A significant number of the current therapeutic agents including antihistamines, skeletal muscle relaxants, antiarrhymics, and p-adreno receptor blocking agents, as well as endogenous bioactive amines (e.g., catecholamines, dopamine, hista-mine, and choline) are organic cations (OC). Based on the fact that many of these organic cations are polar and positively charged at physiological pH, membrane bound transporters are required to enhance their intestinal uptake and absorption in an acidic environment. The OC transporter family has been identified as one of the main classes of transporters that act in this fashion. It should be noted that most of the literature dealing with the function of OC transporters have been conducted in the liver and kidney (Katsura and Inui, 2003; Koepsell et al., 2003; Koepsell and Endou, 2004; Sai and Tsuji, 2004; Steffansen et al., 2004), while few discuss the role of OC transporters in the GI tract. The results pertaining to the expression and functional significance of OC transporters in the GI tract are summarized here.
Organic cation transporter 1 (OCT1) was the first member of the OC family identified and was cloned from rat kidney by Grundemann et al. (1994). Subsequently, other organic cation transporters (OCT2-3), as well as the more distantly related carnitine and organic cation transporters (OCTN1-3) have been cloned and characterized (Koepsell et al., 2003; Koepsell and Endou, 2004; You, 2004). Although they share some common structural features, OCTs and OCTNs are considered distinct subfamilies within the OCT family with each member having been isolated from multiple species. Similar to OATs, the OCT and OCTN isoforms represent 12 a-helix TMD protein, which contain a large glycosylated extracellular loop between TMD 1 and 2, and a large intracellular loop carrying phosphorylation sites between TMD 6 and 7 (You, 2004). Since OCT and OCTN family members have sequence homology to the OAT family (Koepsell and Endou, 2004; Miyazak et al., 2004; You, 2004), OATs, OCTs and OCTNs, together with other uncharacterized, yet homologous orphan transporters (e.g. BOCT, brain-type organic cation transporter; ORCTL, organic cation transporter like; UST, unknown solute transporter; etc.) comprise a transporter superfamily, referred to as the organic ion transporter family SLC22A (Miyazak et al., 2004; http://www.bioparadigms.org/slc/SLC22.htm).
126.96.36.199 The Substrate Specificity of Organic Cation Transporters
OCT1-3 are polyspecific transporters capable of transporting various organic cations (Table 7.7), including model compounds such as tetraethylammonium (TEA) and N-methylquinine, as well as other xenobiotics including 1-methyl-4-phenylpyridium (MPP+), acyclovir and ganciclovir, metformin and phenformin, memantine, as well as quinidine (Koepsell et al., 2003; Koepsell and Endou, 2004; You, 2004). Endogenous substrates of the OCTs include the monoamine neurotransmitters acetylcholine, dopamine, serotonin, histamine, choline, and physiological compounds such as creatinine, guanidine, and thiamine (Koepsell et al., 2003; Koepsell and Endou, 2004; You, 2004). Although organic cations are clearly the preferred ligands of the OCTs, several uncharged or anionic compounds are known to be substrates of these transporters (Table 7.7). For example, hOCT2 is partially responsible for the transport of cimetidine, a weak base (Barendt and Wright, 2002), while both hOCT1 and hOCT2 mediate the transport of anionic prostaglandins (Kimura et al., 2002). Although the substrate and inhibitor specificities of OCT1-3 overlap extensively, there are distinct differences in affinity and maximal transport rates among different OCT isoforms and species, which have been summarized by Koepsell et al. (2003). For example, the IC50s of hOCT2 (16 |M) andhOCT3 (14 |M) to desipramine, an antidepressant, were one order of magnitude higher than that of hOCT3 (5.4 |M) (Gorboulev et al., 1997; Zhang et al., 1998; Wu et al., 2000b). In contrast, IC50 of hOCT2 (>100 |M) to a Blocker Prazosin was much higher when compared to hOCT1 (1.8 |M) and hOCT3 (13 |M) (Hayer-Zillgen et al., 2002).
