Abstract
Previous inhibition studies with taurocholate and cardiac glycosides suggested the presence of separate uptake systems for small “type I” (system1) and for bulky “type II” (system2) organic cations in rat hepatocytes. To identify the transport systems involved in type I and type II organic cation uptake, we compared the organic cation transport properties of the rat and human organic cation transporter 1 (rOCT1; hOCT1) and of the organic anion-transporting polypeptides 2 and A (rat Oatp2; human OATP-A) in cRNA-injectedXenopus laevis oocytes. Based on characteristiccis-inhibition patterns of rOCT1-mediated tributylmethylammonium and Oatp2-mediated rocuronium uptake, rOCT1 and Oatp2 could be identified as the organic cation uptake systems1 and 2, respectively, in rat liver. While hOCT1 exhibited similar transport properties as rOCT1, OATP-A- but not Oatp2-mediated rocuronium uptake was inhibited by the OATP-A substrateN-methyl-quinidine. The latter substrate was also transported by rOCT1 and hOCT1, demonstrating distinct organic cation transport activities for rOCT1 and Oatp2 and overlapping organic cation transport activities for hOCT1 and OATP-A. Finally, the data demonstrate that unmethylated quinidine is transported by rOCT1, hOCT1, and OATP-A at pH 6.0, but not at pH 7.5, indicating that quinidine requires a positive charge for carrier-mediated uptake into hepatocytes. In conclusion, the studies demonstrate that in rat liver the suggested organic cation uptake systems1 and 2 correspond to rOCT1 and Oatp2, respectively. However, the rat-based type I and II organic cation transporter classification cannot be extended without modification from rat to human.
Hepatic clearance of organic cations is a major pathway of xenobiotic elimination from the systemic circulation. It has been estimated that at least 50% of the presently available therapeutic agents have a (partly) cationic character (Groothuis and Meijer, 1996). The positive charge is either due to quaternary ammonium groups in the molecule or to tertiary amine groups, which are protonated to a large extent at physiological pH (Meijer et al., 1990). In isolated rat hepatocytes and in isolated perfused rat liver, two uptake systems for organic cations have been proposed (Steen et al., 1992; Groothuis and Meijer, 1996). Uptake system1 transports relatively small “type I” organic cations such as tetraethylammonium (TEA) (Moseley et al., 1992), tributylmethylammonium (TBuMA) (Steen et al., 1991, 1992; Moseley et al., 1996), procainamide ethobromide (PAEB), and its azido analog azidoprocainamide methoiodide (APM) (Mol et al., 1992). Uptake system2 transports more bulky “type II” organic cations, includingd-tubocurarine, metocurine as well as the steroidal muscle relaxants vecuronium (Mol et al., 1988) and rocuronium (ORG 9426) (Steen et al., 1992; Proost et al., 1997). While the rat liver organic cation uptake system1 can be inhibited by type II organic cations, it is not sensitive to cardiac glycosides and taurocholate (Steen et al., 1992; Oude Elferink et al., 1995). In contrast, the rat liver organic cation uptake system2 is insensitive to type I substrates, but it is inhibited by cardiac glycosides and taurocholate (Steen et al., 1992;Oude Elferink et al., 1995). Hence, uptake system2 seems to represent a multispecific organic solute uptake system that recognizes bulky amphipathic compounds independent of their charge (Groothuis and Meijer, 1996).
Rat hepatocytes express the organic cation transporter 1 (rOCT1;gene symbol Slc22a1) (Grundemann et al., 1994; Meyer-Wentrup et al., 1998) and the organic anion-transporting polypeptide 2 (Oatp2;Slc21a5) (Reichel et al., 1999) at their blood-faced basolateral (sinusoidal) plasma membrane domain. rOCT1 mediates charge-selective transport of relatively water-soluble small organic cations such as TEA, 1-methyl-4-phenylpyridinium, and choline (Koepsell, 1998) and, thus, could qualify for the organic cation uptake system1 in rat liver. Oatp2 exhibits a wide substrate preference and can mediate uptake of anionic bile salts, uncharged cardiac glycosides (e.g., digoxin) and type II organic cations such asN-(4,4-azo-n-pentyl)-21-deoxyajmalinium (APDA) and rocuronium (Reichel et al., 1999; van Montfoort et al., 1999), indicating that Oatp2 might represent the multispecific organic cation uptake system2 in rat liver (van Montfoort et al., 1999).
