Palaiologos G, Hertz L, Schousboe A
Department of Biochemistry A. Panum Institute, University of Copenhagen, Denmark.
Neurochem Res. 1989 Apr;14(4):359-66. doi: 10.1007/BF01000039.
Evoked release of glutamate and aspartate from cultured cerebellar granule cells was studied after preincubation of the cells in tissue culture medium with glucose (6.5 mM), glutamine (1.0 mM), D[3H] aspartate and in some cases aminooxyacetate (5.0 mM) or phenylsuccinate (5.0 mM). The release of endogenous amino acids and of D-[3H] aspartate was measured under physiological and depolarizing (56 mM KCl) conditions both in the presence and absence of calcium (1.0 mM), glutamine (1.0 mM), aminooxyacetate (5.0 mM) and phenylsuccinate (5.0 mM). The cellular content of glutamate and aspartate was also determined. Of the endogenous amino acids only glutamate was released in a transmitter fashion and newly synthesized glutamate was released preferentially to exogenously supplied D-[3H] aspartate, a marker for exogenous glutamate. Evoked release of endogenous glutamate was reduced or completely abolished by respectively, aminooxyacetate and phenylsuccinate. In contrast, the release of D-[3H] aspartate was increased reflecting an unaffected release of exogenous glutamate and an increased "psuedospecific radioactivity" of the glutamate transmitter pool. Since aminooxyacetate and phenylsuccinate inhibit respectively aspartate aminotransferase and mitochondrial keto-dicarboxylic acid transport it is concluded that replenishment of the glutamate transmitter pool from glutamine, formed in the mitochondrial compartment by the action of glutaminase requires the simultaneous operation of mitochondrial keto-dicarboxylic acid transport and aspartate aminotransferase which is localized both intra- and extra-mitochondrially. The purpose of the latter enzyme apparently is to catalyze both intra- and extra-mitochondrial transamination of alpha-ketoglutarate which is formed intramitochondrially from the glutamate carbon skeleton and transferred across the mitochondrial membrane to the cytosol where transmitter glutamate is formed.(ABSTRACT TRUNCATED AT 250 WORDS)
在用葡萄糖（6.5 mM）、谷氨酰胺（1.0 mM）、D-[3H]天冬氨酸预孵育培养的小脑颗粒细胞后，研究了谷氨酸和天冬氨酸的诱发释放，在某些情况下还加入了氨基氧乙酸（5.0 mM）或苯琥珀酸（5.0 mM）。在有钙（1.0 mM）、谷氨酰胺（1.0 mM）、氨基氧乙酸（5.0 mM）和苯琥珀酸（5.0 mM）存在和不存在的情况下，在生理和去极化（56 mM KCl）条件下测量内源性氨基酸和D-[3H]天冬氨酸的释放。还测定了谷氨酸和天冬氨酸的细胞含量。在内源性氨基酸中，只有谷氨酸以递质方式释放，新合成的谷氨酸比外源提供的D-[3H]天冬氨酸（外源谷氨酸的标记物）优先释放。氨基氧乙酸和苯琥珀酸分别使内源性谷氨酸的诱发释放减少或完全消除。相反，D-[3H]天冬氨酸的释放增加，反映出外源谷氨酸的释放未受影响，且谷氨酸递质池的“假特异性放射性”增加。由于氨基氧乙酸和苯琥珀酸分别抑制天冬氨酸转氨酶和线粒体酮二羧酸转运，得出结论：由谷氨酰胺酶作用在线粒体区室中形成的谷氨酰胺补充谷氨酸递质池需要线粒体酮二羧酸转运和天冬氨酸转氨酶同时运作，后者位于线粒体内外。后一种酶的目的显然是催化α-酮戊二酸的线粒体内外转氨作用，α-酮戊二酸由谷氨酸碳骨架在线粒体内形成，并穿过线粒体膜转移到胞质溶胶中，在那里形成递质谷氨酸。（摘要截短于250字）
