Impaired activity of the extraneuronal monoamine transporter system known as uptake-2 in Orct3/Slc22a3-deficient mice

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 [(3)H]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 [(3)H]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.

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.

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.

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.

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.

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.

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.