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Molecular and Cellular Biology, July 2001, p. 4188-4196, Vol. 21, No. 13
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.13.4188-4196.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Impaired Activity of the Extraneuronal Monoamine
Transporter System Known as Uptake-2 in
Orct3/Slc22a3-Deficient Mice
Ronald
Zwart,1
Sandra
Verhaagh,1
Marije
Buitelaar,1
Corrie
Popp-Snijders,2 and
Denise P.
Barlow1,*
Department of Molecular Genetics (H5), The
Netherlands Cancer Institute, 1066 CX
Amsterdam,1 and Department of
Endocrinology, Free University, 1007 MB
Amsterdam,2 The Netherlands
Received 23 February 2001/Returned for modification 15 March
2001/Accepted 13 April 2001
 |
ABSTRACT |
Two uptake systems that control the extracellular concentrations of
released monoamine neurotransmitters such as noradrenaline and
adrenaline have been described. Uptake-1 is present at presynaptic nerve endings, whereas uptake-2 is extraneuronal and has been identified in myocardium and vascular and nonvascular smooth muscle cells. The gene encoding the uptake-2 transporter has recently been
identified in humans (EMT), rats (OCT3),
and mice (Orct3/Slc22a3). To generate an
in vivo model for uptake-2, we have inactivated the mouse
Orct3 gene. Homozygous mutant mice are viable and
fertile with no obvious physiological defect and also show no
significant imbalance of noradrenaline or dopamine. However,
Orct3-null mice show an impaired uptake-2 activity as
measured by accumulation of intravenously administered
[3H]MPP+ (1-methyl-4-phenylpyridinium). A
72% reduction in MPP+ levels was measured in hearts of
both male and female Orct3 mutant mice. No significant
differences between wild-type and mutant mice were found in any other
adult organ or in plasma. When [3H]MPP+ was
injected into pregnant females, a threefold-reduced MPP+
accumulation was observed in homozygous mutant embryos but not in their
placentas or amniotic fluid. These data show that Orct3 is the principal component for uptake-2 function in the adult heart and
identify the placenta as a novel site of action of uptake-2 that acts
at the fetoplacental interface.
 |
INTRODUCTION |
The catecholamines adrenaline,
noradrenaline, dopamine, and the tryptophan derivative serotonin
function as neurotransmitters of the monoaminergic neurons and as
hormones in the control of physiological processes like glucose storage
and metabolism, thermoregulation, and blood pressure. Changes in
synaptic concentration or temporary availability of monoamines are
associated with mental dysfunction, neuropsychiatric disorders, and
drug addiction (10). Furthermore, altered plasma
concentrations can result in physiological dysfunction (28). Tight control of the levels of synaptic and
circulating catecholamines is thus essential for proper neuronal
signaling and maintenance of internal homeostasis.
Two uptake systems that clear extracellular monoamines have been
described. The neuronal uptake-1 system is present at presynaptic nerve
endings and mediates the reuptake of released monoamines from the
synaptic cleft. Uptake-1 is a high-affinity and
Na+- and Cl
-dependent
system mediated by the noradrenaline (Net), dopamine (Dat), and
serotonin (Sert) transporter proteins (reviewed in reference
1). Targeted inactivation experiments with mice have shown
that the uptake-1 transporter proteins are a target for antidepressant
and psychostimulatory treatments and are pivotal in the control of
synaptic catecholamine concentrations and prevention of neurobehavioral
changes (3, 14, 37).
The extraneuronal uptake-2 system was originally discovered in
myocardial cells of the rat heart but has also been identified in
vascular and nonvascular smooth muscle cells like those in the uterus,
as well as in human central nervous system glial and kidney carcinoma
cell lines (19, 24, 26, 31, 33). Uptake-2 can be
discriminated from uptake-1 in substrate specificity and transport kinetics (reviewed in reference 33). In
addition, corticosteroids,
-haloalkylamines, and O-methylated
catecholamines are inhibitors of uptake-2 but not of uptake-1. The
cyanine derivative disprocynium 24 (D24) was isolated as a highly
potent uptake-2 inhibitor in vitro (23). However, the
application of D24 in vivo to study uptake-2 was revealed to be
limited, as it was shown previously that D24 blocks not only uptake-2
but also other transport mechanisms that clear catecholamines (8,
11).
Recently, molecular identification of the uptake-2 transporter protein
has been reported. Called extraneuronal monoamine transporter (EMT) in
humans and organic cation transporter 3 (OCT3) in rats, the protein is
predicted to contain a 12-transmembrane domain structure (18,
36). The mouse homolog of the EMT and OCT3 genes, called Orct3 (locus name Slc22a3), was
isolated from the critical region of the natural embryonic lethal mouse
mutant tw73 and shown to be tightly
linked to the closely related Orct1/Slc22a1 and
Orct2/Slc22a2 organic cation transporter genes
(35). This physical linkage is conserved in humans and
suggests that these genes have evolved from a common ancestor. Further
evidence for this comes from in vitro studies in which it has been
shown that the Orct1, Orct2, and Orct3 proteins can all transport
catecholamines and the neurotoxin MPP+
(1-methyl-4-phenylpyridinium) (4, 16-18). However,
transport inhibition studies have shown that only Orct3 is sensitive to all uptake-2 antagonists, including O-methylisoprenaline,
with nearly identical kinetics (15, 18). In mice, the
Orct1, Orct2, and Orct3 genes have
clearly distinct expression profiles. Orct1 is expressed in
liver, kidney, and intestine, whereas Orct2 expression is
restricted to the kidney and brain (21, 27). In contrast, Orct3 expression is seen in a wide range of tissues. The
highest levels of expression are found in skeletal muscle and in the
heart and uterus, for which uptake-2 activity has been described
previously (33, 35). Similarly, high expression of the
human homolog is found in aorta, prostate, adrenal gland, skeletal
muscle, and liver (35). During mouse embryonic
development, Orct3 is expressed in the early
postimplantation embryo (38). At later stages, expression is restricted to the labyrinth layer of the placenta, in
which Orct3 is coexpressed with the gene for the
monoamine oxidase A (Maoa) metabolizing enzyme
(34). Thus, both the in vitro studies and the expression
data have provided strong evidence that Orct3 is the
molecular component of the extraneuronal monoamine transport (uptake-2) system.
