ca2+ entryinto the cell, and TMB-8, an inhibitor of Ca2+ release fromintracellular stores. The presence in mucosal and serosal sidesof either 20 µmol⋅L–1 A23187 or 0.3 mmol⋅L–1 TMB-8 pro-voked a significant increase in the mucosal to serosal mannitolflux (Fig. 4); the effect was not enhanced with time.

Effect of the PKC activator TPAIn the presence of the PKC-activating phorbol ester TPA(1 mmol⋅L–1) in both mucosal and serosal mediums, a signifi-cant increase was obtained in the mucosal to serosal mannitolflux, from 0.284 ± 0.02 µmol⋅cm–2⋅60 min–1, n = 17, withoutTPA, to 0.392 ± 0.02 µmol⋅cm–2⋅60 min–1, n = 28, with TPA,p < 0.01. This rise in mannitol flux became significant onlyafter the 1st h of exposure, being increased during the 2nd h(data not shown).

Discussion

Paracellular mannitol permeability and, therefore, occluding-junction structure seem to be dependent on cytoskeletal activ-ity. This is suggested by the fact that both cytochalasin E, anagent that disrupts microfilament organization, and colchicine,a microtubule modifier, increase intestinal mannitol perme-ability. The enhancing effect appears during the first20–40 min of exposure to the cytoskeletal inhibitors, it is quan-titatively similar independent of which side, mucosal or se-rosal, they are on and it remains constant with time. Becausechanges in paracellular permeability appear after a relativelyshort delay, a relation between cytoskeleton and tight junctionsis suggested.

Exposure of intestinal epithelium to cytochalasin D resultsin a decrease in epithelial electrical resistance and an increasein mannitol permeability (Madara et al. 1986), through altera-tion of enterocyte TJ structure with contraction of the apicalperijunctional ring, and leads to a reduction of the number ofstrands within the TJ (Madara 1987). In confluent monolayersof Madin Darby canine kidney (MDCK) cells, addition of cy-tochalasin B produces a decrease in the electrical resistanceafter about 20 min (Meza et al. 1980), an effect also observedwith cytochalasin D (Stevenson and Begg 1994). In TJs andcytoskeleton interactions, the ZO-1 junctional protein couldpresumably be involved (Ballard et al. 1995; Stevenson et al.1988; Stevenson and Begg 1994).

Oral colchicine administration to rats induces an increasein the lactulose/mannitol urinary ratio, as a result of increasedlactulose permeation (Fradkin et al. 1995). Effects of col-chicine on the brush-border cytoskeleton, probably indirect,have been reported (Keller et al. 1985). In MDCK cells, col-chicine causes a profound disruption of microtubules, as wellas a decrease in transepithelial electrical resistance (Meza et al.1980). The colchicine effects on the paracellular pathway havebeen related by some authors (Nassar et al. 1991; Ohya andOgura 1993) to direct alterations in cytosolic Ca2+ levels.

Fig. 2. Influence of colchicine on mannitol fluxes across ratjejunum in vitro. (Top panel) Effect of 0.5 mmol⋅L–1 colchicine onboth mucosal and serosal sides on total J m-s and J s-m. *p < 0.05;**p < 0.01. (Bottom panel) Effect of colchicine present in eithermucosal or serosal medium on J m-s (µmol⋅cm–2⋅60 min–1) overtime. The increasing effect is statistically significant (p < 0.05)from the beginning. Values in parentheses, number ofdeterminations.

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Fig. 3. Effect of the increase of cAMP cytosolic level on mucosal to serosal mannitol flux. Tested agents (10 mmol⋅L–1 theophylline or1 mmol⋅L–1 dibutyryl-cAMP) were present in both mucosal and serosal medium. Except where indicated, the effect is statistically significant.J m-s (µmol·cm–2·60 min–1) over time.

Fig. 4. Influence of changes in extracellular or cytosolic Ca2+ levels on mannitol permeability in rat jejunum in vitro. Changes (suppression ofCa2+) and additions (0.3 mmol⋅L–1 TMB-8, 10 µmol⋅L–1 or 20 µmol⋅L–1 A23187) were carried out in both mucosal and serosal medium.J m-s (µmol·cm–2·60 min–1) over time. The lower concentration of A23187 is ineffective. In the other cases, the increasing effect issignificantly (p < 0.05) observed from the first period.

