The Regulation of OATPsOatps

The regulation of OATP/Oatp isoform expression and the functional kinetics of each transporter can occur at both the transcriptional and posttranscriptional levels (Hagenbuch and Meier, 2003). Several studies have investigated the tran-scriptional regulation of OATP/Oatp isoforms and various control elements were identified (Rausch-Derra et al., 2001; Staudinger et al., 2001; Guo et al., 2002b). Increased Oatp2 mRNA and protein expression were observed from livers of rats treated with pregnenolone-16a-carbonitrile (PCN) (Rausch-Derra et al., 2001). Pregnane X receptors (PXR) were suggested to play a major role in this PCN induction. Oatp2 expression was strongly induced by PCN treatment in the PXR+/+ mice, but not in the PXR ' mice (Staudinger et al., 2001). Furthermore, several PXR response elements have been identified on the rat Oatp2 promoter (Guo et al., 2002b). Since PXR is one of the key regulators of cytochrome P450 3A (CYP3A) (Luo et al., 2004), concomitant PXR-dependent upregulation of OATP/Oatp and CYP3A in response to stimuli represents an important mechanism in the hepatic detoxification of both bile salts and xenobiotics (Staudinger et al., 2001, Xie et al., 2001). Hepatic expression of the human OATP-C gene may be dependent on a liver-enriched transcriptional factor, hepatocyte nuclear factor 1a (HNF-1a) (Jung et al., 2001). Coexpression of HNF-1a stimulated OATP-C promoter activity by 30-fold in HepG2 and 49-fold in HeLa cells. Similarly, promoters of human OATP8 and mouse Oatp4 were also responsive to HNF-1 a (Jung et al., 2001). In addition, OATP8 mRNA levels were induced by ligand of FXR/BAR (Farnesoid X receptor/Bile acid receptor), but not PXR or LXR (Liver X receptor) (Jung et al., 2002), suggesting a FXR-mediated OATP8 gene regulation. An inverted hexa-nucleotide repeat motif (IR-1 element) in the promoter region of OATP-8 was suggested to be the bile acid response element (Jung et al., 2002), where targeted mutation abolished the inducibility of OATP8 (Jung etal., 2002).

The functional regulation of rat Oatp1 and Oatp2 isoforms was also determined to occur at the posttranslational level. Glavy et al. (2000) demonstrated that serine phosphorylation of rat Oatp1 reduces uptake of BSP by 85% in the presence of extracellular ATP in cultured rat hepatocytes. The activation of protein kinase C (PKC), but not PKA, significantly suppressed estrone-3-sulfate uptake in Oatp1 expressing oocytes in a concentration- and time-dependent manner, while pretreatment with specific PKC inhibitor partially reversed this suppression (Guo and Klaassen, 2001). Similarly, PKC activators suppressed Oatp2 mediated digoxin transport and the downregulation effect was completely abolished by PKC inhibitors (Guo and Klaassen, 2001), demonstrating that Oatp2 is also regulated at the protein level by PKC. It is clear that increased attention needs to be focused on studying the mechanistic role of phosphorylation pathways in regulating OATP/Oatp isoform function.

Ontogenic expression patterns of OATP/Oatp isoforms have also been investigated. It appears that the rat Oatp family members follow a similar temporal pattern with regard to the developmental regulation of their mRNAs. For example, low hepatic expression of rat Oatp2 (Guo et al., 2002a) and Oatp4 (Li et al., 2002) was observed in newborn rats, with a gradual increase shown during postnatal development. Expression of rat Oatp1 mRNA and protein in the choroid plexus were not observed until 15 days postnatal, and were at adult levels by 30 days (Angeletti et al., 1998). Rat Oatp5 expression in the kidney could not be found during the first 3 weeks after birth (Choudhuri et al., 2001). Unlike rat Oatp iso-forms, mouse hepatic rPGT (Oatp2a1) was expressed at adult levels at birth, while renal Oatp1, Oatp5, and Oatp-D were expressed at lower level at birth versus at 6 weeks of age (Cheng et al., 2005). Other renal mouse Oatp isoforms followed a similar age-dependent expression pattern to their rat orthologues while mouse hepatic mRNA expression of Oatp1, Oatp2, Oatp4, and Oatp5 elevated gradually after birth and reached observed maximum adult levels by 6 weeks of age (Cheng etal., 2005).

