Expression and functional activity of nucleoside transporters in human choroid plexus

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.

Molecular Biology of Nucleoside Transporters and their Distributions and Functions in the Brain

“…Endothelial cells of the BBB and BCSFB epithelial cells of the choroid plexus interface with the peripheral blood supply and have key roles in CNS nucleoside and nucleobase homeostasis [1,95,98,107,110]. Reviewed in Section 3.7, these cells exhibit multiple NTs on both brain-and bloodfacing membrane surfaces, and bi-directional transcellular movement of nucleosides and nucleobases across the BBB and BCSFB has been demonstrated.…”

“…Rabbit choroid plexus tissue slices were also shown to possess a separate sodium-dependent mechanism for nucleobase transport [106]. Isolated portions of human lateral ventricle choroid plexus have been investigated by quantitative RT-PCR (hENT3 > hENT1, hENT2, hCNT3 >> hCNT1, hCNT2) and by inosine uptake (hCNT3 plus hENT2) [107].…”

Transport Mechanisms of Nucleosides and Nucleoside Analogues ReverseTranscriptase Inhibitors in the Brain

“…Human CP samples took up radiolabelled inosine by both concentrative and equilibrative processes. However, the equilibrative uptake was mediated only by hENT2, while hENT1 transport activity was absent, which could suggested either that this protein was absent in the human CP or that it was confined to the basolateral side of the CP epithelium (which did not come in contact with uptake buffer in this study) (Redzic et al, 2010a). Thus, several lines of evidences suggest that polarized distribution of nucleoside transporters at the BBB and in the CP epithelium, with CNTs located on sides facing brain extracellular fluids (ISF and CSF) and equlibrative transport across the opposite, blood facing side, may play an important role in the removal of nucleosides from brain fluids.…”

“…Human choroid plexus expressed abundantly hENT3 mRNA (Redzic et al, 2010a). However, so far there are no data demonstrating functional role of this transporter in the brain.…”

“…The remaining adenosine uptake at apical cell surfaces that was sodium-independent was NBMPRinsensitive, thus could be attributed to rENT2 ( Figure 2B) (Redzic et al, 2005). Expression of nucleoside transporters at transcript level and nucleoside uptake was studied in isolated portions of human lateral ventricle choroid plexuses that were obtained during neurosurgery (Redzic et al, 2010a). Quantitative RT-PCR revealed that transcripts for hENT1-3 and hCNT3 were present with mRNA for hENT3 being the most abundant.…”