Apical side

In contrast to previous in vivo models, this in vitro model provides the possibility of dissociating experimentally two important processes of intestinal absorption cellular uptake and secretion. Under conditions mimicking the postprandial state (taurocholate/oleic acid supplementation), differentiated Caco-2 cells were able to (1) take up carotenoids at the apical sides and incorporate them into CMs and (2) secrete them at the basolateral sides associated with CM fractions. Using this approach, the extent of absorption of P-carotene through Caco-2 cell monolayers after 16 hr of incubation was 11.2%, a value falling within the in vivo range (9 to 22%). ° - Of the total amount of P-carotene secreted, 78% was associated with the two CM fractions and 10% with the VLDL fraction. ... 

The absorption of short-chain weak acids in the rat intestine, as a function of pH, does not appear to conform to the pH partition hypothesis [44]. Similar anomalies were found with weak bases [77]. The apparent pKa values observed in the absorp-tion-pH curve were shifted to higher values for acids and to lower values for bases, compared with the true pKa values. Such deviations could be explained by the effect of an acid layer on the apical side of cells, the so-called acid pH microclimate [44,70,73,76-84],... 

TABLE 7.2 Microclimate pH on the Apical Side of Epithelial Cells in the GIT in Rats... 

Epithelial cells express but do not apically localize Pins, and do not express Insc. We have previously shown that ectopically expressed Insc localizes to the apical cortex in wild-type epithelial cells (Kraut et al 1996). Interestingly, ectopic Insc expression causes Pins, which is normally localized to the lateral cortex, to localize to the apical cortex. Conversely, apical localization of ectopically expressed Insc is dependent on pins. Insc ectopically expressed in Pins- epithelial cells does not localize as an apical crescent it adopts a cytoplasmic distribution which is enriched towards the apical side of the cell during interphase and is undetectable during mitosis, presumably due to rapid degradation. This instability of ectopically expressed Insc may be why the 90° rotation in the mitotic spindles which occurs as a consequence of Insc ectopic expression in the wild-type epithelial cells no longer occurs when Insc is expressed in Pins-embryos. These results indicate that Insc is necessary and sufficient for the recruitment of Pins to the apical cortex of wild-type epithelial cells. 

The Caco-2 cell line was isolated from a human colon carcinoma, and has been characterized as one of the best in vitro models of intestinal epithelium. Indeed, in contrast to other intestinal cell lines, Caco-2 cells are able to constitute a homogenous monolayer and to spontaneously differentiate into polarized cells, highly similar to human mature enterocytes, after approximately 2 weeks of culture. Furthermore, the Caco-2 cells present microvillosities at the apical side and have a high transmembrane resistivity, which confirms the fact that the cells are confluent and link to one another via gap junctions. Finally, they can absorb different compounds, express many enzymes involved in intestinal metabolic pathways (Pinto et al. 1983, Musto et al. 1995, Salvini et al. 2002), and give reproducible in vitro results consistent with results obtained in in vivo studies (Artursson and Karlsson 1991). 

Tab. 3.2. Microclimate pH on the apical side of epithelial cells in the gastrointestinal (Gl) tract in rats [32].

Drug absorption experiments are easy to perform in the cell culture model. The drug is added to the apical (mucosal) side, and the appearance of the drug on the basolateral side (serosal) is followed by time. The model also permits experiments to be carried out in the reverse direction, i.e., from the basolateral side to the apical side. 

Fig. 5.5. Example of using the Caco-2 monolayers to evaluate paracellular transport of an unknown compound. Two markers, mannitol and propranolol are added together with the unknown compound to the apical side and transport of each of the molecules is measured both in the absence and in the...

It is also important to predict the in vivo biliary excretion clearance in humans, and for this purpose MDCK II cell lines expressing both uptake and efflux transporters may be used (Fig. 12.3) [92, 93]. It has been shown that MRP2 is expressed on the apical membrane, whereas OATP2 and 8 are expressed on the basolateral membrane after cDNA transfection (Fig. 12.3) [92, 93]. The transcellular transport across such double-transfected cells may correspond to the excretion of ligands from blood into bile across hepatocytes. Indeed, the vectorial transport from the basal to apical side was observed for pravastatin only in OATP2- and MRP2-expressing... 

