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Uneven distribution of nucleoside transporters and intracellular enzymatic degradation prevent transport of intact [14C] adenosine across the sheep choroid plexus epithelium as a monolayer in primary culture.

ABSTRACT: BACKGROUND:Efflux transport of adenosine across the choroid plexus (CP) epithelium might contribute to the homeostasis of this neuromodulator in the extracellular fluids of the brain. The aim of this study was to explore adenosine transport across sheep CP epithelial cell monolayers in primary culture. METHODS:To explore transport of adenosine across the CP epithelium, we have developed a method for primary culture of the sheep choroid plexus epithelial cells (CPEC) on plastic permeable supports and analysed [14C] adenosine transport across this cellular layer, [14C] adenosine metabolism inside the cells, and cellular uptake of [14C] adenosine from either of the chambers. The primary cell culture consisted of an enriched epithelial cell fraction from the sheep fourth ventricle CP and was grown on laminin-precoated filter inserts. RESULTS AND CONCLUSION:CPEC grew as monolayers forming typical polygonal islands, reaching optical confluence on the third day after the seeding. Transepithelial electrical resistance increased over the time after seeding up to 85 +/- 9 Omega cm2 at day 8, while permeability towards [14C] sucrose, a marker of paracellular diffusion, simultaneously decreased. These cells expressed some features typical of the CPEC in situ, including three nucleoside transporters at the transcript level that normally mediate adenosine transport across cellular membranes. The estimated permeability of these monolayers towards [14C] adenosine was low and the same order of magnitude as for the markers of paracellular diffusion.However, inhibition of the intracellular enzymes, adenosine kinase and adenosine deaminase, led to a significant increase in transcellular permeability, indicating that intracellular phosphorylation into nucleotides might be a reason for the low transcellular permeability. HPLC analysis with simultaneous detection of radioactivity revealed that [14C] radioactivity which appeared in the acceptor chamber after the incubation of CPEC monolayers with [14C] adenosine in the donor chamber was mostly present as [14C] hypoxanthine, a product of adenosine metabolic degradation. Therefore, it appears that CPEC in primary cultures act as an enzymatic barrier towards adenosine. Cellular uptake studies revealed that concentrative uptake of [14C] adenosine was confined only to the side of these cells facing the upper or apical chamber, indicating uneven distribution of nucleoside transporters.

SUBMITTER: Redzic ZB

PROVIDER: S-EPMC1450313 | biostudies-literature | 2006 Mar

REPOSITORIES: biostudies-literature

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Uneven distribution of nucleoside transporters and intracellular enzymatic degradation prevent transport of intact [14C] adenosine across the sheep choroid plexus epithelium as a monolayer in primary culture.

Redzic Zoran B ZB Isakovic Aleksandra J AJ Misirlic Dencic Sonja T ST Popadic Dusan D Segal Malcolm B MB

Cerebrospinal fluid research 20060329

<h4>Background</h4>Efflux transport of adenosine across the choroid plexus (CP) epithelium might contribute to the homeostasis of this neuromodulator in the extracellular fluids of the brain. The aim of this study was to explore adenosine transport across sheep CP epithelial cell monolayers in primary culture.<h4>Methods</h4>To explore transport of adenosine across the CP epithelium, we have developed a method for primary culture of the sheep choroid plexus epithelial cells (CPEC) on plastic per ...[more]

PMID: 16571111

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Project description:BACKGROUND:Human equilibrative nucleoside transporters (hENTs) 1-3 and human concentrative nucleoside transporters (hCNTs) 1-3 in the human choroid plexus (hCP) play a role in the homeostasis of adenosine and other naturally occurring nucleosides in the brain; in addition, hENT1, hENT2 and hCNT3 mediate membrane transport of nucleoside reverse transcriptase inhibitors that could be used to treat HIV infection, 3'-azido-3'-deoxythymidine, 2'3'-dideoxycytidine and 2'3'-dideoxyinosine. This study aimed to explore the expression levels and functional activities of hENTs 1-3 and hCNTs 1-3 in human choroid plexus. METHODS:Freshly-isolated pieces of lateral ventricle hCP, removed for various clinical reasons during neurosurgery, were obtained under Local Ethics Committee approval. Quantification of mRNAs that encoded hENTs and hCNTs was performed by the hydrolysis probes-based reverse transcription real time-polymerase chain reaction (RT-qPCR); for each gene of interest and for 18 S ribosomal RNA, which was an endogenous control, the efficiency of PCR reaction (E) and the quantification cycle (Cq) were calculated. The uptake of [(3)H]inosine by the choroid plexus pieces was investigated to explore the functional activity of hENTs and hCNTs in the hCP. RESULTS:RT-qPCR revealed that the mRNA encoding the intracellularly located transporter hENT3 was the most abundant, with E(-Cq )value being only about 40 fold less that the E(-Cq )value for 18 S ribosomal RNA; mRNAs encoding hENT1, hENT2 and hCNT3 were much less abundant than mRNA for the hENT3, while mRNAs encoding hCNT1 and hCNT2 were of very low abundance and not detectable. Uptake of [(3)H]inosine by the CP samples was linear and consisted of an Na(+)-dependent component, which was probably mediated by hCNT3, and Na(+)-independent component, mediated by hENTs. The latter component was not sensitive to inhibition by S-(4-nitrobenzyl)-6-thioinosine (NBMPR), when used at a concentration of 0.5 muM, a finding that excluded the involvement of hENT1, but it was very substantially inhibited by 10 muM NBMPR, a finding that suggested the involvement of hENT2 in uptake. CONCLUSION:Transcripts for hENT1-3 and hCNT3 were detected in human CP; mRNA for hENT3, an intracellularly located nucleoside transporter, was the most abundant. Human CP took up radiolabelled inosine by both concentrative and equilibrative processes. Concentrative uptake was probably mediated by hCNT3; the equilibrative uptake was mediated only by hENT2. The hENT1 transport activity was absent, which could suggest either that this protein was absent in the CP cells or that it was confined to the basolateral side of the CP epithelium.

