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▼ Part one of this two-part article1 outlined sev-eral relevant anatomical, biochemical andphysiological properties of the gastrointestinal(GI) tract related to oral drug delivery. In that re-view, the barrier properties of the small intestineand large intestine (colon) were described in re-lation to the uptake of molecules across intestinalepithelial cells through transcellular pathways.Ananalogy was drawn between cobblestones heldin place with mortar and the transcellular andparacellular pathways of the intestine. The cob-blestones represent the cells and the surface areapossible for the events associated with transcellu-lar transport.The mortar between adjacent stonesis likened to structures present between adjacentcells, which serve to hold the cells together andimpede the paracellular movement of solutes.The surface area of this region represents a smallfraction of the total area of the potential absorp-tive surface of the intestine, and is indicative ofthe lower transport capacity of this route com-pared with that of transcellular transport. As pre-viously discussed1, however, the potential formacromolecular transport via transcellular trans-port pathways appears to be limited.

For the oral delivery of macromolecules, selec-tively increasing the permeability of the paracellu-lar barrier may provide an opportunity to circum-vent the difficulties associated with delivering a

therapeutic by transcellular transport, such as in-tercellular degradation. The primary barrier ofthe paracellular route is a structure known as thetight junction (TJ) complex, which exists at theapical neck where adjacent intestinal epithelialcells are observed to be very closely opposed2

(Fig. 1). The TJ complex completely circumnavi-gates each epithelial cell to form a continuousseal that segregates the apical and basolateralmembrane components. This barrier has beensuggested to have size- and charge-selectiveproperties.This review describes current levels ofunderstanding of the structure and function of TJcomponents, and assesses possible means to ma-nipulate these components in order to enhanceparacellular permeability.

Structural relationships of the TJ complexAlthough evidence has been presented to supporta role for lipids in the TJ3, recent studies havedramatically expanded and modified levels ofunderstanding of the TJ by identifying severalprotein components localized to this structure4.Furthermore, several potential interactions be-tween these newly-identified proteins have beensuggested (Fig. 1). Starting at the plasma mem-brane, several transmembrane proteins are nowthought to participate in the extracellularcell–cell contacts between adjacent epithelialcells at the TJ. Occludin was the first to be iden-tified5. Recently, two other proteins with similarcell–cell contact interactions have been identi-fied6, and designated as claudin-1 and claudin-2.Expression of either claudin-1 or claudin-2 ap-pears to establish intramembrane strands charac-teristic of TJ structures. Co-expression studieswith occludin suggests claudins to be the pri-mary TJ seal, with occludin possibly providing a coordinating and/or modulatory function.Another transmembrane protein, the junctionaladhesion molecule (JAM) with features of the

Regulation of the intestinal epithelialparacellular barrierAnn L. Daugherty and Randall J. Mrsny

Ann L. Daughertyand Randall J. Mrsny

Drug Delivery/Biology GroupDepartment of Pharmaceutical

Research and DevelopmentGenentech, Inc.

1 DNA WaySouth San Francisco

CA 94080USA

tel: 11 650 225 2198fax: 11 650 225 1418

e-mail: masi@gene.com

reviews research focus

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Paracellular transport of orally-administered drugs, the passage of

molecules between adjacent intestinal epithelial cells, is impeded by

a range of structural and functional features found in the intestine.

An increased knowledge of the mechanisms that govern the paracel-

lular barrier will enable the pharmaceutical scientist to design novel

and rational formulations and delivery platforms that will improve

the oral bioavailability of therapeutic molecules, particularly proteins

and peptides, which would be taken-up by the paracellular pathway.

immunoglobulin superfamily, has also been localized to theTJ7. Although it is unclear how the extracellular domains ofthese TJ proteins might interact to form a seal to paracellularsolute transport, freeze-fracture images suggest that claudin-1,rather than claudin-2, may provide the primary TJ barrier com-ponent of the intestinal epithelium8.