Members of the OCTN subfamily have differential abilities to interact with a variety of organic cation drugs, as well as carnitine (Koepsell and Endou, 2004; You, 2004). For example, TEA is a substrate for rat, mouse, and human OCTN1 (Tamai et al., 1997, Wu et al., 2000a) and OCTN2 (Wu et al., 1998, 1999; Tamai et al., 1998; Friedrich et al., 2003), but not for OCTN3, at least for mouse isoform (Tamai et al., 2000b). Rat OCTN1 and hOCTN1 exhibit a very low affinity for carnitine (Tamai et al., 1997; Wu et al., 2000a), but mOCTN1 mediates significant transport of carnitine (Tamai et al., 2000b), suggesting a species difference of substrate specificity. In addition, OCTN2 from all species (Wu et al., 1998; Tamai et al., 2000b; Friedrich et al., 2003), and mOCTN3 (Tamai et al., 2000b) exhibit medium and very high affinity for carnitine, respectively.
188.8.131.52 Organic Cation Transporter Mediated Transport
Several common transport properties have been identified for all OCTs and independent from subtype or species. OCTs translocate organic cations and other compounds in an electrogenic manner, which has been shown for the rat isoforms rOCT1, rOCT2, and rOCT3 (Busch et al., 1996; Nagel et al., 1997; Kekuda et al., 1998; Okuda et al., 1999), and for the human transporters hOCT1 and hOCT2 (Busch et al., 1998; Gorboulev et al., 1997). In addition, OCTs medicated transport is independent from Na+ and H+ ions (Busch et al., 1996; Gorboulev et al., 1997; Kekuda et al., 1998). Driving force for substrate transport is provided by the substrate concentration gradient and the membrane potential (Busch et al., 1996, 1998; Gorboulev et al., 1997; Kekuda et al., 1998; Okuda et al., 1999). As such, OCTs are able to translocate substrates across the plasma membrane in either direction (Busch etal., 1996,1998; Nagel etal., 1997; Kekuda et al., 1998).
In contrast to OCTs, OCTN mediated transport mechanism depends largely on the isoform and substrate tested. Human and rat OCTN1 work as H+/organic cation antiporters that mediate transport of tetraethylammonium (TEA) and other organic cations (Tamai et al., 1997; Wu et al., 1998). However, mOCTN1 mediate carnitine transport in a Na+/dependent manner (Tamai et al., 2000b). OCTN2 is a Na+/carnitine cotransporter with a high affinity for carnitine (Tamai et al., 1998, 2001; Wu et al., 1999; Wagner et al., 2000). It can also function alternatively as a polyspecific cation uniporter in a Na+-independent manner (Tamai et al., 1998, 2001; Wu et al., 1999; Wagner et al., 2000). In the presence of Na+, hOCTN2 could transport short-chain acyl esters of carnitine (Ohashi et al., 1999) as well as zwitterions, e.g., cephaloridine (Ganapathy et al., 2000). Furthermore, OCTN2 mediated transport is electrogenic and pH-dependent (Wagner et al., 2000). OCTN3 was found only in mice (Tamai et al., 2000b). In contrast to OCTN2 from different species, mOCTN3 transports carnitine independently from Na+ and demonstrates less affinity for organic cation compared to OCTN1 and OCTN2 (Tamai et al., 2000b).
184.108.40.206 The Expression of Organic Cation Transporters
The tissue distribution and membrane localization of the OC family of proteins have been studied using different approaches. In each case, it has been demonstrated that the respective isoforms have different tissue expression patterns that can vary with species (Koepsell et al., 2003; You, 2004). In general, OCT iso-forms are mainly expressed in the liver or kidney, and may also be found, to a less extent, in the heart, skeletal muscle, placenta, and small intestine. hOCT1 is mainly expressed in the liver, whereas hOCT2 is mainly found in the kidney
(Gorboulev et al., 1997). hOCTNl and hOCTN2 are both abundantly expressed in the kidney, skeletal muscle, placenta, prostate, and heart (Tamai et al., 1997, 1998; Wu et al., 1999), with hOCTN2 also being expressed at low level in the liver (Tamai et al., 1998). Although hOCTN1 is expressed strongly in the kidney (Tamai et al., 1997), rOCTN1 is present principally in the liver (Wu et al., 2000a). For a more detailed description of the tissue distribution of OCT and OCTN iso-forms, the reader is directed to the reviews by Koepsell et al. (2003) and You (2004). The following sections will focus on the expression of OCTs and OCTNs in the GI tract.