Project description:BackgroundAlthough fibroblast growth factor (Fgf) signalling plays crucial roles in several developing and mature tissues, little information is currently available on expression of Fgf2 during early choroid plexus development and whether Fgf2 directly affects the behaviour of the choroid plexus epithelium (CPe). The purpose of this study was to investigate expression of Fgf2 in rodent and human developing CPe and possible function of Fgf2, using in vitro models. The application of Fgf2 to brain in vivo can affect the whole tissue, making it difficult to assess specific responses of the CPe.MethodsExpression of Fgf2 was studied by immunohistochemistry in rodent and human embryonic choroid plexus. Effects of Fgf2 on growth, secretion, aggregation and gene expression was investigated using rodent CPe vesicles, a three-dimensional polarized culture model that closely mimics CPe properties in vivo, and rodent CPe monolayer cultures.ResultsFgf2 was present early in development of the choroid plexus both in mouse and human, suggesting the importance of this ligand in Fgf signalling in the developing choroid plexus. Parallel analysis of Fgf2 expression and cell proliferation during CP development suggests that Fgf2 is not involved in CPe proliferation in vivo. Consistent with this observation is the failure of Fgf2 to increase proliferation in the tri-dimensional vesicle culture model. The CPe however, can respond to Fgf2 treatment, as the diameter of CPe vesicles is significantly increased by treatment with this growth factor. We show that this is due to an increase in cell aggregation during vesicle formation rather than increased secretion into the vesicle lumen. Finally, Fgf2 regulates expression of the CPe-associated transcription factors, Foxj1 and E2f5, whereas transthyretin, a marker of secretory activity, is not affected by Fgf2 treatment.ConclusionFgf2 expression early in the development of both human and rodent choroid plexus, and its ability to modulate behaviour and gene expression in CPe, supports the view that Fgf signalling plays a role in the maintenance of integrity and function of this specialized epithelium, and that this role is conserved between rodents and humans.

Project description:The choroid plexus (CP) consists of specialized ependymal cells and underlying blood vessels and stroma producing the bulk of the cerebrospinal fluid (CSF). CP epithelial cells are considered the site of the internal blood-cerebrospinal fluid barrier, show epithelial characteristics (basal lamina, tight junctions), and express aquaporin-1 (AQP1) apically. In this study, we analyzed the expression of aquaporins in the human CP using immunofluorescence and qPCR. As previously reported, AQP1 was expressed apically in CP epithelial cells. Surprisingly, and previously unknown, many cells in the CP epithelium were also positive for aquaporin-4 (AQP4), normally restricted to ventricle-lining ependymal cells and astrocytes in the brain. Expression of AQP1 and AQP4 was found in the CP of all eight body donors investigated (3 males, 5 females; age 74-91). These results were confirmed by qPCR, and by electron microscopy detecting orthogonal arrays of particles. To find out whether AQP4 expression correlated with the expression pattern of relevant transport-related proteins we also investigated expression of NKCC1, and Na/K-ATPase. Immunostaining with NKCC1 was similar to AQP1 and revealed no particular pattern related to AQP4. Co-staining of AQP4 and Na/K-ATPase indicated a trend for an inverse correlation of their expression. We hypothesized that AQP4 expression in the CP was caused by age-related changes. To address this, we investigated mouse brains from young (2 months), adult (12 months) and old (30 months) mice. We found a significant increase of AQP4 on the mRNA level in old mice compared to young and adult animals. Taken together, we provide evidence for AQP4 expression in the CP of the aging brain which likely contributes to the water flow through the CP epithelium and CSF production. In two alternative hypotheses, we discuss this as a beneficial compensatory, or a detrimental mechanism influencing the previously observed CSF changes during aging.

Dataset Information

Uneven distribution of nucleoside transporters and intracellular enzymatic degradation prevent transport of intact [14C] adenosine across the sheep choroid plexus epithelium as a monolayer in primary culture.

Publications

Uneven distribution of nucleoside transporters and intracellular enzymatic degradation prevent transport of intact [14C] adenosine across the sheep choroid plexus epithelium as a monolayer in primary culture.

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