The extraneuronal monoamine transporter Slc22a3/Orct3 co-localizes with the Maoa metabolizing enzyme in mouse placenta

Monoamine clearance is a combined function of uptake mechanisms in the plasma membrane with intracellular metabolizing enzymes. Two different uptake mechanisms have been described. Uptake(1) is located in presynaptic neurones, whereas uptake(2) is extraneuronal. Recently, the Slc22a3/Orct3 gene was identified as the extraneuronal monoamine transporter. In mouse embryonic development Orct3 expression is restricted to the placenta, which is also a site of expression of neuronal transporters. We have used RNA blots and in situ hybridization to examine the expression of Orct3 and other members of the monoamine uptake and metabolizing pathways in mouse placenta. The results show that Orct3 expression overlaps that of the monoamine metabolizing enzyme Maoa in the labyrinth layer of the placenta with an expression pattern distinct from that of the neuronal transporters Slc6a2/Net and Slc6a4/Sert.     

Genetic analysis of the organic cation transporter genes Orct2/Slc22a2 and Orct3/Slc22a3 reduces the critical region for the t haplotype mutant t w73 to 200 kb

Here we report an analysis of two candidate genes for the t ^w73 implantation mutation. The t ^w73 gene maps to a 20-cM region of mouse Chromosome (Chr) 17 known as the t-complex, which exists in a wild-type and t haplotype form in present-day mice. The t haplotype variants contain several mutant alleles affecting male fertility and embryonic viability and offer the opportunity to identify genes critical for these processes. t ^w73 homozygous embryos are defective in trophoblast production and fail to implant adequately, with death occurring at approximately 7.5 days post coitum (pc). Two recently described organic cation transporter genes, Slc22a2 (Orct2) and Slc22a3 (Orct3), fulfill criteria predicted for t ^w73 candidate genes, since both map to the previously defined 500-kb t ^w73 minimal region and both are also expressed in 7.5 days pc post-implantation embryos. The genomic locus of the Orct2 gene appears similar in wild-type and t ^w73 chromosomes. In contrast, the genomic locus of Orct3 is amplified and displays an altered expression profile in all t haplotype variant chromosomes tested. In addition, Orct3 shows a t ^w73 specific polymorphism. To test whether either Orct2 or Orct3 is involved in the t ^w73 phenotype, we have performed a genetic rescue experiment using YAC transgenes overexpressing Orct2, and genetic complementation with an allele in which the Orct3 gene was inactivated by homologous recombination. The results eliminate both Orct2 and Orct3 as candidates and further reduce the critical region containing the t ^w73 mutant from 500 kb to 200 kb. Received: 8 February 2001 / Accepted: 1 May 2001     

Impaired organic anion transport in kidney and choroid plexus of organic anion transporter 3 (Oat3 (Slc22a8)) knockout mice

To begin to develop in vivo model systems for the assessment of the contributions of specific organic anion transporter (OAT) family members to detoxification, development, and disease, we carried out a targeted disruption of the murine organic anion transporter 3 (Oat3) gene. Surviving Oat3(-/-) animals appear healthy, are fertile, and do not exhibit any gross morphological tissue abnormalities. No Oat3 mRNA expression was detected in kidney, liver, or choroid plexus (CP) of Oat3(-/-) mice. A distinct phenotype manifested by a substantial loss of organic anion transport capacity in kidney and CP was identified. Uptake sensitive to inhibition by bromosulfophthalein or probenecid was observed for taurocholate, estrone sulfate, and para-aminohippurate in renal slices from wild-type mice, whereas in Oat3(-/-) animals transport of these substances was greatly reduced. No discernable differences in uptake were observed between hepatic slices from wild-type and Oat3(-/-) littermates, suggesting Oat3 does not play a major role in hepatic organic anion uptake. Cellular accumulation of fluorescein was reduced by approximately 75% in CP from Oat3(-/-) mice. However, capillary accumulation of fluorescein-methotrexate was unchanged, indicating the effects of Oat3 loss are restricted to the entry step and that Oat3 is localized to the apical membrane of CP. These data indicate a key role for Oat3 in systemic detoxification and in control of the organic anion distribution in cerebrospinal fluid.     

