Boado R J, Pardridge W M
Department of Medicine, UCLA School of Medicine, 90024.
J Neurosci Res. 1994 Nov 1;39(4):430-5. doi: 10.1002/jnr.490390410.
Hypertension has been linked to opening of the blood-brain barrier and may be related to the expression of the smooth muscle alpha-actin gene in contractile cells at the brain microvasculature. However, the cellular origin (i.e., endothelial cells, pericytes, smooth muscle cells) of the alpha-actin mRNA in the brain microvasculature is not clearly identified. Therefore, we investigated the abundance of actin mRNA by Northern blot analysis in isolated brain microvessels and in brain microvascular endothelial or pericytes in tissue culture. All samples showed the characteristic 2.1 kb transcript corresponding to cytoplasmic beta and gamma isoform mRNA. The 1.7 kb transcript corresponding to smooth muscle alpha-actin was detected in freshly isolated bovine brain microvessels, in primary cultures of brain microvascular pericytes, or endothelial cells; the latter cultures contain both endothelial cells and pericytes. The alpha-actin mRNA was absent in a cloned bovine brain endothelial cell line. The relative abundance of the alpha/(beta + gamma) actin transcript ratio was: cultured pericytes > freshly isolated microvessels > endothelial primary. The cellular distribution of the smooth muscle alpha-actin immunoreactive protein was studied by immunocytochemistry in cytospun/methanol-fixed isolated bovine brain microvessels with a monoclonal antibody directed to the amino-terminal decapeptide of the smooth muscle alpha-actin isoform. This antibody reacted strongly with precapillary arterioles of isolated microvessels, whereas no immunostaining was observed in either capillary endothelial cells or in pericytes. In conclusion, the alpha-actin mRNA is expressed in brain microvascular pericytes in tissue culture, but the immunoreactive alpha-actin protein is not expressed in brain microvascular pericytes in vivo. These data suggest that either 1) alpha-actin gene expression is induced in capillary pericytes in tissue culture or 2) alpha-actin mRNA in brain capillary pericytes in vivo is subject to translational repression resulting in no detectable alpha-actin protein under normal conditions.
高血压与血脑屏障的开放有关，可能与脑微血管收缩细胞中平滑肌α-肌动蛋白基因的表达有关。然而，脑微血管中α-肌动蛋白mRNA的细胞来源（即内皮细胞、周细胞、平滑肌细胞）尚未明确鉴定。因此，我们通过Northern印迹分析研究了分离的脑微血管以及组织培养中的脑微血管内皮细胞或周细胞中肌动蛋白mRNA的丰度。所有样品均显示出对应于细胞质β和γ同工型mRNA的特征性2.1 kb转录本。在新鲜分离的牛脑微血管、脑微血管周细胞或内皮细胞的原代培养物中检测到对应于平滑肌α-肌动蛋白的1.7 kb转录本；后者的培养物包含内皮细胞和周细胞。在克隆的牛脑内皮细胞系中未检测到α-肌动蛋白mRNA。α/（β + γ）肌动蛋白转录本比率的相对丰度为：培养的周细胞＞新鲜分离的微血管＞内皮原代细胞。通过免疫细胞化学方法，使用针对平滑肌α-肌动蛋白同工型氨基末端十肽的单克隆抗体，研究了平滑肌α-肌动蛋白免疫反应性蛋白在细胞涂片/甲醇固定的分离牛脑微血管中的细胞分布。该抗体与分离微血管的毛细血管前小动脉强烈反应，而在毛细血管内皮细胞或周细胞中均未观察到免疫染色。总之，α-肌动蛋白mRNA在组织培养的脑微血管周细胞中表达，但免疫反应性α-肌动蛋白蛋白在体内的脑微血管周细胞中不表达。这些数据表明，要么1）组织培养中的毛细血管周细胞中α-肌动蛋白基因表达被诱导，要么2）体内脑毛细血管周细胞中的α-肌动蛋白mRNA受到翻译抑制，导致在正常条件下无法检测到α-肌动蛋白蛋白。
