Glutamate triggers intracellular Ca2+ oscillations and nitric oxide release by inducing NAADP- and InsP3- dependent Ca2+ release in mouse brain endothelial cells

Abstract

The neurotransmitter glutamate increases cerebral blood flow (CBF) by activating postsynap- tic neurons and presynaptic glial cells within the neurovascular (NVU) unit. Glutamate does so by causing an increase in intracellular Ca2+ concentration ([Ca2+]i) in the target cells, which activates the Ca2+/Calmodulin-dependent NO synthase (NOS) to release NO. It is unclear whether brain endothelial cells also sense glutamate through an elevation in [Ca2+]i and NO production. The pre- sent 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 MCPG, 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 inositol-1,4,5-trisphosphate-sensitive receptors and NAADP-gated two-pore channel 1 (TPC1). Constitutive SOCE mediated Ca2+ entry during ongoing Ca2+ oscilla- tions. Finally, glutamate evoked a robust, although delayed increase in NO levels, which was blocked by pharmacologically inhibition of the accompanying intracellular Ca2+ wave. 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.