Deficiency in the organic cation transporters 1 and 2 (Oct1/Oct2 [Slc22a1/Slc22a2]) in mice abolishes renal secretion of organic cations

The polyspecific organic cation transporters 1 and 2 (Oct1 and -2) transport a broad range of substrates, including drugs, toxins, and endogenous compounds. Their strategic localization in the basolateral membrane of epithelial cells in the liver, intestine (Oct1), and kidney (Oct1 and Oct2) suggests that they play an essential role in removing noxious compounds from the body. We previously showed that in Oct1(-/-) mice, the hepatic uptake and intestinal excretion of organic cations are greatly reduced. Since Oct1 and Oct2 have extensively overlapping substrate specificities, they might be functionally redundant. To investigate the pharmacologic and physiologic roles of these proteins, we generated Oct2 single-knockout and Oct1/2 double-knockout mice. Oct2(-/-) and Oct1/2(-/-) mice are viable and fertile and display no obvious phenotypic abnormalities. Absence of Oct2 in itself had little effect on the pharmacokinetics of tetraethylammonium (TEA), but in Oct1/2(-/-) mice, renal secretion of this compound was completely abolished, leaving only glomerular filtration as a TEA clearance mechanism. As a consequence, levels of TEA were substantially increased in the plasma of Oct1/2(-/-) mice. This study shows that Oct1 and Oct2 together are essential for renal secretion of (small) organic cations. A deficiency in these proteins may thus result in increased drug sensitivity and toxicity.     

The localisation of the extraneuronal monoamine transporter (EMT) in rat brain

The extraneuronal monoamine transporter plays an important role in the inactivation of monoamine transmitters. A basal extraneuronal tissue expression of this transporter has been reported, but it is also expressed in CNS glia. As little is known about the expression pattern and the function of the extraneuronal monoamine transporter in the brain, we performed a detailed investigation. Firstly, a northern blot analysis of different rat organs revealed that the transporter is strongly expressed in placenta, lung and heart and less prominently in the whole brain, brain stem, intestine, testis, epididymis, stomach, kidney and skeletal muscle. It was not expressed in cerebellum, liver and embryo. Using an in situ hybridization to the rat brain, we detected a marked and highly confined expression of the extraneuronal monoamine transporter in the area postrema, but in no other brain areas. These findings were confirmed by polyclonal antibodies against rat extraneuronal monoamine transporter showing an intensive signal in the area postrema, although a few cells in the cerebellum and the brain stem also showed a signal. Additionally, a partly overlapping expression pattern of the monoamine oxidase-B was detected. Summarizing, we firstly describe a marked and highly confined expression of the extraneuronal monoamine transporter in the rat area postrema by in situ hybridisation which may play a role in physiological functions of this circumventricular organ such as emesis, food intake and the regulation of cardiovascular functions.     

Expression and functional characterization of the extraneuronal monoamine transporter in normal human astrocytes

In this study we examined the functional expression of the extraneuronal monoamine transporter (EMT) in normal human astrocytes (NHA). RT-PCR with EMT-specific primers demonstrated the presence of EMT mRNA in NHA. The RT-PCR products were subjected to restriction-site analysis using three different enzymes (HinfI, SacI and BclI). The restriction patterns with the three enzymes were identical and were exactly as expected from the known restriction map of human EMT cDNA. DNA sequencing was performed for the RT-PCR products from NHA. Sequence analysis demonstrated that the sequences of RT-PCR products were identical to that of EMT. The extract of NHA was immunoblotted with anti-EMT polyclonal antibody raised against EMT polypeptides. Western blotting indicated that anti-EMT polyclonal antibody recognized a band of 63 kDa. Immunocytochemical staining using anti-EMT polyclonal antibody in NHA revealed that the plasma membrane, as well as intracellular, perinuclear compartments, presumably endoplasmic reticulum or Golgi membranes, showed a considerable level of immunoreactivity. We examined the time course of temperature-dependent [3H]MPP+ uptake in NHA for 60 min. Temperature-dependent [3H]MPP+ uptake increased in a time-dependent manner for the initial 45 min and almost reached a plateau level (8.70 +/- 0.59 pmol/mg protein) at 60 min. In the presence of 3 micro m decynium22 (D22) (the most potent EMT inhibitor), temperature-dependent [3H]MPP+ uptake was strongly reduced by 61% (3.39 +/- 0.76 pmol/mg protein at 60 min). D22-sensitive [3H]MPP+ uptake was saturable over a MPP+ concentration of 6.25-200 micro m. Km for this process was 78.01 +/- 7.64 micro m and Vmax was 295.4 +/- 12.8 pmol/mg protein/min. D22-sensitive [3H]MPP+ uptake was reduced when the astrocyte membrane potential was depolarized by increasing the concentration of K+ in the uptake buffer or by adding Ba2+ to the uptake buffer. These results provide evidence that the MPP+ transport activity in NHA is potential-sensitive. Moreover, D22-sensitive [3H]MPP+ uptake was independent of extracellular Na+. D22-sensitive [3H]MPP+ uptake was inhibited by D22, various organic cations, steroids and monoamine neurotransmitters. Our results showed that the EMT is functionally expressed in NHA and may also play a key role in the disposition of cationic drugs, neurosteroids, the neurotoxin MPP+ and monoamine neurotransmitters in the brain.     