These data suggest that developmental changes influence the OATP/Oatp family and can significantly influence the substrate pharmacokinetic and pharmaco-dynamic profiles, especially those in the liver and kidney, the two major organs in drug detoxification. For example, newborn rats are more sensitive to ouabain (cardiac glycoside) toxicity due to lower Oatp2 expression in the liver (Guo et al., 2002a), which results in less hepatic uptake and higher blood and tissue levels resulting in ouabain toxicity (Klaassen, 1972; Guo et al., 2002a). Interestingly, pregnenalone-16a-carbonitrile (PCN) protects newborns from cardiac glycoside toxicity by dramatically inducing hepatic sinusoidal Oatp2 mRNA and protein levels in neonatal rats (Guo et al., 2002a) and thus increasing hepatic ouabain clearance. The elucidation of similarities and differences in OATP/Oatp expression among rat, mouse, and human will aid in extrapolation of rodent pharma-cokinetic data to humans.

Gender-specific expression of OATP/Oatp isoforms was also reported. In one study, the protein expression of Oatp2 in the female rat liver was significantly lower than those observed in male rats, while levels of Oatp1 and Oatp4 were comparable (Rost et al., 2005). Rost et al. (2005) further demonstrated that the protein expression of both Oatp1 and Oatp4 was dramatically downregulated after DHEA (dehydroepiandrosterone) treatment in both male and female rats, while Oatp2 expression was only downregulated in male rats. Higher renal expression of Oatp1 in female rats was also inferred from increased urinary excretion of estradiol-17ß-D-glucuronide when contrasted with male rats (Gotoh et al., 2002). It has also been demonstrated that renal Oatp1 expression is stimulated by testosterone and inhibited by estrogens (Lu etal., 1996), while hepatic Oatp 1 expression is not influenced (Simon et al., 1999). These findings suggest that sex hormones may play a role in the regulation of OATP/Oatp isoforms, although further work is required to elucidate the effects in the human intestine.

A number of allelic polymorphisms have been identified for each human OATP isoform (Tirona and Kim, 2002). Mutations may manifest in the promoter region influencing expression or sites impacting tertiary structure and/or substrate interacting regions. Since they may play a critical role in the pharmacokinetics of anionic drugs and other compounds, mutations in these OATP isoforms might alter the handling of certain drugs in the human body and thus enhance (or decrease, depending on the particular situation) toxicity and therapeutic efficacy. For example, SNPs in OATP-C from a population of African- and European Americans have variable pharmacokinetic attributes (Tirona et al., 2001). In vitro assessment of 16 OATP-C alleles revealed that several variants exhibit markedly reduced OATP-C mediated uptake. Additionally, alterations in transport were associated with SNPs imparting amino acid changes within the TMD and also with those modifying extracellular loop 5. Genetic polymorphisms of OATP-B have also been studied in a Japanese population (Nozawa et al., 2002). A SNP of OATP-B, S486F, had an allelic frequency of as high as 10%. This OATP-B variant demonstrated dramatic decreased activity in in vitro transport assay. Given the allelic differentiation arising from SNPs among populations, SNPs in human OATPs represent a heretofore unrecognized factor influencing drug disposition. Expression and functional characterization of allelic variants as to the pharmacokinetics of substrates requires further investigation.

These data demonstrate that genetic, age, and gender dependent variability exists in the expression of OATP/Oatp isoforms. The pharmacogenomic differences may result in significant pharmacokinetic and pharmacodynamic outcomes with administered OATP/Oatp substrates. This is an especially important factor when one considers those substrates that have a narrow therapeutic index. This also ignores the potential confounding idiosyncratic events that may arise from environmental/xenobiotic exposure.

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