To test whether Orct3 has a major role in uptake-2 activity
in any particular organ in vivo, we have generated mice deficient for
Orct3 by homologous recombination in embryonic stem (ES)
cells. These mice are viable and fertile and show no obvious
physiological defect and no significant imbalance of two tested
monoamines, noradrenaline and dopamine. However, using
MPP+ as a substrate, we show that
Orct3 is an essential component for uptake-2 function in the
adult heart and placenta but not in other adult organs. These data
establish the presence of uptake-2 in the heart and identify the
placenta as a novel uptake-2 site of action, where it functions at the
fetoplacental interface.
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MATERIALS AND METHODS |
Orct3 gene targeting.
For constructing the
targeting vector, a 17.5-kb BglII fragment spanning the
first exon of the mouse Orct3 gene was isolated from the
genomic BAC clone 228C21 (Research Genetics, Inc.) (35). To generate plasmid p16BglII, a 1.4-kb fragment from the 3'
end of the subclone was removed using Asp718, followed by
religation. In p16BglII, a 4.3-kb BamHI fragment
containing exon 1, and 0.65 kb of upstream and 2.9 kb of downstream
genomic sequences, was replaced by the pGKneo/pGKtk selection cassette
flanked by loxP sites (9). The targeting vector was
linearized with XhoI, and 50 µg was used in an
electroporation of E14 (129/Ola) mouse ES cells. One hundred ninety-two
individual colonies were screened for homologous recombination by
hybridization with an 0.8-kb Asp718-SpeI 3'
external probe of BglII-digested genomic DNA. Six homologous recombinant clones were identified (i.e., a 3% recombination
frequency). The integrity of the homologous recombination event was
verified with a SalI-BglII 5' external probe in
an SpeI digest (data not shown). The subclonal appearance of
the targeted allele is caused by a contamination of the clones with
surviving nontargeted ES cells (Fig. 1B).
Four individually targeted ES cell clones were transiently transfected
with a CRE expression plasmid to remove the neomycin/tk
selection cassette. Individual clones were picked and analyzed for
CRE-mediated recombination. Upon karyotyping, two independently derived
ES cell clones with the Orct3 (positive or negative)
genotype were used for blastocyst injections to generate chimeric mice.
Both clones resulted in germ line transmission and yielded similar
results. The mutant Orct3 allele was bred into an FVB/N
genetic background for analysis and is available for distribution.

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FIG. 1.
Disruption of the mouse Orct3 gene in ES
cells. (A) Orct3 gene targeting strategy. The top line
is a schematic overview of the Orct3 gene locus. Exon 1 is shown as a gray box. The arrow marks the wild-type
Orct3 promoter and the direction of transcription.
Restriction enzyme sites for Asp718 (A),
BamHI (B), BglII (Bg), and
SpeI (Sp) are indicated. In the targeting construct, a
4.3-kb BamHI genomic fragment containing exon 1 was
replaced by the selection cassette (neo) flanked by loxP
sites (arrowheads). Following homologous recombination, the selection
cassette was removed by CRE-mediated recombination, leaving a single
loxP site in place of the 4.3-kb BamHI
fragment containing exon 1. wt, wild type. (B and C) Genotype analysis
of the targeted ES cell clones (B) and the CRE-transfected ES cells
(C). An 800-bp 3' external Asp718-SpeI
fragment was used as a probe on BglII-digested ES cell
genomic DNA, detecting a 17-kb wild-type (+/+), a 9.5-kb homologous
recombined (+/neo), and a 12.5-kb floxed (+/ ) allele.
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Genomic Southern blot analysis.
ES cell genomic DNA was
isolated by proteinase K digestion (100 µg/ml) in 100 mM Tris (pH
8.0)-5 mM EDTA-200 mM NaCl-0.2% sodium dodecyl sulfate. Five
micrograms of DNA was used for digestion and run on a 0.6% agarose
gel. Upon transfer of the DNA to a Hybond-N+
nylon membrane (Amersham), hybridization was performed under Church
hybridization conditions (6).
Northern blot analysis.
Total RNA of mouse tissues was
isolated by the lithium chloride extraction method (2).
Analysis of the RNAs by Northern blotting was done as described
previously (35). The Orct3 probe was a 2.7-kb
fragment from the 3' end of the Orct3 cDNA; mouse Pai-1 and human glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) cDNAs were used as probes for loading control.
MPP+ transport studies.
The mice used for the
time curve were 7-week-old wild-type FVB/N female mice. Four mice were
injected for the 5- and 10-min points, and three mice were injected for
the 15- and 60-min points. For the adult transport experiment,
homozygous mutant Orct3 or wild-type littermates of a mixed
genetic background (25% 129/Ola, 75% FVB/N) were injected at
the age of 12 weeks. Seven mice of each genotype were used. Timed
matings were performed for the placental transport studies. Three
pregnant mothers were injected at 15.5 days postcoitum (dpc) of
gestation, resulting in a total of eight wild-type and six mutant
embryos. [3H]MPP+
(methyl-4-phenylpyridinium acetate,
N-methyl-3H labeled, 77.5 Ci/mmol) was obtained from NEN Life Science Products, Inc., and
was diluted 1:150 with cold MPP+ iodide (Research
Biochemicals International) to a final concentration of 0.2 mg/ml in
0.9% NaCl. After anesthesia with methoxyflurane (Medical
Developments), animals were injected intravenously in the tail vein
with 1.0 mg of the drug per kg of body weight. The mice were killed at
the indicated time points by axillary bleeding after renewed
anesthesia. Plasma and organs were collected, weighed, and frozen till
further processing. Intestinal contents were separated from intestinal
tissues. For the placental transport studies, placentas, embryos, and
amniotic fluid as well as maternal blood were collected. Tissues were
homogenized in 4% bovine serum albumin, and concentrations of
[3H]MPP+-derived
radioactivity were measured by liquid scintillation counting.
