Tight junction cells

Reduced Culture Time Caco-2 cells are usually grown for at least 20 days to form differentiated monolayers on a porous filter support, forming an apical and a basolateral compartment. The 20-day culture time is required to obtain tight junctions, cell polarity, and an adequate expression of drug efflux mechanisms. For economical reasons, various attempts have... 

Coyne, C.B., and Bergelson, J.M. (2006). Virus-induced Abl and Fyn kinase signals permit coxsackievirus entry through epithelial tight junctions. Cell 124,119-131. 

Specific barriers may serve to limit dmg distribution. The placental barrier is of obvious importance to dmg action in the fetus. Dmg transfers across the placenta primarily by Hpid solubiHty. Hence, this barrier is not particularly restrictive. Similarly, the Hpid solubiHty of a dmg is a primary deterrninant in access to the brain and cerebrospinal fluid. Generally, hydrophilic or charged dmgs can also penetrate to these latter areas, but the result is slow and incomplete. The blood brain barrier is composed of cells having tight junctions which are much less permeable to solutes than are the endotheHal cells of other tissues. 

The blood-brain barrier (BBB) forms a physiological barrier between the central nervous system and the blood circulation. It consists of glial cells and a special species of endothelial cells, which form tight junctions between each other thereby inhibiting paracellular transport. In addition, the endothelial cells of the BBB express a variety of ABC-transporters to protect the brain tissue against toxic metabolites and xenobiotics. The BBB is permeable to water, glucose, sodium chloride and non-ionised lipid-soluble molecules but large molecules such as peptides as well as many polar substances do not readily permeate the battier. 

FIGURE 29-1. The blood-brain barrier selectively inhibits certain substances from entering the interstitial spaces of the brain and spinal fluid. It is thought that certain cells within the brain form tight junctions that prevent or slow the passage of certain substances. Levodopa passes the blood-brain barrier, whereas dopamine is unable to pass. 

There are regional asymmetries in membranes. Some, such as occur at the villous borders of mucosal cells, are almost macroscopicaUy visible. Others, such as those at gap junctions, tight junctions, and synapses, occupy much smaller regions of the membrane and generate correspondingly smaller local asymmetries. 

Fig. 1.—Diagrammatic Representation of the Three Steps in the Taste-cell Transduction. Step 1, interaction of stimulus (S) with membrane-bound receptor (R) to form stimulus-receptor complex (SR) step 2, conformational change (SR) to (SR), brought about by interaction of S with R (this change initiates a change in plasma-membrane conformation of taste cells, probably below the level of the tight junction) and step 3, conformational changes of the membrane result in lowered membrane resistance, and the consequential influx on intracellular ionic species, probably Na. This influx generates the receptor potential which induces synaptic vesicular release to the innervating, sensory nerve, leading to the generator potential.

Nakamuta S, Endo H, Higashi Y, Kousaka A, Yamada H, Yano M, Kido H (2008) Human immunodeficiency virus type 1 gpl20-mediated disruption of tight junction proteins by induction of proteasome-mediated degradation of zonula occludens-1 and -2 in human brain microvascular endothelial cells. J Neurovirol 14 186-195... 

The in vitro system we have been using to study the transepithelial transport is cultured Madin-Darby canine kidney (MDCK) epithelial cells (11). When cultured on microporous polycarbonate filters (Transwell, Costar, Cambridge, MA), MDCK cells will develop into monolayers mimicking the mucosal epithelium (11). When these cells reach confluence, tight junctions will be established between the cells, and free diffusion of solutes across the cell monolayer will be markedly inhibited. Tight junction formation can be monitored by measuring the transepithelial electrical resistance (TEER) across the cell monolayers. In Figure 1, MDCK cells were seeded at 2 X 104 cells per well in Transwells (0.4 p pore size) as described previously. TEER and 14C-sucrose transport were measured daily. To determine 14C-sucrose... 

There are also numerous important variations in the microvasculature bed (i.e., arterioles, capillaries, and venules) that affect permeability. For example, venular portions of the capillaries have thin endothelial cells (170 nm), with frequent interendothelial discontinuities. About 30% of venular junctions are believed to have gaps of about 6 nm. Arterioles, in contrast, have endothelial cells that are linked by the tight junctions and communicating junctions, whereas the capillary endothelium contains... 

