DIFFERENT PATTERNS OF Ca2+ SIGNALING DRIVE ACETYLCHOLINE AND GLUTAMATE INDUCED-NO RELEASE IN MOUSE AND HUMAN BRAIN MICROVASCULAR ENDOTHELIAL CELLS

Acetylcholine (Ach) and glutamate (Glu) are two of the major excitatory neurotransmitters in the brain which increase cerebral blood flow by releasing nitric oxide (NO) from postsynaptic neurons and astrocytes and causing vasorelaxationin adjacent microvessels. An increase in intracellular Ca2+ concentration recruits a multitude of endothelial Ca2+-dependent pathways, such as Ca2+/Calmodulin endothelial NO synthase (eNOS). Surprisingly, the Ca2+-dependent mechanisms whereby Ach induces NO synthesis in brain endothelial cells (ECs) is still unclear. On the other hand, Glu stimulates NMDA receptors to activate eNOS, but it is able to cause a metabotropic increase in intracellular Ca2+ concentration in brain microvascular ECs. The present investigation sought to fill these gaps by analysing murine (bEND5) and human (hCMEC/D3) brain microvascular ECs. Herein, we first demonstrated that Ach induces NO release by triggering two different modes of Ca2+ signals in bEND5 and hCMEC/D3 cells. Of note, endoplasmic reticulum Ca2+ release via inositol-1,4,5-trisphosphate receptors and store-operated Ca2+ entry shapes the Ca2+ response to Ach in both cell types but their different Ca2+ toolkits result in two quite different waveforms, i.e. Ca2+ oscillations vs. biphasic Ca2+ elevation. Whatever its waveform, however, Ach-induced intracellular Ca2+ signals lead to robust NO release in both murine and human brain microvascular ECs. Likewise, we demonstrated for the first time that Glu activated metabotropic intracellular Ca2+ oscillation in bEND5 cells and a biphasic increase in intracellular Ca2+ concentration in hCMEC/D3 cells. We further showed that glutamate-dependent Ca2+ signals drive NO release in both types of cells. This NO signal is delayed as compared to the Ach-induced one and is likely to play a crucial role in the slower vasodilation that often follows brief neuronal activity or that sustains functional hyperemia during persistent synaptic transmission. This information has a potential clinical relevance as the decrease in neuronal activity-induced cortical CBF is involved in a growing number of neurodegenerative disorders, such as Alzheimer’s Disease. Understanding the underlying mechanisms could, therefore, be used in the future as target to rescue local blood perfusion in patients affected by neurodegenerative disorders.