The goals of the current study were 2-fold: 1) to definitely investigate the suggested correspondence of the rat liver organic cation uptake systems1 and 2 with rOCT1 and Oatp2, respectively, using the same model compounds as originally used to establish the type I and II classification of organic cations (Steen et al., 1992); and 2) to compare the organic cation transport specificities of the rOCT1 and Oatp2 with the human transporters hOCT1 (SLC22A1) and OATP-A (previously called OATP; SLC21A3), the latter having the broadest transport activity for type II organic cations of all Oatps/OATPs so far characterized (van Montfoort et al., 1999;Kullak-Ublick et al., 2001). The results support the hypothesis that rOCT1 corresponds to the organic cation uptake system1 and Oatp2 to the organic cation uptake system2 in rat liver. Some cationic drugs such as APDA can be transported both by Oatp2 and rOCT1, indicating at least some overlapping substrate specificity between the two families of membrane transporters. The data also demonstrate differences in the substrate preferences between rat (rOCT1; Oatp2) and human (hOCT1; OATP-A) carriers, indicating that the type I and II classification cannot be extended without modification from rat to human.
Experimental Procedures
Materials.
[14C]Tetraethylammonium (TEA; 5 mCi/mmol) was obtained from PerkinElmer Life Science Products (Boston, MA). [3H]Quinidine (QD; 20 Ci/mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO). [14C]Rocuronium (54 mCi/mmol) and unlabeled rocuronium were kind gifts of Organon International BV (Oss, The Netherlands).N-(4,4-Azo-n-pentyl)-21-deoxy[21-3H]ajmalinium (APD-ajmalinium, APDA; 1.2 Ci/mmol),N-(4,4-azo-n-pentyl)-quinuclidine (APQ; 2.5 Ci/mmol), and unlabeled APQ were synthesized as described (Müller et al., 1994). [3H]Tributylmethylammonium (TBuMA; 85 Ci/mmol) was synthesized according to Neef et al. (1984). Unlabeled TBuMA was obtained from Fluka (Buchs, Switzerland). [3H]N-Methyl-quinine (NMQ, 85 Ci/mmol) and [3H]N-methyl-quinidine (NMQD; 85 Ci/mmol) were synthesized and characterized as described (van Montfoort et al., 1999). [3H]Azidoprocainamide methoiodide (APM; 85 Ci/mmol) and unlabeled APM were synthesized according to Mol et al. (1992). Radiochemical purity of the not commercially available substrates was determined by thin-layer chromatography and exceeded 99%. All other chemicals were of analytical grade and readily available from commercial sources.
Uptake Studies in Xenopus laevis Oocytes.
