Purine receptors and Ca2+ signalling in the human blood–brain barrier endothelial cell line hCMEC/D3
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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.
KeywordsP2 receptors G-Protein Neurovascular unit Gene silencing siRNA
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.
- 15.King BF, Townsend-Nicholson A (2003) Nucleotide and nucleoside receptors. Tocris Rev 23:1–12Google Scholar
- 17.Abbracchio MP, Burnstock G, Boeynaems JM, Barnard EA, Boyer JL, Kennedy C, Knight GE, Fumagalli M, Gachet C, Jacobson KA, Weisman GA (2006) International Union of Pharmacology LVIII: update on the P2Y G protein-coupled nucleotide receptors: from molecular mechanisms and pathophysiology to therapy. Pharmacol Rev 58:281–341PubMedCrossRefGoogle Scholar
- 24.Weksler BB, Subileau EA, Perrière N, Charneau P, Holloway K, Leveque M, Tricoire-Leignel H, Nicotra A, Bourdoulous S, Turowski P, Male DK, Roux F, Greenwood J, Romero IA, Couraud PO (2005) Blood–brain barrier-specific properties of a human adult brain endothelial cell line. FASEB J 19:1872–1884PubMedGoogle Scholar
- 30.Brunschweiger A, Müller CE (2006) P2 receptors activated by uracil nucleotides—an update. Curr Med Chem 12:763–771Google Scholar
- 34.Ullmann H, Meis S, Hongwiset D, Marzian C, Wiese M, Nickel P, Communi D, Boeynaems JM, Wolf C, Hausmann R, Schmalzing G, Kassack MU (2005) Synthesis and structure activity relationships of suramin-derived P2Y11 receptor antagonists with nanomolar potency. J Med Chem 48:7040–7048PubMedCrossRefGoogle Scholar
- 39.Burnstock G (2006) Purinergic signalling—an overview. Novartis Found Symp 276:46–48Google Scholar
- 41.Hillmann P, Ko GY, Spinrath A, Raulf A, von Kügelgen I, Wolff SC, Nicholas RA, Kostenis E, Höltje HD, Müller CE (2009) Key determinants of nucleotide-activated G protein-coupled P2Y2 receptor function revealed by chemical and pharmacological experiments, mutagenesis and homology modeling. J Med Chem 52:2762–2775PubMedCrossRefGoogle Scholar