Kishimoto K, Matsumura K, Kataoka Y, Morii H, Watanabe Y
Department of Neuroscience, Osaka Bioscience Institute, Suita-shi, Japan.
Neuroscience. 1999;92(3):1061-77. doi: 10.1016/s0306-4522(99)00051-2.
Ca2(+)-sensitive 85,000 mol. wt cytosolic phospholipase A2 plays an essential role in the selective and stimulus-dependent release of arachidonic acid from membrane phospholipids. Cytosolic phospholipase A2-catalysed lipid mediators including arachidonic acid and its metabolites have been suggested to be involved in a variety of neuronal functions in the CNS. Since the cellular localization of cytosolic phospholipase A2 is still controversial and obscure, we tried an improved method of rapid processing of each specimens and succeeded in obtaining intense signals of cytosolic phospholipase A2 messenger RNA in the normal rat brain by northern blot analysis and in situ hybridization. Northern blot analysis showed the abundant distribution of cytosolic phospholipase A2 messenger RNA in most regions of the brain, with intense signals observed in the pineal gland and pons. Macroautoradiographs prepared after in situ hybridization with three different antisense riboprobes gave essentially similar patterns of localization; significant signals were widely detected in the gray matter of various regions, i.e. the olfactory bulb, cerebral cortex, hippocampus, amygdala, several thalamic and hypothalamic nuclei and cerebellum. Microautoradiographs showed that most of the intense signals were predominant in neurons, and that faint signals were from glial cells and other non-neuronal cells in the choroid plexus, inner surface cells of veins and the leptomeninges. In addition, the cycloheximide treatment increased the cytosolic phospholipase A2 messenger RNA level in the same cell populations originally possessing messenger RNA signals. Predominant expression of cytosolic phospholipase A2 messenger RNA in neurons may provide the basis for the contribution of cytosolic phospholipase A2-catalysed lipid mediators to a variety of neurotransmission and synaptic functions in the CNS.
分子量为85,000的Ca2+敏感型胞质磷脂酶A2在从膜磷脂中选择性和刺激依赖性释放花生四烯酸的过程中起着至关重要的作用。据推测，胞质磷脂酶A2催化的脂质介质，包括花生四烯酸及其代谢产物，参与中枢神经系统的多种神经元功能。由于胞质磷脂酶A2的细胞定位仍存在争议且尚不明确，我们尝试了一种改进的各标本快速处理方法，并通过Northern印迹分析和原位杂交成功在正常大鼠脑中获得了胞质磷脂酶A2信使RNA的强信号。Northern印迹分析显示，胞质磷脂酶A2信使RNA在大脑的大多数区域分布丰富，在松果体和脑桥中观察到强信号。用三种不同的反义核糖探针进行原位杂交后制备的宏观放射自显影片给出了基本相似的定位模式；在各个区域的灰质中广泛检测到显著信号，即嗅球、大脑皮层、海马体、杏仁核、几个丘脑和下丘脑核以及小脑。微观放射自显影片显示，大多数强信号主要存在于神经元中，而微弱信号来自脉络丛、静脉内表面细胞和软脑膜中的神经胶质细胞及其他非神经元细胞。此外，环己酰亚胺处理增加了原本具有信使RNA信号的相同细胞群体中的胞质磷脂酶A2信使RNA水平。神经元中胞质磷脂酶A2信使RNA的主要表达可能为胞质磷脂酶A2催化的脂质介质对中枢神经系统中多种神经传递和突触功能的贡献提供基础。