Apical Side = Donor Basolateral Side = Receiver... 

Apical Side = Receiver Basolateral Side = Donor... 

The extent to which the cell line supports appropriate expression of the cDNA. The level of expression achieved is determined by interactions of the vector/ expressed protein with the cell. These interactions include the strength of the promoter (weaker promoters can be compensated for by using a vector which is present at high copy number), the adequacy of the selective agent (not all agents are toxic to all cells), the stability of the expressed protein (some proteins may be rapidly degraded in some cells), and whether the expressed protein exerts any deleterious effects on the viability of host cells (some efflux transporters could deplete the cell of essential components). Finally, transporters must be expressed in a polarized manner in the host cell (i.e., preferentially on either the basolateral or apical side of the cell). 

The intention to study transport processes at pulmonary epithelia, however, raised two particular problems (i) the apical side of these epithelia is typically in contact with air rather than with a liquid and (ii) in order to maximize the surface area, the lungs have a complex treelike structure, ending in millions of tiny alveolar bubbles. The total surface area of the human alveolar epithelium is almost half of that of the intestines (100-120 m2), with its macroscopic appearance resembling a sponge, and it is virtually impossible to use such a tissue for transport experiments in a diffusion-chamber setup. 

Hepatocytes make up 60-70% of the total number of liver cells. They have a well-organized intracellular structure with huge numbers of cell organelles to maintain the high metabolic profile. At the apical side or canalicular membrane the cell is specialized for the secretion of bile components. There are several ATP-dependent transport carriers located on this side of the membrane, which transport bile salts, lipids and xenobiotics into the canaliculus. On the sinusoidal side, the cells specialize in uptake and secretion of a wide variety of components. To increase the surface of the membrane for this exchange with the bloodstream, the sinusoidal domain of the membrane is equipped with irregular microvilli. The microvilli are embedded into the fluid and matrix components of the space of Disse and are in close contact with the sinusoidal blood because of the discontinuous and fenestrated SECs. To facilitate its metabolic functions numerous membrane transport mechanisms and receptors are situated in the membrane. 

As indicated earlier in this chapter, these transporter proteins are found in a variety of cell types but especially in those organs exposed to chemicals from the environment (e.g., gastrointestinal tract), excretory organs (e.g., kidney), and sensitive organs (e.g., brain). The proteins are usually found on the luminal side of epithelial cells in organs of exposure, such as the small intestine, which allows the cells to pump out the potentially hazardous chemical. In sensitive organs such as the brain, the transporters are on that side of cells that will allow chemicals to be pumped back into the blood or interstitial fluid. In organs of excretion, such as the kidney, the transporters are located on the apical side of cells such as the proximal convoluted tubular cells. 

Beside membrane transporters such as PepTl and PepT2, which act as absorptive systems, there are transporters like P-gp and the MRP 15, which transport certain drugs actively back into the intestinal lumen. These efflux pumps are located in several tissues including liver, kidney, brain, and intestine [90,91]. In the intestine, efflux systems are predominantly located at the apical side of the epithelial cells. Lipophilic drugs are usually absorbed by the transcellular route so that they are mostly affected by these systems. Interestingly, the intracellular occurring CYP3A metabolizes compounds to substrates that are eliminated by P-gp [92],... 

The setup of this membrane permeability study involves the use of culture inserts that contain Caco-2 cells grown as epithelial layers. A drug candidate is delivered to the apical side of the cell mono-layer (donor) and allowed to incubate for approximately 60 min. Samples from the apical and basolateral (recipient) side are collected for analysis by LC/MS/MS. Membrane permeability is expressed as the percentage of substrate transported across the monolayer from the apical to the basolateral side. 

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