Acetylcholine induces intracellular Ca2+ oscillations and nitric oxide release in mouse brain endothelial cells

Abstract

Basal forebrain neurons increase cortical blood flow by releasing acetylcholine (Ach), which stimulates endothelial cells (ECs) to produce the vasodilating gasotransmitter, nitric oxide (NO). Surprisingly, the mechanism whereby Ach induces NO synthesis in brain microvascular ECs is unknown. An increase in intracellular Ca²⁺ concentration recruits a multitude of endothelial Ca²⁺-dependent pathways, such as Ca²⁺/calmodulin endothelial NO synthase (eNOS). The present investigation sought to investigate the role of intracellular Ca²⁺ signaling in Ach-induced NO production in bEND5 cells, an established model of mouse brain microvascular ECs, by conventional imaging of cells loaded with the Ca²⁺-sensitive dye, Fura-2/AM, and the NO-sensitive fluorophore, DAF-DM diacetate. Ach induced dose-dependent Ca²⁺ oscillations in bEND5 cells, 300 μM being the most effective dose to generate a prolonged Ca²⁺ burst. Pharmacological manipulation revealed that Ach-evoked Ca²⁺ oscillations required metabotropic muscarinic receptor (mAchR) activation and were patterned by a complex interplay between repetitive ER Ca²⁺ release via inositol-1,4,5-trisphosphate receptors (InsP₃Rs) and store-operated Ca²⁺ entry (SOCE). A comprehensive real time-polymerase chain reaction analysis demonstrated the expression of the transcripts encoding for M3-mAChRs, InsP₃R1 and InsP₃R3, Stim1-2 and Orai2. Next, we found that Ach-induced NO production was hindered by L-NAME, a selective NOS inhibitor, and BAPTA, a membrane permeable intracellular Ca²⁺ buffer. Moreover, Ach-elicited NO synthesis was blocked by the pharmacological abrogation of the accompanying Ca²⁺ spikes. Overall, these data shed novel light on the molecular mechanisms whereby neuronally-released Ach controls neurovascular coupling in blood microvessels.

Keywords: Acetylcholine; Ca(2+) signaling; Intracellular Ca(2+) oscillations; Mouse brain microvascular endothelial cells; Nitric oxide.