Statistical analysis.
All values are given as means ± standard deviations (SD). The two-tailed unpaired Student t
test was used to assess the significance of differences between data
sets. Differences were considered to be statistically significant when
P was <0.01.
Orct3 antiserum.
Three polyclonal antibodies were raised in
rabbits: (i) one against a C-terminal peptide at amino acids 524 to
542, synthesized onto a lysine tree; (ii) one against a fusion protein
consisting of the large extracellular loop between TM1 and TM2 fused to
maltose binding protein; and (iii) one against a similar fusion
protein, which contained the large intracellular loop between TM6 and
TM7. These regions are the least conserved among the Orct1,
Orct2, and Orct3 family members. Only the first antigen
gave an antiserum which recognized the Orct3 protein in lysates from
cells transfected with the wild-type Orct3 gene in a Western
blot assay. However, this antiserum was unable to detect Orct3 in
placental or adult organ lysates. The Orct3 gene is heavily
glycosylated in vivo, and Western blotting produces bands only if
transfected cells are treated with tunicamycin (5 µg/ml).
HPLC analysis of noradrenaline and dopamine.
E12.5 embryos
and placentas with a wild-type genotype and an Orct3-null
genotype were obtained from heterozygous matings. The samples were
prepared as described previously (32). Noradrenaline and
dopamine concentrations were measured by high-performance liquid
chromatography (HPLC) (Gynkotec; Separations) with
electrochemical detection (Antec Leyden) using
3,4-dihydroxybenzylamine as an internal standard. Protein
concentrations were determined as described previously
(32), and noradrenaline and dopamine concentrations were
expressed per milligram of protein.
 |
RESULTS |
Orct3 gene targeting.
To inactivate the
Orct3 gene, a two-step targeting approach was used. First, a
4.3-kb genomic BamHI fragment that contains the complete
first exon of the Orct3 gene and 650 bp of upstream sequences was replaced with a pGKneo/pGKtk selection cassette flanked
by loxP sites (Fig. 1A). Successful homologous recombination was
monitored by Southern blotting with a 3' external probe, detecting a
17-kb wild-type (+/+) BglII fragment and a 9.5-kb fragment
of the recombined allele (+/neo [Fig. 1B]). Subsequently, four
independently targeted clones were transiently transfected with a
CRE-recombinase expression vector to remove the pGKneo/pGKtk selection
cassette. CRE-mediated recombination resulted in an Orct3
knockout allele (+/
) that could be identified as a 12-kb
BglII fragment (Fig. 1C). Thus, the resulting
Orct3 knockout allele lacked exon 1 and contained a single
loxP site replacing a 4.3-kb genomic BamHI fragment (Fig. 1A).
Two independently targeted and CRE-recombined ES cell clones were used
for blastocyst injections to generate chimeric mice,
and both ES clones
passed through the germ
line.
The Orct3 targeted allele is a complete null.
It was
anticipated that removal of the first exon and 650 bp of upstream
sequences would result in the complete absence of Orct3 gene
expression. To test this, RNA was isolated from various organs of adult
wild-type and Orct3 homozygous knockout mice and analyzed by
Northern blotting (Fig. 2A). Wild-type
mice show high expression in heart and skeletal muscle. No
Orct3 expression was detected in these tissues of homozygous
mutant mice. Also, Orct3 expression was absent in brain
tissue, which shows low levels of expression in wild-type mice. In
placentas of homozygous mutant embryos, no Orct3 expression
was detected at 11.5 days of gestation (Fig. 2B). At later time points,
however, expression of an aberrant Orct3 transcript was
observed. Figure 2B shows expression of this aberrant transcript, which
is approximately 700 bp shorter than the wild-type transcript, in 12.5- to 17.5-dpc placentas. Reverse transcription-PCR with different
sets of intron-spanning Orct3 oligonucleotide primers
confirmed the presence of an Orct3 transcript in the
homozygous mutant placentas and showed that exons 3 to 11 of the
Orct3 gene were contained within this mRNA (data not shown
and map in Fig. 3A). A 5'
rapid-amplification-of-cDNA-ends experiment was performed with
Orct3-specific oligonucleotides on homozygous mutant
placenta RNA. A unique 158-bp sequence that is not present in the
wild-type Orct3 transcript was identified. This sequence was
mapped to intron 2 of the Orct3 gene and contains a
consensus splice donor sequence by which it splices to exon 3 of the
Orct3 gene (data not shown). The 158-bp sequence does not
reintroduce an ATG translation start codon, which was removed with exon
1, into the mutant transcript. The first in-frame ATG codon is within
the fourth transmembrane domain, and if it were used, the predicted
protein would contain only 8 of the 12 transmembrane domains of the
wild-type protein. Transient overexpression of the mutant RNA in
transfected cell lines confirmed that the short transcript is incapable
of generating a protein product (Fig. 3). These results show that the
targeting strategy resulted in the complete absence of Orct3
gene expression in adult mice and in early placentas, whereas at later
stages in embryonic development an aberrant, noncoding Orct3
mRNA was expressed in the placenta.

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FIG. 2.