rOCT1 cDNA was subcloned into the pRSSP vector (Busch et al., 1996) and linearized with Mlu I. hOCT1 cDNA was subcloned into the pOG1 vector (Gorboulev et al., 1997) and linearized withNotI. cRNA was synthesized with the Ambion mMessage mMachine In Vitro Transcription kit using SP6 RNA polymerase for rOCT1 and T7 RNA polymerase for hOCT1 (Ambion, Austin, TX). In vitro synthesis of Oatp2- and OATP-A-cRNA was performed as described (Kullak Ublick et al., 1995; Noe et al., 1997). X. laevis oocytes were prepared (Hagenbuch et al., 1996) and cultured overnight at 18°C. Healthy oocytes were microinjected with 50 nl of water without and with 10 ng of rOCT1-, 10 ng of hOCT1-, 5 ng of Oatp2-, or 2.5 ng of OATP-A-cRNA and cultured for 3 days in a medium containing 88 mM NaCl, 2.4 mM NaHCO3, 1 mM KCl, 0.3 mM Ca(NO3)2, 0.41 mM CaCl2, 0.82 mM MgSO4, 0.05 mg/ml gentamycin, and 15 mM HEPES (pH 7.6). Tracer uptake studies were performed in a NaCl-containing uptake medium (100 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES-Tris, pH 7.5 or 10 mM 4-morpholinoethanesulfonic acid-Tris, pH 6.0). The oocytes were washed in the uptake medium and then incubated at 25°C in 100 μl of the uptake medium containing the indicated substrate concentrations. Water-injected oocytes were used as controls for unspecific uptake of the substrate. After the indicated time intervals, uptake was stopped by addition of 6 ml of ice-cold uptake medium. The oocytes were washed twice with 6 ml of ice-cold uptake medium. Subsequently, each oocyte was dissolved in 0.25 ml of 10% SDS and 4 ml of scintillation fluid (Ultima Gold; Canberra Packard, Zürich, Switzerland) and the oocyte-associated radioactivity determined in a Tri-Carb 2200 CA liquid scintillation analyzer (Canberra Packard). Determination of kinetic uptake parameters was performed by a nonlinear curve-fitting program (Systat 8.0; SPSS Inc., Chicago, IL) using a simple Michaelis-Menten model:v = (Vmax · [S])/(Km + [S]).
Statistical Analysis.
Uptake results are given as means ± S.D. Statistical significance of transport differences between the various oocyte groups was determined by the Student's ttest (Systat 8.0; SPSS Inc.).
Results
To identify rOCT1 as the type I organic cation uptake system (system1) and Oatp2 as the type II organic cation uptake system (system2) of rat liver, we first performed a series ofcis-inhibition studies in rOCT1- and Oatp2-expressingX. laevis oocytes using TBuMA as a type I and rocuronium as a type II substrate. As illustrated in Fig.1A, rOCT1-mediated TBuMA uptake was inhibited by APM (a type I compound) and rocuronium (a type II compound), but not by taurocholate, ouabain, and K-strophantoside. Thiscis-inhibition pattern is characteristic for the rat liver organic cation uptake system1 (Steen et al., 1992), which therefore corresponds to rOCT1. The latter was also inhibited by QD as previously described (Koepsell et al., 1999) as well as by its methylated derivative NMQD (Fig. 1A). In contrast, Oatp2-mediated rocuronium uptake was insensitive to the type I compounds TBuMA and APM, but was strongly inhibited by taurocholate, ouabain, and K-strophantoside (Fig.1B). This cis-inhibition pattern is characteristic for the rat liver organic cation uptake system2 (Steen et al., 1992), which therefore corresponds to Oatp2. No significant inhibition of Oatp2-mediated rocuronium uptake was found for QD and NMQD (Fig. 1B), which is consistent with the previous observation that NMQD is not transported by Oatp2 (van Montfoort et al., 1999).
Next, we evaluated whether the characteristic cis-inhibition pattern of rOCT1 and Oatp2 is also valid for the human transporters hOCT1 and OATP-A. As illustrated in Fig.2A, hOCT1-mediated TBuMA uptake showed qualitatively a similar cis-inhibition pattern as rOCT1-mediated TBuMA uptake (Fig. 1A), indicating that hOCT1 and rOCT1 represent orthologous gene products with similar type I organic cation transport properties in rat and human liver. In contrast, OATP-A-mediated rocuronium uptake was inhibited by APM, taurocholate, K-strophantoside, QD, and NMQD, but not by TBuMA and ouabain (Fig. 2B). This cis-inhibition pattern is clearly different compared with Oatp2 (Fig. 1B). While part of these differences can be explained by distinct transport activities [e.g., NMQD is a transport substrate of OATP-A, but not of Oatp2 (van Montfoort et al., 1999)] and/or substrate affinities [e.g., Km values for ouabain transport: OATP-A, ∼5.5 mM; Oatp2, ∼470 μM (Bossuyt et al., 1996; Noe et al., 1997)] of OATP-A and Oatp2, the inhibitory effect of the type I organic cation APM on OATP-A-mediated rocuronium uptake is surprising since APM has been shown to be not transported by OATP-A (van Montfoort et al., 1999). Hence, the data indicate that the rat-based classification of type I and type II organic cations and its association with distinct organic cation carriers cannot be applied to the human situation without modification.
