Modulation of intestinal paracellular permeability by intracellular mediators and cytoskeleton
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Modulation of intestinal paracellular
permeability by intracellular mediators and
M. Prez, 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 molL1) or colchicine (0.5 mmolL1), when present in mucosal,serosal, or in both mediums, significantly increase unidirectional mannitol fluxes. Exposure of mucosal and serosal intestinalsurface to 10 mmolL1 theophylline or 1 mmolL1 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 molL1) or by 0.3 mmolL1 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.
Rsum: On a examin le rle des inhibiteurs cytosquelettiques (cytochalasine E et colchicine) et des mdiateursintracellulaires (AMPc, Ca2+ et protine kinase C) dans la rgulation de la permabilit paracellulaire au mannitol dans lejjunum de rat maintenu dans des chambres de type Ussing. La prsence des inhibiteurs cytochalasine E (20 molL1) oucolchicine (0,5 mmolL1) dans les milieux sreux ou muqueux, ou dans les deux milieux, a significativement augment lesflux unidirectionnels de mannitol. Par milieux, on entend le milieu situ du ct muqueux, le milieu situ du ct sreux, ou lemilieu situ la fois du ct sreux et muqueux. Lexposition de la surface intestinale sreuse et muqueuse 10 mmolL1 dethophylline ou 1 mmolL1 dun analogue de lAMP cyclique pour augmenter lAMPc intracellulaire stimule lapermabilit paracellulaire. Dans un milieu physiologique sans Ca2+, la permabilit passive augmente considrablement. Lavariation des taux de Ca2+ cytosoliques par lemploi de lionophore Ca2+ A23187 (20 molL1) ou de TMB-8 (0,3 mmolL1),un inhibiteur de la libration de Ca2+ des rserves intracellulaires, stimule le flux de mannitol. Laddition de lester de phorbol,TPA, qui active la protine kinase C (PKC), induit aussi une forte augmentation de la permabilit intestinale au mannitol.Ces rsultats indiquent que les mdiateurs intracellulaires (Ca2+, AMPc et PKC) pourraient rguler les jonctions tanches et,par consquent, la permabilit paracellulaire pithliale, et ce probablement en modulant lactivit du cytosquelette.
Mots cls : cytosquelette, pithlium intestinal, mdiateurs intracellulaires, permabilit paracellulaire, jonction tanche.[Traduit par la Rdaction]
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 latterbarrier 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 andOBrien 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; Prez et al. 1993; Turner and
Received August 21, 1996.
M. Prez, A. Barber,1 and F. Ponz.Departamento deFisiologa y Nutricin, Universidad de Navarra,31008 Pamplona, Spain.
1 Author for correspondence.
Can. J. Physiol. Pharmacol. 75: 287292 (1997)
1997 NRC Canada
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.
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 400600 g andfasted for 24 h were anesthetized with sodium pentobarbital(60 mgkg1, 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 37C. The buff-er solution consisted of 140 mmolL1 NaCl, 10 mmolL1 KHCO3,2.4 mmolL1 K2HPO4, 0.4 mmolL1 KH2PO4, 1.2 mmolL1 CaCl2,and 1.2 mmolL1 MgCl2H2O, at pH 7.4, and both mucosal and se-rosal media contained 20 mmolL1 D-mannitol. [14C]Mannitol(0.5 Ci; 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-L 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 hotside (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 byStudents 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 GBqmmol1) was from Du Pont (Mississauga, Ont.).
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 molL1 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