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 (U73122 and 2-APB) showed a PLC-IP₃ cascade-mediated Ca2+ release, indicating that the nucleotide-induced Ca2+ signal was mainly related to P2Y_{2, 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.