Based on their physicochemical properties as permanently charged bulky quaternary ammonium compounds it has been suggested that NMQ and NMQD represent new type II organic cations (van Montfoort et al., 1999). However, the overall structure of these compounds shows a spatial separation of the single cationic group (surrounded by aliphatic moieties as in TBuMA and PAEB) from an aromatic structure as has been considered as being typical for type I compounds (Meijer et al., 1990;Groothuis and Meijer, 1996). Even APDA exhibits these structural features to some extent. The latter consideration is supported by the findings that NMQD inhibits rOCT1- and hOCT1-mediated TBuMA uptake rather than Oatp2-mediated rocuronium uptake (Figs. 1 and 2). Therefore, we tested next organic cation transport in rOCT1- and hOCT1-expressing oocytes. As illustrated in Fig.3, and consistent with thecis-inhibition data, rOCT1 and hOCT1 mediated not only transport of the type I compounds TBuMA, APM, and APQ but also of the previously supposed type II substrates NMQ, NMQD, and also but to a lesser extent, ADPA. The only type II organic cation not significantly transported by rOCT1 and hOCT1 was rocuronium. Time course experiments showed linear uptake rates for NMQ, NMQD, TBuMA, and APM for at least 30 min (data not shown). Therefore, kinetic uptake measurements were performed at 15 min. They showed saturation kinetics as indicated in Fig. 4 for hOCT1. Similar kinetic features were also obtained for rOCT1 with all apparentKm values given in Table1. These data confirm the affinity of rOCT1 and hOCT1 for the established type I organic cations TBuMA, APM, and APQ. However, since rOCT1 and hOCT1 also transport the rather bulky organic cations NMQ, NMQD, and APDA, the data indicate that this physicochemical property alone should not be used to predict the types of carrier involved in hepatic organic cation uptake. In this study, only rocuronium showed entirely the expected behavior as a type II organic cation, i.e., exclusive and taurocholate/cardiac glycoside inhibitable transport by Oatp2 and OATP-A, respectively. Other examples of type II compounds are d-tubocurarine, metocurine, hexafluronium, and vecuronium. A general feature of these agents is that 1) the cationic groups are not clearly separated from the aromatic moieties or other bulky ring structures, and 2) that they contain a second quaternary or tertiary amine structure and consequently can form bivalent cationic molecules.
The quaternary ammonium compounds used in this study are permanently positively charged model compounds and with the exception of rocuronium not used as drugs. Therefore, we also investigated transport of the drug quinidine, which is a tertiary amine and shown to inhibit rOCT1, hOCT1, and OATP-A (Figs. 1 and 2). Quinidine is a base with a pKa of 8.5 (Notterman et al., 1986), which means that at pH 7.5 about 10% of the quinidine molecules are protonated, while at pH 6.0 almost all molecules are positively charged. It can be seen in Fig. 5 that there is no significant quinidine transport by OATP-A-, rOCT1-, or hOCT1-cRNA-injected X. laevis oocytes at pH 7.5. At pH 6.0, however, transport of quinidine becomes detectable since the unspecific uptake into water injected oocytes is markedly reduced. These findings suggest that quinidine molecules need a positive charge to be transported by OATP-A, rOCT1, and hOCT1. In the unprotonated form, quinidine most probably enters the oocytes by passive diffusion as can be seen by the large unspecific uptake into water-injected oocytes at pH 7.5 (Fig. 5).