Altered visual function in monocarboxylate transporter 3 (Slc16a8) knockout mice

To meet the high-energy demands of photoreceptor cells, the outer retina metabolizes glucose through glycolytic and oxidative pathways, resulting in large-scale production of lactate and CO(2). Mct3, a proton-coupled monocarboxylate transporter, is critically positioned to facilitate transport of lactate and H(+) out of the retina and could therefore play a role in pH and ion homeostasis of the outer retina. Mct3 is preferentially expressed in the basolateral membrane of the retinal pigment epithelium and forms a heteromeric complex with the accessory protein CD147. To examine the physiological role of Mct3 in the retina, we generated mice with a targeted deletion in Mct3 (slc16A8). The overall retinal histology of 4- to 36-wk-old Mct3(-/-) mice appeared normal. In the absence of Mct3, expression of CD147 was lost from the basolateral but not apical RPE. The saturated a-wave amplitude (a(max)) of the scotopic electroretinogram (ERG) was reduced by approximately twofold in Mct3(-/-) mice relative to wild-type mice. A fourfold increase in lactate in the retina suggested a decrease in outer-retinal pH. In single-cell recordings from superfused retinal slices, saturating amplitudes of single rod photocurrents (J(max)) were comparable indicating that Mct3(-/-) mouse photoreceptor cells were inherently healthy. Based on these data, we hypothesize that disruption of Mct3 leads to a potentially reversible decrease in subretinal space pH, thereby reducing the magnitude of the light suppressible photoreceptor current.     

Altered aminergic neurotransmission in the brain of organic cation transporter 3-deficient mice

Organic cation transporters (OCTs) are carrier-type polyspecific permeases known to participate in low-affinity extraneuronal catecholamine uptake in peripheral tissues. OCT3 is the OCT subtype most represented in the brain, yet its implication in central aminergic neurotransmission in vivo had not been directly demonstrated. In a detailed immunohistochemistry study, we show that OCT3 is expressed in aminergic pathways in the mouse brain, particularly in dopaminergic neurons of the substantia nigra compacta, non-aminergic neurons of the ventral tegmental area, substantia nigra reticulata (SNr), locus coeruleus, hippocampus and cortex. Although OCT3 was found mainly in neurons, it was also occasionally detected in astrocytes in the SNr, hippocampus and several hypothalamic nuclei. In agreement with this distribution, OCT3/Slc22a3-deficient mice show evidence of altered monoamine neurotransmission in the brain, with decreased intracellular content and increased turnover of aminergic transmitters. The behavioral characterization of these mutants reveal subtle behavioral alterations such as increased sensitivity to psychostimulants and increased levels of anxiety and stress. Altogether our data support a role of OCT3 in the homeostastic regulation of aminergic neurotransmission in the brain.     

Cloning of the mouse organic cation transporter 2 gene, Slc22a2, from an enhancer-trap transgene integration locus

A novel mouse gene, associated with the enhancer-trap mutation TKZ736, has been cloned and sequenced. It encodes a polyspecific transmembrane transporter with 12 putative transmembrane domains, that shares significant homology with the mouse organic cation transporter 1 (Oct1/Slc22a1) called Lx1. Like Oct1/Slc22a1/Lx1, this gene maps to the proximal part of Chromosome (Chr) 17, but shows a different expression pattern from Oct1/Slc22a1/Lx1. The gene identified here is predominantly expressed in the kidney and ureter, but no expression is detectable in liver. Sequence comparisons suggest that this novel gene most likely represents the mouse homolog of the rat organic cation transporter 2 gene. The genomic DNA flanking the 3′ transgene integration site in the enhancer-trap mutation TKZ736 encodes the second exon of the Oct2/Slc22a2 gene. Received: 6 July 1998 / Accepted: 15 October 1998     

Deletion of multispecific organic anion transporter Oat1/Slc22a6 protects against mercury-induced kidney injury

