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.     

Regulation and developmental expression of the divalent metal-ion transporter in the rat brain.

Divalent metal ion transporter 1 (DMT1) is a recently identified metal-ion transporter that appears to mediate the absorption of iron in the intestine. DMT1 mRNA is also present in discrete areas of the brain. In this study, we examined the expression of DMT1 mRNA in developing rat brain. DMT1 mRNA was found by in situ hybridization in the striatum, cortex, hippocampus and cerebellum. During development, DMT1 mRNA was found in Purkinje and granule cells in the cerebellum at post-natal day (PND) 14 and PND 30. DMT1 mRNA was also expressed in the external granular layer of the cerebellum at PND 14. No change in the level of DMT1 mRNA was observed by Northern analysis in the cerebellum at different ages between PND 1 and 21. DMT1 was found by Northern analysis in cultures of rat astrocytes. Activation of protein kinase C increased the expression of DMT1 in kidney epithelial cells but not astrocytes from newborn rats. Because DMT1 is expressed in a wide variety of types of cells, we suggest that it plays an important role in metal homeostasis in the brain.     

Expression of neuronal nitric oxide synthase variant, nNOS-mu, in rat brain.

Neuronal nitric oxide synthase (nNOS) is alternatively spliced. An nNOS splice variant form, nNOS-mu, was first found to be selectively expressed in rat skeletal muscle and heart. To date, the expression of nNOS-mu in the brain has not been well characterized. The aim of this study was to determine whether nNOS-mu is expressed in rat brain, and whether nNOS-mu exhibits a specific expression pattern. To analyze the expression of nNOS-mu, we generated a monoclonal antibody that is specific for nNOS-mu. An immunoblot analysis using this antibody showed that nNOS-mu is expressed in the rat brain at a measurable level, which was 10.3% of total nNOSs. In rat brain, the nNOS-mu expression was high in the mesencephalon and the cerebellum. nNOS-mu was immunohistochemically localized in neurites and perikarya of large neurons. In the cerebellum, granule cells showed marked staining, while weak staining was detected in basket and stellate cells. This expression pattern is different from that described for nNOS and suggests that nNOS-mu plays unique roles in different neurons.     

The Na+-dependent l-ascorbic acid transporter SVCT2 expressed in brainstem cells, neurons, and neuroblastoma cells is inhibited by flavonoids

Ascorbic acid (AA) is best known for its role as an essential nutrient in humans and other species. As the brain does not synthesize AA, high levels are achieved in this organ by specific uptake mechanisms, which concentrate AA from the bloodstream to the CSF and from the CSF to the intracellular compartment. Two different isoforms of sodium–vitamin C co-transporters (SVCT1 and SVCT2) have been cloned. Both SVCT proteins mediate high affinity Na⁺-dependent l -AA transport and are necessary for the uptake of vitamin C in many tissues. In the adult brain the expression of SVCT2 was observed in the hippocampus and cortical neurons by in situ hybridization; however, there is no data regarding the expression and distribution of this transporter in the fetal brain. The expression of SVCT2 in embryonal mesencephalic neurons has been shown by RT-PCR suggesting an important role for vitamin C in dopaminergic neuronal differentiation. We analyze SVCT2 expression in human and rat developing brain by RT-PCR. Additionally, we study the normal localization of SVCT2 in rat fetal brain by immunohistochemistry and in situ hybridization demonstrating that SVCT2 is highly expressed in the ventricular and subventricular area of the rat brain. SVCT2 expression and function was also confirmed in neurons isolated from brain cortex and cerebellum. The kinetic parameters associated with the transport of AA in cultured neurons and neuroblastoma cell lines were also studied. We demonstrate two different affinity transport components for AA in these cells. Finally, we show the ability of different flavonoids to inhibit AA uptake in normal or immortalized neurons. Our data demonstrates that brain cortex and cerebellar stem cells, neurons and neuroblastoma cells express SVCT2. Dose-dependent inhibition analysis showed that quercetin inhibited AA transport in cortical neurons and Neuro2a cells.     