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Our data indicate that addition of 10 mmol⋅L–1 theophyl-line, a phosphodiesterase inhibitor, or of 1 mmol⋅L–1

dibutyryl-cAMP, a stable permeable cAMP analogue,increases passive permeability. In both cases, an increase inintracellular cAMP level has to be expected, which may causethe TJ opening. These results could correlate with those ob-tained by other authors, in MDCK cells (Balda et al. 1991),jejunum (Barnett et al. 1978), or colon (Argenzio and Whipp1983), who found a decrease in transepithelial electrical resis-tance and an increase in ionic conductance. Discrepant datahave been reported, however, in Necturus gallbladder (Duffeyet al. 1981) and endothelium (Wolbrug et al. 1994), but thisdisagreement may be due to differences in these epithelial re-sponses to cAMP. The cAMP action has been ascribed to PKAactivation, which induces cytoskeletal protein phosphorylationin relation to occluding junctions (Balda et al. 1991).

Our results emphasize the critical importance of extracel-lular Ca2+ for paracellular permeability. Suppression of Ca2+

at both intestinal wall sides results in a dramatic increase inmannitol flux, which is enhanced over time and may be ex-plained by the extracellular Ca2+ role in maintaining TJ func-tional structure. In fact, extracellular calcium depletion leadsto rapid destruction of the junctional complex in several epi-thelia (Gonzalez-Mariscal et al. 1990; Nassar et al. 1991;Pitelka et al. 1983). In Necturus gallbladder (Palant et al.1983), a decrease in transepithelial electrical resistance and adisruption of junctional morphology by exposure to Ca2+-freebathing media has been observed, an effect that appears within5–15 min after Ca2+ chelation by EGTA, becoming greaterwhen Ca2+ is absent in both mucosal and serosal media. How-ever, in A6 monolayer cells, serosal Ca2+ but not mucosal wasrequired for the TJ integrity (Jovov et al. 1994).

Cytosolic Ca2+ levels are also important for paracellularpermeability. The increase in Ca2+ entry into the cells by ad-dition of the Ca2+ ionophore A23187 is followed by an in-crease in epithelial permeability to mannitol. In perfusedisolated rat liver a similar increase in paracellular permeabilityhas been reported (Kan and Coleman 1988). In Necturus gall-bladder epithelium (Palant et al. 1983) and in MDCK cells(Kan and Coleman 1988), A23187 addition elicits a rapid de-crease in the transepithelial resistance.

On the other hand, when TMB-8, an inhibitor of Ca2+ exitfrom intracellular stores, is added to the medium, a significantincrease in the mannitol flux is also observed, which suggeststhat a diminution in Ca2+ intracellular level may lead again toTJ opening. Altered cytoskeletal organization in conjunctionwith increased permeability has been related to the phospho-rylation of myosin regulatory light chain accomplished bymyosin light chain kinase, which in turn is activated by intra-cellular Ca2+ spikes (Turner and Madara 1995). In MDCKcells, the intracellular Ca2+ chelant BAPTA-AM decreasesjunctional electrical resistance (Stuart et al. 1994).

Furthermore PKC seems to participate in the regulation ofintestinal paracellular permeability. Addition of TPA, a PKCintracellular activator, induces a substantial increase in the mu-cosal to serosal mannitol flux across jejunum after about a60-min delay. This effect is consistent with the decrease intransepithelial resistance and changes in cytoskeleton and TJstructure due to TPA in the MDCK cells (Palant et al. 1983)and in the LLC-PK renal epithelial cell line (Mullin andO’Brien 1986), actions that enhance epithelial permeability to

various solutes. In the intestinal Caco-2 cells, TPA additionalso decreases transepithelial resistance and increases manni-tol permeation, but addition of staurosporine, a PKC inhibitor,does not modify either permeability or junctional resistance(Stenson et al. 1993). An early event in response to exposureof different epithelial cells to TPA seems to be cytoskeletalreorganization (Hedberg et al. 1994; Mullin and McGinn1988), which suggests diacylglycerol and PKC involvement inthe functional structure of tight junctions.

In summation, the experiments conducted in rat jejunumepithelium show that different agents, such as extracellularCa2+, cytoskeleton inhibitors, cAMP, Ca2+ cytosolic levelmodifiers, and a PKC activator, induce substantial changes inparacellular permeability to mannitol, which seem to be re-lated to changes in tight junction structure and function. Re-sults emphasize the physiological control of the paracellularpathway in various ways, and support the hypothesis that theenterocyte cytoskeletal system is involved in the tight junctionregulation by intracellular mediators.

Acknowledgements

This work was partially supported by a grant from theGobierno de Navarra (Spain).

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