Discussion
The present study identifies rOCT1 as the type I organic cation uptake system (system1) and Oatp2 as the type 2 organic cation uptake system (system2) in rat liver. This conclusion is based on the typicalcis-inhibition of rOCT1-mediated TBuMA uptake by type I and type II cations, but not by taurocholate and cardiac glycosides (Fig.1A) (Steen et al., 1992), and of Oatp2-mediated rocuronium uptake by taurocholate and cardiac glycosides, but not by type I cations (Fig.1B) (Steen et al., 1992). While the transport properties of hOCT1 were similar to rOCT1 (Fig. 2A), the cis-inhibition patterns of Oatp2- and OATP-A-mediated rocuronium uptake were different in part in that OATP-A, but not Oatp2, was inhibited by the type I organic cation APM, QD, and NMQD (Fig. 2B). Thus, the clear-cut associations between transport of type I and type II organic cations by rOCT1 and Oatp2, respectively, is not entirely valid for hOCT1 and OATP-A, which supports the concept that the human OATP-A is not the orthologous gene product of the rat Oatp2 (Kullak-Ublick et al., 2001). Furthermore, studies in isolated human hepatocytes showed that rocuronium uptake could be inhibited by K-strophantoside and by the type I organic cation PAEB, but not by taurocholate (Olinga et al., 1998). These findings are an additional indication for species differences between organic cation uptake systems of rat and human liver. Since none of the so-far-identified human hepatic OATPs [i.e., OATP-B (SLC21A9), OATP-C (SLC21A6), and OATP8 (SLC2A8)] was able to transport organic cations, a human ortholog of rat Oatp2 remains to be identified (Kullak-Ublick et al., 2001). It might also be possible that the transport functions of rat and human OATPs have evolved differently since in contrast to rat Oatp2 no human OATP can transport both rocuronium and digoxin. In humans, rocuronium is transported by OATP-A (van Montfoort et al., 1999), while digoxin is transported by OATP8 (Kullak-Ublick et al., 2001).
The concept of rOCT1 corresponding to uptake system1 and Oatp2 to uptake system2 is valid for the same substrates and inhibitors that were used originally in rat hepatocytes (Steen et al., 1992). Other substrates have to be classified with caution. NMQ, NMQD, and APDA could, at first sight, be viewed upon as type II organic cations because of their bulky structure (van Montfoort et al., 1999). According to the proposed concept their uptake into rat liver is supposed to be mediated by Oatp2 rather than by rOCT1. However, while Oatp2 solely mediates the transport of the real type II organic cation rocuronium, APDA is only to some extent accommodated by OATP-A and Oatp2, respectively, whereas NMQ and NMQD even seem pure rOCT1-mediated type I compounds (van Montfoort et al., 1999). Therefore, a positive charge and a bulky structure alone are not sufficient to predict the putative uptake system. As NMQ and NMQD are mainly taken up by rOCT1, which corresponds to uptake system1, they should be reclassified as type I organic cations despite their bulky structure. It is possible that rOCT1 and hOCT1 recognize a single cationic group spatially separated from a flat (aromatic) ring structure that can be recognized in APDA, NMQ, NMQD, and PAEB. The type I agents TBuMA and APQ also have a single cationic group but lack an aromatic moiety. The only true type II substrate used in this study that is consistently transported only by Oatps is the steroidal muscle relaxant rocuronium. It shares a potential dicationic nature with previously categorized type II compounds (Meijer et al., 1990) in combination with a sufficient lipophilicity. The latter aspect and the presence of a permanently charged ammonium group as well as a second tertiary amine function may render APDA a mixed type I/type II substrate.