Claudin-1 and claudin-2 have only minor intracellular do-mains6 and thus it is difficult to imagine how these proteinscould establish interactions with other TJ components requiredfor the regulatory control of TJ function. The intracellular do-main of occludin is substantially greater however, and has beenshown to interact with zona occludin-1 (ZO-1).The amino ter-minal half of ZO-1 contains multiple domains, including an al-ternatively spliced region termed a. It is known that ZO-1a1 isexpressed in more stable TJ structures, whereas ZO-1 a2 is ob-served in more dynamic TJ structures. Intestinal TJ complexescontain ZO-1 a1. Binding studies have suggested that ZO-1 interacts with at least two similar proteins, ZO-2 (Ref. 9) and

ZO-3 (Ref. 10). Other intracellular proteins, such as cingulin,the 7H6 antigen, symplekin, and AF-6 (Ref. 11), have also beenlocalized to the TJ, as has been recently reviewed4, but their in-teractions with other components unique to the TJ have yet tobe elucidated.

Tight junction structures are closely associated with anothercell–cell contact system, the adherens junction (AJ). Multiple AJcontacts can be observed along the lateral border of intestinalepithelial cells, holding adjacent cells in close, but not tight, ap-proximation.The AJ is composed of a, b and g catenins com-plexed to the transmembrane protein E-cadherin (Fig. 1). E-cadherin forms divalent cation-dependent associations betweenadjacent epithelial cells12.The complex of TJ and AJ structures atthe apical neck of enterocytes act to anchor cytoskeletal com-ponents. Interactions between the cytoskeleton and occludinhave been suggested to occur through spectrin, which acts as alinker between ZO-1 and actin.A complex of proteins, which in-cludes radixin, vinculin and a-actinin, act to tether a cytoskeletal

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Figure 1. Model depicting the structural components and potential modulating factors of the apical junctional complex of the intestinal epithelium. The complex, composed of the tight junction and adherens junctions, is defined by a series of integral membrane proteins, which interact with cytoskeletal components through proteins with multiple binding domains.

structure known as the perijunctional actin–myosin II ring tothe cadherin–catenin complex (Fig. 1). It is quite possible thatthere are substantial differences in the composition and regu-lation of the cytoskeletal structures that associate with TJ struc-tures to those that tether AJ structures. The establishment andmaintenance of AJ structures appears to be important in the de-velopment of nascent, functional TJ complexes13.

Functional interactions of TJ componentsAgents that are known to affect the TJ and thereby alter para-cellular permeability as determined by a decrease in transep-ithelial electrical resistance are summarized in Tables 1–3.Ethylenediamine tetraacetic acid (EDTA), phytic acid and citricacid have been widely used to open the paracellular route be-tween adjacent epithelial cells, presumably through their abil-ity to chelate divalent cations such as Ca21 and Mg21. The in-crease in paracellular permeability induced by the addition ofthe Ca21 chelator EGTA to several cultured cell lines was seento be polarized, however14. Epithelial permeability increaseddramatically when EGTA was added to the basolateral side andeven more so when the addition was made to both the basolat-eral and apical sides, whereas no increase was observed withonly apical application14. At present, however, there is no datato suggest that chelator-induced permeability increases are dueto a direct action on extracellular TJ protein–protein interac-tions. Rather, disruption of extracellular divalent cation associ-ations at AJ structures between E-cadherins may be an initialand indirect action of chelators, which induce a destabilizationof the TJ complex. Another in vivo study, in which the targetedexpression of a dominant negative mutant for E-cadherin ofthe intestinal mucosa of mice produced a paracellular leak15,

supports this putative relationship. Although the actions ofchelators can be readily demonstrated on intestinal epithelialcell lines in vitro, the application of such an agent in vivo cannoteasily overcome the very large reservoir of divalent cations ofthe body, unless the chelator is applied at a particular site at ex-ceptionally high levels.

