Confocal imaging of organic anion transport in intact rat choroid plexus.
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We used confocal microscopy and quantitative image analysis to follow the movement of the fluorescent organic anion fluorescein (FL) from bath to cell and cell to blood vessel in intact rat lateral choroid plexus. FL accumulation in epithelial cells and underlying vessels was rapid, concentrative, and reduced by other organic anions. At steady state, cell fluorescence exceeded bath fluorescence by a factor of 3-5, and vessel fluorescence exceeded cell fluorescence by a factor of approximately 2. In cells, FL distributed between diffuse and punctate compartments. Cell and vessel accumulation of FL decreased when metabolism was inhibited by KCN, when bath Na(+) was reduced from 130 to 26 mM, and when the Na(+) gradient was collapsed with ouabain. Cell and vessel accumulation increased by >50% when 1-10 microM glutarate was added to the bath. Finally, transport of FL and carboxyfluorescein (generated intracellularly from carboxyfluorescein diacetate) from cell to blood vessel was greatly diminished when medium K(+) concentration ([K(+)]) was increased 10-fold. These results 1) validate a new approach to the study of choroid plexus function, and 2) indicate a two-step mechanism for transepithelial organic anion transport: indirect coupling of uptake to Na(+) at the apical membrane and electrical potential-driven efflux at the basolateral membrane. (+info)
Expression and functional characterization of rat organic anion transporter 3 (rOat3) in the choroid plexus.
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We reported previously that an efficient efflux system for benzylpenicillin (PCG) is located on the choroid plexus (CP). In this study, we investigated the involvement of rat organic anion transporter 1 (rOat1; Slc22a6) and rOat3 (Slc22a8) in the uptake of PCG and p-aminohippurate (PAH) by the CP. Western blot analysis indicates the expression of rOat3, but not rOat1, on the CP, and immunohistochemical staining shows that rOat3 is localized on the brush border membrane of the choroid epithelial cells. PCG and PAH were found to be taken up by isolated rat CP, with K(m) values of 111 and 354 microM, respectively. A mutual inhibition study suggests that the same transporter is responsible for the uptake of PCG and PAH by isolated rat CP. This was confirmed by examining the effect of organic anions and cimetidine on their uptake. Estradiol-17beta-glucuronide and cimetidine were found to be selective inhibitors of rOat3. The inhibition constants of the inhibitors including estradiol-17beta-glucuronide and cimetidine were comparable for the uptake of PAH and PCG by isolated rat CP. In addition, these values were also comparable with those for rOat3, but not with those for rOat1. These results suggest that rOat3 is mainly responsible for the uptake of PCG and PAH by isolated rat CP, and it functions as one of the detoxification systems on the CP by removing its substrates from the cerebrospinal fluid. (+info)
Cloning, chromosomal organization and expression analysis of Neurl, the mouse homolog of Drosophila melanogaster neuralized gene.
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The Drosophila neuralized (neur) gene belongs to the neurogenic group of genes involved in regulating cell-cell interactions required for neural precursor development. neur mutant phenotypes include strong overcommitment to neural fates at the expense of epidermal fates. The human neuralized homolog (NEURL) has been recently determined and found to map to chromosome 10q25.1 within the region frequently deleted in malignant astrocytomas. Because of its potential importance in developmental processes, we analyzed the structure of the mouse homolog, Neurl, and its expression pattern in embryonic tissues. Neurl activity is detected from early developmental stages in several tissues and organs including neural tissues, limbs, the skeletal system, sense organs and internal organs undergoing epithelial-mesenchymal interactions. Neurl encodes a polypeptide associated with the plasma membrane but also detected in the cytoplasm. Similarly to the Drosophila gene, mammalian neuralized may code for an important regulatory factor. (+info)
Cellular localization of the diazepam binding inhibitor in glial cells with special reference to its coexistence with brain-type fatty acid binding protein.
