Elhusseiny A, Cohen Z, Olivier A, Stanimirović D B, Hamel E
Laboratory of Cerebrovascular Research, Montreal Neurological Institute, McGill University, Montréal, Quebec, Canada.
J Cereb Blood Flow Metab. 1999 Jul;19(7):794-802. doi: 10.1097/00004647-199907000-00010.
Acetylcholine is an important regulator of local cerebral blood flow. There is, however, limited information available on the possible sites of action of this neurotransmitter on brain intraparenchymal microvessels. In this study, a combination of molecular and functional approaches was used to identify which of the five muscarinic acetylcholine receptors (mAChR) are present in human brain microvessels and their intimately associated astroglial cells. Microvessel and capillary fractions isolated from human cerebral cortex were found by reverse transcriptase-polymerase chain reaction to express m2, m3, and, occasionally, m1 and m5 receptor subtypes. To localize these receptors to a specific cellular compartment of the vessel wall, cultures of human brain microvascular endothelial and smooth muscle cells were used, together with cultured human brain astrocytes. Endothelial cells invariably expressed m2 and m5 receptors, and occasionally the m1 receptor; smooth muscle cells exhibited messages for all except the m4 mAChR subtypes, whereas messages for all five muscarinic receptors were identified in astrocytes. In all three cell types studied, acetylcholine induced a pirenzepine-sensitive increase (62% to 176%, P<0.05 to 0.01) in inositol trisphosphate, suggesting functional coupling of m1, m3, or m5 mAChR to a phospholipase C signaling cascade. Similarly, coupling of m2 or m4 mAChR to adenylate cyclase inhibition in endothelial cells and astrocytes, but not in smooth muscle cells, was demonstrated by the ability of carbachol to significantly reduce (44% to 50%, P<0.05 to 0.01) the forskolin-stimulated increase in cAMP levels. This effect was reversed by the mAChR antagonist AFDX 384. The results indicate that microvessels are able to respond to neurally released acetylcholine and that mAChR, distributed in different vascular and astroglial compartments, could regulate cortical perfusion and, possibly, blood-brain barrier permeability, functions that could become jeopardized in neurodegenerative disorders such as Alzheimer's disease.
乙酰胆碱是局部脑血流量的重要调节因子。然而，关于这种神经递质在脑实质内微血管上可能的作用位点，目前可用信息有限。在本研究中，采用分子和功能相结合的方法来确定五种毒蕈碱型乙酰胆碱受体（mAChR）中哪些存在于人类脑微血管及其紧密相连的星形胶质细胞中。通过逆转录聚合酶链反应发现，从人类大脑皮质分离出的微血管和毛细血管部分表达m2、m3，偶尔也表达m1和m5受体亚型。为了将这些受体定位到血管壁的特定细胞区室，使用了人类脑微血管内皮细胞和平滑肌细胞培养物以及培养的人类脑星形胶质细胞。内皮细胞总是表达m2和m5受体，偶尔也表达m1受体；平滑肌细胞除了m4 mAChR亚型外，均表现出所有亚型的信息，而在星形胶质细胞中鉴定出了所有五种毒蕈碱受体的信息。在所有研究的三种细胞类型中，乙酰胆碱均诱导肌醇三磷酸水平出现哌仑西平敏感的升高（62%至176%，P<0.05至0.01），表明m1、m3或m5 mAChR与磷脂酶C信号级联存在功能偶联。同样，在乙酰胆碱能显著降低（44%至50%，P<0.05至0.01）福司可林刺激的cAMP水平升高的实验中，证明了m2或m4 mAChR在内皮细胞和星形胶质细胞中与腺苷酸环化酶抑制偶联，但在平滑肌细胞中未出现这种偶联。这种作用被mAChR拮抗剂AFDX 384逆转。结果表明，微血管能够对神经释放的乙酰胆碱作出反应，并且分布在不同血管和星形胶质区室的mAChR可能调节皮质灌注，也可能调节血脑屏障通透性，而在诸如阿尔茨海默病等神经退行性疾病中，这些功能可能会受到损害。
