McKinnon E, Hargittai P T, Grossfeld R M, Lieberman E M
Department of Physiology, East Carolina University School of Medicine, Greenville, North Carolina 27858, USA.
Glia. 1995 Jul;14(3):198-208. doi: 10.1002/glia.440140305.
Two of the key enzymes involved in glutamate metabolism, glutaminase and glutamine synthetase, were quantitatively localized to axons and glia of the crayfish giant nerve fiber by immunocytochemistry and electron microscopy of antibody-linked gold microspheres. In Western blots, rabbit antisera for glutamine synthetase and glutaminase specifically recognized crayfish polypeptides corresponding approximately in size to subunits of purified mammalian brain enzymes. Glutamine synthetase immunoreactivity was found to be 11 times greater in the adaxonal glial cells than in the axon. Glutaminase immunoreactivity was found in somewhat greater concentration (2.5:1) in glia as compared to axoplasm. Glutamate immunoreactivity also was evaluated and found to be present in high concentration in both glia and axons, as might be expected for an important substrate of cellular metabolism. Using radiolabeled substrates it was demonstrated that glutamine and glutamate were interconverted by the native enzymes in the intact crayfish giant nerve fiber and that the formation of glutamine from glutamate occurred in the axoplasm-free nerve fiber, the cellular component of which is primarily periaxonal glia. The results of this investigation provide immunocytochemical and metabolic evidence consistent with an intercellular glutamine cycle that modulates the concentration of periaxonal glutamate and glutamine in a manner similar to that described for perisynaptic regions of the vertebrate central nervous system. These findings further corroborate previous electrophysiological evidence that glutamate serves as the axon-to-glial cell neurochemical signal that activates glial cell mechanisms responsible for periaxonal ion homeostasis.
通过免疫细胞化学以及抗体连接金微球的电子显微镜技术，参与谷氨酸代谢的两种关键酶——谷氨酰胺酶和谷氨酰胺合成酶，被定量定位到小龙虾巨神经纤维的轴突和神经胶质细胞中。在蛋白质免疫印迹分析中，针对谷氨酰胺合成酶和谷氨酰胺酶的兔抗血清特异性识别出小龙虾中大小与纯化的哺乳动物脑酶亚基大致相当的多肽。发现轴突周围神经胶质细胞中的谷氨酰胺合成酶免疫反应性比轴突中的高11倍。与轴浆相比，神经胶质细胞中谷氨酰胺酶免疫反应性的浓度略高（2.5:1）。还对谷氨酸免疫反应性进行了评估，发现其在神经胶质细胞和轴突中均以高浓度存在，这对于细胞代谢的重要底物来说是可以预期到的。使用放射性标记底物证明，在完整的小龙虾巨神经纤维中，谷氨酰胺和谷氨酸可被天然酶相互转化，并且从谷氨酸形成谷氨酰胺发生在无轴浆的神经纤维中，其细胞成分主要是轴突周围神经胶质细胞。本研究结果提供了免疫细胞化学和代谢证据，与一种细胞间谷氨酰胺循环一致，该循环以类似于脊椎动物中枢神经系统突触周围区域所描述的方式调节轴突周围谷氨酸和谷氨酰胺的浓度。这些发现进一步证实了先前的电生理证据，即谷氨酸作为轴突到神经胶质细胞的神经化学信号，激活负责轴突周围离子稳态的神经胶质细胞机制。
