Galea E, Reis D J, Feinstein D L
Department of Neurology and Neuroscience, Cornell University Medical College, New York, New York.
J Neurosci Res. 1994 Feb 15;37(3):406-14. doi: 10.1002/jnr.490370313.
In primary cultures of rat astroglial cells exposure to bacterial endotoxin lipopolysaccharide (LPS) causes induction of a Ca(2+)-independent form of the nitric oxide synthase (iNOS) enzyme. We have now cloned the mRNA encoding astroglial iNOS using a combination of cDNA library screening and polymerase chain reaction (PCR) amplification with degenerate oligonucleotides directed against conserved regions of all NOS enzymes. The sequence of astroglial iNOS cDNA is highly similar to the mouse macrophage sequence, having an overall homology of 92% at the DNA level and 93% at the protein level. As in other NOSs, canonical binding sites for redox cofactors are present. The 3'-untranslated region displays 4 consensus AU-pentamers, 2 polyadenylation sites, and terminates in a stretch of 17 adenosine residues. In situ hybridization studies with LPS-treated astrocyte cultures demonstrated the presence of iNOS mRNA in the majority of astroglial cells, identified by antibody staining to the glial fibrillary acidic protein (GFAP). PCR analysis showed that LPS stimulated synthesis of astrocyte iNOS mRNA, which was detected as early as 2 hr after exposure to LPS, peaked at 4 hr, and slowly declined over the next 20 hr. These results confirm that astrocytes can express iNOS and provide tools for the subsequent analysis of iNOS gene expression in rodent brain.
在大鼠星形胶质细胞的原代培养物中，暴露于细菌内毒素脂多糖（LPS）会导致诱导出一种不依赖钙离子的一氧化氮合酶（iNOS）。我们现在使用cDNA文库筛选和聚合酶链反应（PCR）扩增相结合的方法，用针对所有一氧化氮合酶保守区域的简并寡核苷酸克隆了编码星形胶质细胞iNOS的mRNA。星形胶质细胞iNOS cDNA的序列与小鼠巨噬细胞序列高度相似，在DNA水平上总体同源性为92%，在蛋白质水平上为93%。与其他一氧化氮合酶一样，存在氧化还原辅因子的典型结合位点。3'非翻译区有4个一致的AU五聚体、2个聚腺苷酸化位点，并以一段17个腺苷残基结尾。对用LPS处理的星形胶质细胞培养物进行原位杂交研究表明，通过对胶质纤维酸性蛋白（GFAP）的抗体染色鉴定，大多数星形胶质细胞中存在iNOS mRNA。PCR分析表明，LPS刺激星形胶质细胞iNOS mRNA的合成，在暴露于LPS后2小时即可检测到，4小时达到峰值，并在接下来的20小时内缓慢下降。这些结果证实星形胶质细胞可以表达iNOS，并为随后分析啮齿动物脑中iNOS基因表达提供了工具。
