Expression of Transporters

The identities and roles of many drug transporters are discussed in other chapters in this volume and are not extensively reintroduced here. The goal is to develop a comprehensive panel of cells expressing individual, functional transporters as research reagents. To simplify data interpretation, the set of transporters should be expressed in the same host cell line and the abundance of functional proteins in the cell line should be known relative to the corresponding in vivo values. However, useful mechanistic data can be obtained from less comprehensive systems. 

As stated earlier, there are many drug transporters expressed in the intestine [27, 28]. Although the expression levels of some transporters have been examined, the protein levels remain to be further defined and the methods need to be standardized. The export proteins MDR1, MRP1, MRP2, and BCRP have been of particular interest because they are expressed at relatively high levels in the intestine [27, 28] and are known to function to efHux drugs. 

That protein expression level ratio is not critical in determining transport rank order was further confirmed by the good correlation between the transport activity in human and rat double-transfected cell lines, and close prediction of in vivo biliary clearance from in vitro results obtained from double-transfected cell line [36]. 

An alternative method to overcome this issue is to perform uptake studies by using membrane vesicles prepared from cDNA-expressing and control cells [5]. Vesicle preparation and vesicle assays are, however, labor intensive. BCRP, MRPs, and BSEP inside-out vesicles prepared from insect cells by using baculovirus system are currently available from BD Biosciences, Solvo, and GenoMembrane. 

A clear priority remains to expand the panel of intestinal efflux transporters that are expressed individually in modified cell lines. These research tools will be instrumental in identifying and validating selective probe transporter substrates and inhibitors. The availability of such probes will allow for a better understanding of the influence of transporters on in vivo pharmacokinetics. A similar set of probes has been instrumental in increasing our understanding of the role that cytochrome P450 plays in human pharmacokinetics and in avoiding issues associated with these enzymes. 

In addition, the availability of specific probes for transporters will allow the generation of data to create transporter structure activity models for the transporters [23], and this provide the ability to design rationally around any transporter-related 

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It is well known that chemical compo.sition of rhizosphere solution can affect plant growth. Particularly, uptake of nutrients may be considerably influenced by the ionic concentration of the rhizosphere solution (40). Despite the difficulty of defining the exact concentration of ions in the rhizosphere surrounding each root (or even root portion), it has been unequivocally demonstrated that plants have evolved mechanisms to cope with the uneven distribution of ions in the root surrounding in order to provide adequate supply of each essential nutrient (41). These mechanisms include expression of transporter genes in specific root zones or cells and synthesis of enzymes involved in the uptake and assimilation of nutrients (40,43). Interestingly, it has been shown that specific isoforms of the H -ATPase are expressed in the plasma membrane of cell roots it has been proposed that the expression of specific isoforms in specific tissues is relevant to nutrient (nitrate) acquisition (44) and salt tolerance (45). 

The rat intestinal cell line IEC-18 has been evaluated as a model to study small intestinal epithelial permeability. This cell line forms very leaky monolayers with TER of 50 n cm2 and permeability to mannitol of 8 x 10-6 cm s 1. The IEC-18 model was proposed to be a better model than the Caco-2 monolayers for evaluating the small intestinal paracellular permeation of hydrophilic molecules. However, the leakier paracellular pathway is related to the poor differentiation level of the cells and an undeveloped paracellular barrier lacking peri-junctional actin-belt. In addition, due to the poor differentiation the cells have minute expression of transporters and are therefore not useful for studies of carrier-mediated transport [82, 84]... 

Fig. 12.3. Transcellular transport of pravastatin across MDCK II cells. Upper panel schematic diagram illustrating the expression of transporters in MDCK II cells after transfection of OATP2 and MRP2 cDNAs. Lower...

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

Anderle P, Rakhmanova V, Woodford K, Zerangue N, Sadee W (2003) Messenger RNA expression of transporter and ion channel genes in undifferentiated and differentiated Caco-2 cells compared to human intestines. Pharm Res 20 3-15. 

