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 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.

Keywords: Ca2+ oscillations; endothelial cells; glutamate; neurovascular coupling (NVC); nitric oxide.