12 - substrates uptake transporters

In cell lines, the organic anion transporters (OAT and OATP) have been identified and cloned into cells of kidney origin such as LLC-PK1, MDCK, HK-2, and Caco-2 [129]. The most well-known uptake transporters, which transport the substrate over the membrane into the organism are the amino acid- [35, 42, 139] and oligopeptide-carriers (PepTl and PepT2) [139-142]. These two transporter families are abundantly expressed in the small intestine of most animals, and can therefore be involved in the absorption process of pharmaceutical drugs. The PepTl is expressed in the cell lines Caco-2 and HT-29 [140-142]. 

Both active and passive transport occur simultaneously, and their quantitative roles differ at different concentration gradients. At low substrate concentrations, active transport plays a major role, whilst above the concentration of saturation passive diffusion is the major transport process. This very simple rule can be studied in an experimental system using cell culture-based models, and the concentration dependency of the transport of a compound as well as asymmetric transport over the membrane are two factors used to evaluate the presence and influence of transporters. Previous data have indicated that the permeability of actively absorbed compounds may be underestimated in the Caco-2 model due to a lack of (or low) expression of some uptake transporters. However, many data which show a lack of influence of transporters are usually derived from experiments... 

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]. 

Organic anions have frequently been implicated as substrates for transporters in the sinusoidal membrane of the liver. This was illustrated for a series of TxRAs, where hepatic uptake was identified as the rate-determining step in the clearance process [22]. A representative compound from this series, UK-147,535 (Figure 9.3), was progressed to clinical trials [23]. It is thus possible to contrast clearance of this compound between a number of species including man (Figure 9.4). 

Stimulation of active H+ extrusion from roots (Cesco, 1995 Pinton et al., 1997 Table 9.1) and transmembrane potential hyperpolarization (Slesak and Jurek, 1988) indicated the involvement of the PM H+-ATPase in the increased nutrient uptake generally observed in the presence of humic substances. Direct proof of an interaction between humic molecules and the PM H+-ATPase has been obtained by Vara-nini et al. (1993), who demonstrated that low-molecular-weight (<5kDa) humic molecules at concentrations compatible with those present in the rhizosphere can stimulate the phospho-hydrolytic activity of this enzyme in isolated PM vesicles (Table 9.1). Further proof of the action of humic molecules on PM FT-ATPase activity and on nutrient uptake mechanisms was obtained when studying the effect of these molecules on NO3 uptake. Transport of this nutrient is a substrate-inducible process and involves FT co-transport. At higher uptake rates, the levels and activity of root PM FT-ATPase increased (Santi et al., 1995). The short-term (4h) contact... 

Figure 15.2. Location of xenobiotic transporters in selected barrier and excretory tissues. For simplicity, the tissues are arranged along a structure representing the vascular space. Arrows indicated direction of transport under normal conditions. This figure is not meant to be comprehensive not all transporters expressed in a tissue are shown. Transporters driven by ATP pump substrates out of cells (efflux). Other transporters are capable of supporting substrate uptake or efflux. Which of these processes predominates depends on available driving forces—for example, substrate concentration gradient and the capability to couple transport to sources of potential energy.

Outliers in these models, as well in biological assays, are due to several reasons which encompass issues related to transporters in the cells which could be efflux or uptake transporters. We have tried to address the problem using MI Fs to understand P-glycoprotein (PGP) efflux, especially as it pertains to substrates. 

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