Tissue expression of OCT1 isoforms indicates that rat OCT1 (Grundemann et al., 1994, Zhang et al., 1997a) and OCT3 (Kekuda et al., 1998) mRNA are expressed in the small intestine at a relatively low and high level, respectively. With respect to the human variants, hOCT1 is expressed to a much lower extent in the human small intestine (Zhang et al., 1997b), while hOCT3 expression in the human small intestine has not been confirmed. Although rOCT2 is predominantly expressed in the rat kidney (Okuda et al., 1996), hOCT2 is also expressed in the human small intestine, as detected by RT-PCR (Gorboulev et al., 1997). A recent study using Oct1-/- mice further suggests the basolateral localization of mOCT1 in the mouse intestine (Fig. 7.2) (Jonker et al., 2001; Jonker and Schinkel, 2004). However, direct immunolocalization data are not currently available.
The mRNA expression of the rOCTN1 (Wu et al., 2000a) and hOCTN2 (Tamai et al., 1998; Wu et al., 1998) were also detected in intestinal enterocytes, the latter one being very weak. To date, no further studies on the intestinal protein expression of OCTNs have been reported.
220.127.116.11 The Regulation of Organic Cation Transporters
The expression and function of OCT family members have been suggested to be regulated via subtype, species, and tissue-specific parameters. The short-term regulation of basolateral and apical OCTs in different experimental systems is well documented (Ciarimboli and Schlatter, 2005). For rOCT1, hOCT1, hOCT2, and hOCT3, regulation has been associated with phosphorylation/dephosphorylation of the transporter, which can result in changes in the substrate affinity (Ciarimboli and Schlatter, 2005). For example, after stable transfection in HEK293 cells, rOCT1-mediated organic cation transport was stimulated by protein kinase C (PKC), PKA, and endogenous tyrosine kinase activation (Mehrens et al., 2000). Furthermore, it was determined that at least one of these phosphorylation sites is PKC dependent. PKC mediated phosphorylation in these sites leads to a confor-mational change at the substrate binding site, and thus results in alteration of the substrate transport including TEA and quinine (Mehrens et al., 2000). Therefore, the potential phosphorylation sites may play an important role in the regulation of transporter activity, especially the S286 residue, which is conserved in almost all OCTs (Ciarimboli and Schlatter, 2005).
A gender dependent difference was observed when it was shown that renal expression of rOCT2 was significantly increased in male versus female rats (Urakami et al., 1999, 2000). Furthermore, these studies demonstrated that testosterone increased rOCT2, while estradiol resulted in a moderate decrease in the expression of rOCT2 (Urakami et al., 1999, 2000), suggesting the role of sex hormone regulation. In contrast, there was no gender difference observed in the renal expression of rOCT1 or rOCT3. In a separate ontogenic study, it was determined that rat renal OCT1, OCTN1, and OCTN2 mRNA levels increased gradually from infants to adults (Slitt et al., 2002). Since OCT1 and OCTN1/2 mediate the renal clearance of organic cations, substrates of these isoforms may be excreted more slowly in infants and children in contrast to adults (Slitt et al., 2002). Similarly, hOCTN1 mRNA was strongly expressed in the fetus when contrasted to the adult liver (Tamai et al., 1997), which indicates that the potential increased fetal hepatic toxicity. Clearly, the current data suggest that the gender-and age-dependent effects on the expression of OCT isoforms need to be considered when evaluating the overall pharmacokinetics and potential toxicity of OCT substrates that have a narrow therapeutic index.
Similar to the OATP/Oatp family, SNPs have been identified for several iso-forms of human OCT and OCTN family (Koepsell and Endou, 2004). Some are associated with decreased transporter activity. In a population of 57 Caucasians, 25 SNPs within the hOCT1 gene were detected and further analyzed (Kerb et al., 2002). Eight SNPs resulted in single amino acid substitutions. Out of these, three SNPs (Arg61Cys, Cys88Arg, and Gly401Ser) affected in vitro organic cation transport. Uptake of MPP+ by Arg61Cys variant, the most frequent mutant (16%), was dramatically decreased (50%) compared to wild-type hOCT1. A follow-up study by Shu et al. (2003) screened 15 protein-altering variants of hOCT1 in Xenopus oocytes. It was demonstrated that predicted mutations occurring in the evolutionarily conserved regions of the gene had a more significant impact on the hOCT1 function. In a separate investigation on a heterogeneous collection of 247 patients, 28 hOCT2 variants were identified with eight (four occurring at a frequency >1% in ethnic populations) causing a nonsynonomous amino acid change (Leabman et al., 2002). Interestingly, the pharmacogenomic analysis of hOCT3 revealed no polymorphisms that resulted in an amino acid composition change (Lazar et al., 2003). The net impact of these OCT isoform variants in mediating the intestinal absorption of xenobiotics has yet to be determined.
