Muscarinic M5 receptors trigger acetylcholine‐induced Ca2+ signals and nitric oxide release in human brain microvascular endothelial cells. Issue 4 (7th September 2018)

• Record Type: Journal Article
• Authors: Zuccolo, Estella; Laforenza, Umberto; Negri, Sharon; Botta, Laura; Berra‐Romani, Roberto; Faris, Pawan; Scarpellino, Giorgia; Forcaia, Greta; Pellavio, Giorgia; Sancini, Giulio; Moccia, Francesco
• Is Part Of: Journal of cellular physiology. Volume 234:Issue 4(2019:Apr.)
• Page Start: 4540
• Page End: 4562
• Publication Date: 2018-09-07
• Subjects: acetylcholine -- Ca2+ signaling -- hCMEC/D3 -- M5 muscarinic receptors -- nitric oxide; Physiology -- Periodicals; Cell physiology -- Periodicals; 571.6
• DOI: 10.1002/jcp.27234
• Languages: English
• ISSNs: 0021-9541

Abstract

Abstract: 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 Ca 2+ ‐dependent manner in hCMEC/D3 cells, a widespread model of human brain microvascular endothelial cells. Ach induced a dose‐dependent increase in intracellular Ca 2+ concentration ([Ca 2+ ] 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 Ca 2+ response to Ach was mediated by InsP3 R3, TPC1–2, and store‐operated Ca 2+ 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 Ca 2+ buffer. Moreover, the pharmacological blockade of the Ca 2+ 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 Ca 2+ signal via M5‐mAchRs. Abstract : Acetylcholine induces Ca 2+ ‐dependent nitric oxide release in human brain microvascular endothelial cells by binding to M5 muscarinic receptors. This finding sheds novel light on the mechanism by which acetylcholine triggers neurovascular coupling in the brain.