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The diazepam binding inhibitor (DBI) was originally isolated from the brain as an intrinsic ligand of the benzodiazepine binding site on the type-A gamma-aminobutyric acid receptor (GABA(A) receptor). Its wide-spread distribution in non-neural tissues outside the brain suggests that DBI has various functions other than GABA-mediated neurotransmission. Since DBI is identical with the acyl-CoA binding protein, which has the ability to bind long chain acyl-CoA esters, the major function of DBI may possibly be related to lipid metabolism. This idea was supported by our previous study showing the consistent coexpression of DBI and fatty acid binding proteins (FABPs) in epithelia throughout the gastrointestinal tract. The present histochemical study focused on the distribution of DBI in neural tissues, and revealed a definite existence of DBI in non-neuronal supporting cells in both the central and peripheral nervous systems. In the brain, intense immunoreactivity for DBI was detected in the cerebellar Bergmann glia, olfactory ensheathing glia, subgranular layer of the dentate gyrus, and retinal Muller cells. In the peripheral nervous system, satellite cells in sensory/autonomic ganglia, Schwann cells, and sustentacular cells in the adrenal medulla were immunoreactive to a DBI antibody. Moreover, the colocalization of DPI and brain-type FABP (B-FABP) was observed in most of the non-neuronal supporting cells mentioned above, indicating that DBI and B-FABP are cooperatively involved in the energy metabolism of astrocytes and related cells, which are thought to support neuronal development and functions. (+info)
Unusual small choroid plexus cyst obstructing the foramen of monroe: case report.
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This is a case report of unusual case of choroid plexus cyst at the right foramen of Monro in the anterior third ventricle that caused unilateral obstructive hydrocephalus. The value of small-FOV thin-section MR imaging in the diagnosis of small lesions of the foramen of Monroe is demonstrated. The immunohistochemical findings in choroid epithelial cysts in comparison with those of other types of cysts at this location are discussed. (+info)
Synaptic multiprotein complexes associated with 5-HT(2C) receptors: a proteomic approach.
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Membrane-bound receptors such as tyrosine kinases and ionotropic receptors are associated with large protein networks structured by protein-protein interactions involving multidomain proteins. Although these networks have emerged as a general mechanism of cellular signalling, much less is known about the protein complexes associated with G-protein-coupled receptors (GPCRs). Using a proteomic approach based on peptide affinity chromatography followed by mass spectrometry and immunoblotting, we have identified 15 proteins that interact with the C- terminal tail of the 5-hydroxytryptamine 2C (5-HT(2C)) receptor, a GPCR. These proteins include several synaptic multidomain proteins containing one or several PDZ domains (PSD95 and the proteins of the tripartite complex Veli3-CASK-Mint1), proteins of the actin/spectrin cytoskeleton and signalling proteins. Coimmunoprecipitation experiments showed that 5-HT(2C) receptors interact with PSD95 and the Veli3-CASK-Mint1 complex in vivo. Electron microscopy also indicated a synaptic enrichment of Veli3 and 5-HT(2C) receptors and their colocalization in microvilli of choroidal cells. These results indicate that the 5-HT(2C) receptor is associated with protein networks that are important for its synaptic localization and its coupling to the signalling machinery. (+info)
Impaired organic anion transport in kidney and choroid plexus of organic anion transporter 3 (Oat3 (Slc22a8)) knockout mice.
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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. (+info)
Role of PEPT2 in peptide/mimetic trafficking at the blood-cerebrospinal fluid barrier: studies in rat choroid plexus epithelial cells in primary culture.
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Recent studies have established the functional and molecular presence of a high-affinity peptide transporter, PEPT2, in whole tissue rat choroid plexus. However, the precise membrane location and directionality of PEPT2-mediated transport is uncertain at present. In this study, we examined the transport kinetics of a model dipeptide, glycylsarcosine (GlySar), along with the protein expression of PEPT2 using primary cell cultures of choroidal epithelium from neonatal rats. GlySar accumulation and transepithelial transport were 3 to 4 times higher when introduced from the apical as opposed to the basal side of the monolayers. GlySar apical uptake was also stimulated by an inwardly directed proton gradient. The uptake of GlySar was inhibited by di/tripeptides, carnosine, and alpha-amino cephalosporins but was unaffected by amino acids, cephalosporins lacking an alpha-amino group, and organic anions and cations. The Michaelis constant (K(m)) of GlySar was 59.6 microM for apical uptake and 1.4 mM for basal uptake; this is consistent with the high-affinity properties of PEPT2 at the apical membrane. Immunoblot analyses and immunofluorescent confocal microscopy demonstrated the presence of PEPT2, but not PEPT1, in rat choroid plexus epithelial cells. Moreover, PEPT2 was present in the apical and subapical regions of the cell but was absent in the basolateral membrane. These findings demonstrate, for the first time, that PEPT2 protein is present at the apical membrane of choroidal epithelial cells and that it is functionally active at this membrane surface. The results suggest that PEPT2 may have a role in the efflux of peptides and/or mimetics from cerebrospinal fluid to the blood. (+info)