25.4.1

Transport Pathways

Throughout the GI tract, transport of electrolytes, solutes, and water across epithelia occurs across both a transcellular and paracellular pathways ( Fig. 25.1 ). The transcellular route for hydrophilic molecules, for example, Na ⁺ , Cl ⁻ , and glucose, is governed by the profile of membrane pumps, carrier, and channels expressed in a particular cell type. The passive movement across the lipid component of the membrane is very limited for charged and hydrophilic molecules. For example, the electrical resistance across model lipid membrane bilayers is in the range of 10 ⁶ –10 ⁹ ohm × cm ² whereas the resistance across real membranes in the GI tract is 3–4 orders of magnitude less, reflecting facilitated conductance through protein-based channels ( Table 25.1 ). The profile of conductance proteins differs among epithelia explaining their unique functions. Individual transporters also show a polarized distribution to either the apical or basolateral membrane surface as the basis for directional transport. For example, the apical H ⁺ -K ⁺ ATPase of gastric parietal cells is responsible for secreting hydrochloric acid within the stomach. Na ⁺ -dependent bile acid transporters are positioned on the hepatocyte’s sinusoidal surface and the apical-luminal surface in the ileum to produce the enterohepatic circulation of bile salts. The cystic fibrosis transmembrane regulator (CFTR), a chloride channel, is positioned on the apical surface of biliary, pancreatic, and intestinal surfaces to bring about luminal Cl ⁻ secretion which is followed by Na ⁺ and water secretion.

Equivalent electrical circuit model of the intestinal epithelial cell layer. Only resistive elements are shown. Series resistance across the transcellular pathway is the sum of individual resistance across the apical ( R _ap ) and basolateral membranes ( R _bl ). These are in parallel with resistances of the TJ ( R _TJ ) plus the lateral intercellular space ( R _LIS ). The R _LIS is small, the membrane resistances are usually high and the epithelial resistance is governed by resistance of the TJ.

Primary transcellular transport is “active,” powered by ATP hydrolysis to move ions against an electrical or concentration gradient. The prime example is the ubiquitously expressed Na ⁺ -K ⁺ -ATPase, which moves three Na ⁺ ions out the basolateral surface in exchange for two K ⁺ ions, with the net effect being to generate an inwardly directed Na ⁺ and outwardly directed K ⁺ gradient and negative intracellular electrical potential. The high membrane conductance for K ⁺ and its exit from the cell further enhances the intracellular negative electrical potential. These electrical and chemical gradients are then used in “secondary” active transport to couple energetically unfavorable uphill movement of nutrients, such as glucose or amino acids, to the downhill movement of Na ⁺ through, for example, the Na ⁺ -coupled glucose cotransporter (SGLT-1) of the jejunum. As a final generalization, the characteristics of transcellular transport are highly regulated by short-term signals, for example, hormone-stimulated bicarbonate secretion from pancreatic ducts, and long-term transcriptional control, for example, aldosterone-stimulated expression of the Na ⁺ -K ⁺ -ATPase or electrogenic sodium channel ENAC. We outline these features of transcellular transport before proceeding with a detailed discussion of the paracellular pathway, to highlight the sharp distinction to paracellular transport, which is passive, nonrectifying and does not appear to be as highly regulated at least by physiologic stimuli. The transcellular and paracellular transport can be coordinated via intracellular signaling and actin cytoskeleton to enhance the total transport.

25.4.2

The Apical Junction Complex

The paracellular barrier to material movement coincides with continuous cell-cell contacts located at the apical end of their lateral surfaces ( Figs. 25.2 and 25.3 ). The earliest histological description of what we now refer to as the “apical junction complex” comes from the late 19th century. When sections of small intestine were stained with vital dyes a distinct intercellular density was observed between cells at the apical end of the lateral space. The English literature referred to this as the terminal bar ; other names reveal an assumed role in intercellular adhesion, e.g., “Schlussleiten,” and “bandelettes de fermeture.” The first speculation about a barrier function is attributed to Bonnet. After examining several different GI tissues obtained from an executed man, he concluded that the terminal bar was a general feature of all epithelia and might play a role in segregating the distinct fluid compositions found in different regions of the GI tract.

Junction types within the apical junction complex between intestinal epithelial cells. Left: Two columnar epithelial cells with apical brush border typical of the small intestine. A thick band of peri-junctional actin and myosin filaments connected to the tight and adherens junctions are typical of intestinal epithelial cells. Center: The “terminal bar” or apical junction complex is amplified revealing a series of intercellular contacts including the tight and adherens junctions, desmosomes, and gap junctions. Tight and adherens junctions are linked to the actin cytoskeleton and desmosomes to intermediate filaments. Right: TJ contacts are further magnified revealing rows of claudin strands adhering between adjacent cells to seal the paracellular space.

(A) Freeze-fracture electron microscopic replica of the TJ region of mouse jejunum, showing the interconnected network on claudin-based strands crossing the membrane. Continuous rows of claudins from adjacent cells adhere and seal the paracellular space. Above the barrier contact zone a few apical microvilli are visible. (B) Transmission electron micrograph of the apical junction complex region of two adjacent mouse mammary epithelial cells, rotated at 90° around a vertical axis to the image in (A). Lanthanum hydroxide (black) was added to the basolateral side; it freely diffuses through the intercellular space until it is partially blocked at the TJ from reaching the apical side. The TJ is recognized as a region of very close cell-cell apposition. Microvilli are seen on the apical surfaces. (C) Immunofluorescent microscopic localization of the TJ protein ZO-1 in mouse distal colon. The epithelial surface is at the top and two crypts are visible descending toward the bottom of the image. ZO-1 at the TJ is visible at the apical end of the lateral cell contacts, from crypt to surface. Tangential sectioning in the crypt reveals the continuous circumferential location of TJs around each cell. Bar, 10 μm.

(Image C is courtesy of J. Holmes.)

With the first ultrastructural images of intestinal epithelia in 1963, the apical junction complex was revealed as a set of morphologically distinct junction types ( Fig. 25.2 ). Each of these functions in cell-cell adhesion and signal transduction and provide links to the cytoskeleton. The TJ is invariably the most apical. It appears along the apical to basal axis, in transverse sections, as a series of close cell-to-cell contacts, or “kisses.” In freeze fracture images ( Fig. 25.3 A), the contacts are revealed as continuous rows of transmembrane protein particles. Actin filaments terminate on the plasma membrane directly at the contacts and participate in the regulation of the TJ barrier, ^, and are known to bind the peripheral membrane scaffolding proteins ZO-1 and cingulin ( Table 25.2 ). Below this is the adherens junction, location of the intercellular adhesion molecule cadherin and its cytoplasmic binding partner beta-catenin and extensive attachments to a ring of peri-junctional actin filaments. The importance of cadherin in adhesion and maintaining the differentiated cell phenotype are underscored by its frequent mutation as a final step facilitating metastasis of colon cancer. Beta-catenin has a second role in the nucleus where it signals cell growth and adenoma formation unless degraded by interacting with the adenomatous polyposis coli protein (APC). Human mutations in APC leave beta-catenin free to signal cell growth and transformation into adenomatous polyps. Below the adherens junctions are desmosomes, whose transmembrane proteins, while homologous to cadherin, are linked to intermediate filaments not actin. Desmosomes serve to protect the alimentary epithelia from shear-induced damage. Gap junctions that are made up of intercellular channels formed by six subunits of connexins that are bundled together in homomeric or heteromeric fashion and allow transfer of small metabolites and second messengers like Ca ⁺⁺ and IP3, between adjacent cells for coordination of epithelial functions like secretion and exocytosis.

