Georgopoulos A, Svarna R, Palaiologos G
Laboratory of Biological Chemistry, Medical School, University of Athens, Greece.
Neurochem Res. 1995 Jan;20(1):45-9. doi: 10.1007/BF00995151.
To study the effect of agents interfering with the biosynthesis and/or the K(+)-evoked Ca(2+)-dependent release of neurotransmitter glutamate, rat cerebral slices were preincubated with Krebs-Ringer-HEPES-glucose-glutamine buffer (KRH buffer), loaded with D-[3H]aspartate and superfused with the preincubation medium in the presence or in the absence of Ca2+. The difference in radioactivity release divided by the basal release per min under the two conditions represented the K(+)-evoked Ca(2+)-dependent release. The agents used were: 1) Aminooxyacetic acid (AOAA), the inhibitor of transaminases, 2) Leucine (Leu), the inhibitor of phosphate activated glutaminase (PAG), 3) NH4+, the inhibitor of PAG, 4) Phenylsuccinic acid (Phs), the inhibitor of the mitochondrial ketodicarboxylate carrier, 5) ketone bodies, the inhibitors of glycolysis, 6) the absence of glutamine, the substrate of PAG. The results show that Leu, NH4+, Phs and the absence of Gln significantly increase the K(+)-evoked Ca(2+)-dependent release of radioactivity by 64%, 200%, 95% and 147% respectively, indicating that these agents are inhibitors of the K(+)-evoked Ca(2+)-dependent release of glutamate. Ketone bodies and AOAA had no effect. These results indicate that the major if not the exclusive biosynthetic pathway of neurotransmitter glutamate in rat cerebral cortex is through the PAG reaction and support a model for the pathway followed by neurotransmitter glutamate i.e. glutamate formed outside the inner mitochondrial membrane has to enter the mitochondrial matrix or is formed within it from where it can be extruded to supply the transmitter pool in exchange of GABA.
为研究干扰神经递质谷氨酸生物合成和/或钾离子诱发的钙离子依赖性释放的药物的作用，将大鼠脑片用Krebs-Ringer-HEPES-葡萄糖-谷氨酰胺缓冲液（KRH缓冲液）预孵育，加载D-[3H]天冬氨酸，并在有或无钙离子存在的情况下用预孵育培养基进行灌流。两种条件下放射性释放的差异除以每分钟的基础释放量代表钾离子诱发的钙离子依赖性释放。所用药物如下：1）转氨酶抑制剂氨基氧乙酸（AOAA）；2）磷酸激活谷氨酰胺酶（PAG）抑制剂亮氨酸（Leu）；3）PAG抑制剂NH4+；4）线粒体酮二羧酸载体抑制剂苯琥珀酸（Phs）；5）糖酵解抑制剂酮体；6）PAG底物谷氨酰胺缺失。结果显示，Leu、NH4+、Phs和谷氨酰胺缺失分别使钾离子诱发的钙离子依赖性放射性释放显著增加64%、200%、95%和147%，表明这些药物是钾离子诱发的钙离子依赖性谷氨酸释放的抑制剂。酮体和AOAA无作用。这些结果表明，大鼠大脑皮层中神经递质谷氨酸的主要（若非唯一）生物合成途径是通过PAG反应，并支持神经递质谷氨酸所遵循途径的模型，即线粒体内膜外形成的谷氨酸必须进入线粒体基质或在其中形成，然后从那里挤出以供应递质池来交换GABA。
