Winter H C, Ueda T
Department of Biological Chemistry, Medical School, University of Michigan, Ann Arbor 48109.
Neurochem Res. 1993 Jan;18(1):79-85. doi: 10.1007/BF00966925.
A variety of naturally occurring amino acids, their isomers, and synthetic analogs were tested for their ability to inhibit uptake of [3H]glutamate into presynaptic vesicles from bovine cerebral cortex. Strongest inhibition (Ki < 1mM) was observed for trans-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD) and erythro-4-methyl-L-glutamic acid (MGlu), while 4-methylene-L-glutamic acid (MeGlu) was only moderately inhibitory (Ki = approximately 3mM), indicating that the synaptic vesicle glutamate translocator has higher affinity for trans-ACPD and MGlu than for glutamate. A few other amino acids, e.g., 4-hydroxyglutamic acid, S-carboxyethyl cysteine, and 5-fluorotryptophan, were slightly inhibitory; all L- and DL-isomers of protein amino acids and longer chain acidic amino acids were without measurable inhibition. Potassium tetrathionate and S-sulfocysteine exhibited strong to moderate noncompetitive or irreversible inhibition. Inhibition by t-ACPD, MGlu, or MeGlu was competitive with glutamic acid. Each of these competitive inhibitors was also taken up by the vesicle preparation in an ATP-dependent manner, as indicated by their being recovered unchanged from filtered vesicles. Similar results were obtained with reconstituted vesicles, while glutamate uptake by partially purified rat synaptosomes was inhibited only by MGlu. These results indicate that the glutamate translocator of presynaptic vesicles has stringent structural requirements distinct from those of the plasma membrane translocator and the metabotropic type of postsynaptic glutamate receptor. They further suggest possible structural requirements of pharmacologically significant compounds that can substitute for glutamic acid in the presynaptic side of glutamatergic synapses, thus serving to moderate or control glutamate excitation and associated excitotoxic effects in these neurons.
对多种天然存在的氨基酸、它们的异构体以及合成类似物进行了测试，以考察其抑制[3H]谷氨酸摄取到牛大脑皮质突触前囊泡中的能力。反式-1-氨基环戊烷-1,3-二羧酸（t-ACPD）和赤式-4-甲基-L-谷氨酸（MGlu）表现出最强的抑制作用（Ki < 1mM），而4-亚甲基-L-谷氨酸（MeGlu）仅具有中等抑制作用（Ki约为3mM），这表明突触囊泡谷氨酸转运体对反式ACPD和MGlu的亲和力高于对谷氨酸的亲和力。其他一些氨基酸，如4-羟基谷氨酸、S-羧乙基半胱氨酸和5-氟色氨酸，具有轻微的抑制作用；蛋白质氨基酸的所有L-和DL-异构体以及长链酸性氨基酸均无明显抑制作用。连四硫酸钾和S-磺基半胱氨酸表现出强到中等的非竞争性或不可逆抑制作用。t-ACPD、MGlu或MeGlu的抑制作用与谷氨酸具有竞争性。这些竞争性抑制剂中的每一种也以ATP依赖的方式被囊泡制剂摄取，这可从它们在过滤后的囊泡中未发生变化地被回收得到证明。用重组囊泡也得到了类似的结果，而部分纯化的大鼠突触体对谷氨酸的摄取仅被MGlu抑制。这些结果表明，突触前囊泡的谷氨酸转运体具有严格的结构要求，这与质膜转运体和代谢型突触后谷氨酸受体的结构要求不同。它们进一步提示了在谷氨酸能突触的突触前侧可替代谷氨酸的具有药理学意义的化合物的可能结构要求，从而有助于调节或控制这些神经元中的谷氨酸兴奋及相关的兴奋性毒性作用。