Viewed by freeze-fracture election microscopy ( Fig. 25.3 A), the TJ barrier coincides with a network of transmembrane strands. In unfixed tissue, the strands often appear as rows of individual particles, now known to be a family of transmembrane adhesion molecules claudins. The Latin root, claudere , means to close. Rows of claudins from each cell meet in the intercellular space forming adhesive contacts and a semipermeable seal. The complexity (number and cross-linking) of strands differs among various tissues. It was long thought that the number of strands correlated with the resistance of the barrier. Consistent with this, in the small intestine, the complexity of strands increases at the crypt-to-villus transition. Since discovery of the barrier forming proteins, this structure-function correlation has been called into question; the molecular species of claudin in a particular junction appear be an important determinant. However, composition of other transmembrane TJ proteins is also likely to be important in the regulation and development of TJ barrier function. The specific proteins that affect paracellular flux of solutes of varying size need continuing clarification.

25.4.3

Barrier Properties: Resistance, Flux, and Permselectivity

Early electron microscopic studies ( Fig. 25.3 B) interpreted the close membrane apposition of adjacent cells at TJ contact points as membrane fusion, and even suggested convergence of the outer leaflets of the lipid bilayer. Supported by studies showing the inability of electron-dense proteins, such as hemoglobin and colloidal lanthanum, to pass through the TJs, these analyses led to the popular view of the TJ as an absolute barrier to paracellular flux. Although commonly thought of as an impermeant seal, it might be more appropriate to compare TJs to sieves. However, the characteristics of the selectively permeable TJ barrier vary widely among different tissues, within different cell types of a single tissue, and in response to physiological and pathophysiological stimuli ( Table 25.1 ). Thus, while the paracellular barrier is most often assessed by electrical conductance, TER, or transepithelial flux of small fluid phase markers, such as mannitol or polyethylene glycol, measurement of only one or two of these parameters provides an incomplete picture of overall barrier function.

25.4.3.3

Size Selectivity

In addition to charge selectivity, the TJ discriminates between solutes on the basis of size. This property has been recognized for over 30 years, but, for the most part, detailed analysis has not been possible due to the limited number of probes available. This obstacle has been overcome using mixtures of polyethylene glycol oligomers with hydrodynamic radii ranging from ~ 3 to ~ 7 Å. The relationship between size and apparent permeability of these probes changed sharply at radii of ~ 4 Å, indicating a size-restricted population of paracellular pores. This is similar to the ~ 6 Å pores reported within small intestinal villous epithelium and may reflect the same structure. Notably, recent data have shown that claudin-2 expression, which enhances paracellular flux of monovalent cations, also increases flux of polyethylene glycol oligomers with radii less than 4 Å. Thus, it appears that a claudin-based class of paracellular channels defines overall charge selectivity as well as size selectivity of this route, which has been termed the pore pathway.

Despite the claudin-2-based cutoff at ~ 4 Å radii, larger polyethylene glycol oligomers are still able to traverse epithelial monolayers, albeit at much lower rates than smaller solutes. The magnitude of this component of paracellular flux does not vary substantially with solute radius, suggesting that these molecules traverse a size nonrestricted route that is distinct from the claudin-based pore pathway. The route of large solute flux has been referred to as the leak pathway and may correlate with the 50–60 Å channels described within crypt epithelium. Enhanced leak pathway flux is the means by which IFN-γ and TNF-α increase paracellular permeability, and it has, therefore, been proposed that leak pathway regulation is primarily associated with pathologic processes. While this may be true, the converse is not, as some inflammatory mediators, for example, IL-13, which enhances claudin-2 expression, increases flux across the pore pathway without affecting the leak pathway. Based on recent studies, occludin-dependent regulation of leak pathway is now clearly recognized. Transient occludin depletion in mature Caco-2 cell monolayers was shown to cause a progressive size-dependent increase in paracellular flux rate (three- to fourfold increase for urea (molecular radius 2.9 Å), five- to sixfold increase for mannitol (molecular radius 4.1 Å), ∼ 25-fold increase for inulin (molecular radius 15 Å), ~ 25-fold increase for 10-kDa dextran (molecular radius 23 Å), and ~ 45-fold increase for 70-kDa dextran (molecular radius 36 Å) ( Fig. 25.4 ). These findings were supported by another study showing charge-nonselective reduction in macromolecular flux with stable knockdown of occludin in Caco-2BBe cells. In the same study, C-terminal occludin/ELL domain was shown to be critical for anchoring and exchange of occludin at the TJ. Although occludin knockdown has been shown to have differential effect on the expression of claudins, possible due to different approaches and heterogenicity of cell lines, the contribution of claudins to the noncharge selective, large-size leak pathway in the absence of occludin is unclear.

Graph of molecular radius of paracellular markers vs. relative increase in flux rate following occludin siRNA transfection (relative correlation coefficient, r = 0.98).

(From Al-Sadi R, Khatib K, Guo S, Ye D, Youssef M, Ma T. Occludin regulates macromolecule flux across the intestinal epithelial tight junction barrier. Am J Physiol Gastrointest Liver Physiol 2011; 300 :G1054–G1064, with permission.)

Myosin light chain kinase (MLCK), which causes contraction of peri-junctional actomyosin, is also a key regulator of TJ permeability. Close association of TJ with the cytoskeleton described by Madara was later shown to be the basis for cytoskeletal control of tight-junction permeability. Under physiological conditions, MLCK-induced increase in pore pathway permeability during Na ⁺ glucose cotransport is associated with condensation of peri-junction actomyosin. Under physiopathological conditions, such as TNF-α-mediated increase in TJ permeability, the enhancement in leak pathway permeability is associated with MLCK-dependent occludin endocytosis. While MLCK activation do not affect pore pathway directly, in vivo secondary immune activation in constitutively active MLCK expressing mice has been shown to increase claudin-2 expression and alter TJ Na ⁺ /Cl ⁻ selectivity in IL-13-dependent way. Also, proinflammatory cytokines are known to increase leak pathway permeability in MLCK transcription and activation-dependent way, as discussed in detail, later in this chapter.