Disruption of the extracellular interactions between mol-ecules directly involved in the TJ structure would be an obviousway to increase paracellular transport, although these associ-ations are not yet completely understood. What has beenshown in vitro is that the addition of a peptide, identical to the33 amino acids of the second extracellular loop of occludin, toan epithelial cell monolayer resulted in the reversible opening

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Table 1. Time course of action of some agents capable oftight junction disruption in vitro

Agent Approximate response time

Insulin 3–4 daysInsulin-like growth factor-1, 4 daysor -2 (IGF-1 or IGF-2)

Tumour necrosis factor (TNFa) 90 minInterferon-gamma (IFN-g) 2–3 daysCytochalasin B or D 20 minC. difficile toxin A 6–8 hATP depletion 30 minProtein kinase A inhibition 1 hProtein kinase C activation 2 hTyrosine phosphorylation 30 minCa2+ chelators 10 minCa2+ ionophores 30 minNa+-linked nutrient uptake 20 min

Table 2. Agents that increase paracellular permeability ofepithelial cells in vitro

Agent Classification Putative mechanism of action

Epithelial permeability Serum protein(s) Induces basolateral factor (Ref. 53) cytoskeletal construction

A23187 (Ref. 48) Ionophore Increases cytoplasmic Ca2+

Cyclic AMP Second messenger Phosphorylation events(Ref. 54)

H7, PMB (Ref. 49) PKC inhibitors Decreases phospholipase CMastoparan Tetradecapeptide Increases apical Cl– and K+

(Ref. 39) channelsOncostatin M Cytokine Increases basolateral (Ref. 55) permeability

PZ-pro-leu-gly- Collagenase Stimulates transepithelialpro-D-arg (Ref. 56) substrate Na+ flux

IFN-g (Ref. 57) Cytokine UnknownTNF (Ref. 58) Cytokine UnknownC. difficile toxin Bacterial toxin Perturbs cytoskeleton(Ref. 59)

ZOT (Ref. 25) Bacterial toxin Perturbs cytoskeleton

Table 3. Factors that decrease paracellular permeability ofepithelial cells in vitro

Agent Classification Putative mechanism of action

Phlorizin (Ref. 60) Glucose transport Reduces Na+ uptakeinhibitor

Choline (Ref. 60) Replacement ion Reduces Na+ uptakefor Na+

diC8, TPA (Ref. 49) PKC stimulators Protein phosphorylationTRH (Ref. 49) Thyroid hormone Increased phospholipase COncostatin (Ref. 55) Cytokine Decreased apical transport

of the paracellular route16. Similar addition of a 33 amino acidpeptide representing the first extracellular loop or a random-ized sequence of the second extracellular-loop peptide failed toaffect the TJ. Taking advantage of this observation for an in vivoapplication to enhance paracellular permeability would be limited by the difficulty in delivering and maintaining such apeptide in the intestinal lumen, given the overwhelming proteolytic capacity of this site. Further, as claudin-1, and notoccludin, may establish the primary TJ barrier of the intestinalepithelium8, this approach might prove unsuccessful in vivo.Similarly, randomly methylated b-cyclodextrin17, sodiumcaprate18 and long-chain acylcarnitines19 have been shown toopen the intestinal paracellular route. The mechanisms bywhich these compounds disrupt TJ function are currently amatter for debate; it is possible that they may also act directlyon the TJ. Finally, modulation of TJ components, such as oc-cludin, do not appear to correlate directly with paracellular per-meability properties20.

The cytoskeletal ring at the apical neck of intestinal epithelialcells acts to coordinate and stabilize the TJ protein arrays at themembrane surface. C3 exotoxin isolated from Clostridium difficileor Clostridium botulinum compromises the functional integrity ofTJ structures by disrupting this actin ring. This toxin ADP ribo-sylates a small GTP-binding protein, Rho, whose function is re-quired for the stabilization of actin filaments in the perijunc-tional cytoskeletal ring21. Stabilization of the actin ring by Rhoappears to be in a tightly-controlled balance, because Rho ac-tivity can also disrupt TJ structures22. Because the downstreamsignalling of Rho kinase can affect the ERM proteins, ezrin,radixin, and moesin23, disruption of cytoskeletal interactions ofAJ structures may also lead to an opening of the paracellularpathway. In addition to Rho, heterotrimeric G proteins and pro-tein kinase C isoforms localize to TJ structures24, which mayprovide important controlling functions related to TJ function.