The primary site of mercury-induced injury is the kidney due to uptake of the reactive Hg(2+)-conjugated organic anions in the proximal tubule. Here, we investigated the in vivo role of Oat1 (organic anion transporter 1; originally NKT (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471-6478)) in handling of known nephrotoxic doses of HgCl(2). Oat1 (Slc22a6) is a multispecific organic anion drug transporter that is expressed on the basolateral aspects of renal proximal tubule cells and that mediates the initial steps of elimination of a broad range of endogenous metabolites and commonly prescribed pharmaceuticals. Mercury-induced nephrotoxicity was observed in a wild-type model. We then used the Oat1 knock-out to determine in vivo whether the renal injury effects of mercury are mediated by Oat1. Most of the renal injury (both histologically and biochemically as measured by blood urea nitrogen and creatinine) was abolished following HgCl(2) treatment of Oat1 knock-outs. Thus, acute kidney injury by HgCl(2) was found to be mediated mainly by Oat1. Our findings raise the possibility that pharmacological modulation of the expression and/or function of Oat1 might be an effective therapeutic strategy for reducing renal injury by mercury. This is one of the most striking phenotypes so far identified in the Oat1 knock-out. (Eraly, S. A., Vallon, V., Vaughn, D. A., Gangoiti, J. A., Richter, K., Nagle, M., Monte, J. C., Rieg, T., Truong, D. M., Long, J. M., Barshop, B. A., Kaler, G., and Nigam, S. K. (2006) J. Biol. Chem. 281, 5072-5083).     

Creatine transporter (CrT; Slc6a8) knockout mice as a model of human CrT deficiency

Mutations in the creatine (Cr) transporter (CrT; Slc6a8) gene lead to absence of brain Cr and intellectual disabilities, loss of speech, and behavioral abnormalities. To date, no mouse model of CrT deficiency exists in which to understand and develop treatments for this condition. The purpose of this study was to generate a mouse model of human CrT deficiency. We created mice with exons 2–4 of Slc6a8 flanked by loxP sites and crossed these to Cre:CMV mice to create a line of ubiquitous CrT knockout expressing mice. Mice were tested for learning and memory deficits and assayed for Cr and neurotransmitter levels. Male CrT^−/y (affected) mice lack Cr in the brain and muscle with significant reductions of Cr in other tissues including heart and testes. CrT^−/y mice showed increased path length during acquisition and reversal learning in the Morris water maze. During probe trials, CrT^−/y mice showed increased average distance from the platform site. CrT^−/y mice showed reduced novel object recognition and conditioned fear memory compared to CrT^+/y. CrT^−/y mice had increased serotonin and 5-hydroxyindole acetic acid in the hippocampus and prefrontal cortex. Ubiquitous CrT knockout mice have learning and memory deficits resembling human CrT deficiency and this model should be useful in understanding this disorder.     

Impaired ubiquitin-proteasome system activity in the synapses of Huntington's disease mice

Huntington's disease (HD) is caused by the expansion of a polyglutamine tract in the N-terminal region of huntingtin (htt) and is characterized by selective neurodegeneration. In addition to forming nuclear aggregates, mutant htt accumulates in neuronal processes as well as synapses and affects synaptic function. However, the mechanism for the synaptic toxicity of mutant htt remains to be investigated. We targeted fluorescent reporters for the ubiquitin-proteasome system (UPS) to presynaptic or postsynaptic terminals of neurons. Using these reporters and biochemical assays of isolated synaptosomes, we found that mutant htt decreases synaptic UPS activity in cultured neurons and in HD mouse brains that express N-terminal or full-length mutant htt. Given that the UPS is a key regulator of synaptic plasticity and function, our findings offer insight into the selective neuronal dysfunction seen in HD and also establish a method to measure synaptic UPS activity in other neurological disease models.     

Impaired hepatic insulin signalling in PON2-deficient mice: a novel role for the PON2/apoE axis on the macrophage inflammatory response

