Polarized expression, of transporters

MDCK Dog kidney epithelial cells Polarized cells with low intrinsic expression of transporters Suitable cell fine for transfections... 

Philp, N.J., et al. 2003. Polarized expression of monocarboxylate transporters in human retinal pigment epithelium and ARPE-19 cells. Invest Ophthalmol Vis Sci 44 1716. 

Schroeter S, Levey Al, Blakely RD (1997) Polarized expression of the antidepressant-sensitive serotonin transporter in epinephrine-synthesizing chromaffin cells of the rat adrenal gland. Mol Cell Neurosci 9 170-184... 

An additional in vitro approach that can provide valuable insight into the possible influence on BSEP inhibition of cooperative interactions with other transporters is by studying vectorial transport of probe substrates using transfected ceUs cultured as confluent monolayers in transwell devices. This approach enables quantification of both basal-to-apical transport through the cells and apical efflux. In these studies it is important to use polarized epithelial cells that express distinct apical and basolateral sinusoidal plasma membrane domains and that also exhibit a low level of endogenous expression of transporters other than BSEP that may mediate apical probe substrate efflux. Appropriate cells include dog kidney-derived MDCK cell lines and porcine kidney-derived LLC-PKl cells. It is also necessary to ensure that basolateral solute carriers are present in the cells that mediate probe substrate uptake. This may require coexpression of BSEP and relevant uptake transporter(s). MDCK cell lines... 

Maulen, N. R Henriquez, E. A. Kempe, S. Caicamo, J. G. Schmid-Kotsas, A. Bachem, M. Grunert, A. Bustamante, M. E. Nualart, E Vera, J. C. 2003. Upregulation and polarized expression of the sodium-ascorbic acid transporter SVCTl in post-confluent differentiated CaCo-2 cells. J. Biol. Chem. 278 9035-9041. 

Caco-2 cells and ezetimibe, a potent inhibitor of chloresterol absorption in humans, it was reported that (1) carotenoid transport was inhibited by ezetimibe up to 50% and the extent of that inhibition diminished with increasing polarity of the carotenoid molecule, (2) the inhibitory effects of ezetimibe and the antibody against SR-BI on P-carotene transport were additive, and (3) ezetimibe may interact physically with cholesterol transporters as previously suggested - and also down-regulate the gene expression of three surface receptors, SR-BI, NPCILI, and ABCAl. 

A. H., Meijer, D. K., Heterologous expression of various P-glycoproteins in polarized epithelial cells induces directional transport of small (type 1) and bulky (type 2) cationic drugs, J. Pharmacol. Exp. Ther. 1998, 286, 321— 327. 

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). 

Some drugs with low intrinsic permeability achieve acceptable oral bioavailability because they are substrates for uptake transporters, which normally function in nutrient uptake. The most prominent example is the peptide transporter, PepTl, which is active toward peptidomimetic antibiotics such as cephalexin, the antiviral agent valacyclovir [24] and other drugs. PepTl is natively expressed in Caco-2 cells, and adenovirus transduction has been used to increase PepTl expression levels [25]. However, the expression of PepTl was not polarized in this system and this expressed system appears to be of limited value as an improved screening model. PepTl has also been expressed in Chinese hamster ovary cells and a variety of other mammalian systems [26, 27]. 

The presence of a transporter can be assessed by comparing basolateral-to-apical with apical-to-basolateral transport of substrates in polarized cell monolayers. If P-gp is present, then basolateral-to-apical transport is enhanced and apical-to baso-lateral transport is reduced. Transport experiments are in general performed with radioactively labeled compounds. Several studies have been performed with Caco-2 cell lines (e.g. Ref. [85]). Since Caco-2 cells express a number of different transporters, the effects measured are most probably specific for the ensemble of transporters rather than for P-gp alone. P-gp-specific transport has been assayed across confluent cell layers formed by polarized kidney epithelial cells transfected with the MDR1 gene [86], Figure 20.11 shows experimental data obtained with these cell lines. A rank order for transport called substrate quality was determined for a number of compounds [86]. The substrate quality is a qualitative estimate, but nevertheless allows an investigation of the role of the air/water (or lipid/water) partition coefficient, log Kaw, for transport as seen in Fig. 20.11(A). For most of the compounds, a linear correlation is observed between substrate quality and log Kaw- However, four compounds are not transported at all despite their distinct lipophilicity. A plot of the substrate quality as a function of the potential of a... 

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... 

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