Laboratory of General Physiology, Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Via Forlanini 6, 27100, Pavia, Italy.
Research Center, Salahaddin University, Erbil, Kurdistan-Region of Iraq, Iraq.
Cell Mol Life Sci. 2020 Jun;77(11):2235-2253. doi: 10.1007/s00018-019-03284-1. Epub 2019 Aug 31.
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 Ca entry and stimulate neuronal nitric oxide (NO) synthase to release NO, thereby triggering NVC. Recent work suggested that endothelial Ca signals could underpin NVC by recruiting the endothelial NO synthase. For instance, acetylcholine induced intracellular Ca 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 Ca signals and NO release in hCMEC/D3 cells. Glutamate induced a dose-dependent increase in intracellular Ca concentration ([Ca]) 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 Ca response to glutamate was inhibited by their pharmacological blockade with, respectively, CPCCOEt and MTEP hydrochloride. The Ca response to glutamate was initiated by endogenous Ca release from the endoplasmic reticulum and endolysosomal Ca store through inositol-1,4,5-trisphosphate receptors and two-pore channels, respectively, and sustained by store-operated Ca entry. In addition, glutamate induced robust NO release that was suppressed by pharmacological blockade of the accompanying increase in [Ca]. These data demonstrate for the first time that glutamate may induce metabotropic Ca signals in human brain microvascular endothelial cells. The Ca 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.
神经血管耦合（NVC）是一种机制，通过该机制，神经元活动的增加会导致局部脑血流（CBF）增加，以确保激活区域的局部氧气和营养供应。兴奋性神经递质谷氨酸门控突触后 N-甲基-D-天冬氨酸受体（NMDAR）介导细胞外 Ca 内流，并刺激神经元一氧化氮合酶（NOS）释放一氧化氮（NO），从而触发 NVC。最近的工作表明，内皮细胞 Ca 信号可能通过募集内皮型一氧化氮合酶来支撑 NVC。例如，乙酰胆碱通过激活 hCMEC/D3 细胞中的毒蕈碱 5 型受体（广泛用于人脑微血管内皮细胞的模型）引起细胞内 Ca 信号，随后通过激活毒蕈碱 5 型受体（muscarinic 5 receptors）释放 NO。在此，我们试图评估谷氨酸是否也会在 hCMEC/D3 细胞中引发代谢型 Ca 信号和 NO 释放。谷氨酸诱导细胞内 Ca 浓度 ([Ca]) 呈剂量依赖性增加，该增加被 α-甲基-4-羧基苯甘氨酸阻断，并被反式-1-氨基-1,3-环戊烷二羧酸模拟，分别阻断和激活组 1 代谢型谷氨酸受体（mGluRs）。因此，hCMEC/D3 表达 mGluR1 和 mGluR5，谷氨酸对 [Ca] 的反应被其药理学阻断剂 CPCCOEt 和 MTEP 盐酸盐抑制。谷氨酸对 [Ca] 的反应是由内质网和内溶酶体 Ca 库通过肌醇 1,4,5-三磷酸受体和双孔通道分别释放内源性 Ca 引起的，并通过储存操作的 Ca 内流维持。此外，谷氨酸诱导强烈的 NO 释放，该释放被伴随的 [Ca] 增加的药理学阻断所抑制。这些数据首次表明，谷氨酸可能在人脑微血管内皮细胞中诱导代谢型 Ca 信号。谷氨酸引起的 Ca 反应可能支持神经元活动期间的 NVC，从而增强脑微血管内皮细胞在调节 CBF 中的新兴作用。
