0022-3565/01/2981-110 –115$3.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics
JPET 298:110–115, 2001
Vol. 298, No. 1
3765/911050
Printed in U.S.A.
Comparison of “Type I” and “Type II” Organic Cation Transport
by Organic Cation Transporters and Organic AnionTransporting Polypeptides
JESSICA E. VAN MONTFOORT, MICHAEL MÜLLER, GENY M. M. GROOTHUIS, DIRK K. F. MEIJER,
HERMANN KOEPSELL, and PETER J. MEIER
Division of Clinical Pharmacology and Toxicology, Department of Medicine, University Hospital, Zürich, Switzerland (J.E.v.M., P.J.M.);
Department of Pharmacokinetics and Drug Delivery, Groningen University Institute for Drug Exploration, Groningen, The Netherlands (J.E.v.M.,
G.M.M.G., D.K.F.M.); Sub-department of Human Nutrition and Epidemiology, Nutrition, Metabolism, and Genomics Group, Wageningen
University and Research Center, Wageningen, The Netherlands (M.M.); and Department of Anatomy, University of Würzburg, Würzburg,
Germany (H.K.)
This paper is available online at http://jpet.aspetjournals.org
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-injected Xenopus laevis oocytes.
Based on characteristic cis-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,
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;
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 [J Hepatol 32 (Suppl 2):118].
OATP-A- but not Oatp2-mediated rocuronium uptake was inhibited by the OATP-A substrate N-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.
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, including d-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 tauro-
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)-21deoxyajmalinium; 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.
110
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Received January 16, 2001; accepted March 16, 2001
Hepatic Uptake of “Type I” and “Type II” Organic Cations
Experimental Procedures
14
Materials. [ C]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)-21deoxy[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 pu-
rity 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 with NotI. 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 acidTris, pH 6.0). The oocytes were washed in the uptake medium and
then incubated at 25°C in 100 ␮l 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 䡠 [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 t test (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 of cis-inhibition studies in rOCT1- and Oatp2-expressing X. laevis oocytes using TBuMA as a type I and rocuronium as a type II substrate. As illustrated in Fig. 1A, rOCT1mediated TBuMA uptake was inhibited by APM (a type I
compound) and rocuronium (a type II compound), but not by
taurocholate, ouabain, and K-strophantoside. This cis-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, Oatp2mediated 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 Oatp2mediated rocuronium uptake was found for QD and NMQD
(Fig. 1B), which is consistent with the previous observation
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cholate (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 as N-(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.
111
112
van Montfoort et al.
Fig. 2. cis-Inhibition pattern of TBuMA and rocuronium uptake mediated
by hOCT1 (A) and OATP-A (B), respectively. X. laevis oocytes were
injected with 10 ng of hOCT1 or 2.5 ng of OATP-A cRNA. After 3 days in
culture, uptakes of [3H]TBuMA (35 ␮M) by hOCT1 (A) and of [14C]rocuronium (25 ␮M) by OATP-A (B) were measured at 30 min (under Experimental Procedures) in the absence (control) and presence of the indicated
compounds (all 200 ␮M, except ouabain 2 mM). Uptake rates of waterinjected oocytes were subtracted from cRNA-injected oocytes for each
substrate. Data are expressed as means ⫾ S.D. of uptake measurements
into 10 oocytes. ⴱ, significant uptake inhibition (Student’s t test, p ⬍ 0.01)
compared with controls.
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 ␮M (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 the cis-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,
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Fig. 1. cis-Inhibition pattern of TBuMA and rocuronium uptake mediated
by rOCT1 (A) and Oatp2 (B), respectively. X. laevis oocytes were injected
with 10 ng of rOCT1 or 5 ng of Oatp2 cRNA. After 3 days in culture,
uptakes of [3H]TBuMA (35 ␮M) by rOCT1 (A) and of [14C]rocuronium (25
␮M) by Oatp2 (B) were measured at 30 min (under Experimental Procedures) in the absence (control) and presence of the indicated compounds
(all 200 ␮M, except ouabain 2 mM). Uptake rates of water-injected
oocytes were subtracted from cRNA-injected oocytes for each substrate.
Data are expressed as means ⫾ S.D. of uptake measurements into 10
oocytes. ⴱ, significant uptake inhibition (Student’s t test, p ⬍ 0.01) compared with controls.
