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

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.