Hepatic glucose metabolism is strongly influenced by oxidative stress and pro-inflammatory stimuli. PON2 (paraoxonase 2), an enzyme with undefined antioxidant properties, protects against atherosclerosis. PON2-deficient (PON2-def) mice have elevated hepatic oxidative stress coupled with an exacerbated inflammatory response from PON2-deficient macrophages. In the present paper, we demonstrate that PON2 deficiency is associated with inhibitory insulin-mediated phosphorylation of hepatic IRS-1 (insulin receptor substrate-1). Unexpectedly, we observed a marked improvement in the hepatic IRS-1 phosphorylation state in PON2-def/apoE (apolipoprotein E)(-/-) mice, relative to apoE(-/-) mice. Factors secreted from activated macrophage cultures derived from PON2-def and PON2-def/apoE(-/-) mice are sufficient to modulate insulin signalling in cultured hepatocytes in a manner similar to that observed in vivo. We show that the protective effect on insulin signalling in PON2-def/apoE(-/-) mice is directly associated with altered production of macrophage pro-inflammatory mediators, but not elevated intracellular oxidative stress levels. We further present evidence that modulation of the macrophage inflammatory response in PON2-def/apoE(-/-) mice is mediated by a shift in the balance of NO and ONOO(-) (peroxynitrite) formation. Our results demonstrate that PON2 plays an important role in hepatic insulin signalling and underscores the influence of macrophage-mediated inflammatory response on hepatic insulin sensitivity.     

Impaired flow-dependent control of vascular tone and remodeling in P2X4-deficient mice

The structure and function of blood vessels adapt to environmental changes such as physical development and exercise. This phenomenon is based on the ability of the endothelial cells to sense and respond to blood flow; however, the underlying mechanisms remain unclear. Here we show that the ATP-gated P2X4 ion channel, expressed on endothelial cells and encoded by P2rx4 in mice, has a key role in the response of endothelial cells to changes in blood flow. P2rx4(-/-) mice do not have normal endothelial cell responses to flow, such as influx of Ca(2+) and subsequent production of the potent vasodilator nitric oxide (NO). Additionally, vessel dilation induced by acute increases in blood flow is markedly suppressed in P2rx4(-/-) mice. Furthermore, P2rx4(-/-) mice have higher blood pressure and excrete smaller amounts of NO products in their urine than do wild-type mice. Moreover, no adaptive vascular remodeling, that is, a decrease in vessel size in response to a chronic decrease in blood flow, was observed in P2rx4(-/-) mice. Thus, endothelial P2X4 channels are crucial to flow-sensitive mechanisms that regulate blood pressure and vascular remodeling.     

Identification and functional characterization of uric acid transporter Urat1 (Slc22a12) in rats

Uric acid transporter URAT1 contributes significantly to reabsorption of uric acid in humans to maintain a constant serum uric acid (SUA) level. Since alteration of SUA level is associated with various diseases, it is important to clarify the mechanism of change in SUA. However, although expression of mRNA of an ortholog of URAT1 (rUrat1) in rats has been reported, functional analysis and localization have not been done. Therefore, rat rUrat1 was functionally analyzed using gene expression systems and isolated brush-border membrane vesicles (BBMVs) prepared from rat kidney, and its localization in kidney was examined immunohistochemically. Uric acid transport by rUrat1 was chloride (Cl-) susceptible with a Km of 1773μM. It was inhibited by benzbromarone and trans-stimulated by lactate and pyrazinecarboxylic acid (PZA). Cl- gradient-susceptible uric acid transport by BBMVs showed similar characteristics to those of uric acid transport by rUrat1. Moreover, rUrat1 was localized at the apical membrane in proximal tubular epithelial cells in rat kidney. Accordingly, rUrat1 is considered to be involved in uric acid reabsorption in rats in the same manner as URAT1 in humans. Therefore, rUrat1 may be a useful model to study issues related to the role of human URAT1.     

Impaired host defense to infection and Toll-like receptor 2-independent killing of Borrelia burgdorferi clinical isolates in TLR2-deficient C3H/HeJ mice

To investigate the role of Toll-like receptor 2 (TLR2)-mediated signaling in host innate defense and development of Lyme disease, the pathogenicity of Borrelia burgdorferi sensu stricto clinical isolates representing two distinct genotypes (RST1 and RST3A) was assessed in TLR2(-/-) C3H/HeJ mice. All TLR2(-/-) mice infected with a B. burgdorferi RST1 isolate developed severe arthritis. The numbers of spirochetes in heart, joint and ear biopsy specimens were significantly higher in TLR2(-/-) mice than in wild-type mice similarly infected as determined by real-time quantitative polymerase chain reaction. Interestingly, despite the higher spirochete levels in heart tissues, milder carditis was observed in TLR2(-/-) than in wild-type mice infected with this RST1 isolate (P=0.02). By contrast, no positive cultures were obtained from any of the blood and tissue specimens from TLR2(-/-) mice inoculated with two RST3A clinical isolates. The data suggest that there is impaired host innate defense against infection and TLR2-independent killing of B. burgdorferi clinical isolates in TLR2-deficient C3H/HeJ mice.