Muscarinic M5 receptors trigger acetylcholine‐induced Ca 2+ signals and nitric oxide release in human brain microvascular endothelial cells
| Autor: | Roberto Berra-Romani, Estella Zuccolo, Laura Botta, Giorgia Pellavio, Greta Forcaia, Giorgia Scarpellino, Sharon Negri, Francesco Moccia, Pawan Faris, Umberto Laforenza, Giulio Sancini |
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| Přispěvatelé: | Zuccolo, E, Laforenza, U, Negri, S, Botta, L, Berra-Romani, R, Faris, P, Scarpellino, G, Forcaia, G, Pellavio, G, Sancini, G, Moccia, F |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Physiology Clinical Biochemistry Vasodilation hCMEC/D3 M5 muscarinic receptor Nitric oxide 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine BAPTA nitric oxide BIO/09 - FISIOLOGIA Muscarinic acetylcholine receptor medicine Receptor Ca2+ signaling Basal forebrain Chemistry Cell Biology STIM2 acetylcholine Cell biology 030104 developmental biology 030220 oncology & carcinogenesis Acetylcholine medicine.drug |
| Zdroj: | Journal of Cellular Physiology. 234:4540-4562 |
| ISSN: | 1097-4652 0021-9541 |
| DOI: | 10.1002/jcp.27234 |
| Popis: | Basal forebrain neurons control cerebral blood flow (CBF) by releasing acetylcholine (Ach), which binds to endothelial muscarinic receptors to induce nitric (NO) release and vasodilation in intraparenchymal arterioles. Nevertheless, the mechanism whereby Ach stimulates human brain microvascular endothelial cells to produce NO is still unknown. Herein, we sought to assess whether Ach stimulates NO production in a Ca2+ -dependent manner in hCMEC/D3 cells, a widespread model of human brain microvascular endothelial cells. Ach induced a dose-dependent increase in intracellular Ca2+ concentration ([Ca2+ ]i ) that was prevented by the genetic blockade of M5 muscarinic receptors (M5-mAchRs), which was the only mAchR isoform coupled to phospholipase Cβ (PLCβ) present in hCMEC/D3 cells. A comprehensive real-time polymerase chain reaction analysis revealed the expression of the transcripts encoding for type 3 inositol-1,4,5-trisphosphate receptors (InsP3 R3), two-pore channels 1 and 2 (TPC1-2), Stim2, Orai1-3. Pharmacological manipulation showed that the Ca2+ response to Ach was mediated by InsP3 R3, TPC1-2, and store-operated Ca2+ entry (SOCE). Ach-induced NO release, in turn, was inhibited in cells deficient of M5-mAchRs. Likewise, Ach failed to increase NO levels in the presence of l-NAME, a selective NOS inhibitor, or BAPTA, a membrane-permeant intracellular Ca2+ buffer. Moreover, the pharmacological blockade of the Ca2+ response to Ach also inhibited the accompanying NO production. These data demonstrate for the first time that synaptically released Ach may trigger NO release in human brain microvascular endothelial cells by stimulating a Ca2+ signal via M5-mAchRs. |
| Databáze: | OpenAIRE |
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