Chapter 24 Cloning and Functional Heterologous Expression of Transporters... 

In addition to screening molecules for intestinal absorption, Caco-2 cells have also been used to study mechanisms of drug transport. For many compounds, intestinal permeation involves a transporter to either aid or limit transepithelial transport. The value of Caco-2 cells in this type of studies is due to the fact that these cells express various membrane transporters relevant to drug absorption.1719-23,28,30 However, when interpreting results of studies that involve carrier-mediated transport, discretion, and scaling factors may be required because of the difference in expression level of transporters between in vitro and in vivo systems.12 Another important consideration in carrier-mediated transport studies is that some transport systems in Caco-2 cells may achieve maximal expression level at different days in culture.17,21,38,74 Thus, validation of Caco-2 cells for mechanistic studies should include the identification of the time for optimal expression of transporters as well as the qualitative evaluation of the transporters to establish that they are representative of the native intestinal transporters. 

Lou, Y., Vitalis, T. Z., Basha, G., Cai, B., Chen, S. S., Choi, K. B., Jeffries, A. P., Elliott, W. M., Atkins, D., Seliger, B., and Jefferies, W. A., 2005, Restoration of the expression of transporters associated with antigen processing in lung carcinoma increases tumor-specific immune responses and survival, Cancer Res. 65 7926-7933. 

M. R. Ballestero, M. J. Monte, O. Briz, F. Jimenez, F. Gonzalez-San Martin and J. J. G. Marin, Expression of transporters potentially involved in the targeting of cytostatic bile-acid derivatives to colon cancer and polyps, Biochem. Pharmacol., 2006, 72, 729. 

Studies were undertaken to quantify transporters and examine regional expression in the intestine. mRNA levels in the gut were studied by Englund et al. [35]. Nine transporters were examined and eight were shown to have significant regional differences in expression. In addition, up to a 20-fold difference in expression was observed for certain transporters between intestinal tissue and Caco-2 cells. Expression of transporters in cell models compared to normal tissue can be markedly different, depending on the age of the cells, passage number and culture conditions [35]. Cell models may under- or overexpress transporters. In addition cell lines may express transporters which may not be relevant in vivo. 

In addition it was also suggested that cell lines that have limited expression of transporters (e.g., 2/4/Al) are of use in understanding passive permeability. Saturation of transporters or inhibition can also be used to understand the contribution of passive permeation and active transport. 

Like some of the CYP isozymes, the expression of some transporters is inducible. Induction of the expression of transporters in response to chemical inducers has been primarily studied in the in vitro models using cell lines derived from animals... 

This expression of transport is provided by Eq. 3.30 (where D has been replaced by Dr for generality). It demonstrates that v and U have equivalent—mathematically indistinguishable—roles in component transport and thus equivalent roles in the evolution of component concentration profiles. A decrease in either v or U can be exactly offset by a gain in the other one of the pair, since only their sum, v + Uy enters the equation. 

Phosphorus is predominantly absorbed as inorganic phosphate in the upper small intestine. Phosphate is transported into the epithelial cells by co-transport with sodium expression of transport is enhanced by vitamin D. 

The third cell-based approach concerns sandwich-cultured rat or human hepatocytes (SCH) [80], which closely mimic the hepatic environment in terms of expression of transporters and metabolizing enzymes. In the SCH model, hepatocytes are cultured in a sandwich configuration between two layers of gelled matrix to form intact bile canaliculi [81]. The advantage of this model is that both hepatic uptake and biliary excretion can be studied. 

Another technical limitation of Caco-2 cells is the long culture time required to obtain full differentiation of the cells. It takes 3 weeks to obtain fully differentiated cell monolayers of Caco-2 cells on filter inserts [1, 116, 121]. It has recently been suggested that 2 weeks of culture on filters is sufficient for obtaining a full expression of transporters and integrity [23], but these claims require solid experimental confirmation. 

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