Morley P, Small D L, Murray C L, Mealing G A, Poulter M O, Durkin J P, Stanimirovic D B
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada.
J Cereb Blood Flow Metab. 1998 Apr;18(4):396-406. doi: 10.1097/00004647-199804000-00008.
Excitatory amino acids can modify the tone of cerebral vessels and permeability of the blood-brain barrier (BBB) by acting directly on endothelial cells of cerebral vessels or indirectly by activating receptors expressed on other brain cells. In this study we examined whether rat or human cerebromicrovascular endothelial cells (CEC) express ionotropic and metabotropic glutamate receptors. Glutamate and the glutamate receptor agonists N-methyl-d-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), and kainate failed to increase [Ca2+]i in either rat or human microvascular and capillary CEC but elicited robust responses in primary rat cortical neurons, as measured by fura-2 fluorescence. The absence of NMDA and AMPA receptors in rat and human CEC was further confirmed by the lack of immunocytochemical staining of cells by antibodies specific for the AMPA receptor subunits GluR1, GluR2/3, and GluR4 and the NMDA receptor subunits NR1, NR2A, and NR2B. We failed to detect mRNA expression of the AMPA receptor subunits GluR1 to GluR4 or the NMDA receptor subunits NR1(1XX); NR1(0XX), and NR2A to NR2C in both freshly isolated rat and human microvessels and cultured CEC using reverse transcriptase polymerase chain reaction (RT-PCR). Cultured rat CEC expressed mRNA for KA1 or KA2 and GluR5 subunits. Primary rat cortical neurons were found to express GluR1 to GluR3 and NR1, NR2A, and NR2B by both immunocytochemistry and RT-PCR and KA1, KA2, GluR5, GluR6, and GluR7 by RT-PCR. Moreover, the metabotropic glutamate receptor agonist 1-amino-cyclopentyl-1S, 3R-dicorboxylate (1S,3R-trans-ACPD), while eliciting both inositol trisphosphate and [Ca2+]i increases and inhibiting forskolin-stimulated cyclic AMP in cortical neurons, was unable to induce either of these responses in rat or human CEC. These results strongly suggest that both rat and human CEC do not express functional glutamate receptors. Therefore, excitatory amino acid-induced changes in the cerebral microvascular tone and BBB permeability must be affected indirectly, most likely by mediators released from the adjacent glutamate-responsive cells.
兴奋性氨基酸可通过直接作用于脑血管内皮细胞或间接激活其他脑细胞上表达的受体，来改变脑血管张力和血脑屏障（BBB）的通透性。在本研究中，我们检测了大鼠或人类脑微血管内皮细胞（CEC）是否表达离子型和代谢型谷氨酸受体。谷氨酸以及谷氨酸受体激动剂N-甲基-D-天冬氨酸（NMDA）、α-氨基-3-羟基-5-甲基异恶唑-4-丙酸（AMPA）和海人藻酸均未能增加大鼠或人类微血管及毛细血管CEC中的[Ca2+]i，但通过fura-2荧光检测发现，它们能在原代大鼠皮层神经元中引发强烈反应。用针对AMPA受体亚基GluR1、GluR2/3和GluR4以及NMDA受体亚基NR1、NR2A和NR2B的特异性抗体对细胞进行免疫细胞化学染色，结果显示大鼠和人类CEC中不存在NMDA和AMPA受体，进一步证实了上述结果。使用逆转录聚合酶链反应（RT-PCR），我们未能在新鲜分离的大鼠和人类微血管及培养的CEC中检测到AMPA受体亚基GluR1至GluR4或NMDA受体亚基NR1(1XX)、NR1(0XX)以及NR2A至NR2C的mRNA表达。培养的大鼠CEC表达KA1或KA2以及GluR5亚基的mRNA。通过免疫细胞化学和RT-PCR发现，原代大鼠皮层神经元表达GluR1至GluR3以及NR1、NR2A和NR2B，通过RT-PCR发现其表达KA1、KA2、GluR5、GluR6和GluR7。此外，代谢型谷氨酸受体激动剂1-氨基环戊基-1S,3R-二羧酸（1S,3R-反式-ACPD）虽然能增加皮层神经元中的肌醇三磷酸和[Ca2+]i，并抑制福斯可林刺激的环磷酸腺苷，但在大鼠或人类CEC中均无法诱导这些反应。这些结果强烈表明，大鼠和人类CEC均不表达功能性谷氨酸受体。因此，兴奋性氨基酸诱导的脑微血管张力和BBB通透性变化必定是间接影响的，很可能是由相邻的谷氨酸反应性细胞释放的介质所介导。
