RESUMEN
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 Ca2+ concentration ([Ca2+ ]i ) in the target cells, which activates the Ca2+ /Calmodulin-dependent nitric oxide (NO) synthase to release NO. It is unclear whether brain endothelial cells also sense glutamate through an elevation in [Ca2+ ]i and NO production. The current study assessed whether and how glutamate drives Ca2+ -dependent NO release in bEND5 cells, an established model of brain endothelial cells. We found that glutamate induced a dose-dependent oscillatory increase in [Ca2+ ]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 Ca2+ oscillations were triggered by rhythmic endogenous Ca2+ mobilization and maintained over time by extracellular Ca2+ entry. Pharmacological manipulation revealed that glutamate-induced endogenous Ca2+ release was mediated by InsP3 -sensitive receptors and nicotinic acid adenine dinucleotide phosphate (NAADP) gated two-pore channel 1. Constitutive store-operated Ca2+ entry mediated Ca2+ entry during ongoing Ca2+ oscillations. Finally, glutamate evoked a robust, although delayed increase in NO levels, which was blocked by pharmacologically inhibition of the accompanying intracellular Ca2+ signals. Of note, glutamate induced Ca2+ -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 Ca2+ oscillations and NO release have the potential to impact on neurovascular coupling in the brain.