25.5.1

Transmembrane Proteins and TJ Stability

The transmembrane proteins of TJs fall into three main groups: the single transmembrane domain proteins [e.g., JAM (junctional adhesion molecule), Crb3 (Crumbs protein homolog 3), CAR (coxsackievirus and adenovirus receptor), etc.]; the triple transmembrane domain protein, Bves (blood vessel epicardial substance); and the four-transmembrane domain proteins of the claudin and TAMP (tight junction-associated MARVEL proteins) families, which include occludin, tricellulin, and MarvelD3. A large number of transmembrane proteins serving diverse functions are found at TJs. Given the restricted, though, presumably, friendly confines of the TJ, it has been assumed that these proteins are embedded in a densely packed array that restricts their mobility. Together with the identification of multiple sites of interaction between TJ proteins, this led to the presumption that these proteins are tightly anchored and immobile. While possible, this conclusion would seem at odds with data demonstrating rapid regulation of TJ structure and function and, potentially, with the discovery that TJ membrane microdomains are enriched in glycolipids and cholesterol, i.e., membrane rafts, in which membrane fluidity is greater than more abundant phospholipid membrane domains.

The stability or mobility of proteins within the TJ has been studied using fluorescent-tagged TJ proteins. Importantly, these fluorescent fusion proteins were carefully validated and demonstrated to (1) colocalize and cofractionate with their endogenous, unlabeled counterparts ; (2) not interfere with TJ function ; and (3) complement TJ protein deficiencies in mice and in cell lines. Thus, one can conclude that their behaviors with regard to dynamic behavior are related to, and likely the same as, those of endogenous TJ proteins. Consistent with the conclusion that TJ-associated proteins are stably anchored, EGFP-claudin-1 was found to have a mobile fraction of only ~ 25%. While the hypothesis that such anchoring depends on the interaction of claudin-1 with ZO-1 (discussed below), anchoring of a truncated EGFP-claudin construct lacking the ZO-1 binding domain was similar to that of full-length EGFP-claudin although, as discussed below, trafficking to the TJ was severely impaired. Such data in epithelia were also consistent with the observation that, in fibroblasts, claudin-1-EGFP, which forms TJ strand-like fibrils despite blockade of ZO-1 binding by the C-terminal EGFP, demonstrated little fluorescent recovery after photobleaching. In contrast to claudin-1, occludin displayed a mobile fraction of ~ 75%. Occludin mobility occurred by diffusion within the plasma membrane and was dependent on membrane fluidity, as it could be inhibited by cholesterol extraction, with methyl-β-cyclodextrin, or reduced temperature, but was not an energy-dependent process. In contrast, while the mobile fraction of the peripheral membrane or plaque protein ZO-1 was similar to that of occludin, it was recovered by energy-dependent exchange with a cytosolic ZO-1 pool and was unaffected by cholesterol extraction or reduced temperature. While the functional significance of these observations is poorly understood at present, the data do show that protein interactions at the TJ are labile and that, despite the densely packed nature of the TJ, the raft-like biophysical properties of TJ membranes support free diffusion of TJ proteins.

Data including the observation that claudin-1-EGFP forms TJ-like strands in fibroblasts, which lack endogenous TJs, suggest that claudins form the core of TJ strands in epithelia. Claudins are the only junction-associated proteins that have been found to form such strands, although occludin can incorporate into claudin-based strands. Other transmembrane proteins, such as JAM-1, CAR, and connexin, are located around, but not incorporated into the strands. Beside varying stability of various TJ proteins within the plasma membrane, various modes of intracellular transport such as clathrin-mediated transport, caveolar transport, and micropinocytosis, as well as signaling molecules such as small GTPaseRabs and their adaptors have been shown to regulate biogenesis and function of TJs.

25.5.2

PDZ Containing Scaffolding Proteins

The interface between the transmembrane proteins and most cytoplasmic components is formed by a set of scaffolding proteins with multiple PDZ domains. PDZ domains, named for the proteins PSD-95, discs large, and ZO-1, are protein-binding modules that recognize target sequences at the extreme C-termini of transmembrane proteins. They are well characterized for their ability to cluster signal transduction complexes at specialized membrane contacts, such as synapses and TJs. PDZ-proteins at the TJ include the ZO-MAGUK proteins ZO-1, ZO-2, and ZO-3; the MAGUK relatives MAGI-1, MAGI-2, and MAGI-3; a protein with 13 PDZ called MUPP-1, and several of the polarity components cited below including PAR-3, PAR-6, PALS1, PATJ, scribble, and Dlg. Most of these proteins contain several PDZ domains and bind the tails of claudins and JAMs. Some, like the ZO-MAGUKs have other protein-binding modules, including SH3 and enzymatically inactive guanylate kinase (GuK) domains, which are known to bind additional targets including α-catenin and occludin. In addition, ZO-1 has a well-defined actin-binding domain as well as less well-characterized U5, U6, and ZU5 domains.

Although much work remains, several functions of ZO-1 in TJ assembly and regulation are recognized. ZO-1 recruitment to nascent adherens junctions requires the U5 motif, likely as a result of U5-dependent interactions with α-catenin, AVRCF, and AF-6/afadin. However, it is equally possible that ZO-1 and ZO-2 are involved in recruitment of other proteins to the nascent adherens junction. For example, mammary epithelial (Eph4) cells deficient in both ZO-1 and ZO-2 have a marked delay in adherens junction assembly, as do MDCK cells lacking only ZO-1. ZO-1 is able to rescue this defect. Data regarding ZO-2 are less clear, as one study showed that knockdown in MDCK cells delayed E-cadherin, ZO-1, and occludin recruitment to the plasma membrane, while a separate study found no effect of ZO-2 knockdown. Thus, while differences between labs make it difficult to precisely compare the results, these data do suggest that ZO-1 and ZO-2 have overlapping, but distinct functions. This is consistent with the observation that knockout of either ZO-1 or ZO-2 results in embryonic lethality in mice as well as the association of PDZ1 domain ZO-2, but not ZO-1, mutations with familial hypercholanemia in human patients.

Following adherens junction assembly, ZO-1 and ZO-2 direct polymerization of claudin proteins into the TJ strands. The interaction between claudin proteins and the first PDZ domain of ZO-1 and ZO-2 is important in the first phase of this process, as claudins are not recruited to the junctional complex in EPh4 cells that lack both ZO proteins and, as noted above, claudin proteins in which the PDZ binding motif are either mutated or blocked are trafficked to TJs inefficiently. However, the SH3-U5-GuK-U6 region is also critical to regulation of claudin polymerization. Although the means by which claudin polymerization is controlled requires further study, the U6 motif does appear to negatively regulate this process, as U6 deletion results in aberrant strand assembly.

