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Group 1 metabotropic glutamate receptors trigger glutamate-induced intracellular Ca2+ signals and nitric oxide release in human brain microvascular endothelial cells

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Abstract

Neurovascular coupling (NVC) is the mechanism whereby an increase in neuronal activity causes an increase in local cerebral blood flow (CBF) to ensure local supply of oxygen and nutrients to the activated areas. The excitatory neurotransmitter glutamate gates post-synaptic N-methyl-d-aspartate receptors to mediate extracellular Ca2+ entry and stimulate neuronal nitric oxide (NO) synthase to release NO, thereby triggering NVC. Recent work suggested that endothelial Ca2+ signals could underpin NVC by recruiting the endothelial NO synthase. For instance, acetylcholine induced intracellular Ca2+ signals followed by NO release by activating muscarinic 5 receptors in hCMEC/D3 cells, a widely employed model of human brain microvascular endothelial cells. Herein, we sought to assess whether also glutamate elicits metabotropic Ca2+ signals and NO release in hCMEC/D3 cells. Glutamate induced a dose-dependent increase in intracellular Ca2+ concentration ([Ca2+]i) that was blocked by α-methyl-4-carboxyphenylglycine and phenocopied by trans-1-amino-1,3-cyclopentanedicarboxylic acid, which, respectively, block and activate group 1 metabotropic glutamate receptors (mGluRs). Accordingly, hCMEC/D3 expressed both mGluR1 and mGluR5 and the Ca2+ response to glutamate was inhibited by their pharmacological blockade with, respectively, CPCCOEt and MTEP hydrochloride. The Ca2+ response to glutamate was initiated by endogenous Ca2+ release from the endoplasmic reticulum and endolysosomal Ca2+ store through inositol-1,4,5-trisphosphate receptors and two-pore channels, respectively, and sustained by store-operated Ca2+ entry. In addition, glutamate induced robust NO release that was suppressed by pharmacological blockade of the accompanying increase in [Ca2+]i. These data demonstrate for the first time that glutamate may induce metabotropic Ca2+ signals in human brain microvascular endothelial cells. The Ca2+ response to glutamate is likely to support NVC during neuronal activity, thereby reinforcing the emerging role of brain microvascular endothelial cells in the regulation of CBF.

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Acknowledgements

This research was funded by: Italian Ministry of Education, University and Research (MIUR): Dipartimenti di Eccellenza Program (2018–2022)—Dept. of Biology and Biotechnology “L. Spallanzani”, University of Pavia (F.M.), and by Fondo Ricerca Giovani from the University of Pavia (F.M.). P.S.F. was supported by MAECI (Ministero degli Affari Esteri e della Cooperazione Internazionale).

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Supplementary Fig.

 1. TRPC7 activation did not induce Ca2+and NO signals in hCMEC/D3 cells. A. 1-oleoyl-2-acetyl-sn-glycerol (OAG; 100 μM) failed to increase the [Ca2+]i in hCMEC/D3 cells. The Ca2+ tracing is representative of 63 cells from three independent experiments. B. OAG (100 μM) did not increase DAF-FM fluorescence in hCMEC/D3 cells, while glutamate (100 μM) elicited NO release. C. Bar histogram shows the mean ± SE of the percentage of hCMEC/D3 cells displaying NO release in the presence of OAG and glutamate. The asterisk indicates p < 0.05. NR = No response. D. Bar histogram shows the mean ± SE of the amplitude of OAG- and glutamate-induced NO release in hCMEC/D3 cells. The asterisk indicates p < 0.05. NR = No Response. (TIFF 353 kb)

Supplementary Fig.

 2. The Orai1 specific inhibitor S66 triggers intracellular Ca2+oscillations in hCMEC/D3 cells. The specific Orai1 inhibitor S66 (20 μM) was administered following recovery of the intracellular Ca2+ response to glutamate (100 μM) under 0Ca2+ conditions to assess its effect on the subsequent restoration of extracellular Ca2+ levels. However, S66 immediately elicited repetitive Ca2+ spikes which prevented further examination of its effect on SOCE. This trace is representative of 71 recordings from three independent experiments. (TIFF 93 kb)

Supplementary Fig.

 3. The Orai1 specific inhibitor BTP-2 impairs SOCE in hCMEC/D3 cells. A. Pharmacological depletion of the ER Ca2+ pool with the SERCA inhibitor CPA (10 μM) under 0Ca2+ conditions resulted in robust SOCE upon Ca2+ restitution to the bath. B. BTP-2 (20 µM), a selective inhibitor of Orai1, attenuated CPA-induced SOCE in hCMEC/D3 cells. C. Bar histogram shows the mean ± SE of the percentage of cells displaying SOCE under control (Ctrl) conditions and upon treatment with BTP-2 (20 µM, 20 min). D. Bar histogram shows the mean ± SE of SOCE amplitude under control (Ctrl) conditions and upon treatment with BTP-2 (20 µM, 20 min). The asterisk indicates p < 0.05. (TIFF 995 kb)

Supplementary Fig.

 4. The mGluR5 specific agonist CHPG reliably induces intracellular Ca2+signals in hCMEC/D3 cells. CHPG (25 μM), a selective CHPG agonist, induced an increase in [Ca2+]i in hCMEC/D3 cells. The trace was representative of 65 cells from three independent experiments. (TIFF 195 kb)

Supplementary Fig.

 5. BTP-2 did not affect glutamate-induced NO release in hCMEC/D3 cells. A. Glutamate (100 μM) induced a robust increase in DAF-FM fluorescence both in control (Ctrl) conditions and in the presence of BTP-2 (20 μM, 20 min). B. Bar histogram shows the mean±SE of the percentage of cells showing glutamate-induced NO release in the absence (Ctrl) and presence of BTP-2. C. Bar histogram shows the mean±SE of the amplitude of glutamate-induced NO release in the absence (Ctrl) and presence of BTP-2. (TIFF 1359 kb)

Supplementary Fig.

 6. Pyr6 and La3+did not affect glutamate-induced NO release in hCMEC/D3 cells. A. Glutamate (100 μM) induced a robust increase in DAF-FM fluorescence both in control (Ctrl) conditions and in the presence of Pyr6 (10 μM, 10 min) plus La3+ (100 μM, 20 min). B. Bar histogram shows the mean±SE of the amplitude of glutamate-induced NO release in the absence (Ctrl) and presence of Pyr6 plus La3+. (TIFF 585 kb)

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Negri, S., Faris, P., Pellavio, G. et al. Group 1 metabotropic glutamate receptors trigger glutamate-induced intracellular Ca2+ signals and nitric oxide release in human brain microvascular endothelial cells. Cell. Mol. Life Sci. 77, 2235–2253 (2020). https://doi.org/10.1007/s00018-019-03284-1

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