Beczkowska I W, Gracy K N, Pickel V M, Inturrisi C E
Department of Pharmacology, Cornell University Medical College, New York, NY 10021, U.S.A.
Neuroscience. 1997 Aug;79(3):855-62. doi: 10.1016/s0306-4522(97)00034-1.
Retinoic acid treatment of NT-era2/cl.D1 (NT2) cells, a human teratocarcinoma cell line, yields 95% pure cultures of terminally differentiated neuronal cells. Concomitant with their terminal differentiation into neurons, NT2 cells are induced by retinoic acid to express neuronal N-methyl-D-aspartate receptor channels, which are fully functional. We determined the effects of retinoic acid-induced differentiation of NT2 cells on the levels of N-methyl-D-aspartate, delta opioid and mu opioid receptor messenger RNAs. RNA levels were measured using quantitative solution hybridization assays. The riboprobes were complementary to major portions of the coding regions of the N-methyl-D-aspartate, delta opioid and mu opioid receptor complementary DNAs. After four weeks of exposure to 10 microM retinoic acid, followed by four weeks of treatment with mitotic inhibitors (1 microM of cytosine arabinoside, 10 microM of fluorodeoxyuridine and 10 microM of uridine) the levels of N-methyl-D-aspartate receptor messenger RNA in differentiated NT2-N cells increased 10-fold, delta opioid receptor messenger RNA increased three-fold, and mu opioid receptor messenger RNA increased four-fold. Northern blot analysis revealed two transcripts for the N-methyl-D-aspartate receptor messenger RNA (4.2 and 4.4 kb) and two transcripts for delta opioid receptor messenger RNA (7.0 and 11.0 kb). To determine whether the increases in messenger RNAs were accompanied by an increased synthesis of the respective proteins, we examined the immunoperoxidase localization of N-methyl-D-aspartate receptor and delta opioid receptor antisera. N-Methyl-D-aspartate receptor-like immunoreactivity was seen within the cell bodies as well as on the processes of the retinoic acid-differentiated cells. Although delta opioid receptor-like immunoreactivity was detected within the soma of isolated cells prior to retinoic acid treatment, the apparent number of these labelled cells and their ramified processes were markedly enhanced following retinoic acid differentiation. These results demonstrate parallels between the inducible expression of the N-methyl-D-aspartate and opioid receptor messenger RNAs and proteins during the acquisition of the fully differentiated neuronal phenotype in cultured NT2 cells. Retinoic acid-differentiated NT2 cells express increased levels for the N-methyl-D-aspartate, delta opioid and mu opioid receptor messenger RNAs, providing the opportunity to study the interactions among these receptor systems in human terminally differentiated neuronal cells in culture.
视黄酸处理人畸胎瘤细胞系NT-era2/cl.D1（NT2细胞）可产生95%纯度的终末分化神经元细胞培养物。在NT2细胞向神经元终末分化的同时，视黄酸诱导其表达具有完整功能的神经元N-甲基-D-天冬氨酸受体通道。我们测定了视黄酸诱导NT2细胞分化对N-甲基-D-天冬氨酸、δ阿片样物质和μ阿片样物质受体信使核糖核酸水平的影响。使用定量溶液杂交分析法测量核糖核酸水平。核糖探针与N-甲基-D-天冬氨酸、δ阿片样物质和μ阿片样物质受体互补脱氧核糖核酸编码区的主要部分互补。在暴露于10微摩尔视黄酸四周后，再用有丝分裂抑制剂（1微摩尔阿糖胞苷、10微摩尔氟脱氧尿苷和10微摩尔尿苷）处理四周，分化后的NT2-N细胞中N-甲基-D-天冬氨酸受体信使核糖核酸水平增加了10倍，δ阿片样物质受体信使核糖核酸增加了3倍，μ阿片样物质受体信使核糖核酸增加了4倍。Northern印迹分析显示N-甲基-D-天冬氨酸受体信使核糖核酸有两种转录本（4.2和4.4千碱基），δ阿片样物质受体信使核糖核酸有两种转录本（7.0和11.0千碱基）。为了确定信使核糖核酸的增加是否伴随着各自蛋白质合成的增加，我们检测了N-甲基-D-天冬氨酸受体和δ阿片样物质受体抗血清的免疫过氧化物酶定位。在视黄酸分化细胞的细胞体以及突起上可见N-甲基-D-天冬氨酸受体样免疫反应性。虽然在视黄酸处理前在分离细胞的胞体中检测到了δ阿片样物质受体样免疫反应性，但在视黄酸分化后这些标记细胞及其分支突起的明显数量显著增加。这些结果表明，在培养的NT2细胞获得完全分化的神经元表型过程中，N-甲基-D-天冬氨酸和阿片样物质受体信使核糖核酸及蛋白质的可诱导表达之间存在平行关系。视黄酸分化的NT2细胞中N-甲基-D-天冬氨酸、δ阿片样物质和μ阿片样物质受体信使核糖核酸水平升高，为研究这些受体系统在培养的人终末分化神经元细胞中的相互作用提供了机会。