Gene expression analysis in Orct3 mutant
mice. (A) Northern blot analysis of Orct3 expression in
different organs of wild-type and homozygous mutant adult mice. GAPDH
was used as a loading control. (B) Orct3 expression in
wild-type and homozygous mutant mouse placentas at different stages of
development. The 3.5-kb wild-type and 2.8-kb aberrant transcripts are
indicated. Pai-1 hybridization was used as a loading
control.
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FIG. 3.
The knockout (KO)-specific 2.8-kb transcript is not
translated. (A) Exon map of the knockout-specific RNA 2.8-kb transcript
and the wild-type (wt) mRNA. The 2.8-kb transcript lacks exons 1 and 2 but contains a novel exon (gray box labeled 1') from intron 2 spliced onto exons 3 to 11 (there are 11 exons in wild-type
Orct3). The first in-frame ATG codon is within the
fourth transmembrane domain, and if it were used, the predicted protein
would contain only 8 of the 12 transmembrane domains of the wild-type
protein. The position of the 4.3-kb deleted sequence that spans the
Orct3 promoter is indicated by the dotted line. (B) RNase protection
assay (RPA) of transiently transfected human embryonic kidney 293 cells. Four constructs were used: Orct3, wild-type
Orct3/Slc22a3 cDNA; Orct3myc, wild-type
Orct3 cDNA with a Myc tag inserted into a
HindIII site located six codons before the translation
stop; Orct3KO, the knockout-specific cDNA; and Orct3KOmyc, the
knockout-specific cDNA containing a Myc tag inserted into a
HindIII site located six codons before the translation
stop. All constructs were driven by a cytomegalovirus promoter and
enhancer and generated abundant RNA. 13.5 dpc, wild-type placental RNA
that serves as a control for Orct3 production; nt,
nontransfected control cells. (C) Western blotting using antiserum
raised to an Orct3 peptide as described in Materials and
Methods. Only the wild-type cDNA is translated; the wild-type
Orct3 Myc-tagged protein cannot be recognized by the
Orct3 antiserum, because the Myc tag is inserted in the
epitope recognized by the antiserum. (D) Western blotting of the same
samples using an anti-Myc antiserum; only the wild-type Myc-tagged
protein is recognized.
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Orct3-null mice are viable and show no obvious
phenotype.
Mice heterozygous for the two independent
Orct3 null alleles were bred to establish two independent
Orct3 knockout mouse lines that behaved similarly in all
tests (see Materials and Methods). Mice homozygous for the targeted
allele appeared in normal Mendelian ratios (data not shown), indicating
that Orct3 is dispensable for embryonic development.
Orct3-null mice appear normal in stature and have a normal
life span (the oldest mice in the colony are now 15 to 16 months). Both
male and female Orct3-null mice are fertile with normal
breeding behavior. In addition, female mice show normal maternal
nurturing behavior and have reared the same sizes of litters as did
wild-type mice. Finally, the Orct3-null mice show no
abnormal behavior under routine housing and handling, indicating a
degree of tolerance to normal stress. Histological examination of
Orct3
/
placentas,
which are the highest Orct3-expressing organs at any stage,
and of the heart, which is the highest Orct3-expressing organ in adult mice, similarly revealed no cellular or structural alterations (data not shown). The heart in Orct3-null mice
was of a normal size range, color, and appearance. In addition, the weight of the heart in Orct3-null mice was unchanged from
that of wild-type mice (Table 1).
Impaired uptake-2 activity in adult Orct3-deficient
mice.
To study uptake-2 in Orct3-deficient mice, we
designed a protocol measuring uptake-2-mediated accumulation of the
neurotoxin MPP+ after intravenous injection.
MPP+ has been described elsewhere as a good
uptake-2 substrate and is not subject to metabolism in vivo, in
contrast to the monoamines (22, 25). Monoamines are
converted into antagonistic metabolites for uptake-2, which complicates
their use as substrates in the analysis of uptake-2 activity in vivo
(33). To measure a maximal effect in primary uptake, we
first determined the temporal curve of
[3H]MPP+ accumulation.
Wild-type FVB/N female mice were injected intravenously with 1.0-mg/kg
[3H]MPP+, and
MPP+ concentrations in the heart, liver, and
plasma were determined 5, 10, 15, and 60 min after injection (Fig.
4). In both heart and liver, an increase
in MPP+ concentration was seen up to 15 min. At
later time points, the MPP+ levels decreased in
both organs, following the rapid decline in MPP+
plasma concentrations (Fig. 4). Based on these data in subsequent studies, the primary uptake of
[3H]MPP+ was determined
10 min after intravenous injection.

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FIG. 4.
Time curve of MPP+ accumulation in heart and
liver tissues of wild-type female mice. Levels of MPP+ in
the heart ( ) and liver ( ) (plotted on the primary
y axis in nanograms per gram of tissue) are indicated at
different time points after intravenous injection. The MPP+
plasma levels are shown by the dashed line ( ) and plotted on the
secondary y axis in nanograms per milliliter.
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Uptake-2 has been particularly well defined in the heart, which has
high levels of
Orct3 gene expression, whereas the liver
is
one of the few organs that is completely lacking
Orct3 gene
expression in mice. To analyze the consequences of a loss of
Orct3 for uptake-2 activity, MPP
+
concentrations were determined in plasma, heart, liver, and other
organs of wild-type and
Orct3 mutant male and female mice
(Fig.
5). In wild-type female hearts,
MPP
+ concentrations reached 2,415 ± 331 ng/g of tissue, whereas the
levels in
Orct3 mutant hearts
were only 674 ± 137 ng/g (Fig.
5A).
This reduction is nearly
fourfold (72%) with a
P value of <0.0001.
In males, an
identical reduction (72%;
P < 0.0001) in
MPP
+ accumulation was measured in the heart (Fig.
5B). The reduced
uptake in the heart does not reflect differences in
concentrations
of MPP
+ in plasma, as those were
comparable between wild-type and mutant
animals (Fig.