OCTs polymorphism may also contribute to the toxicity of drugs. Influence of OCT1 on a severe drug side effect, lactic acidosis, was observed in Oct1~/-mice treated with the biguanide analog, phenformin and metformin. Biguanides are substrates of OCTs, and can accumulate in the plasma due to reduced hepatic clearance caused by OCT transport (Wang et al., 2003; Jonker and Schinkel, 2004). Biguanides exhibit their antidiabetic effects partially through the inhibition of the mitochondrial respiration, leading to reduced glucose, which subsequently leads to an accumulation of plasma lactic acid (Owen et al., 2000; Wang et al., 2003). Deletion of the mOCT1 isoform was demonstrated to decrease the excretion of biguanides and promote inhibition of the mitochondrial respiration, i.e., lactic acidosis (Wang et al., 2003; Jonker and Schinkel, 2004). It is hypothesized that hOCT1 is largely responsible for the potentially fatal lactic acidosis, and may contribute to the removal of phenformin from the market (Wang et al.,
2003; Jonker and Schinkel, 2004; Koepsell, 2004). Other OCT knockout studies are well reviewed by Koepsell (2004) and Jonker and Schinkel (2004).
Defect mutations in hOCTN1 and hOCTN2 have also been demonstrated (Koepsell and Endou, 2004). Kawasaki et al. (2004) identified two SNPs in hOCTN1 of the Japanese population. One of them almost completely abrogated the TEA transport activity in stable transfected HEK293 cells. This result suggests that mutations in hOCTN1 might affect its physiological function and/or the pharmacological characteristics of its substrates. For example, decreased renal secretion of organic cations mediated by hOCTN1 might increase the nephrotoxic potential of relevant substrates.
Mutations in hOCTN2 lead to a recessive hereditary disorder called "primary systemic carnitine deficiency (SCD)" (Nezu et al., 1999; Lahjouji et al., 2001). This potentially lethal disease is characterized by progressive infantile-onset cardiomyopathy, skeletal myopathy, hypoketotic hypoglycemic encephalopathy, and extremely low plasma and tissue carnitine concentrations (Tein et al., 1990; Stanley et al., 1991; Pons et al., 1997). Nonsense or missense mutations in hOCTN2 have been demonstrated to cause low or nonfunctional carnitine transporters. Therefore, defect of carnitine reabsorption in kidney and carnitine uptake in cardiac muscles and other organs leads to systemic carnitine depletion, which lead to the inhibition of (3-oxidation of fatty acids (Tein, 2003). Patients with SCD are treated by oral administration of carnitine. Due to the potential drug-drug interaction, mediation with carnitine drugs interacting with hOCTN2 in patients with partial defects of hOCTN2 is suggested to be avoided or supplemented with higher dose of carnitine (Koepsell and Endou, 2004).
Mutations in hOCTNs might increase susceptibility to Crohn's disease. Peltekova et al. (2004) first identified that a missense mutation in hOCTN1 gene and a transversion in the promoter region of hOCTN2, which form haplo-type related with an increase in the prevalence of Crohn's disease. The resulting amino acid change in hOCTN1 reduced its affinity and capacity for transporting carnitine. It, however, enhanced the affinity and uptake of other xenobiotics (Peltekova et al., 2004). The hOCTN2 promoter mutation occurred in the heat shock transcription factor binding element region and altered transcription factor binding affinity (Peltekova et al., 2004). Therefore, these variants alter transcription and transporter functions of the OCTNs and interact with variants in another gene associated with Crohn's disease, CARD15, to increase risk of Crohn's disease. The increased risk observed in this patient population was later confirmed by Torok et al. (2005).
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