Glutamate triggers intracellular Ca 2+ oscillations and nitric oxide release by inducing NAADP- and InsP 3 -dependent Ca 2+ release in mouse brain endothelial cells

Estella Zuccolo, Dlzar A. Kheder, Dmitry Lim, Angelica Perna, Francesca Di Nezza, Laura Botta, Giorgia Scarpellino, Sharon Negri, Simona Martinotti, Teresa Soda, Greta Forcaia, Laura Riboni, Elia Ranzato, Giulio Sancini, Luigi Ambrosone, Egidio D’Angelo, Germano Guerra, Francesco Moccia

Research output: Contribution to journalArticlepeer-review


The neurotransmitter glutamate increases cerebral blood flow by activating postsynaptic neurons and presynaptic glial cells within the neurovascular unit. Glutamate does so by causing an increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) in the target cells, which activates the Ca 2+ /Calmodulin-dependent nitric oxide (NO) synthase to release NO. It is unclear whether brain endothelial cells also sense glutamate through an elevation in [Ca 2+ ] i and NO production. The current study assessed whether and how glutamate drives Ca 2+ -dependent NO release in bEND5 cells, an established model of brain endothelial cells. We found that glutamate induced a dose-dependent oscillatory increase in [Ca 2+ ] i , which was maximally activated at 200 μM and inhibited by α-methyl-4-carboxyphenylglycine, a selective blocker of Group 1 metabotropic glutamate receptors. Glutamate-induced intracellular Ca 2+ oscillations were triggered by rhythmic endogenous Ca 2+ mobilization and maintained over time by extracellular Ca 2+ entry. Pharmacological manipulation revealed that glutamate-induced endogenous Ca 2+ release was mediated by InsP 3 -sensitive receptors and nicotinic acid adenine dinucleotide phosphate (NAADP) gated two-pore channel 1. Constitutive store-operated Ca 2+ entry mediated Ca 2+ entry during ongoing Ca 2+ oscillations. Finally, glutamate evoked a robust, although delayed increase in NO levels, which was blocked by pharmacologically inhibition of the accompanying intracellular Ca 2+ signals. Of note, glutamate induced Ca 2+ -dependent NO release also in hCMEC/D3 cells, an established model of human brain microvascular endothelial cells. This investigation demonstrates for the first time that metabotropic glutamate-induced intracellular Ca 2+ oscillations and NO release have the potential to impact on neurovascular coupling in the brain.

Original languageEnglish
Pages (from-to)3538-3554
Number of pages17
JournalJournal of Cellular Physiology
Issue number4
Publication statusPublished - Apr 1 2019


  • Ca oscillations
  • endothelial cells
  • glutamate
  • neurovascular coupling (NVC)
  • nitric oxide

ASJC Scopus subject areas

  • Physiology
  • Clinical Biochemistry
  • Cell Biology


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