Details
| Original language | English |
|---|---|
| Pages (from-to) | 71-80 |
| Number of pages | 10 |
| Journal | Purinergic signalling |
| Volume | 8 |
| Issue number | 1 |
| Publication status | Published - Mar 2012 |
Abstract
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.
Keywords
- G-Protein, Gene silencing, Neurovascular unit, P2 receptors, siRNA
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Molecular Biology
- Neuroscience(all)
- Cellular and Molecular Neuroscience
- Biochemistry, Genetics and Molecular Biology(all)
- Cell Biology
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In: Purinergic signalling, Vol. 8, No. 1, 03.2012, p. 71-80.
Research output: Contribution to journal › Article › Research › peer review
}
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 -