Abstract
BackgroundHuman 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.MethodsFreshly-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 [3H]inosine by the choroid plexus pieces was investigated to explore the functional activity of hENTs and hCNTs in the hCP.ResultsRT-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 [3H]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 μM, a finding that excluded the involvement of hENT1, but it was very substantially inhibited by 10 μM NBMPR, a finding that suggested the involvement of hENT2 in uptake.ConclusionTranscripts 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.
Highlights
Human equilibrative nucleoside transporters 1-3 and human concentrative nucleoside transporters 1-3 in the human choroid plexus 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
Thioinosine (NBMPR) and their subcellular localizations. These processes are mediated by the proteins hENT1, hENT2 [2] and hENT3, which is predominantly located intracellularly, in lysosomes [3] and mitochondria [4], human placental cells expressed this protein in the cell membrane [4]
The results in this study indicate that human choroid plexuses (CPs) expressed three equilibrative nucleoside transporter family members, hENTs 1-3, but only one type of concentrative nucleoside transporter, hCNT3; the uptake of inosine by freshly isolated CP pieces was found to be mediated primarily by just two transporter types, hENT2 and hCNT3
Summary
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. Nucleosides play key roles as precursors for nucleotide synthesis by salvage pathways in a number of human tissues Their cellular uptake and release are dependent on the activity of one or more members of two families of membrane proteins, the human equilibrative nucleoside transporters (hENTs) and the human concentrative thioinosine (NBMPR) and their subcellular localizations. These processes are mediated by the proteins hENT1, hENT2 [2] (with hENT1 being 1000-fold more sensitive to NBMPR inhibition than hENT2 [2]) and hENT3, which is predominantly located intracellularly, in lysosomes [3] and mitochondria [4], human placental cells expressed this protein in the cell membrane [4]. Efflux transport of adenosine across the BCSFB depends primarily on three factors: the surface area between the CP epithelium and the CSF, which is expanded by the presence of microvilli on the apical side of the epithelium and complex interdigitations between the lateral walls of the epithelial cells [8]; the presence of nucleoside transporters in the CP epithelium; and the concentration gradient for this nucleoside across that epithelial layer
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