SVCT2 Is Expressed by Cerebellar Precursor Cells,Which Differentiate into Neurons in Response to Ascorbic Acid

Ascorbic acid (AA) is a known antioxidant that participates in a wide range of processes, including stem cell differentiation. It enters the cell through the sodium-ascorbate co-transporter SVCT2, which is mainly expressed by neurons in the adult brain. Here, we have characterized SVCT2 expression in the postnatal cerebellum in situ, a model used for studying neurogenesis, and have identified its expression in granular precursor cells and mature neurons. We have also detected SVCT2 expression in the cerebellar cell line C17.2 and in postnatal cerebellum-derived neurospheres in vitro and have identified a tight relationship between SVCT2 expression and that of the stem cell-like marker nestin. AA supplementation potentiates the neuronal phenotype in cerebellar neural stem cells by increasing the expression of the neuronal marker β III tubulin. Stable over-expression of SVCT2 in C17.2 cells enhances β III tubulin expression, but it also increases cell death, suggesting that AA transporter levels must be finely tuned during neural stem cell differentiation.     

cDNA cloning of putative rat acetyl-CoA transporter and its expression pattern in brain

Rat acetyl-CoA transporter gene (Acatn) encodes a hydrophobic multi-transmembrane protein involved in the O-acetylation of gangliosides. O-acetylated gangliosides have been found to play important roles in the embryonic development of the nervous system. We have isolated rat Acatn cDNA by PCR cloning. The amino acid sequence of rat Acatn exhibited 92% and 96% homology with human and mouse sequences, respectively. The mRNA was expressed in brain at all developmental stages. Acatn expression was higher in embryonic and postnatal rats than in adult rats. Cellular localization of Acatn mRNA in adult rat brain was also analyzed by in situ hybridization. Acatn mRNA expression was detected in the neuronal cells of cerebellum, hippocampus, hypothalamus, cortex, olfactory bulb, and dorsal and ventral anterior olfactory nucleus in adult rat brain.     

Molecular targets for steroids in airway vascular smooth muscle

The actions of norepinephrine (NE) released from airway sympathetic nerves are partially terminated by the extraneuronal catecholamine uptake. Because various steroid hormones inhibit extraneuronal uptake, it could be responsible for the airway vasoconstriction caused by inhaled glucocorticosteroids (GSs) in vivo. Using bronchial arteries obtained from donor lungs rejected for transplantation, we showed that a plasma membrane-associated transporter is responsible for NE uptake by airway vascular smooth muscle. We identified this transporter, namely the extraneuronal monoamine transporter (EMT), by demonstrating its function and mRNA expression. Furthermore, we showed that the rapid, nongenomic inhibitory GS effect on EMT is likely mediated through the activation of specific K+ channels in the plasma membrane. We believe that our studies identified new molecular targets for GSs in modulating noradrenergic control of airway vascular tone.     

Lgr5 Marks Post-Mitotic,Lineage Restricted Cerebellar Granule Neurons during Postnatal Development

Wnt signaling regulates self-renewal and fate commitment of stem and progenitor cells in development and homeostasis. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) is a co-receptor for Wnt signaling that marks highly proliferative stem and progenitor cells in many epithelial tissue types. Wnt signaling instructs neural developmental and homeostatic processes; however, Lgr5 expression in the developing and adult brain has not been characterized. Here we report that Lgr5 is expressed in the postnatal cerebellum during the maturation and synaptogenesis of cerebellar granule neurons (CGNs), processes controlled by Wnt signaling. Using a transgenic reporter mouse for in vivo Lgr5 expression analysis and lineage tracing, we reveal that Lgr5 specifically identified CGNs and was restricted temporally to the CGN maturation phase within the internal granule layer, but absent in the adult brain. Cells marked by Lgr5 were lineage restricted, post-mitotic and long-lived. The ligand for Lgr5, R-spondin, was secreted in a paracrine fashion that evolved during the maturation of CGNs, which coincided with the Lgr5 expression pattern. Our findings provide potential new insight into the critical regulation of Wnt signaling in the developing cerebellum and support a novel role for Lgr5 in the regulation of post-mitotic cells.     

