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Purinergic Signalling

, Volume 8, Issue 1, pp 71–80 | Cite as

Purine receptors and Ca2+ signalling in the human blood–brain barrier endothelial cell line hCMEC/D3

  • Willem Bintig
  • Daniela Begandt
  • Barbara Schlingmann
  • Linda Gerhard
  • Maria Pangalos
  • Lutz Dreyer
  • Natalija Hohnjec
  • Pierre-Olivier Couraud
  • Ignacio A. Romero
  • Babette B. Weksler
  • Anaclet NgezahayoEmail author
Original Article

Abstract

The expression and physiology of purine receptors of the human blood–brain barrier endothelial cells were characterised by application of molecular biological, gene-silencing and Ca2+-imaging techniques to hCMEC/D3 cells. Reverse transcription polymerase chain reaction showed the expression of the G-protein-coupled receptors P2Y2-, P2Y6-, P2Y11- as well as the ionotropic P2X4-, P2X5- and P2X7-receptors. Fura-2 ratiometry revealed that adenosine triphosphate (ATP) or uridine triphosphate (UTP) mediated a change in the intracellular Ca2+ concentration ([Ca2+]i) from 150 to 300 nM in single cells. The change in [Ca2+]i corresponded to a fourfold to fivefold increase in the fluorescence intensity of Fluo-4, which was used for high-throughput experiments. Pharmacological dissection using different agonists [UTPγS, ATPγS, uridine diphosphate (UDP), adenosine diphosphate (ADP), BzATP, αβ-meATP] and antagonist (MRS2578 or NF340) as well as inhibitors of intracellular mediators (U73122 and 2-APB) showed a PLC-IP3 cascade-mediated Ca2+ release, indicating that the nucleotide-induced Ca2+ signal was mainly related to P2Y2, 6 and 11 receptors. The gene silencing of the P2Y2 receptor reduced the ATP- or UTP-induced Ca2+ signal and suppressed the Ca2+ signal mediated by P2Y6 and P2Y11 more specific agonists like UDP (P2Y6), BzATP (P2Y11) and ATPγS (P2Y11). This report identifies the P2Y2 receptor subtype as the main purine receptor involved in Ca2+ signalling of the hCMEC/D3 cells.

Keywords

P2 receptors G-Protein Neurovascular unit Gene silencing siRNA 

Notes

Acknowledgments

The authors thank Prof. Dr. Helge Küster and his team for discussion on the manuscript. The work was supported by the NANOTOME project (Biophotonik III) and by Boehringer Ingelheim International.

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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Willem Bintig
    • 1
  • Daniela Begandt
    • 1
  • Barbara Schlingmann
    • 1
    • 2
  • Linda Gerhard
    • 1
  • Maria Pangalos
    • 1
  • Lutz Dreyer
    • 1
  • Natalija Hohnjec
    • 3
  • Pierre-Olivier Couraud
    • 4
    • 5
    • 6
  • Ignacio A. Romero
    • 7
  • Babette B. Weksler
    • 8
  • Anaclet Ngezahayo
    • 1
    • 2
    Email author
  1. 1.Institute of BiophysicsLeibniz University HannoverHannoverGermany
  2. 2.Center of Systemic Neurosciences (ZSN)HannoverGermany
  3. 3.Institute for Plant Genetics, Unit IV-Plant GenomicsLeibniz University HannoverHannoverGermany
  4. 4.INSERM, U1016Institut CochinParisFrance
  5. 5.CNRS, UMR8104ParisFrance
  6. 6.Université Paris DescartesParisFrance
  7. 7.Department of Biological SciencesThe Open UniversityWalton HallUK
  8. 8.Weill Medical College of Cornell UniversityNew YorkUSA

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