Tight junction polarized cells

Saitou M, Fujimoto K, Doi Y, Itoh M, Fujimoto T, Furuse M, Takano H, Noda T, and Tsukita S [1998] Occludin-deficient embryonic stem cells can differentiate into polarized epithelial cells bearing tight junctions. J Cell Biol 141 397-408... 

Fig. 2.9 Liver cell and sinusoidal cells with organelles and polarized membrane compartments hepatocytes (H), sinusoids (S), Disse s space (D), erythrocytes (ER), endothelial cells (E), Kupffer cells (K), Ito cells (I), microvilli (MV), canahculus (BC), nucleolus (N), tight junctions (tj), cell nucleus (CN), mitochondria (M), smooth endoplasmic reticulum (SER), rough endoplasmic reticulum (RER), Golgi apparatus (GA), lysosomes (L), peroxisomes (P), ribosomes (R), microfilaments (ME) (modified from L. Cossel) (s. figs. 2.16-2.18)...

An intestinal epithelial cell, like all epithelial cells, is said to be polarized because the apical and basolateral domains of the plasma membrane contain different sets of proteins. These two plasma-membrane domains are separated by the tight junctions between cells. The apical portion of the plasma membrane, which faces the Intestinal lumen, is specialized for absorption of sugars, amino acids, and other molecules that are produced from food by various digestive enzymes. Numerous fingerlike projections (100 nm in diameter) called microvilli greatly increase the area of the apical surface and thus the number of transport proteins it can contain, enhancing the cell s absorptive capacity. 

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. 

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. 

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). 

The oral administration of large proteins and peptides is limited due to their low membrane permeability. These compounds are mainly restricted to the para-cellular pathway, but because of their polar characteristics and their size the pore of the tight junctional system is also highly restrictive. An additional transcellular pathway has therefore been suggested for these peptides, i.e., the transcytotic pathway, which involves a receptor-mediated endocytosis in Caco-2 cells [126],... 

CNS development are employed for the same purpose in the PNS. PNS microglia-like cells, like microglia in the CNS, are bone-marrow-derived and have a similar repertoire of responses to activation [2], Both oligodendroglia and Schwann cells speed axonal action potential propagation by assembling and maintaining myelin. Capillary endothelial cells linked by tight junctions restrict entry of polar molecules into the PNS, as into the CNS [3],... 

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... 

MDCK Madin-Darby canine kidney (MDCK) cells have received attention as an alternative to Caco-2 cells for permeability measurements. When grown under standard culture conditions, MDCK cells develop tight junctions and form monolayers of polarized cells. The main advantage over Caco-2 cells is the shorter culture time to confluence (3-5 days). The transep-ithelial electrical resistance of MDCK cells is lower than that of Caco-2 cells and thus, closer to the TEER of the small intestine in vivo. The permeability coefficients of hydrophilic compounds are usually lower in Caco-2 cells than in MDCK cells, which is consistent with the lower TEER values for MDCK cell monolayers. The nonhuman (canine) and nonintestinal (renal) origin of MDCK cells is considered as a disadvantage. They have low expression levels of transporter proteins and low metabolic activity [34], MDCK cells that are stably transfected with P-gp/MDRl are often proposed as an alternative for Caco-2 cells to study bidirectional transport of compounds and, more... 

The blood-brain barrier forms the interface between the bloodstream and the brain parenchyma and thus controls the passage of endogenous substances and xenobiotics into and out of the central nervous system. Brain microvessels exhibit a variety of unique structural features, such as an extremely tight endothelium without fenestration, a very low rate of pinocytosis, tight junctions between endothelial cells excluding paracellular permeability, and a series of polarized transport proteins. The following chapter describes the structural and functional characteristics of the blood-brain barrier with emphasis on transport proteins, as well as in vitro techniques, which allow studying this complex barrier in the brain. 

There are several cell monolayer models that are frequently used for the evaluation of drug permeability and absorption potential (Table 18.1). For a more detailed discussion, please refer to Chap. 8. Caco-2 cells (adenocarcinoma cells derived from colon) are the most extensively characterized and frequently used of the available cell lines [5-9], A unique feature of Caco-2 cells is that they undergo spontaneous enterocyte differentiation in cell culture. Unlike intestinal enterocytes, Caco-2 cells are immortalized and replicate rapidly into confluent monolayers. When the cells reach confluency during culture on a semi-porous membrane, they start to polarize and form tight junctions, creating an ideal system for permeability and transport studies. During the past decade, use of... 

Figure 26.5 Immunofluorescent staining for transformed airway epithelial cells with antibodies specific for (A) keratin 18 (a marker of epithelial cells) and (B) ZO-1 (marker of tight junction formation). Both markers indicate that the cells have retained epithelial characteristics after transformation. The staining for the presence of ZO-1 at the periphery of the cells indicates that the cells have not lost their polarity and can form tight monolayers that will generate a transepithelial resistance. This is a particularly attractive feature for the analysis of ion transport and transcellular transport of macromolecules. ZO-1 is also found in the nucleus and can also be detected by the anti-ZO-1 antibody.

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