Mis Katarina, Mars Tomaz, Jevsek Marko, Brank Martina, Zajc-Kreft Katarina, Grubic Zoran
Laboratory for Molecular Neurobiology, Institute of Pathophysiology, School of Medicine, University of Ljubljana, Ljubljana, Slovenia.
J Histochem Cytochem. 2003 Dec;51(12):1633-44. doi: 10.1177/002215540305101207.
In spite of intensive investigations, the roles of acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BuChE; EC 3.1.1.8) in the central nervous system (CNS) remain unclear. A role recently proposed for BuChE as an explanation for survival of AChE knockout mice is compensation for AChE activity if it becomes insufficient. Neuronal contribution of both enzymes to the cholinesterase pool in the neuromuscular junction has also been suggested. These proposals imply that BuChE expression follows that of AChE and that, in addition to AChE, BuChE is also expressed in alpha-motor neurons. However, these assumptions have not yet been properly tested. Histochemical approaches to these problems have been hampered by a number of problems that prevent unambiguous interpretation of results. In situ hybridization (ISH) of mRNAs encoding AChE and BuChE, which is the state-of-the-art approach, has not yet been done. Here we describe rapid nonradioactive ISH for the localization of mRNAs encoding AChE and BuChE. Various probes and experimental conditions had been tested to obtain reliable localization. In combination with RT-PCR, ISH revealed that, in rat spinal cord, cells expressing AChE mRNA also express BuChE mRNA but in smaller quantities. alpha-Motor neurons had the highest levels of both mRNAs. Virtual absence of transcripts encoding AChE and BuChE in glia might reflect a discrepancy between mRNA and enzyme levels previously reported for cholinesterases.
尽管进行了深入研究，但乙酰胆碱酯酶（AChE；EC 3.1.1.7）和丁酰胆碱酯酶（BuChE；EC 3.1.1.8）在中枢神经系统（CNS）中的作用仍不清楚。最近有人提出，BuChE的作用是在AChE活性不足时补偿其活性，以此解释AChE基因敲除小鼠的存活情况。也有人提出这两种酶在神经肌肉接头处对胆碱酯酶池的神经元贡献。这些观点意味着BuChE的表达与AChE的表达一致，并且除了AChE之外，BuChE也在α运动神经元中表达。然而，这些假设尚未得到充分验证。解决这些问题的组织化学方法受到许多问题的阻碍，这些问题妨碍了对结果的明确解释。作为目前最先进的方法，对编码AChE和BuChE的mRNA进行原位杂交（ISH）尚未开展。在此，我们描述了用于定位编码AChE和BuChE的mRNA的快速非放射性ISH方法。我们测试了各种探针和实验条件以获得可靠的定位。结合逆转录聚合酶链反应（RT-PCR），ISH显示，在大鼠脊髓中，表达AChE mRNA的细胞也表达BuChE mRNA，但表达量较少。α运动神经元中这两种mRNA的水平最高。神经胶质细胞中编码AChE和BuChE的转录本几乎不存在，这可能反映了先前报道的胆碱酯酶mRNA水平与酶水平之间的差异。