5A and B). In the
liver, no difference was found
in MPP
+
accumulation between wild-type and
Orct3 mutant mice. As
Orct3 is not expressed in the liver, this result indicates
that other
systems in addition to the
Orct3 transporter
mediate MPP
+ uptake and that the activity of
these systems is not affected
by a deletion of
Orct3 (Fig.
5A and B). A total of 12 additional
organs of wild-type and
Orct3 mutant mice were analyzed for a
difference in
MPP
+ uptake (Fig.
5C and D). A similar
distribution of MPP
+ was seen in males and
females, with some sex-specific differences
in adrenal glands and
skeletal muscle. The highest accumulation
was detected in adrenal
glands, and only very low MPP
+ concentrations
were measured in the brain, which is due to the
inability of
MPP
+ to cross the blood-brain barrier
(
29). Taken together, these
data show that in adult mice
Orct3 deficiency results in a specific
impairment of
uptake-2 activity in the heart.

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FIG. 5.
MPP+ transport in adult
Orct3-deficient mice. (A and B) Concentrations of
MPP+ in the heart and liver (plotted on the primary
y axis in nanograms per gram of tissue) of adult
wild-type (black bars) and Orct3 mutant (white bars)
females (A) and males (B). The levels in plasma are also shown and are
plotted on the secondary y axis in nanograms per
milliliter. The asterisk indicates a statistically significant
difference. P values were <0.0001 for both male and
female hearts. (C and D) MPP+ distribution in different
organs of adult wild-type (black bars) and Orct3 mutant
(white bars) female (C) and male (D) mice. small int., small
intestine.
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Orct3 transports MPP+ at the fetoplacental
interface.
We next addressed the role of Orct3 in the
placenta. Pregnant females of an Orct3 heterozygous cross
were injected at 15.5 dpc with 1.0 mg of
[3H]MPP+ per kg. The
MPP+ concentrations were determined in embryos,
placentas, and amniotic fluid 10 min after injection (Fig.
6). The highest levels of
MPP+ were detected in the placenta, intermediate
levels were detected in the embryo, and the lowest levels were detected
in the amniotic fluid. In embryos, a threefold reduction in
MPP+ accumulation, from 64.7 ± 22.7 ng/g in
wild-type mice to 20.4 ± 4.4 ng/g in mutants, was detected
(P < 0.001). However, no differences in
[3H]MPP+ accumulation
were found in placentas and amniotic fluid of both groups. Since
Orct3 is not expressed in embryonic tissue, these data
indicate that Orct3 is the rate-limiting step in
MPP+ transport from the placenta to the fetus but
does not play a major role in placental uptake from the maternal
circulation. These results identify the placenta as a novel uptake-2
site of action.

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FIG. 6.
Placental MPP+ transport. MPP+
distribution in wild-type (black bars) and Orct3 mutant
(white bars) placentas (plotted on the primary y axis)
and embryos and amniotic fluid (plotted on the secondary
y axis). The asterisk indicates a statistically
significant difference. The P value was <0.001 for
embryos.
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Noradrenaline and dopamine levels in Orct3-null
mice.
Embryos and placentas at 12.5 dpc with a wild-type and an
Orct3-null genotype were obtained from a heterozygous
mating. Noradrenaline and dopamine steady-state levels were measured by
HPLC. Table 2 shows that, although both
noradrenaline and dopamine levels are reduced by approximately 50% in
Orct3-null embryos, the results are not statistically
significant (P = 0.1015). Samples with the same
genotype and obtained from the same litter showed a very large
variation in noradrenaline and dopamine steady-state levels for which
there is no current explanation. An independent examination of
monoamine levels in heart tissue has also found a large variation among
animals with the same genotype, with no significant difference between
wild-type and Orct3-null mice (B. Giros and S. Gautron, personal communication)
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DISCUSSION |
In this report, we have described the targeted
inactivation of the mouse extraneuronal monoamine transporter gene
Orct3, which has been shown to function as uptake-2. Removal
of exon 1 abolished transcription in tissues of adult mice but resulted
in an aberrant noncoding transcript in the placenta. We have
analyzed uptake-2 activity in Orct3-null mice using the
neurotoxin MPP+ as a substrate and identified a
transport phenotype in the adult heart and in the embryonic placenta.
Despite the large differences in MPP+ transport
in embryos and adult hearts, an overt physiological or neural phenotype
in Orct3 mutant mice that may be associated with altered
extracellular monoamine concentrations was not observed. In addition,
no significant differences in the steady-state levels of noradrenaline
and dopamine could be detected in either embryos or placentas.
Targeted inactivation of the Orct3 gene was performed by
deletion of a 4.3-kb genomic fragment that contains the
Orct3 gene promoter and exon 1. The Orct3
promoter consists of two closely linked transcriptional start sites
that were mapped 150 and 170 bp upstream of the beginning of the CpG
island in which exon 1 is embedded and were fully removed by the
deletion (data not shown). Orct3 expression was completely
abolished in all tissues of adult homozygous mutant mice. Later-stage
placentas from 12.5 dpc to term, however, express an aberrant,
noncoding transcript. This transcript is not driven by the wild-type
Orct3 gene promoter, as it was fully included in the
targeted deletion. These data indicate that deletion of the wild-type
transcriptional start sites activated an ectopic promoter sequence to
drive expression of the aberrant transcript. Alternatively, deletion of
the wild-type promoter may have fortuitously joined together a DNA
sequence that can drive expression of the aberrant transcript. The
expression, however, of the aberrant nontranslated transcript is tissue
specific and temporally restricted in development.