Augmentation of paracellular permeabilityThe GI tract is constantly exposed to a large spectrum of po-tentially pathogenic agents. Its selective permeability proper-ties provide a great deal of initial protection, but a number ofpathogenic agents have acquired mechanisms capable of modi-fying or breaching this epithelial barrier.The mechanisms usedby these pathogens to override protective barriers provide aninsight into the structure–function relationships of the intesti-nal TJ. Vibrio cholerae releases a zona occludins toxin (ZOT),which reversibly modifies actin polymerization. Zona oc-cludins toxin appears to increase TJ permeability to macromol-ecules through modification of a protein kinase C pathway25.In fact, activation of protein kinase C can increase the paracel-lular transport of macromolecules26. Infection of MDCK cellsby Salmonella typhimurium shows a rapid disruption of the epi-

thelial barrier, which may be because of similar stimulation ofsignalling pathways that alter the cytoskeletal structure27.Some pathogens can directly disrupt the paracellular barrierfunction following interaction with epithelial cells. For exam-ple, Mycoplasma pulmonis can produce a twofold increase in theparacellular permeability of tracheal epithelial cells28. Themechanism behind this observation has not yet been de-scribed, but other mechanisms of augmenting paracellular per-meability are better understood2.

Although some pathogens and/or their associated toxinscan directly cause a disruption of the intestinal paracellularbarrier properties, intestinal infections can frequently trigger acascade of signalling events, which lead to the release of cyto-kines and lymphokines during the ensuing inflammatory andimmune responses. Tumour necrosis factor alpha (TNFa) andinterferon-gamma (INF-g) released by a range of white cellshave been shown to diminish the paracellular barrier proper-ties of the intestinal mucosa29.The actions of TNFa and INF-gon the paracellular properties of polarized monolayers of intestinal epithelial cells in vitro occurs over a time-frame ofhours-to-days, consistent with response events of intestinal in-fections. Other cytokines, such as IL-4 and IL-13, also dimin-ish barrier function and increase the paracellular flux ofmacromolecules across intestinal tissue30. Just as the immunesystem can influence the paracellular permeability of the in-testinal epithelial mucosa, several growth factors can also alterenterocyte TJ function. Hepatocyte growth factor/scatter factor(HGF) is an important response-chemokine involved in repairfollowing intestinal injury31. One of its early actions on intactintestinal epithelia adjacent to an intestinal mucosa wound isto de-differentiate columnar epithelial cells to squamous cellsthat move and cover over the wound32. In order to affect thiscell phenotype change, epithelial cells separate from one an-other by losing cell–cell contacts, including TJ structures. Acti-vation of the HGF receptor, c-met, results in the activation of atyrosine kinase cascade, which acts in a similar manner asother non-receptor tyrosine kinases, such as c-yes and c-src,which appear to be associated with TJ structures (Fig. 1). Theactions of cytokines and growth factors on TJ function are con-sistent with a role for phosphorylation events in the control ofTJ function.

Nutrient-enhanced augmentation of paracellular permeabilityTwo primary functions of the intestine are digestion and ab-sorption of food stuffs to obtain nutrients, cofactors and vita-mins. Based upon a series of observations, a physiological para-digm of maximizing the absorptive capacity of the intestineduring peak nutrient loads has been proposed33. In general,glucose and amino acids, such as tryptophan, are absorbed

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from the intestinal lumen through Na1-coupled transporters,which control this process (Fig. 2). When these transportersare functioning at maximum capacity, a large amount of Na1

enters the epithelial cytosol with a large influx of water.Presumably, subsequent events occur in response to the cellswelling that results from the uptake of Na1 and water.Efflux of Na1 occurs through several mechanisms, includingexchange for K1 (via the Na1, K1-ATPase) and Ca21 (via aNa1–Ca21 antiporter). In response to increased cytosolic Ca21

levels, myosin light-chain kinase is activated and thenphosphorylated, resulting in contraction of the apical actinring. Contraction of the actin ring may lead to a slight openingof the paracellular pathway by producing a strain on the cyto-skeleton associated with the TJ.This ‘tug’ at the TJ structure in re-sponse to cell swelling presumably leads to the enhanced trans-port of sugars and oligopeptides through the paracellular route.

