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

摘要

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 ( 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.