To study the role of Orct3 in uptake-2 function in vivo, we
performed transport studies using the neurotoxin
MPP+ as a substrate. MPP+
has been described elsewhere in the etiology of
1,2,3,6-methylphenyltetrahydropyridine (MPTP)-induced Parkinson's
disease (reviewed in reference 10). To exert its
neurotoxic effect on the central catecholaminergic neurons, MPTP
requires conversion to MPP+ by monoamine oxidase
B (Maob) in glial cells. Subsequently, MPP+ has
to leave the glial cells, upon which it is transported into the
catecholaminergic neurons by the neuronal dopamine uptake system.
Previously, it has been shown by in vitro cell culture that
MPP+ is able to make use of the extraneuronal
monoamine transport system to exit human glial cells (24).
In this report, we have shown that Orct3 transports
MPP+ in vivo in mice, raising the possibility
that Orct3 is an important mediator of MPTP neurotoxicity.
In addition to the effect in the central nervous system, it has been
reported that systemic injection of MPP+ or MPTP
results in severe depletion of heart noradrenaline concentrations (12). The cardiac depletion is resistant to desipramine
and GBR12909, suggesting that there is no involvement of the uptake-1 transporters Net and Dat (13, 20). Our transport studies
show reduced MPP+ uptake in hearts of
Orct3-deficient mice, which suggests that Orct3
might be involved in the action of
MPP+-MPTP-mediated depletion of cardiac noradrenaline.
Uptake-2 has been identified in various organs, like the heart and
uterus. Both organs express high levels of Orct3. However, our results identified a major effect of Orct3 deficiency on
MPP+ uptake in only adult hearts.
MPP+ accumulation was reduced by 72% in
Orct3 mutant hearts in males and females. This reduction is
identical, despite the fact that nearly twofold-lower levels of
MPP+ were measured in wild-type males than in the
females (Fig. 5A and B). This indicates that the relative contribution
of uptake-2 in MPP+ transport is not different
between males and females. A second difference in the amount of
MPP+ accumulation was found in hearts of inbred
FVB/N females and wild-type females of a mixed genetic background (Fig.
4 and 5A). The difference in MPP+ accumulation
might be explained by a differential contribution of different genetic
backgrounds. However, there is also an age difference between the two
cohorts (7 and 12 weeks, respectively), which may be an important
contributor. In the uterus, a nearly twofold reduction in
MPP+ accumulation was observed between wild-type
and Orct3
/
females, but this was not statistically significant (Fig. 5C). In pigs,
it was demonstrated that uptake-1 and uptake-2 activity in the uterine
artery vary during the estrous cycle (7). Since the mice
in our experiments were not synchronized, this could explain the large
variation seen for the uterus that may potentially mask an effect of
Orct3 deficiency. Other Orct3-expressing organs showed no difference in MPP+ uptake. This might
be caused by a functional redundancy with other transporter genes.
Orct3 is only moderately expressed in the kidney, for
instance, whereas the closely related organic cation transporter gene
Orct2 is found at high levels (21). As
Orct2 is also capable of MPP+
transport, expression of this gene could contribute significantly to
the total MPP+ accumulation in the kidney
(17). Similarly, the Orct1 gene is highly
expressed in the liver, which may account for the
MPP+ uptake measured in this tissue (Fig. 4 and
5).
The placental MPP+ transport studies show that
Orct3 transports MPP+ between the placenta and fetus, but
not to the maternal circulation, and identify the placenta as a novel
uptake-2 site of action (Fig. 6). It has been reported previously that
during development embryos show a high monoamine turnover compared with
any other physiological condition seen for adults (30).
For sheep, it has been determined that nearly 50% of the total in
utero monoamine clearance was mediated by the placenta. Only part of
the placental activity was assigned to the action of neuronal monoamine
transporters, as was determined by inhibition mediated by cocaine,
which is a neuronal monoamine transporter antagonist (5).
Using RNA in situ hybridization, we have recently described the
cellular expression pattern of Orct3 and the
monoamine-degrading enzyme gene Maoa in the mouse placenta.
The two genes are expressed in a similar pattern in the labyrinth layer
(34), indicating that they could form a functional
monoamine degradation pathway. This interpretation is supported by the
transport studies presented here that have identified an
Orct3-mediated uptake-2 activity at the fetoplacental interface.
The MPP+ transport studies show that
Orct3 is an essential component in vivo for the transport
activity of the extraneuronal monoamine clearance system known as
uptake-2. Transport defects in
Orct3-deficient mice were observed in embryonic development. However, despite a broad expression pattern in adult animals, an
essential function is seen in only one adult organ, the heart. Surprisingly, despite significant differences in
MPP+ uptake, the Orct3-null mice show
no overt neural or physiological dysfunction as embryos or adults that
may indicate a monoamine imbalance. In addition, we have been unable to
identify a significant difference in the levels of two tested
monoamines in Orct3-null embryos that show a 65% reduction
in MPP+ levels. Thus, the functional significance
of the role of the Orct3 gene in monoamine transport remains
unclear. These results do not dismiss a function for the
Orct3 gene in extraneural monoamine transport, but they
indicate the possibility that its role is more complex than has
been predicted.
 |
ACKNOWLEDGMENTS |
R. Zwart and S. Verhaagh contributed equally to this work.
We thank C. Brouwer and J. Vink for help with the ES cell work in the
gene targeting experiment. K. van Veen is acknowledged for the
blastocyst injections, N. Bosnie and T. Maidment are acknowledged for
taking good care of the mice, and J. W. Jonker is acknowledged for
help with the MPP+ transport studies. We thank B. Giros and
S. Gautron for sharing their unpublished data; A. H. Schinkel,
J. W. Jonker, and all members of the lab for critical discussions
and reading of the manuscript; and A. Berns for constant help and support.
