Dunlop J, Grieve A, Schousboe A, Griffiths R
Department of Biochemistry and Microbiology, University of St. Andrews, Fife, Scotland, U.K.
J Neurochem. 1991 Oct;57(4):1388-97. doi: 10.1111/j.1471-4159.1991.tb08305.x.
In primary cultures of mouse cerebral cortex neurons, sulphur-containing excitatory amino acids (SAAs; namely, L-cysteine sulphinate, L-cysteate, L-homocysteine sulphinate, L-homocysteate, S-sulphocysteine) at concentrations ranging from 0.1 microM to 1 mM evoked a saturable release of gamma-[3H]aminobutyric acid ([3H]GABA) in the absence of any other depolarizing agent. All SAAs exhibited essentially similar potency (EC50, 100-150 microM) in releasing [3H]GABA although a variable profile of maximal stimulatory effect was observed when compared with basal release. The intracellular accumulation of the lipophilic cation, [3H]tetraphenylphosphonium, was significantly reduced in the presence of all SAAs, thus verifying a depolarization of the neuronal plasma membrane. SAA-stimulated release of [3H]GABA was shown to comprise two distinct components, calcium-dependent and calcium-independent, which occur after activation of N-methyl-D-aspartate (NMDA) and non-NMDA receptors. Thus, all SAA-evoked responses were antagonized by the selective, competitive NMDA-receptor antagonist, 3-[(+/-)-2-carboxypiperazin-4-yl]propyl-1-phosphonic acid (IC50 range, greater than 50 microM) and the non-NMDA-receptor antagonist, 6,7-dinitroquinoxalinedione (IC50 range, 5-50 microM). Removal of magnesium ions from the superfusion medium caused a significant potentiation of SAA-evoked responses without having any effect on basal levels of [3H]GABA efflux, a result consistent with an involvement of NMDA-receptor activation. Calcium-independent release (i.e., that release remaining in the presence of 1 mM cobalt ions) was a distinct component but of smaller magnitude. Using 500 microM excitatory amino acid agonist concentrations, this component of release was (1) markedly attenuated by 15 microM SKF-89976-A, a non-transportable inhibitor of the GABA carrier, and (2) abolished when choline ions replaced sodium ions in the superfusion medium or when in the presence of excitatory amino acid receptor antagonists. These observations are clearly consistent with a receptor-mediated, depolarization-induced reversal of the GABA carrier.
在小鼠大脑皮质神经元的原代培养物中，浓度范围为0.1微摩尔至1毫摩尔的含硫兴奋性氨基酸（SAA；即L-半胱氨酸亚磺酸盐、L-半胱酸盐、L-高半胱氨酸亚磺酸盐、L-高半胱酸盐、S-磺基半胱氨酸）在没有任何其他去极化剂的情况下能引起γ-[3H]氨基丁酸（[3H]GABA）的饱和释放。所有SAA在释放[3H]GABA方面表现出基本相似的效力（EC50，100 - 150微摩尔），尽管与基础释放相比，观察到最大刺激效应的变化情况有所不同。在所有SAA存在的情况下，亲脂性阳离子[3H]四苯基鏻的细胞内积累显著减少，从而证实了神经元质膜的去极化。SAA刺激的[3H]GABA释放显示由两个不同的成分组成，即钙依赖性和钙非依赖性成分，它们在N-甲基-D-天冬氨酸（NMDA）和非NMDA受体激活后发生。因此，所有SAA诱发的反应都被选择性竞争性NMDA受体拮抗剂3-[(±)-2-羧基哌嗪-4-基]丙基-1-膦酸（IC50范围，大于50微摩尔）和非NMDA受体拮抗剂6,7-二硝基喹喔啉二酮（IC50范围，5 - 50微摩尔）所拮抗。从灌流培养基中去除镁离子导致SAA诱发的反应显著增强，而对[3H]GABA流出的基础水平没有任何影响，这一结果与NMDA受体激活的参与一致。钙非依赖性释放（即在1毫摩尔钴离子存在下仍存在的释放）是一个不同的成分，但幅度较小。使用500微摩尔兴奋性氨基酸激动剂浓度时，该释放成分（1）被15微摩尔SKF-89976-A（一种GABA载体的非转运性抑制剂）显著减弱，（2）当灌流培养基中的胆碱离子取代钠离子时或在存在兴奋性氨基酸受体拮抗剂时被消除。这些观察结果显然与受体介导的、去极化诱导的GABA载体逆转一致。