Different distributions of selenoprotein W and thioredoxin during postnatal brain development and embryogenesis

Whereas the levels of other selenoproteins in the brain decrease when selenium is deficient, the level of selenoprotein W (Se-W) is maintained, suggesting that it has a critical role in the brain. Previously, we reported that Se-W is a GSH-dependent antioxidant [Jeong et al. (2002)]. In this study, the expression of Se-W and thioredoxin (Trx) in the brain and during embrynic development was analyzed by an in situ hybridization technique. Se-W mRNA was highly expressed in the cortex, dentate gyrus, and hippocampus of postnatal rat brains, and in the spinal cord and brain of developing embryos. In contrast, Trx mRNA was highly expressed in the cerebellum, olfactory bulb, and dentate gyrus of postnatal rat brains, and in the liver, telencephalon, and back muscle of developing embryos. Thus these two antioxidant proteins have different and non-overlapping expression patterns. The distribution of Se-W suggests that it plays an important role as an antioxidant in the developing brain and embryo.     

Autoradiographic localization of peptide YY and neuropeptide Y binding sites in the medulla oblongata

Peptide YY is a highly potent emetic when given intravenously in dogs. We hypothesized that the area postrema, a small brain stem nucleus that acts as a chemoreceptive trigger zone for vomiting and lies outside the blood-brain barrier, might have receptors that PYY would bind to, in order to mediate the emetic response. We prepared [125I]PYY and used autoradiography to show that high affinity binding sites for this ligand were highly localized in the area postrema and related nuclei of the dog medulla oblongata. Furthermore, the distribution of [125I]PYY binding sites in the rat medulla oblongata was very similar to that in the dog; the distribution of [125I]PYY binding sites throughout the rat brain was seen to be similar to the distribution of [125I]NPY binding sites.     

The organic cation transporters (OCT1, OCT2, EMT) and the plasma membrane monoamine transporter (PMAT) show differential distribution and cyclic expression pattern in human endometrium and early pregnancy decidua

The non-neuronal monoamine transporters (OCT1, OCT2, EMT, and PMAT) play a key role in the clearance of monoamines from extracellular compartments. In a previous report we described endometrial distribution and cyclic variation of the vesicular monoamine transporter (VMAT2) mRNA and the neuronal norepinephrine transporter (NET) mRNA. In the present study we used in situ hybridization, real-time PCR and immunohistochemistry to reveal tissue distribution and cyclic variation of mRNA for the non-neuronal monoamine transporters in the human endometrium and early pregnancy decidua. We found that non-neuronal monoamine transporters are predominantly expressed in the stroma. The plasma membrane monoamine transporter (PMAT) mRNA expression peaked in the proliferative phase, whereas the extra-neuronal monoamine transporter (EMT) mRNA expression peaked in the secretory phase. The organic cation transporter 2 (OCT2) mRNA expression was exclusively detected in few scattered stromal cells and OCT1 mRNA was not detected at all. Our present results demonstrate that PMAT, EMT, and OCT2 transporters are expressed in the endometrial stroma and can potentially regulate reuptake of monoamines in general and histamine in particular. Taken together with our previous finding of VMAT2 mRNA in epithelial cells, we suggest a paracrine interaction between stromal and epithelial cells, which may modulate certain steps of the reproductive process.     

Postnatal Development and Possible Ligand Function of the CNS-Specific Membrane Glycoprotein CNSgp 130

CNSgp 130 is a CNS-specific membrane glycoprotein present in large amounts in the adult mammalian CNS. Using immunohistological techniques, we demonstrated that CNSgp130 is not detectable in the rat cerebellum at birth, and does not appear in the cerebellum until the tenth day of postnatal life. It is expressed first in the white matter of the cerebellar folia, and subsequently (by day 14) it is expressed also in the molecular layer. Expression in the granular layer is not seen until the 18th day of postnatal life, by which time the adult pattern of expression is established. CNSgp130 is also not detectable in the cerebrum at birth. However, it is expressed weakly but diffusely in the cerebrum by the fourth day of life. By the 10th day, there is strong expression in the cerebrum, in marked contrast to its virtual absence from the cerebellum at this stage. By quantitative absorption analysis, CNSgp 130 was undetectable on the day of birth, and increased steadily to 80% of adult values by the 22nd day of postnatal life. Binding studies with pure CNSgp130 demonstrated a Pronase-sensitive ligand in adult chicken brain. This ligand was absent from neonatal rat brain and non-CNS tissues.     