This work was supported by a grant from the Dutch Cancer Society.
 |
FOOTNOTES |
*
Corresponding author. Present address: ÖAW
Institute of Molecular Biology, Billrothstrasse 11, A-5020 Salzburg,
Austria. Phone: 43 662 63 961 14. Fax: 43 662 63 961 40. E-mail:
dbarlow{at}imb.oeaw.ac.at.
 |
REFERENCES |
| 1.
|
Amara, S. G., and M. J. Kuhar.
1993.
Neurotransmitter transporters: recent progress.
Annu. Rev. Neurosci.
16:73-93[Medline].
|
| 2.
|
Auffray, C., and F. Rougeon.
1980.
Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA.
Eur. J. Biochem.
107:303-314[Medline].
|
| 3.
|
Bengel, D.,
D. L. Murphy,
A. M. Andrews,
C. H. Wichems,
D. Feltner,
A. Heils,
R. Mossner,
H. Westphal, and K. P. Lesch.
1998.
Altered brain serotonin homeostasis and locomotor insensitivity to 3, 4-methylenedioxymethamphetamine ("Ecstasy") in serotonin transporter-deficient mice.
Mol. Pharmacol.
53:649-655[Abstract/Free Full Text].
|
| 4.
|
Breidert, T.,
F. Spitzenberger,
D. Grundemann, and E. Schomig.
1998.
Catecholamine transport by the organic cation transporter type 1 (OCT1).
Br. J. Pharmacol.
125:218-224[CrossRef][Medline].
|
| 5.
|
Bzoskie, I.,
I. Blount,
K. Kashiwai,
J. Humme, and J. F. Padbury.
1997.
Placental norepinephrine transporter development in the ovine fetus.
Placenta
18:65-70[CrossRef][Medline].
|
| 6.
|
Church, G. M., and W. Gilbert.
1984.
Genomic sequencing.
Proc. Natl. Acad. Sci. USA
81:1991-1995[Abstract/Free Full Text].
|
| 7.
|
Dynarowicz, I.,
T. Trapkowski, and T. Pirus.
1993.
Uptake-1, Uptake-2 and the release of [3H]-noradrenaline in uterine artery of pigs during the oestrous cycle.
Arch. Vet. Pol.
33:259-267[Medline].
|
| 8.
|
Eisenhofer, G.,
R. McCarty,
K. Pacak,
H. Russ, and E. Schomig.
1996.
Disprocynium24, a novel inhibitor of the extraneuronal monoamine transporter, has potent effects on the inactivation of circulating noradrenaline and adrenaline in conscious rat.
Naunyn-Schmiedeberg's Arch. Pharmacol.
354:287-294[Medline].
|
| 9.
|
Fassler, R., and M. Meyer.
1995.
Consequences of lack of beta 1 integrin gene expression in mice.
Genes Dev.
9:1896-1908[Abstract/Free Full Text].
|
| 10.
|
Feldman, R. S.,
J. S. Meyer, and L. F. Quenzer.
1997.
Principles of neuropsychopharmacology.
Sinauer Associates Inc., Sunderland, Mass.
|
| 11.
|
Friedgen, B.,
R. Wolfel,
H. Russ,
E. Schomig, and K. H. Graefe.
1996.
The role of extraneuronal amine transport systems for the removal of extracellular catecholamines in the rabbit.
Naunyn-Schmiedeberg's Arch. Pharmacol.
354:275-286[Medline].
|
| 12.
|
Fuller, R. W., and S. K. Hemrick-Luecke.
1986.
Depletion of norepinephrine in mouse heart by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mimicked by 1-methyl-4-phenylpyridinium (MPP+) and not blocked by deprenyl.
Life Sci.
39:1645-1650[CrossRef][Medline].
|
| 13.
|
Fuller, R. W.,
S. K. Hemrick-Luecke, and D. W. Robertson.
1988.
Comparison of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium (MPP+) effects on mouse heart norepinephrine.
Biochem. Pharmacol.
37:3343-3347[CrossRef][Medline].
|
| 14.
|
Giros, B.,
M. Jaber,
S. R. Jones,
R. M. Wightman, and M. G. Caron.
1996.
Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter.
Nature
379:606-612[CrossRef][Medline].
|
| 15.
|
Grundemann, D.,
J. Babin-Ebell,
F. Martel,
N. Ording,
A. Schmidt, and E. Schomig.
1997.
Primary structure and functional expression of the apical organic cation transporter from kidney epithelial LLC-PK1 cells.
J. Biol. Chem.
272:10408-10413[Abstract/Free Full Text].
|
| 16.
|
Grundemann, D.,
S. Koster,
N. Kiefer,
T. Breidert,
M. Engelhardt,
F. Spitzenberger,
N. Obermuller, and E. Schomig.
1998.
Transport of monoamine transmitters by the organic cation transporter type 2, OCT2.
J. Biol. Chem.
273:30915-30920[Abstract/Free Full Text].
|
| 17.
|
Grundemann, D.,
G. Liebich,
N. Kiefer,
S. Koster, and E. Schomig.
1999.
Selective substrates for non-neuronal monoamine transporters.
Mol. Pharmacol.
56:1-10[Abstract/Free Full Text].
|
| 18.
|
Grundemann, D.,
B. Schechinger,
G. A. Rappold, and E. Schomig.
1998.
Molecular identification of the corticosterone-sensitive extraneuronal catecholamine transporter.
Nat. Neurosci.
1:349-351[CrossRef][Medline].
|
| 19.
|
Iversen, L. L.
1965.
The uptake of catechol amines at high perfusion concentrations in the rat isolated heart: a novel catechol amine uptake process.
Br. J. Pharmacol.
25:18-33.
|
| 20.
|
Kujacic, M., and A. Carlsson.
1994.
Effects of MPP+ on catecholamine levels in adrenal glands and heart of rats.
Naunyn-Schmiedeberg's Arch. Pharmacol.
350:245-251[Medline].
|
| 21.
|
Mooslehner, K. A., and N. D. Allen.