Figure 2.5 Schematic of the structure of epithelial cells, based on several literature sources [55,63,69,73,74,76,78,79]. The tight junctions and the basement membrane appear to be slightly ion-selective (lined with some negatively charged groups) [75,76,79]. [Avdeef, A., Curr. Topics Med. Chem., 1, 277-351 (2001). Reproduced with permission from Bentham Science Publishers, Ltd.]...

The membrane surface facing the lumen is called the apical surface, and the membrane surface on the side facing blood is called the basolateral surface. The intestinal cells are joined at the tight junctions [63,75]. These junctions have pores that can allow small molecules (MW < 200 Da) to diffuse through in aqueous solution. In the jejunum, the pores are 7-9 A in size. In the ileum the junctions are tighter, and pores are 3-4 A in size (i.e., dimensions of mannitol) [63]. 

Many structural components of the tight junctions (TJs) have been defined since 1992 [85-97]. Lutz and Siahaan [95] reviewed the protein structural components of the TJ. Figure 2.7 depicts the occludin protein complex that makes the water pores so restrictive. Freeze-fracture electronmicrographs of the constrictive region of the TJ show net-like arrays of strands (made partly of the cytoskeleton) circumscribing the cell, forming a division between the apical and the basolateral... 

Bhat, M. Toledo-Velasquez, D. Wang, L. Y. Malanga, C. J. Ma, J. K. H. Rojanasakul, Y., Regulation of tight junction permeability by calcium mediators and cell cytoskeleton in rabbit tracheal epithelium, Pharm. Res. 10, 991-997 (1993). 

Hung TJ, Kemphues KJ 1999 PAR-6 is a conserved PDZ domain-containing protein that colocalizes with PAR-3 in Caenorhabditit elegant embryos. Development 126 127—135 Izumi Y, Hirose T, Tamai Y et al 1998 An atypical PKC directly associates and colocalizes at the epithelial tight junction with ASIP, a mammalian homologue of Caenorhabditit elegant polarity protein PAR-3. J Cell Biol 143 95-106... 

While both paracellular and passive transcellular pathways are available to a solute, the relative contribution of each to the observed transport will depend on the properties of the solute and the membrane in question. Generally, polar membrane-impermeant molecules diffuse through the paracellular route, which is dominated by tight junctions (Section III.A). Exceptions include molecules that are actively transported across one or both membrane domains of a polarized cell (Fig. 2). The tight junction provides a rate-limiting barrier for many ions, small molecules, and macromolecules depending on the shape, size, and charge of the solute and the selectivity and dimensions of the pathway. 

Figure 2 Comparison of intestinal epithelial cells in culture and in situ. (A) Human colon Caco-2 cells grown in culture for 16 days on a semiporous filter. (B) Epithelial layer of rat jejunum. AP, apical or luminal membrane B, basal or abluminal membrane BM, basement membrane G, goblet cell LS, lateral space mv, microvilli Nu, nucleus TJ, tight junction. Bars equal 10 pm. 

Figure 3 A hydrophobic permeant must negotiate through a complex series of diffu-sional and thermodynamic barriers as it penetrates into a cell. The lipid and protein compositions and charge distribution of the inner and outer leaflets of the membrane lipid bilayer can play limiting roles, particularly at the tight junction. Depending upon the permeant s characteristics, it may remain within the plasma membrane or enter the cytoplasm, possibly in association with cytosolic proteins, and partition into cytoplasmic membranes.

All of these characteristics can be under the regulation of the cell and influenced by the cell culture conditions. The age of the cell monolayer in culture can have a profound impact on the quality of the barrier. In monolayers with actively dividing cells, resistance increases with time in culture as tight junctions form (see Fig. 15, Section III.C.4). Resistance reaches a plateau, then decreases as cell viability declines (Section III.C.4). Time in culture may also be a factor in the expression of polarity, which is related to tight junction formation as well as the state of differentiation of the cells (e.g., differential gene expression). 

Figure 6 An electron micrograph of intercellular junctions between two human colon Caco-2 cells in culture. D, desmosome LS, lateral space mv, microvilli ZA, zonula adherens ZO, zonula occludens (i.e., tight junction). Bar equals 200 nm.

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