In addition to the accumulation of data defining a role for ZO proteins in TJ assembly, recent work has defined a role of these proteins, particularly ZO-1 in barrier regulation. ZO-1, but not ZO-2, knockdown in MDCK layers increased paracellular permeability of large solutes, i.e., the leak pathway. Although charge selectivity was not examined, ZO-1 knockdown did not affect paracellular flux of small polyethylene glycols, suggesting that pore pathway barrier function was intact. ZO-1-dependent leak pathway maintenance did not require protein regions beyond the U6 domain, including the actin-binding region (ABR), as ZO-1 mutants truncated just after the U6 domain or in which the ABR was deleted internally were both able to restore barrier function. A following study has found that while ZO-1 PDZ1 is important for the normal organization of both the TJ and the AJC cytoskeleton, the SH3 domain and the U5 motif are required to recruit ZO-1 to the AJC, which is crucial for normal TJ and cytoskeletal organization. The PDZ2 domain is necessary for establishment of the ZO-1, occludin, and claudin-2 association and resultant normal permeability. This study suggests that the coordination of multiple ZO-1 domains is required for normal TJ structure and function. In addition to linking occludin and claudins, ZO-1 is able to connect to F-actin via an ABR located within the C-terminal half of the protein. Recent data indicate that the ABR is required for physiological, MLC-dependent barrier regulation. In this respect, MLCK activity has been shown to regulate the leak pathway in part by energy-dependent mechanical tension induced pulling apart of the TJ barrier. In vitro and in vivo analyses have shown that MLCK inhibition enhances TJ barrier function and is associated with stabilization of ZO-1, as assessed by fluorescence recovery of ZO-1. This ZO-1 stabilization required the ABR. Moreover, either ZO-1 knockdown or dominant negative expression of the free ABR prevented barrier function increases after MLCK inhibition. Together with other data, these results suggest that MLCK-dependent ZO-1 exchange and ABR-dependent ZO-1 anchoring are critical determinants of TJ barrier function and regulate the leak pathway. More recent studies have found that transmembrane TJ proteins were enriched among those binding the N terminus of ZO-1 and cytoskeletal proteins were enriched in among those binding to the C terminus of ZO-1. One such ZO-1-binding protein, F-BAR-domain protein transducer of cdc42-dependent actin activity (TOCA-1; also called formin-binding protein 1-like) has been shown to regulate actin assembly and TJ barrier function via recruiting actin nucleation-promoting factor N-WASP to TJs. Thus, ZO-1 mediates interaction between cytoskeletal elements (regulated by MLCK) and the transmembrane TJ proteins, including occludin and claudins. Also, association between ZO-2, JAM-A, actin filament-binding protein afadin activates small GTPase Rap2c and control apical cytoskeleton contraction.

25.5.3

TJ Associated MARVEL Proteins

Occludin, an ~ 65 kD atetraspan protein, was the first TJ-associated transmembrane protein identified. Although this discovery was greeted with excitement, subsequent understanding of occludin function has been elusive. The role of occludin in TJ assembly was first called into question by the observation that occludin-deficient embryonic cells can differentiate into polarized epithelial cells with structurally and functionally intact TJs. As discussed below, this prompted further studies that resulted in the discovery of claudins. Controversy over the contributions of occludin to TJs was enhanced further by the observation that occludin knockout mice are viable, and did not appear to have GI or renal disease, and as assessed using Ussing chambers, have intact intestinal barrier function. Since intestinal barrier assessment was only performed at 6-weeks of age, it remained unclear as to whether the TJ barrier defects were present at an earlier age but were subsequently compensated by other TJ proteins or associated proteins. Nevertheless, occludin knockout mice do have severe health defects. Males have testicular atrophy and are sterile, while females do not effectively suckle their young. In addition, occludin knockout mice of either gender are small, develop chronic inflammation and hyperplasia of the gastric epithelium with loss of parietal and chief cells, brain calcifications, osteopenia, and salivary gland defects. Subsequent reports have shown that siRNA or microRNA-induced knockdown of occludin caused and increase in intestinal TJ permeability in vitro and in vivo, in Caco-2 monolayers, and in live mice undergoing intestinal perfusion, respectively. As discussed earlier, occludin depletion resulted in a selective and preferential increase in macromolecular flux, suggesting that occludin plays a role in modulation of the leak pathway. Similarly, Buschmann et al. also found that siRNA-induced knockdown of occludin caused an increase in macromolecular flux across the Caco-2 _BBe monolayers. They showed that a 107-amino acid occludin/EEL domain at the C-terminal, which contains binding sites for ZO-1 and actin, was required for the occludin-dependent enhancement of TJ barrier. Occludin deficiency has also been shown to promote ethanol-induced disruption of mouse colonic epithelial barrier function and occludin overexpressing transgenic mice are protected to certain extent against TNF-α-induced increase in leak pathway permeability. Overall, the studies with transient knockdown in mature cell monolayers, stable knockdown in cell monolayers, and exogenous mice intestinal expression have clearly demonstrated a critical role of occludin in intestinal epithelial TJ barrier.

Tricellulin, so named because it is concentrated at tricellular TJs, was the next protein identified as a member of MARVEL family. Occludin and tricellulin both contain tetraspanning MARVEL (MAL and related proteins for vesicle trafficking and membrane link) domains and bind ZO-1 via their carboxy terminal cytoplasmic tail, although the tail is not part of the MARVEL domain. However, in contrast to occludin knockdown in MDCK cells, tricellulin knockdown severely compromises barrier function of Eph4 monolayers. Interestingly, the effects of tricellulin knockdown in Caco-2 cells are less severe.

Tricellulin mutations have been reported in humans and linked to autosomal recessive deafness, likely as a result of defects in function of junctions between supporting and hair cells within the vestibular and cochlear epithelia. Remarkably, these tricellulin mutations, which were most often truncations that disrupted ZO-1 binding, caused deafness without creating a syndrome involving other organs. This limited distribution of disease does not reflect absence of tricellulin expression in other tissues, suggesting that another protein may compensate for tricellulin loss.

Because both occludin and tricellulin contain MARVEL domains, some suggested an evolutionary relationship between these proteins and a third MARVEL domain-containing protein, marvelD3. However, other analyses suggested that this was not the case. The issue was resolved by an evolutionary analysis demonstrating that human MARVEL domain-containing proteins can be segregated into four groups, one of which contains occludin, tricellulin, marvelD3, and their splice variants. This family has, therefore, been referred to as TJ-associated MARVEL proteins (TAMPs). MarvelD3 was found at both bicellular and tricellular TJs of Caco-2 intestinal epithelial monolayers as well as murine jejunum, hepatocytes, and renal tubules. However, while one study found that marvelD3 knockdown enhanced steady-state TER, another found that marvelD3 knockdown delayed TER development but did not affect final TER. This difference may relate to experimental conditions, or siRNA sequences and cell lines; although both studies used Caco-2 cells, the clones employed were clearly different, as the TERs achieved varied significantly. Further, analysis showed that combined knockdown of marvelD3 and either occludin or tricellulin caused a greater delay in TER development than knockdown of either protein individually.

