Bradford S E, Nadler J V
Department of Pharmacology and Cancer Biology, Box 3813, 100B Research Park 2, Research Drive, Duke University Medical Center, Durham, NC 27710, USA.
Neuroscience. 2004;128(4):751-65. doi: 10.1016/j.neuroscience.2004.06.065.
Certain excitatory pathways in the rat hippocampus can release aspartate along with glutamate. This study utilized rat hippocampal synaptosomes to characterize the mechanism of aspartate release and to compare it with glutamate release. Releases of aspartate and glutamate from the same tissue samples were quantitated simultaneously. Both amino acids were released by 25 mM K(+), 300 microM 4-aminopyridine (4-AP) and 0.5 and 1 microM ionomycin in a predominantly Ca(2+)-dependent manner. For a roughly equivalent quantity of glutamate released, aspartate release was significantly greater during exposure to elevated [K(+)] than to 4-AP and during exposure to 0.5 than to 1 microM ionomycin. Aspartate release was inefficiently coupled to P/Q-type voltage-dependent Ca(2+) channels and was reduced by KB-R7943, an inhibitor of reversed Na(+)/Ca(2+) exchange. In contrast, glutamate release depended primarily on Ca(2+) influx through P/Q-type channels and was not significantly affected by KB-R7943. Pretreatment of the synaptosomes with tetanus toxin and botulinum neurotoxins C and F reduced glutamate release, but not aspartate release. Aspartate release was also resistant to bafilomycin A(1), an inhibitor of vacuolar H(+)-ATPase, whereas glutamate release was markedly reduced. (+/-) -Threo-3-methylglutamate, a non-transportable competitive inhibitor of excitatory amino acid transport, did not reduce aspartate release. Niflumic acid, a blocker of Ca(2+)-dependent anion channels, did not alter the release of either amino acid. Exogenous aspartate and aspartate recently synthesized from glutamate accessed the releasable pool of aspartate as readily as exogenous glutamate and glutamate recently synthesized from aspartate accessed the releasable glutamate pool. These results are compatible with release of aspartate from either a vesicular pool by a "non-classical" form of exocytosis or directly from the cytoplasm by an as-yet-undescribed Ca(2+)-dependent mechanism. In either case, they suggest aspartate is released mainly outside the presynaptic active zones and may therefore serve as the predominant agonist for extrasynaptic N-methyl-D-aspartate receptors.
大鼠海马体中的某些兴奋性通路在释放谷氨酸的同时也能释放天冬氨酸。本研究利用大鼠海马体突触体来表征天冬氨酸的释放机制，并将其与谷氨酸的释放机制进行比较。对同一组织样本中的天冬氨酸和谷氨酸释放进行了同步定量分析。两种氨基酸均可被25 mM K⁺、300 μM 4-氨基吡啶（4-AP）以及0.5和1 μM离子霉素以主要依赖Ca²⁺的方式释放。在释放大致等量谷氨酸的情况下，暴露于高浓度[K⁺]时的天冬氨酸释放量显著高于暴露于4-AP时，且暴露于0.5 μM离子霉素时的天冬氨酸释放量高于暴露于1 μM离子霉素时。天冬氨酸的释放与P/Q型电压依赖性Ca²⁺通道的偶联效率较低，并且会被反向Na⁺/Ca²⁺交换抑制剂KB-R7943所降低。相比之下，谷氨酸的释放主要依赖于通过P/Q型通道的Ca²⁺内流，且不受KB-R7943的显著影响。用破伤风毒素以及肉毒杆菌神经毒素C和F对突触体进行预处理可减少谷氨酸的释放，但不影响天冬氨酸的释放。天冬氨酸的释放也对液泡H⁺-ATP酶抑制剂巴弗洛霉素A₁具有抗性，而谷氨酸的释放则显著减少。（±）-苏-3-甲基谷氨酸是一种不可转运的兴奋性氨基酸转运竞争性抑制剂，它不会减少天冬氨酸的释放。尼氟灭酸是一种Ca²⁺依赖性阴离子通道阻滞剂，它不会改变这两种氨基酸的释放。外源性天冬氨酸以及最近由谷氨酸合成的天冬氨酸与外源性谷氨酸以及最近由天冬氨酸合成的谷氨酸一样，都能轻易进入可释放的天冬氨酸池和可释放的谷氨酸池。这些结果与天冬氨酸通过“非经典”形式的胞吐作用从囊泡池中释放，或者通过一种尚未描述的Ca²⁺依赖性机制直接从细胞质中释放的情况相符。无论哪种情况，都表明天冬氨酸主要在突触前活性区之外释放，因此可能作为突触外N-甲基-D-天冬氨酸受体的主要激动剂。
