TY - JOUR

T1 - Purine receptors and Ca 2+ signalling in the human blood-brain barrier endothelial cell line hCMEC/D3

AU - Bintig, Willem

AU - Begandt, Daniela

AU - Schlingmann, Barbara

AU - Gerhard, Linda

AU - Pangalos, Maria

AU - Dreyer, Lutz

AU - Hohnjec, Natalija

AU - Couraud, Pierre Olivier

AU - Romero, Ignacio A.

AU - Weksler, Babette B.

AU - Ngezahayo, Anaclet

N1 - Funding Information: 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.

PY - 2012/3

Y1 - 2012/3

N2 - 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 Ca 2+-imaging techniques to hCMEC/D3 cells. Reverse transcription polymerase chain reaction showed the expression of the G-protein-coupled receptors P2Y 2-, P2Y 6-, P2Y 11-as well as the ionotropic P2X 4-, P2X 5-and P2X 7-receptors. Fura-2 ratiometry revealed that adenosine triphosphate (ATP) or uridine triphosphate (UTP) mediated a change in the intracellular Ca 2+ concentration ([Ca 2+] i) from 150 to 300 nM in single cells. The change in [Ca 2+] 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-IP 3 cascade-mediated Ca 2+ release, indicating that the nucleotide-induced Ca 2+ signal was mainly related to P2Y 2, 6 and 11 receptors. The gene silencing of the P2Y 2 receptor reduced the ATP-or UTP-induced Ca 2+ signal and suppressed the Ca 2+ signal mediated by P2Y 6 and P2Y 11 more specific agonists like UDP (P2Y 6), BzATP (P2Y 11) and ATPγS (P2Y 11). This report identifies the P2Y 2 receptor subtype as the main purine receptor involved in Ca 2+ signalling of the hCMEC/D3 cells.

AB - 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 Ca 2+-imaging techniques to hCMEC/D3 cells. Reverse transcription polymerase chain reaction showed the expression of the G-protein-coupled receptors P2Y 2-, P2Y 6-, P2Y 11-as well as the ionotropic P2X 4-, P2X 5-and P2X 7-receptors. Fura-2 ratiometry revealed that adenosine triphosphate (ATP) or uridine triphosphate (UTP) mediated a change in the intracellular Ca 2+ concentration ([Ca 2+] i) from 150 to 300 nM in single cells. The change in [Ca 2+] 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-IP 3 cascade-mediated Ca 2+ release, indicating that the nucleotide-induced Ca 2+ signal was mainly related to P2Y 2, 6 and 11 receptors. The gene silencing of the P2Y 2 receptor reduced the ATP-or UTP-induced Ca 2+ signal and suppressed the Ca 2+ signal mediated by P2Y 6 and P2Y 11 more specific agonists like UDP (P2Y 6), BzATP (P2Y 11) and ATPγS (P2Y 11). This report identifies the P2Y 2 receptor subtype as the main purine receptor involved in Ca 2+ signalling of the hCMEC/D3 cells.

KW - G-Protein

KW - Gene silencing

KW - Neurovascular unit

KW - P2 receptors

KW - siRNA

UR - http://www.scopus.com/inward/record.url?scp=84857688640&partnerID=8YFLogxK

U2 - 10.1007/s11302-011-9262-7

DO - 10.1007/s11302-011-9262-7

M3 - Article

AN - SCOPUS:84857688640

VL - 8

SP - 71

EP - 80

JO - Purinergic signalling

JF - Purinergic signalling

SN - 1573-9538

IS - 1

ER -