Despite the analyses above, defining specific functions of occludin, tricellulin, and marvelD3 has been challenging. Further, data from occludin knockout animals, patients with tricellulin mutations, and siRNA knockdown cells suggest that a simple knockout/knockdown deletional approach will not be sufficient to dissect functions of the TAMPs. However, the association of occludin internalization with barrier loss induced by actin disruption or TNF core family cytokines show a strong link between acute occludin endocytosis and barrier loss. This was recently explored using a transgenic mouse that expresses functional EGFP-occludin, as well as endogenous occludin, within the intestinal epithelium. While TNF-induced occludin endocytosis depletes the protein from large regions of the intestinal epithelial TJ of wild-type mice, occludin was present throughout the intestinal epithelial TJs of transgenic mice. This maintenance of occludin at TJs of transgenic mice was not due to failure of occludin endocytosis but likely a result of occludin overexpression. The effect of TNF-α on jejunal barrier function was also attenuated, and diarrhea was entirely prevented in EGFP-occludin transgenic mice. In parallel with this in vitro work, an in vitro study in MDCK cells also found that occludin was required for TNF-induced barrier loss. Also, occludin stabilization by hypoxia responsive prolyl hydroxylase domain protein (PHD3) via prevention of the interaction between the E3 ligase Itch and occludin was found to be important for intestinal TJ barrier function. On the other hand, partitioning-defective protein 3 (Par-3) silencing blocked the trafficking of occludin from or through the Golgi complex to the cell surface and delayed TJ barrier development.

The above data suggest an important TJ barrier regulatory role for occludin. However, much work remains, including analysis of protein interactions mediated by occludin and functional significance of the occludin tail hyperphosphorylation that is associated with localization at the TJ and development of barrier function. For example, occludin is a key TJ protein in the claudin-2-independent TJ barrier enhancement in response to serine proteases. Recent studies have demonstrated that the C-terminal occludin tail associates with specific kinases and phosphatases, and that these interactions are able to regulate TJ assembly and barrier function. Moreover, it is now clear that phosphorylation of specific residues within the occludin tail modifies interactions with ZO-1. However, the sites identified are not within the region of occludin responsible for ZO-1 binding, suggesting that phosphorylation induces conformational changes in other regions of the tail. Moreover such a modification of interaction between transmembrane proteins and ZO-1 can also help understand the overlap between leak and pore pathway. For instance, CK2-mediated phosphorylation of occludin phosphorylation at S408 by CK2 enhances its diffusion within the membrane, thereby reducing occludin binding to ZO-1 and claudin-2 and allowing flux across claudin-2 pores. In contrast, CK2 inhibition and occludin dephosphorylation promote occludin- ZO-1-claudin-2 complex that reduces claudin-2-mediated pore function. Definition of these structural changes as well as the means by which they impact protein interactions and barrier function remains an important area of further studies.

25.5.4

Claudins

The discovery of claudins in 1998 significantly advanced our understanding of the TJ barrier. Claudins were first shown to have the ability to form strands and confer cell-to-cell adhesion. Subsequent works clearly link claudins to the selective barrier properties of the TJ ( Table 25.3 ).

Claudins are a family of tetraspan proteins, ranging from 20 to 25 kDa characterized by a conserved amino acid motif in the first extracellular loop (W-GLW-C-C). The human genome contains at least 26 claudin genes. The puffer fish Takifugu contains 56 claudin genes, although this animal has other examples of gene expansions that lack obvious biologic significance. The barrier-forming junctions of invertebrates, septate junctions, are structurally quite different from TJ, thus it was surprising to find claudin-like proteins in both flies and worms and to find that they are required to form epithelial barriers. Drosophila has six claudin sequences. Several of them are expressed at the barrier-forming septate junctions and, in addition to affecting barrier function, appear to regulate size of epithelial tubes. Interestingly, claudin-15 knockout results in mice with mega-intestine and zebrafish with multiple gut lumens, suggesting that claudins regulate epithelial tube development in vertebrates as well as flies.

The first extracellular loops of claudins are highly conserved, ranging from 41 to 55 residues, contain a specific ion-binding site, and appear to make a major contribution to TJ ion selectivity and barrier function. Site-directed mutagenesis of specific residues within the first extracellular loop of claudins can reverse the charge selectivity of the TJ pore, while inclusion of both acidic and basic residues in that region can reduce permeability to both cations and anions. The second loops, which are also highly conserved, range from 10 to 21 residues and appear to be more involved in homotypic and heterotypic adhesion and are also the site of Clostridium perfringins enterotoxin binding in claudins 3 and 4. The claudin cytoplasmic tails range from 21 to 44 residues and are the least well-conserved regions. With the probable exception of claudin- 12, -19a, -21, and -24 to -27, all claudin cytoplasmic tails end in PDZ-binding motifs, which bind PDZ domains in the cytoplasmic scaffolding proteins ZO-1 and MUPP1 and other proteins. PDZ-mediated interactions of claudins with ZO-1 and ZO-2 are required for efficient delivery to the TJ, although this may not be universally true.

The expression of several claudins has been documented within the mouse intestine. Claudins 2, 3, 7, and 15 are the most highly expressed claudins in gastrointestinal tract Many claudins show unique expression patterns along both longitudinal (duodenum-colon) and vertical (crypt-villus) axes. For instance, while claudin-13 was detectable only in the colon, claudin-18 was detectable only in the duodenum and jejunum. Claudin-8 expression increases progressively from small intestine to colon, while expression of claudin-15 decreases from small intestine to colon. Under normal physiological conditions, the expression of claudin-2 is restricted to the deep crypts. Furthermore, expression of at least few claudins is distributed differentially between the apical TJ and the basolateral membrane (see Ref. for a review). As true for many membrane proteins, the expression of claudins is also regulated differentially during development. These spatiotemporal expressions of claudins are thought to confer regional gradients of size and charge selectivity, but the mechanisms and physiological implications are incompletely defined.

It is now clear that claudins are largely responsible for paracellular permeability of small solutes, including ions and water. Although intrinsically, all claudins are barrier forming, some claudins form pores that are similar in size selectivity but are either cationic or anionic charge selective. The outcome of such claudin-based paracellular permeability is probably defined by temporal composition of various claudins at the TJ, their association with cytoplasmic plaque proteins, and their biochemical modification by processes such as sumolylation, phosphorylation, palmitoylation, etc. Based on ultrastructure, electrophysiology, and genetic studies, it is now known that claudin-2 pores are permeable to small cations and water and are exquisitely size-selective. Native or exogeneous claudin-2 expression can explain differences in barrier function in leaky or tight monolayers of several cell lines, under different experimental conditions. In addition to claudin-2, claudin-10b, and claudin-15 are known to form cation pores, while claudin-10a and -17 anion pores. Barrier forming group of claudins include claudin-1, -3, -4, -5, -6, -8, -12, -18, and -19. Further details of claudin function in and beyond epithelial TJ barrier have been discussed in recent reviews.

