Storm-Mathisen J, Ottersen O P, Fu-Long T, Gundersen V, Laake J H, Nordbø G
Med Biol. 1986;64(2-3):127-32.
The immunocytochemical method for demonstrating amino acids makes it possible to study accumulation and depletion of amino acids in individual tissue compartments resulting from experimental manipulations. We have incubated hippocampal slices in oxygenated Krebs solution, containing various additives, under basal conditions and during synaptic release of transmitters evoked by elevated K+ concentrations or by veratrine. Immunoreactivities for glutamate (Glu-LI), aspartate (Asp-LI), glutamine (Gln-LI), gamma-amino-butyrate (GABA-LI) and taurine (Tau-LI) have been demonstrated by specific antibodies after fixation of the slices in glutaraldehyde. Prolonged depolarisation depleted Glu-LI, Asp-LI and Gln-LI from nerve-ending-like structures. GABA-LI was less affected and Tau-LI not affected at all. The depletion of immunoreactivities could be prevented by metabolic precursors of transmitter amino acids, notably glutamine. This effect of glutamine was abolished by inhibiting glutaminase with diazooxonorleucine. Glu-LI, Asp-LI, GABA-LI and Gln-LI accumulated in astroglial cells during conditions of prolonged depolarization-induced release. The accumulation of GABA-LI in glia was strongly increased by inhibition of aminotransferases by aminooxyacetic acid. The described changes in Glu-LI were prevented by low Ca2+/high Mg2+, and promoted when the glial enzyme glutamine synthetase was inhibited by methionine sulfoximine. D-Aspartate, a metabolically inert competitive inhibitor/substrate for high affinity uptake of glutamate, inhibited the accumulation of Glu-LI in glia. The results confirm the biochemically derived theories on metabolic compartmentation in nervous tissue, and add knowledge on the dynamics of the cellular distribution of amino acids. They also indicate the possibilities offered by the present approach for studying metabolism and pharmacology at the cellular level.
用于显示氨基酸的免疫细胞化学方法，使得研究实验操作导致的各个组织区室中氨基酸的积累和消耗成为可能。我们将海马切片在含有各种添加剂的含氧 Krebs 溶液中，于基础条件下以及在由高钾浓度或藜芦碱诱发的递质突触释放期间进行孵育。在将切片用戊二醛固定后，通过特异性抗体显示了谷氨酸（Glu-LI）、天冬氨酸（Asp-LI）、谷氨酰胺（Gln-LI）、γ-氨基丁酸（GABA-LI）和牛磺酸（Tau-LI）的免疫反应性。长时间去极化使神经末梢样结构中的 Glu-LI、Asp-LI 和 Gln-LI 减少。GABA-LI 受影响较小，而 Tau-LI 完全不受影响。递质氨基酸的代谢前体，尤其是谷氨酰胺，可以防止免疫反应性的减少。用重氮氧代正亮氨酸抑制谷氨酰胺酶可消除谷氨酰胺的这种作用。在长时间去极化诱导释放的条件下，Glu-LI、Asp-LI、GABA-LI 和 Gln-LI 在星形胶质细胞中积累。用氨氧基乙酸抑制转氨酶可使胶质细胞中 GABA-LI 的积累显著增加。所述的 Glu-LI 的变化可被低钙/高镁阻止，而当胶质酶谷氨酰胺合成酶被蛋氨酸亚砜胺抑制时则会促进这种变化。D-天冬氨酸是谷氨酸高亲和力摄取的代谢惰性竞争性抑制剂/底物，它抑制了胶质细胞中 Glu-LI 的积累。这些结果证实了关于神经组织中代谢区室化的生化衍生理论，并增加了关于氨基酸细胞分布动态的知识。它们还表明了本方法在细胞水平研究代谢和药理学方面所提供的可能性。