Initial studies evaluating the uptake parameters of glucosefrom the mammalian gut suggested an enhanced uptake path-

way. This finding was disputed by the detection of increasedglucose transporters in response to a high carbohydrate diet34.Although still not univerally accepted, subsequent studies havesupported the possibility of a nutrient-enhanced augmenta-tion of paracellular permeability33. Several elegant in vivo stud-ies have demonstrated the enhanced uptake of peptides via nu-trient-induced increases in paracellular solute transport35. Theaddition of supra-physiological levels of glucose or tryptophan,to the apical side of confluent monolayers of intestinal cell cul-tures, results in an increase in paracellular transport and a de-crease in the trans-epithelial resistance (TEER)36. Manipulationof the Na1-glucose cotransporter (SGCT-1) activity in Caco-2cells by inhibition with phlorizin or by enhancement via SGLT-1 overexpression impacts the TEER and paracellular permeabil-ity37. Substitution with choline to block Na1 uptake inhibitsnutrient-dependent TJ effects. Increased intracellular Na1 re-sults in the activation of stretch-activated Ca21 channels and aninflux of Ca21 (Ref. 37).Artificially increasing intracellular Ca21

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Figure 2. Model depicting the putuative events involved in the increased paracellular permeability of the intestinal epithelium induced by supra-physiological concentrations of nutrients. The influx of sodium ions simultaneous to the uptake of glucose and certain amino acids initiates a series of ion-transport events, leading to calcium-activated phosphorylation events of cytoskeletal elements to produce a contraction that transiently opens theparacellular permeability barrier.

through the actions of the Ca21 ionophore A23187 results inan increase in paracellular transport38. Mastoparan, which ac-tivates apical Cl2 and K1 conductances to produce a decreasein cell volume (cell shrinkage, creating a separation betweenadjacent cells), has also been used to increase the paracellularpermeability of epithelial cell monolayers in vitro39.Expression of the myosin light-chain kinase catalytic subunitresults in increased TJ permeability and conversely, the overex-pression of myosin light-chain kinase also correlates with a decrease in TEER37.Together, these data support the model de-scribed previously and identify several potential targets forpharmacological modulation of paracellular permeability.

Augmentation of paracellular permeability in diseaseIt is clear that the barrier to paracellular permeability is estab-lished, maintained and modulated through the actions of opposing pathways that are capable of opening and closing theTJ structure in a dynamic fashion. A disruption of this balancecan occur in certain disease states. In Ménétrier’s disease, an in-crease in the TJ width from 7.5 to 10.5 nm is observed in thegastric mucosa. The etiology of this disease is unclear, but hasbeen linked to chronic infection or allergic response, possiblyresulting in the continued opening of the TJ, which would cor-relate with the protein loss and edema observed in these pa-tients40. Cystic fibrosis is caused by the disfunction or loss of acAMP-dependent Cl2 channel (CFTR). Because Cl2 conduc-tance of the TJ is increased by cAMP but not by other stimu-lants41, the defect of CF may directly compromise normal TJfunction. Further, the epithelia of CF patients demonstrate in-creased basal Na1 influx through overexpression of the epi-thelial sodium channel (ENaC). Either the decreased CFTRfunction or the increased ENaC function, or both, may drivethe observed decrease in TEER and result in the reduced TJstrand organization found in the epithelia of CF patients42. InTurner’s syndrome, the TJ number is dramatically reduced inthe endometrium of patients, which correlates with a decreasein fertility and other symptoms43. Finally, epithelial cancerstypically have a dramatic reduction in functional TJ and AJstructures and epithelial tumour promotion is accompanied byan increase in the tendency for tight junction leakiness44.