Consistent with the view that claudins regulate TJ permeability, mutations in claudin-16, also known as paracellin-1, prevent paracellular absorption of divalent cations in the renal tubule. This results in the disease familial hypomagnesemia with hypercalciuria and nephrocalcinosis, which is also associated with claudin-19 mutations. Claudin-11 and -14 mutations have also been associated with deafness, claudin-1 mutations have been linked to neonatal sclerosing cholangitis and ichthyosis. Similarly, mice lacking claudin-1 die shortly after birth from rapid evaporative water loss from the skin. They do not live long enough to investigate whether they recreate the biliary pathology. However, a point mutation in the claudin-binding PDZ domain of ZO-2 is the basis of Amish familial hypercholanemia. Claudin-18 knockout mice develop atrophic gastritis shortly after birth due to the paracellular H ⁺ leak in their stomach epithelia and also osteoporosis due to increased bone resorption by osteoclasts. Conditional deletion of claudin-7 in mouse has been shown to increase the paracellular flux of small organic solutes and to induce colonic inflammation. While specific claudin mutations have not been linked to GI disease, inflammatory, and infectious diseases are strongly associated with changes in claudin protein expression and trafficking. For instance, claudin-2 is consistently upregulated in human IBD as well as in animal models of IBD, while claudin-3 and -4 are usually upregulated in gastrointestinal cancers.

25.5.5

JAM and Other Single Transmembrane Domain TJ Proteins

Junctional adhesion molecule-A (JAM-A), a cell adhesion molecule localizing at intercellular junctions is known to play a role in immune cell trafficking across the epithelium and endothelium, cell cycle regulation, and barrier function. In the intestine, epithelial expression of JAM-1, which is also referred to as JAM-A, is reduced in IBD. Moreover, JAM-A knockout animals have intestinal barrier defects and enhanced cell turnover in the absence of exogenous stress as well as greater sensitivity to dextran-sulfate sodium-induced colitis and high saturated fat, fructose, and cholesterol diet-induced steatohepatitis. Increased expression of JAM-A is also associated with the enhancement of TJ barrier function induced by gut acting antibiotics as well as probiotic Bacillus subtilis. Other single transmembrane domain TJ proteins localized to the TJ include the coxsackie virus-adenovirus receptor CAR and Crumbs protein homolog 3 (Crb3). CAR, a cell adhesion molecule and a receptor for type B coxsackievirus and subgroup C adenovirus, regulates E-cadherin stability via promoting its endocytosis. In a study on human preimplantation embryos and embryonic stem cells, expression of soluble CAR was detected in undifferentiated blastomeres while transmembrane CAR was present in epithelial-like cell types, such as the trophectoderm (TE) and the outer layer of hESC colonies, suggesting a role of CAR in early embryogenesis and differentiation. Crumbs protein homolog 3 (Crb3/PATJ) is an apical organizer crucial for the maintenance of epithelial polarity. Crb3 is essential for postnatal viability, and Crb3 knockout E18.5 mouse embryos show impaired differentiation of intestinal apical membrane and fusion of villi apart from cystic kidneys and accumulation of mucosubstances in airways.

25.5.6

Additional Tight Junctional Proteins and Functions

Besides controlling paracellular permeability, TJs are also considered to be a signaling platform for cellular processes including morphogenesis, cell polarity, and differentiation via interactions with actin cytoskeletal elements, kinases and phosphatases, and the intracellular trafficking apparatus. TJ is home to a large number of kinases, including c-src and c-yes, both of which induce junction disassembly. Atypical protein kinase C (PKC) isoforms, including those involved in development of epithelial polarity, are also concentrated within the tight and adherens junctions. Finally, several transcription factors are located at the junction or bind to junction proteins.

TJ proteins also function as receptors for pathogens, interact with pathogen effector molecules, and form an essential step in their pathogenesis. Interactions between CAR and adenoviral capsid protein fiber have been demonstrated to promote adenoviral viral entry and escape, while CAR-type B coxsackievirus interaction leads to occludin internalization dependent coxsackie virus entry. Similarly, claudin-1 and occludin collaborate with at least two other cell surface proteins to facilitate hepatitis C virus entry. Diversity of occludin sequences confer species specificity to hepatitis C virus infection. Further, occludin splice variants and claudin-1 polymorphisms may impact susceptibility of some human hosts to hepatitis C virus. This theme of pathogen receptors being sequestered within the tight and adherens junctions extends beyond coxsackie virus, adenovirus, and hepatitis C. For example, Listeria monocytogenes use E-cadherin as a receptor for infection. Interestingly, E-cadherin is normally inaccessible, and is only exposed and available for L. monocytogenes binding at sites of cell extrusion.

25.6.1

Cytochalasins and the Intestinal TJ Barrier

Cytochalasins have been widely used as pharmacologic agents to examine actin-dependent cellular functions and were among the first agents to be used to study the role of cytoskeletal elements in epithelial TJ barrier modulation. Cytochalasins disrupt actin filaments by several mechanisms including direct severing of actin filaments, inhibition of actin subunit polymerization, and by inducing reactive cellular responses. The cytochalasin disruption of actin microfilaments produces a morphologic alteration in intestinal TJs and an increase in intestinal TJ permeability in both the ex vivo and in vitro intestinal epithelial systems. The cytochalasin D treatment of Ussing chamber mounted guinea pig small intestinal tissue produced a concentration dependent (0.1–20 μg/mL) drop in TER. The drop in small intestinal tissue TER directly correlated with an increase in Na ⁺ and mannitol flux, confirming an increase in intestinal epithelial TJ permeability. The dual-flux studies indicated that the increased permeation through the paracellular pathways fully accounted for the increase in mannitol and Na ⁺ flux. The cytochalasin D increase in intestinal TJ permeability was also accompanied by condensation or aggregation of microfilaments in the peri-junctional acto-myosin ring, especially in the regions of multiple cellular contacts. The condensation of the peri-junctional microfilaments produced a “pulse-string” type contraction of the brush borders of surface intestinal epithelial cells with a bulging or convex appearance of the epithelial surface ( Fig. 25.5 ), a decrease in number of microvilli at the junctional contact areas, and a disturbance in distribution, decrease in number, and loss of mesh-work like organization of TJ strands ( Fig. 25.6 ). These findings suggested that the cytochalasin D-induced increase in intestinal TJ permeability was mediated in part by a contractile-tension generated by contraction of the actin/myosin ring at the level of the TJs. Cytochalasin D appears to affect epithelial TJ actin filaments in two distinct stages: a rapid energy-independent severing of actin filaments into smaller fragments followed by reorganization of actin fragments to form cytoskeleton clumps by an energy-dependent process. The cytochalasin D-induced aggregation of peri-junctional microfilaments, contraction of the peri-junctional actin/myosin ring, alterations in TJ strands, and increase in TJ permeability were inhibited by energy depletion induced by 2,4-dinitrophenol (DNP), indicating the energy requirement in these processes. Similarly, cytochalasin (B and D) treatment of filter-grown intestinal epithelial monolayers Caco-2 and T84 cells also produced an acute drop in TER and an increase in epithelial permeability to paracellular markers mannitol and inulin ( Fig. 25.7 ). In contrast, the disintegration of Caco-2 microtubules with tublin depolymerizing agent colchicine did not have any effect on Caco-2 TJ barrier function or junctional localization of TJ proteins, suggesting that intact microtubules are not required for the acute maintenance of the intestinal epithelial TJ barrier function. The cytochalasin B-induced increase in Caco-2 TJ permeability required metabolic energy and was rapidly reversible (within hours) following cytochalasin B removal, indicating that the cytochalasin effect was not due to a permanent cell damage or cell death but by a rapidly reversible process.