1999.
Cloning of the mouse organic cation transporter 2 gene, Slc22a2, from an enhancer-trap transgene integration locus.
Mamm. Genome
10:218-224[CrossRef][Medline].
|
| 22.
|
Russ, H.,
M. Gliese,
J. Sonna, and E. Schomig.
1992.
The extraneuronal transport mechanism for noradrenaline (Uptake-2) avidly transports 1-methyl-4-phenylpyridinium (MPP+).
Naunyn-Schmiedeberg's Arch. Pharmacol.
346:158-165[CrossRef][Medline].
|
| 23.
|
Russ, H.,
J. Sonna,
K. Keppler,
S. Baunach, and E. Schomig.
1993.
Cyanine-related compounds: a novel class of potent inhibitors of extraneuronal noradrenaline transport.
Naunyn-Schmiedeberg's Arch. Pharmacol.
348:458-465[Medline].
|
| 24.
|
Russ, H.,
K. Staust,
F. Martel,
M. Gliese, and E. Schomig.
1996.
The extraneuronal transporter for monoamine transmitters exists in cells derived from human central nervous system glia.
Eur. J. Neurosci.
8:1256-1264[CrossRef][Medline].
|
| 25.
|
Sayre, L. M.
1989.
Biochemical mechanism of action of the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).
Toxicol. Lett.
48:121-149[CrossRef][Medline].
|
| 26.
|
Schomig, E., and C. L. Schonfeld.
1990.
Extraneuronal noradrenaline transport (Uptake-2) in a human cell line (Caki-1 cells).
Naunyn-Schmiedeberg's Arch. Pharmacol.
341:404-410[Medline].
|
| 27.
|
Schweifer, N., and D. P. Barlow.
1996.
The Lx1 gene maps to mouse chromosome 17 and codes for a protein that is homologous to glucose and polyspecific transmembrane transporters.
Mamm. Genome
7:735-740[CrossRef][Medline].
|
| 28.
|
Shannon, J. R.,
N. L. Flattem,
J. Jordan,
G. Jacob,
B. K. Black,
I. Biaggioni,
R. D. Blakely, and D. Robertson.
2000.
Orthostatic intolerance and tachycardia associated with norepinephrine-transporter deficiency.
N. Engl. J. Med.
342:541-549[Abstract/Free Full Text].
|
| 29.
|
Snijder, S. H.,
R. J. D'Amato,
J. S. Nye, and J. A. Javitch.
1986.
Selective uptake of MPP+ by dopamine neurons is required for MPTP neurotoxicity: studies in brain synaptosomes and PC-12 cells, p. 191-201.
In
S. P. Markey, N. Castagnoli, A. J. Trevor, and I. J. Kopin (ed.), MPTP: a neurotoxin producing a Parkinsonian syndrome. Academic Press, Inc., Orlando, Fla.
|
| 30.
|
Stein, H.,
K. Oyama,
A. Martinez,
B. Chappell, and J. Padbury.
1993.
Plasma epinephrine appearance and clearance rates in fetal and newborn sheep.
Am. J. Physiol.
265:R756-R760[Abstract/Free Full Text].
|
| 31.
|
Streich, S.,
M. Bruss, and H. Bonisch.
1996.
Expression of the extraneuronal monoamine transporter (Uptake-2) in human glioma cells.
Naunyn-Schmiedeberg's Arch. Pharmacol.
353:328-333[CrossRef][Medline].
|
| 32.
|
Thomas, S. A.,
A. M. Matsumoto, and R. D. Palmiter.
1995.
Noradrenaline is essential for mouse fetal development.
Nature
374:643-646[CrossRef][Medline].
|
| 33.
|
Trendelenburg, U.
1988.
The extraneuronal uptake and metabolism of catecholamines.
Handb. Exp. Pharmacol.
90/I:279-319.
|
| 34.
|
Verhaagh, S.,
D. P. Barlow, and R. Zwart.
2001.
The extraneuronal monoamine transporter Slc22a3/Orct3 co-localizes with the Maoa metabolizing enzyme in mouse placenta.
Mech. Dev.
100:127-130[CrossRef][Medline].
|
| 35.
|
Verhaagh, S.,
N. Schweifer,
D. P. Barlow, and R. Zwart.
1999.
Cloning of the mouse and human solute carrier 22a3 (Slc22a3/SLC22A3) identifies a conserved cluster of three organic cation transporters on mouse chromosome 17 and human 6q26-q27.
Genomics
55:209-218[CrossRef][Medline].
|
| 36.
|
Wu, X.,
R. Kekuda,
W. Huang,
Y. J. Fei,
F. H. Leibach,
J. Chen,
S. J. Conway, and V. Ganapathy.
1998.
Identity of the organic cation transporter OCT3 as the extraneuronal monoamine transporter (Uptake-2) and evidence for the expression of the transporter in the brain.
J. Biol. Chem.
273:32776-32786[Abstract/Free Full Text].
|
| 37.
|
Xu, F.,
R. R. Gainetdinov,
W. C. Wetsel,
S. R. Jones,
L. M. Bohn,
G. W. Miller,
Y. M. Wang, and M. G. Caron.
2000.
Mice lacking the norepinephrine transporter are supersensitive to psychostimulants.
Nat. Neurosci.
3:465-471[CrossRef][Medline].
|
| 38.
| Zwart, R., S. Verhaagh, J. de Jong, M. Lyon, and D. P. Barlow. Genetic analysis of the organic cation transporter genes
Orct2/Slc22a2 and Orct3Slc22a3 reduces the
critical region for the t haplotype mutant
tw73 to 200 kb. Mammal. Genome, in
press.
|
Molecular and Cellular Biology, July 2001, p. 4188-4196, Vol. 21, No. 13
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.13.4188-4196.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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