Underlying mechanisms controlling paracellular permeabilityIt is likely that events associated with enhanced intestinal para-cellular permeability, induced by pathogens, cytokines, growthfactors, supra-physiological nutrient concentrations, and cer-tain disease states, occur through common and overlapping in-tracellular pathways. Much of our current understanding ofthese intracellular pathways and events comes from in vitrostudies using synchronized cultures of a single cell type. In re-

ality, the intestinal epithelium is a complex mixture of celltypes (absorptive villus cells, secretory crypt cells and muci-nous goblet cells) in a continuum of growth and differentia-tion stages. Further, epithelial cells are in a continuous dialoguethrough the actions of cytokines and growth factors secretedfrom mesenchymal, inflammatory and immune cells present inthe submucosa or epithelium itself. Because the intestinal ep-ithelia maintains a patent barrier to paracellular permeability,despite the frequent bombardment of pathogens, growth fac-tors and cytokines, it appears that mechanisms and compo-nents that have been identified in vitro are tightly controlled invivo to function in a dynamic and elastic fashion.

Much of the functional control of TJ structural componentsappears to occur through phosphorylation and dephosphos-phorylation events. Serine/threonine phosphatase inhibitors,such as okadaic acid, disrupts TJ function45, whereasserine–threonine kinase inhibitors, such as H7, H8 and stau-rosporine, stabilize TJ function46. Tyrosine phosphatase in-hibitors, such as pervanadate and phenylarsine oxide, disrupt TJfunction47, as does a stimulation of tyrosine phosphorylationby the oncogene v-src48. Other studies have suggested that therole of tyrosine phosphorylation may impact AJ more that TJfunction38. Protein kinase C (PKC) stimulators decrease para-cellular permeability, whereas PKC inhibitors increase paracel-lular permeability49,50. Phosphorylation of serine residues onoccludin appears to increase the formation of TJ structures51,whereas phosphorylation of tyrosine residues on ZO-1 appearsto dismantle TJ structures47. Together, these observations sug-gest the possibility of multiple regulatory pathways controllingthe structure and function of the TJ, possibly through phos-phorylation events of several TJ components.

Several signalling molecules that phosphorylate serine–thre-onine residues or tyrosine residues have been localized to theTJ, including PKC-a, PKC-z, and several heterotrimeric proteinsGa subunits as well as the non-receptor tyrosine kinases c-srcand c-yes (Fig. 1). At least two trafficking proteins, Rab13 andRab3B, may play roles in the localization of some of these com-ponents to the TJ region.There are multiple sites at the TJ wherephosphorylation events could be controlled and where manipu-lation would lead to an opening of the paracellular pathway. Itis likely that specific receptor signalling events associated withAJ interactions lead to events that stimulate and sustain the for-mation of the TJ, whereas activation of receptors by cytokinesor white cell diapedesis may alter this homeostasis and induceTJ disruption.

Conclusions and perspectiveA barrier to random paracellular bi-directional movement is es-sential for the establishment of directional and selective intesti-nal transport pathways. Opening of the intestinal paracellular

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barrier, through a disruption of TJ complexes, can enhance the uptake of orally delivered drugs. It is currentlyunclear, however, what specific health issues may or may notarise from repeated or chronic breaches of this barrier.Complete disruption of TJ structures (common in epithelialcancers) or the transient loss of their function can lead toacute epithelial dysfunction.With an improved understandingof the structure–function relationships of TJ components, itmay be possible to manipulate the TJ in a more dynamic andcontrolled fashion to enable the uptake of many poorly ab-sorbed drugs, such as proteins and peptides at local sites alongthe GI tract without associated traumatic events. Indeed, TJstructures have already been shown to become selectively per-meable to one class of solutes in response to environmentaland cellular stimuli52.

A number of TJ components have recently been identifiedand others will certainly be described in the near future. As theroles and interactions of these components become better understood, it may be possible to selectively open and close theTJ in a manner acceptable for pharmaceutical applications.

AcknowledgementsThe authors wish to thank Tue Nguyen and Jeffrey Cleland fortheir support of this work. James Madara, Asma Nusrat andJerry Turner are acknowledged for their thoughtful discus-sions, and Allison Bruce and Jim Ligos are also thanked fortheir expert artwork.

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