Basolateral side

ENaC mediates Na+ entry from the tubule lumen at the apical membrane and the Na+/K+ ATPase extrudes Na+ at the basolateral side. K+ channels are present on the basolateral and apical membrane. K+ channels at the apical membrane mediate K+ secretion into the tubular lumen. 

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. ... 

Two principal routes of passive diffusion are recognized transcellular (la —> lb —> lc in Fig. 2.7) and paracellular (2a > 2b > 2c). Lateral exchange of phospholipid components of the inner leaflet of the epithelial bilayer seems possible, mixing simple lipids between the apical and basolateral side. However, whether the membrane lipids in the outer leaflet can diffuse across the tight junction is a point of controversy, and there may be some evidence in favor of it (for some lipids) [63]. In this book, a third passive mechanism, based on lateral diffusion of drug molecules in the outer leaflet of the bilayer (3a > 3b > 3c), wih be hypothesized as a possible mode of transport for polar or charged amphiphilic molecules. 

One potential solution to the problem of filter-restricted diffusion of a solute is to use a filter with larger pores. However, there are practical limitations to how porous a filter one can use and still support the monolayer. In some cases, if the pores are too large (e.g., >1-3 pm diameter), the cell can actually migrate through the filter and begin to grow on the basolateral side of the membrane. This results in a poorly defined monolayer and should be avoided whenever possible. 

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. 5.3. Relationship between the oral fraction basolateral Papp values were determined at absorbed in humans and apparent permeability drug concentrations of 10-500 pM at pH 6.5/ coefficients obtained in Caco-2 cell monoalyers 7.4 or 7.4/7.4 on the apical/basolateral sides of at two different pH conditions. Mean apical to the cell monolayers.

For weak acids, e.g., salicylic acid, the dependency on a pH gradient becomes complex since both the passive diffusion and the active transport process will be dependent on the proton concentration in the apical solution [61, 63, 98, 105] and a lowering of the pH from 7.4 to 6.5 will increase the apical to basolateral transport more than 20-fold. Similarly, for weak bases such as alfentanil or cimetidine, a lowering of the pH to 6.5 will decrease the passive transport towards the basolateral side [105]. The transport of the ionizable compound will, due to the pH partition hypothesis, follow the pKa curve. 

Apical Side = Donor Basolateral Side = Receiver... 

Apical Side = Receiver Basolateral Side = Donor... 

For AIC conditions, the apical surface of the epithelial cell layer is exposed to air after the nasal cells reached confluence on the Transwell insert, while the basolateral side is fed with culture fluid. Figure 9.3 shows TEER changes in epithelial cell layers cultured up to 20 days in LCC versus AIC methods [46], In AIC condition (initiated from day 3 after seeding), TEER peaked on day 5 and maintained above the TEER values observed for LCC counterparts. By contrast, TEER observed for LCC conditions peaked on day 2 and declined toward zero by day 15. These data indicate that human nasal epithelial cells at an air interface culture exhibit better electrophysiological characteristics than those cultured by the conventional liquid-covered conditions. 

Compounds can cross biological membranes by two passive processes, transcellu-lar and paracellular mechanisms. For transcellular diffusion two potential mechanisms exist. The compound can distribute into the lipid core of the membrane and diffuse within the membrane to the basolateral side. Alternatively, the solute may diffuse across the apical cell membrane and enter the cytoplasm before exiting across the basolateral membrane. Because both processes involve diffusion through the lipid core of the membrane the physicochemistry of the compound is important. Paracellular absorption involves the passage of the compound through the aqueous-filled pores. Clearly in principle many compounds can be absorbed by this route but the process is invariably slower than the transcellular route (surface area of pores versus surface area of the membrane) and is very dependent on molecular size due to the finite dimensions of the aqueous pores. 

Where a compound is highly lipophilic, membrane binding also retards the permeability [73, 74]. Therefore, it is important to measure the membrane binding at the same time of the permeation assay. In vivo, the blood flow removes the compound from the basolateral side [75]. As a rule of thumb, when log D ct (at assay pH) > 1.5, membrane binding may be an issue [73]. 

The kidneys are susceptible to toxicity from xenobiotics (Fig. 7.1) because they too have a high blood flow. Cells of the tubular nephron face double-sided exposure, to agents in the blood on the basolateral side and in the Altered urine on the luminal side. Proximal tubule cells are generally the site of nephrotoxicity, since these cells have an abundance of cytochrome P450 and can transport organic anions and cations from the blood into the cells, thereby concentrating these chemicals manyfold. 

Vitamin B12. Vitamin B12 is a larger molecule than the other vitamins, and it can be absorbed via the intestine, which involves binding to specialized transport proteins.44 After oral administration, vitamin B12 binds to intrinsic factor (IF) produced from the parietal cells in the stomach and proximal cells in the duodenum. The vitamin B12—IF complex passes down the small intestine until it reaches the ileum, where the complex binds to a specific IF receptor located on the apical membrane of the villous enterocyte. The complex is then internalized via RME, vitamin B12 is released from IF by the action of cathepsin L on IF, and free vitamin B12 consequently forms the complex with transcobal-amin II to be delivered into the basolateral side of the membrane via the transcytotic pathway. 

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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