EphB3 receptor and ligand expression in the adult rat brain

Summary Eph receptors and ligands are two families of proteins that control axonal guidance during development. Their expression was originally thought to be developmentally regulated but recent work has shown that several EphA receptors are expressed postnatally. The EphB3 receptors are expressed during embryonic development in multiple regions of the central nervous system but their potential expression and functional role in the adult brain is unknown. We used in situ hybridization, immunohistochemistry, and receptor affinity probe in situ staining to investigate EphB3 receptors mRNA, protein, and ligand (ephrin-B) expression, respectively, in the adult rat brain. Our results indicate that EphB3 receptor mRNA and protein are constitutively expressed in discrete regions of the adult rat brain including the cerebellum, raphe pallidus, hippocampus, entorhinal cortex, and both motor and sensory cortices. The spatial profile of EphB3 receptors was co-localized to regions of the brain that had a high level of EphB3 receptor binding ligands. Its expression pattern suggests that EphB3 may play a role in the maintenance of mature neuronal connections or re-arrangement of synaptic connections during late stages of development.     

Neural mechanisms of emesis

Emesis is a reflex, developed to different degrees in different species, that allows an animal to rid itself of ingested toxins or poisons. The reflex can be elicited either by direct neuronal connections from visceral afferent fibers, especially those from the gastrointestinal tract, or from humoral factors. Emesis from humoral factors depends on the integrity of the area postrema; neurons in the area postrema have excitatory receptors for emetic agents. Emesis from gastrointestinal afferents does not depend on the area postrema, but probably the reflex is triggered by projections to some part of the nucleus tractus solitarius. As with a variety of other complex motor functions regulated by the brain stem, it is likely that the sequence of muscle excitation and inhibition is controlled by a central pattern generator located in the nucleus tractus solitarius, and that information from humoral factors via the area postrema and visceral afferents via the vagus nerve converge at this point. This central pattern generator, like those for motor functions such as swallowing, presumably projects to the various motor nuclei, perhaps through interneuronal pathways, to elicit the sequential excitation and inhibition that controls the reflex.     

Distinct spatio-temporal expression of ABCA and ABCG transporters in the developing and adult mouse brain

Using in situ hybridization for the mouse brain, we analyzed developmental changes in gene expression for the ATP-binding cassette (ABC) transporter subfamilies ABCA1-4 and 7, and ABCG1, 2, 4, 5 and 8. In the embryonic brains, ABCA1 and A7 were highly expressed in the ventricular (or germinal) zone, whereas ABCA2, A3 and G4 were enriched in the mantle (or differentiating) zone. At the postnatal stages, ABCA1 was detected in both the gray and white matter and in the choroid plexus. On the other hand, ABCA2, A3 and A7 were distributed in the gray matter. In addition, marked up-regulation of ABCA2 occurred in the white matter at 14 days-of-age when various myelin protein genes are known to be up-regulated. In marked contrast, ABCA4 was selective to the choroid plexus throughout development. ABCG1 was expressed in both the gray and white matters, whereas ABCG4 was confined to the gray matter. ABCG2 was diffusely and weakly detected throughout the brain at all stages examined. Immunohistochemistry of ABCG2 showed its preferential expression on the luminal membrane of brain capillaries. Expression signals for ABCG5 and G8 were barely detected at any stages. The distinct spatio-temporal expressions of individual ABCA and G transporters may reflect their distinct cellular expressions in the developing and adult brains, presumably, to regulate and maintain lipid homeostasis in the brain.