Scanning electron micrographs of vehicle control (A, B) and CD-exposed (C, D) (10 ng/mL, 60 min) mucosal sheets. The three villus ridges display smooth surfaces with intermittent linear folds. As seen in B, control villi are covered by polygonal absorptive cells with flat apical surfaces. In contrast, CD-exposed tissues display a cobblestone-like appearance of the villus surfaces (C). Higher magnification (D) shows this cobblestone effect is due to pulse-string contraction of the brush borders of individual absorptive cells resulting in a convex apical absorptive cell surface and flaring of microvilli. Bars, 20 μm.

(From Madara JL, Barenberg D, Carlson S. Effects of cytochalasin D on occluding junctions of intestinal absorptive cells: further evidence that the cytoskeleton may influence paracellular permeability and junctional charge selectivity. J Cell Biol 1986; 102 :2125–36, with permission.)

Freeze-fracture replicas of villus absorptive cell occluding junctions. (A) Control junctions are composed of a net-like mesh of cross-linked strands or grooves. Peri-junctional microvilli are densely aligned above the junction. Bar. 0.1 μm. (Shadow angle, approximately left to right.) (B) Junction exposed to 10 μm/mL CD for 40 min. Junction is composed of an irregular array of strands that underlie occasional broad protrusions of the apical membrane (arrowheads) . Geometric irregularities produced by such protrusions result in a fracture plane which only focally includes the apical-most strand (straight arrows) . Many peri-junctional microvilli are lost and intramembrane particles penetrate into the incompletely isolated intrajunctional compartments (curved arrow) . Bars, 0.1 μm. (Shadow angle, approximately left to right.)

(From Madara JL, Barenberg D, Carlson S. Effects of cytochalasin D on occluding junctions of intestinal absorptive cells: further evidence that the cytoskeleton may influence paracellular permeability and junctional charge selectivity. J Cell Biol 1986; 102 :2125–2136, with permission.)

Effect of Cyto B (5 μg/mL) on Caco-2 epithelial resistance and paracellular permeability. (A) Cyto B (5 μg/mL) effect on Caco-2 epithelial resistance expressed as Ω cm ² . Inset: magnified view of the early time course. (B) Cyto B (5 μg/mL) effect on mucosal-to-serosal flux of paracellular marker mannitol expressed in nmol/cm ² . Values are means ± SE; n = 4.

(From Ma TY, Hoa NT, Tran DD, Bui V, Pedram A, Mills S, Merryfield M. Cytochalasin B modulation of Caco-2 tight junction barrier: role of myosin light chain kinase. Am J Physiol Gastrointest Liver Physiol 2000; 279 :G875–G885, with permission.)

The cytochalasin B-induced increase in Caco-2 TJ permeability also correlated with sequential changes in peri-junctional actin and myosin filaments ( Fig. 25.8 ). In Caco-2 monolayers, actin and myosin filaments are localized in a belt-like manner surrounding the apical junctional area. Cytochalasin treatment produces a rapid energy-independent severing of actin filaments into small fragments (early phase response). Within seconds of cytochalasin B treatment, the peri-junctional actin filaments are severed and become fragmented and are present diffusely in the cytoplasm at the level of the TJs ( Fig. 25.8 B). This early phase fragmentation is then followed by an energy-dependent process in which the severed actin fragments reorganize to form large cytoskeletal aggregates containing actin and myosins filaments (late phase response). By 15–30 min of cytochalasin B treatment, the fragmented actin and myosin filaments coalesce to form large cytoskeletal clumps or “foci” near the peri-junctional areas ( Fig. 25.8 D). Similar effects of cytochalasin D on actin filament breakage and cytoskeletal clump formation (containing actin, myosins, and tropomyosins) were also demonstrated in the African green monkey kidney cells (BSC1cells).

Effect of cytochalasin B (Cyto B) (5 μg/mL) on perijunctional Caco-2 actin microfilaments. Caco-2 F-actin filaments were labeled with fluorescein-conjugated phalloidin. The sequential effect of Cyto B (5 μg/mL) on Caco-2 actin microfilaments at time 0 (A) and 1 (B), 15 (C), and 30 min (D) is shown in the photomicrographs (original magnification, × 80). By 1 min of Cyto B exposure, peri-junctional actin filaments were fragmented and present diffusely throughout the cytoplasm. By 15–30 min of Cyto B exposure, actin fragments coalesced to form large actin clumps or “foci” near the perijunctional areas.

(From Ma TY, Hoa NT, Tran DD, Bui V, Pedram A, Mills S, Merryfield M. Cytochalasin B modulation of Caco-2 tight junction barrier: role of myosin light chain kinase. Am J Physiol Gastrointest Liver Physiol 2000; 279 :G875–G885, with permission.)

