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Zuccolo, Estella; Kheder, Dlzar A; Lim, Dmitry; Perna, Angelica; Nezza, Francesca Di; Botta, Laura; Scarpellino, Giorgia; Negri, Sharon; Martinotti, Simona; Soda, Teresa; Forcaia, Greta; Riboni, Laura; Ranzato, Elia; Sancini, Giulio; Ambrosone, Luigi; D'Angelo, Egidio; Guerra, Germano; Moccia, Francesco
Journal of cellular physiology, 04/2019, Letnik: 234, Številka: 4Journal Article
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 concentration (Ca ) in the target cells, which activates the Ca /Calmodulin-dependent nitric oxide (NO) synthase to release NO. It is unclear whether brain endothelial cells also sense glutamate through an elevation in Ca and NO production. The current study assessed whether and how glutamate drives Ca -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 , 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 oscillations were triggered by rhythmic endogenous Ca mobilization and maintained over time by extracellular Ca entry. Pharmacological manipulation revealed that glutamate-induced endogenous Ca release was mediated by InsP -sensitive receptors and nicotinic acid adenine dinucleotide phosphate (NAADP) gated two-pore channel 1. Constitutive store-operated Ca entry mediated Ca entry during ongoing Ca oscillations. Finally, glutamate evoked a robust, although delayed increase in NO levels, which was blocked by pharmacologically inhibition of the accompanying intracellular Ca signals. Of note, glutamate induced Ca -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 oscillations and NO release have the potential to impact on neurovascular coupling in the brain.
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