Hepatic Uptake of “Type I” and “Type II” Organic Cations
113
Discussion
Fig. 3. Comparison of rOCT1- and hOCT1-mediated uptake of [3H]NMQ,
[3H]NMQD, [3H]APDA, [14C]rocuronium, [3H]TBuMA, [3H]APM, and
[3H]APQ. X. laevis oocytes were injected with 10 ng of rOCT1 (A) or 10 ng
of hOCT1-cRNA (B). After 3 days in culture, uptakes of the indicated
substrates were measured at 2 ␮M (except rocuronium at 14 ␮M) for 30
min in Na⫹ buffer (under Experimental Procedures). The standard substrate TEA was used as positive controls to test the expression of rOCT1
and hOCT1 in the cRNA-injected oocytes. The uptake rates of the waterinjected control oocytes were subtracted from the cRNA-injected oocytes
for each substrate. Data represent the mean ⫾ S.D. from 11 to 15 oocyte
uptake measurements. ⴱ, significantly different from water-injected control oocytes (Student’s t test, p ⬍ 0.001).
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 apparent Km values given in Table 1. 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
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 typical cis-inhibition of rOCT1mediated 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
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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).
114
van Montfoort et al.
TABLE 1
Comparison of apparent Km values between OATP-A, rOCT1, and
hOCT1
X. laevis oocytes were injected with 2.5 ng of OATP-A-cRNA, 10 ng of rOCT1-cRNA,
and 10 ng of hOCT1-cRNA. After 3 days in culture, uptakes of the indicated substrates were measured at 15 min in Na⫹ buffer under Experimental Procedures
containing increasing substrate concentrations. Data are expressed as mean ⫾ S.D.
of 10 to 12 oocyte uptake measurements.
Apparent Km Values
OATP-A
hOCT1
5.1 ⫾ 2.1a
25.6 ⫾ 4.1a
nt
nt
19.5 ⫾ 7.3
11.5 ⫾ 2.1
53.0 ⫾ 13.9
100.9 ⫾ 43.0
rOCT1
␮M
NMQ
NMQD
TBuMA
APM
17.3 ⫾ 4.9
7.4 ⫾ 2.1
34.0 ⫾ 7.7
54.0 ⫾ 15.2
nt, not transported.
a
Taken from van Montfoort et al., 1999.
(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
Fig. 5. pH dependence of OATP-A-, rOCT1-, and hOCT1-mediated uptake of [3H]quinidine. X. laevis oocytes were injected with 2.5 ng of
OATP-A, 10 ng of rOCT1, or 10 ng of hOCT1-cRNA. After 3 days in
culture, uptake of the quinidine was measured at 2 ␮M for 30 min in Na⫹
buffer at pH 7.5 (o) or pH 6.0 (f) (under Experimental Procedures).
NMQD was used as positive control to test the expression of OATP-A,
rOCT1, and hOCT1 in the cRNA-injected oocytes. Data represent the
mean ⫾ S.D. from 10 oocyte uptake measurements. ⴱ, significantly different from water-injected control oocytes (Student’s t test, p ⬍ 0.001).
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 quantita-
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Fig. 4. Saturation kinetics of
NMQ, NMQD, TBuMA, and
APM in hOCT1-cRNA-injected
X. laevis oocytes. X. laevis oocytes were injected with 10 ng of
hOCT1-cRNA. After 3 days in
culture, uptakes of [3H]NMQ
(A), [3H]NMQD (B), [3H]TBuMA
(C), and [3H]APM (D) were measured at 15 min in Na⫹ buffer
(under Experimental Procedures)
containing increasing substrate
concentrations. The dotted lines
represent fitted uptake differences between hOCT1-cRNA (F)
and water-injected oocytes (E).
Data are expressed as mean ⫾
S.D. of 10 to 12 oocyte uptake
measurements.
Hepatic Uptake of “Type I” and “Type II” Organic Cations
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Address correspondence to: Prof. Dr. Peter J. Meier-Abt, Division of Clinical Pharmacology and Toxicology, Department of Medicine, University Hospital, CH-8091 Zürich, Switzerland. E-mail: [email protected]
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tive differences were observed in the inhibition of TBuMA
uptake by APM (rOCT1 ⬎ hOCT1) and NMQD (rOCT1 ⬍
hOCT1). In addition, the apparent Km values 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 larger n-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 apparent Km values of ⬃1.2
and ⬃107 ␮M for TBuMA (Steen et al., 1991) and of ⬃3 and
⬃100 ␮M for APM, respectively (Mol et al., 1992). In the
present study, rOCT1 showed apparent Km values of 34 ␮M
for TBuMA and of 54 ␮M 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.
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