As actin-myosin contraction in smooth muscle and other cell types is mediated by MLCK activation, the possibility that the cytochalasin-induced increase in Caco-2 TJ permeability was also mediated by MLCK-activated actin-myosin interaction was considered. In brief, MLCK catalyzes myosin light chain (MLC) phosphorylation leading to the activation of Mg ⁺⁺ -myosin ATPase, which hydrolyzes ATP to generate the mechanical energy needed for the actin-myosin contraction. The cytochalasin-induced alteration in peri-junctional actin and myosin filaments and increase in Caco-2 TJ permeability were accompanied by an increase in MLCK activity ( Fig. 25.9 ). The inhibition of cytochalasin- induced MLCK activation by MLCK inhibitors (ML-7, ML-9, KT-5926) prevented the late phase cytoskeletal aggregation and the increase in Caco-2 TJ permeability but not the early phase severing of actin filaments, suggesting that MLCK activation mediated the aggregation of the cytoskeletal filaments and the subsequent increase in Caco-2 TJ permeability. The cytochalasin severing of actin filaments has been proposed to be the triggering event for the MLCK activation. Consistent with this possibility, villin induced severing of actin filaments has been shown to induce Mg ⁺⁺ -myosin ATPase activity and actin-myosin interaction. The requirement of metabolic energy and mechanical contraction of actin-myosin filaments in the TJ barrier opening was supported by the studies showing that Mg ⁺⁺ -myosin ATPase inhibitor (2,3-butadione monoxime) and metabolic inhibitors (sodium azide, 2,4-dinitrophenol) inhibit the cytoskeletal clump formation (late-phase response), alteration in junctional localization of ZO-1 proteins and the increase in TJ permeability, but not the severing of the actin filaments (early-phase response). Together, these studies suggested that the cytochalasin-induced increase in intestinal TJ permeability was mediated by cytochalasin-induced activation of MLCK, which causes aggregation of peri-junctional microfilaments and the contraction of the peri-junctional actin-myosin ring, which in turn leads to a pulse-string-type contraction of the cell membrane and centripedal-tension generated opening of the intestinal TJ barrier. The MLCK-dependent retraction of the apical membrane and TJ barrier creates a large channel opening in-between cells and increases paracellular flux of both small and large-sized solutes, consistent with the generation of leak pathway.

Effect of Cyto B on Caco-2 MLCK activity. Caco-2 monolayers were exposed to Cyto B for increasing time periods (0–30 min). Subsequently, Caco-2 monolayers were lysed, and Caco-2 MLCK was immunoprecipitated. The activity of the immunoprecipitated MLCK was determined by in vitro kinetic measurement of MLC phosphorylation. Phosphorylated MLC (P-MLC; ~ 19.5 kDa) was separated by 10% SDS-PAGE, stained with Coomassie blue solution, and autoradiographed. Cyto B produced a time-dependent activation of Caco-2 MLCK with the peak activation occurring between 5 and 10 min after Cyto B exposure.

(From Ma TY, Hoa NT, Tran DD, Bui V, Pedram A, Mills S, Merryfield M. Cytochalasin B modulation of Caco-2 tight junction barrier: role of myosin light chain kinase. Am J Physiol Gastrointest Liver Physiol 2000; 279 :G875–G885, with permission.)

The cytochalsasin D depolymerization of actin also causes a disturbance in junctional localization of TJ protein occludin, characterized by internalization and disassembly of occludin from junctional areas and loss of continuity in occludin junctional localization. Latrunculin A (LatA), an actin depolymerizing agent, has been shown to irreversibly disrupt the actin network and therefore disrupt the TJ barrier irreversibly in kidney derived cell line MDCK, and induced the formation of randomly dispersed actin aggregates. LatA also caused a disturbance in junctional localization of occludin and internalization of occludin in kidney derived cell line MDCK. Live cell imaging coupled with simultaneous recordings of barrier function showed that occludin internalization was temporally related to barrier loss. The latA-induced occludin internalization was mediated by caveolin-1 and dynamin II-dependent endocytosis, and inhibition of caveolae-mediated endocytosis prevented the latA-induced occludin internalization and drop in MDCK TER. These data suggested that latrunculin-induced actin depolymerization induces caveolae-mediated endocytosis of TJ proteins, and that internalization of transmembrane TJ proteins leads to the loss of TJ barrier function. The actin depolymerization-induced activation of MLCK is presumably also involved in the endocytosis of the TJ proteins.

25.6.2

Luminal Osmolarity and Solvent Drag Effect

The relationship between luminal osmolarity, intestinal water flux, and intestinal paracellular permeability has been extensively examined in vivo by recycling perfusion of isolated rat small intestinal segment with perfusate solutions having varying osmolarity. The rate of intestinal absorption of various sized paracellular markers including mannitol, PEG 400, PEG 900, and inulin was linearly related to the increasing concentration of permeability markers, consistent with a passive uptake mechanism. In the in vivo rat intestinal perfusion studies, changing the luminal perfusate pH (from 6.0 to 7.5), varying the unstirred water layer resistance by increasing the luminal flow rate (from 1 to 3 mL/min), or disruption of the mucus layer by treatment with mucolytic agent acetylcysteine did not affect the intestinal flux rates of paracellular markers.

In a leaky or low-resistance epithelia such as the small intestine, junctional or paracellular pathway is the major permeation pathway for the passive water and ionic flux. The passive water flux may be bidirectional, and the direction of the net water flux may be regulated by the differences in osmotic gradient or hydrostatic pressure across the intestinal epithelial barrier. A linear relationship exists between decreasing luminal osmolarity and increasing paracellular water flux. The contribution of solvent drag on the absorption of paracellular markers may be assessed by manipulating the water flux by changing the osmolarity of the luminal perfusate solution. Decreasing the luminal osmolarity from 600 to 225 mOsm results in an osmotic gradient-dependent mucosal-to-serosal water flux ( Fig. 25.10 ). As the water moves rapidly across the paracellular or junctional pathways, hydrophilic solutes in the luminal solution are also carried along via a solvent drag. There is a direct correlation between increasing intestinal water flux and increasing flux of the hydrophilic solutes via the solvent drag ( Fig. 25.11 ).

Relationship between small intestinal luminal osmolarity and water flux.

(From Ma TY, Hollander D, Erickson RA, Truong H, Krugliak P. Is the small intestinal epithelium truly “tight” to inulin permeation? Am J Physiol 1991; 260 :G669–G676, with permission.)

Relationship between jejunal water flux and inulin flux ( n = 3–9 rats). Jejunal water flux was varied by changing osmolarity of luminal perfusate. Plotted values are mean of water and inulin absorption.

(From Ma TY, Hollander D, Erickson RA, Truong H, Krugliak P. Is the small intestinal epithelium truly “tight” to inulin permeation? Am J Physiol 1991; 260 :G669–G676, with permission.)

Solvent drag allows the intestine to absorb all luminal glucose, even when concentrations exceed the capacity of the transcellular transport system; at low concentrations, the transcellular pathway absorbs luminal glucose, but as concentrations increase, a growing fraction of glucose absorption occurs via the paracellular pathway. The relative contribution of diffusive and convective (or solvent drag effect) component to the passive transport of permeability probes and solvent drag reflection coefficient σ _f (an indicator of dependency on solvent drag effect) may be calculated using Fick’s law and modified Kedam-Katchalsky equation of solvent drag effect. Fick’s first law expresses the flux rate J _D at which a given solute diffuses across a semipermeable membrane as diffusional coefficient, P _D .

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