While there were considerable differences in the substrate specificity of Oatp2 and OATP-A in this study, rOCT1 and hOCT1 showed qualitatively similar substrate specificities and inhibition patterns (Figs. 1-3), although some quantitative differences were observed in the inhibition of TBuMA uptake by APM (rOCT1 > hOCT1) and NMQD (rOCT1 < hOCT1). In addition, the apparent Kmvalues for NMQ, NMQD, TBuMA, and APM were comparable (Table 1). hOCT1 showed slightly higher Km values, which is in agreement with previous findings where hOCT1 exhibited a lower affinity than rOCT1 for several other ligands (Koepsell et al., 1999). Furthermore, it has been shown that hOCT1 transports largern-tetraalkylammonium compounds such as tetrapropylammonium and tetrabutylammonium at higher rates than rOCT1 (Dresser et al., 2000). The kinetics of TBuMA and APM has also been determined in isolated rat hepatocytes (Steen et al., 1991; Mol et al., 1992) where for both substrates the presence of high- and low-affinity uptake systems has been suggested with apparentKm values of ∼1.2 and ∼107 μM for TBuMA (Steen et al., 1991) and of ∼3 and ∼100 μM for APM, respectively (Mol et al., 1992). In the present study, rOCT1 showed apparent Km values of 34 μM for TBuMA and of 54 μM for APM. If these values, as obtained in the oocyte system, can be extrapolated to the hepatocyte, they could correspond to the low-affinity system found in isolated rat hepatocytes. In that case, candidate carriers for the supposed high-affinity systems should be identified in the future.
rOCT1, hOCT1, and OATP-A are not only inhibited by the permanently positively charged NMQD but also by the base quinidine (Figs. 1 and 2). However, at pH 7.5 where only about 10% of the quinidine molecules are protonated no carrier-mediated transport of quinidine could be detected (Fig. 5). In contrast, at pH 6.0, where almost all quinidine molecules are positively charged, unspecific diffusion into the oocytes was markedly reduced and uptake of the protonated quinidine by rOCT1, hOCT1, and OATP-A became evident (Fig. 5). This finding is an indication that quinidine is only transported by rOCT1, hOCT1, and OATP-A, when it carries a positive charge. The mechanism of quinidine inhibition of rOCT1, hOCT1, and OATP-A at pH 7.5 is not clear. It has been shown that quinine, which is a diastereomer of quinidine, is a noncompetitive inhibitor of rOCT1 when added at the extracellular site, suggesting an allosteric binding site (Koepsell, 1998). Recent experiments with macropatches of rOCT2 cRNA-injected X. laevis oocytes in which quinine could also be added from the cytoplasmic site showed competitive inhibition, suggesting that quinine inhibits rOCT2 from the intracellular site after crossing the lipid bilayer in its uncharged form (Budimann et al., 2000).
In conclusion, the present study demonstrates that rOCT1 corresponds to the predicted uptake system1 for type I organic cations in rat liver, whereas Oatp2 corresponds to uptake system2 for type II organic cations. However, the results cannot be transferred to human liver without modifications. While the transport properties of rOCT1 and hOCT1 are similar, considerable differences exist between Oatp2 and OATP-A, indicating further that rat Oatps and human OATPs have different transport properties and do not represent orthologous gene products.
Footnotes
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This work was supported by the Swiss National Science Foundation (Grant 3100-045536.95 to P.J.M.). J.E.v.M. was supported by an Ubbo Emmius scholarship of the University of Groningen. A preliminary report of this study was presented at the 35th Annual Meeting of the European Association for the Study of the Liver (EASL) in Rotterdam, April 29–May 3, 2000, and published in abstract form [JHepatol32 (Suppl 2):118].
- Abbreviations:
- TEA
- tetraethylammonium
- TBuMA
- tributylmethylammonium
- PAEB
- procainamide ethobromide
- APM
- azidoprocainamide methoiodide
- rOCT1
- rat organic cation transporter 1
- Oatp2
- rat organic anion-transporting polypeptide 2
- APDA
- N-(4,4-azo-n-pentyl)-21-deoxyajmalinium
- hOCT1
- human organic cation transporter 1
- OATP-A
- human organic anion-transporting polypeptide A
- QD
- quinidine
- APQ
- N-(4,4-azo-n-pentyl)-quinuclidine
- NMQ
- N-methyl-quinine
- NMQD
- N-methyl-quinidine
- Received January 16, 2001.
- Accepted March 16, 2001.
- The American Society for Pharmacology and Experimental Therapeutics