Modulation of glutamate-induced intracellular energy failure in neonatal cerebral cortical slices by kynurenic acid, dizocilpine, and NBQX.
Modulation of glutamate-induced intracellular energy failure in neonatal cerebral cortical slices by kynurenic acid, dizocilpine, and NBQX.
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犬尿酸、地佐环平和 NBQX 对新生儿大脑皮质切片中谷氨酸诱导的细胞内能量衰竭的调节。
DOI：
10.1038/jcbfm.1994.34
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
Chan,PH
中科院分区：
文献类型：
--
作者：
Espanol,MT;Xu,Y;Litt,L;Yang,GY;Chang,LH;James,TL;Weinstein,P;Chan,PH
在活体大脑皮质切片中比较了谷氨酸引起的急性能量衰竭的严重程度和速度。在每个实验中，从新生 Sprague-Dawley 大鼠中获取 80 个活体大脑皮质切片（350 μm 厚），悬浮并灌注在核磁共振 (NMR) 管中，并在 4.7 T 下使用交错 31 P/1 H NMR 波谱进行研究。连续获得的 NMR 谱被确定为 5 分钟平均值。用含有单独谷氨酸或谷氨酸与三种谷氨酸受体拮抗剂之一混合的人工脑脊液（ACSF）灌注切片60分钟：犬尿酸盐、地佐西平（MK-801）和2,3-二羟基-6-硝基-7-氨磺酰苯并（F）喹喔啉（NBQX）。研究了谷氨酸暴露（0.5 至 10 mM）期间高能磷酸盐的剂量依赖性减少，有或没有拮抗剂保护。使用不含谷氨酸、不含拮抗剂的 ACSF 进行 60 分钟冲洗，测量谷氨酸暴露后的能量恢复。可逆和不可逆能量衰竭的特征是细胞内 pH 值的变化以及 ATP、磷酸肌酸 (PCr) 和无机磷酸盐相对浓度的变化。细胞内N-乙酰天冬氨酸和乳酸水平没有观察到变化。还使用 R-(–)-2-氨基-5-磷酸戊酸 (100 μM) 和河豚毒素 (1 mM) 进行了一些特殊研究，以检查该组织模型中谷氨酸受体的特异性。 Dizocilpine (150 μM) 可以最好地改善 2.0 mM 谷氨酸引起的能量衰竭。使用地佐环平时，ATP 最大下降仅为 6 ± 5%，而不是 35 ± 7%。此外，地佐环平诱导的 ATP 水平恢复在谷氨酸暴露 30 分钟后完成，并持续了另外 30 分钟的谷氨酸暴露和 60 分钟的无谷氨酸 ACSF 冲洗。尽管地佐西平没有改变 PCr 的最大下降（至对照的 36 ± 4%），但地佐西平确实使 PCr 水平在谷氨酸暴露 30 分钟后恢复至对照的 7 ± 5% 以内。在谷氨酸暴露的另外 30 分钟内，PCR 水平保持在该值。在清洗期间，PCr 立即升至比对照高出 5 ± 2% 的值，然后在 60 分钟清洗的剩余时间内保持恒定。在施用谷氨酸的前 20 分钟内，犬尿酸 (1.0 mM) 最能改善高能磷酸盐水平。据报道，NBQX (6.0 μM) 可以保护大脑免受缺血性损伤，在谷氨酸暴露期间减少 PCr 消耗，而不影响 ATP 损失。谷氨酸洗脱 60 分钟后，犬尿酸盐（对照的 84 ± 6%）和 NBQX（对照的 84 ± 2%）的 PCr 水平显着高于单独的谷氨酸（对照的 42 ± 6%）（p < 0.001），尽管两种药物的 ATP 水平均未显着改善。我们的大脑切片模型旨在模拟含氧半影组织，急性能量衰竭可能主要发生在神经元中。地佐西平能最好地保持高能磷酸盐水平的原因可能与其阻断N-甲基-d-天冬氨酸受体的机制有关。地佐西平的额外能量保护也可能来自于电压依赖性 Na+通道的部分阻断，这在所使用的浓度下是可能的。
The severity and rapidity of acute, glutamate-induced energy failure were compared in live cerebral cortical slices. In each experiment 80 live cerebral cortical slices (350 μm thick) were obtained from neonatal Sprague–Dawley rats, suspended and perfused in a nuclear magnetic resonance (NMR) tube, and studied at 4.7 T with interleaved31P/1H NMR spectroscopy. NMR spectra, obtained continually, were determined as 5-min averages. Slices were perfused for 60 min with artificial cerebrospinal fluid (ACSF) containing either glutamate alone or glutamate mixed with one of three glutamate-receptor antagonists: kynurenate, dizocilpine (MK-801), and 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline (NBQX). Dose-dependent decreases in high-energy phosphates were studied during glutamate exposure (0.5 to 10 mM), with and without antagonist protection. Energy recovery after glutamate exposures was measured during a 60-min washout with glutamate-free, antagonist-free ACSF. Reversible and irreversible energy failures were characterized by changes in intracellular pH, and by changes in relative concentrations of ATP, phosphocreatine (PCr), and inorganic phosphate. No changes were observed in intracellular levels ofN-acetylaspartate and lactate. Some special studies were also done usingR-(–)-2-amino-5-phosphonovaleric acid (100 μM) and tetrodotoxin (1 mM) to examine glutamate receptor specificity in this tissue model. Dizocilpine (150 μM) best ameliorated the energy failure caused by 2.0 mMglutamate. With dizocilpine the maximum ATP decrease was only 6 ± 5%, instead of 35 ± 7%. Additionally, the dizocilpine-induced recovery of ATP levels, complete after 30 min of glutamate exposure, lasted througout 30 additional min of glutamate exposure and 60 additional min of washout with glutamate-free ACSF. Although dizocilpine did not alter the maximum decrease that occurred in PCr (to 36 ± 4% of control), dizocilpine did cause PCr levels to return to within 7 ± 5% of the control after 30 min of glutamate exposure. PCr levels stayed at this value throughout 30 additional min of glutamate exposure. During the washout period PCr immediately rose to a value 5 ± 2% above the control and then remained constant during the rest of the 60-min washout. During the first 20 min of glutamate administration, kynurenic acid (1.0 mM) best improved the high-energy phosphate levels. NBQX (6.0 μM), reported to protect the brain from ischemic injury, decreased PCr depletion during glutamate exposure without affecting the loss of ATP. After 60 min of glutamate washout, PCr levels with kynurenate (84 ± 6% of control) and NBQX (84 ± 2% of control) were significantly higher (p < 0.001) than with glutamate alone (42 ± 6% of control), although ATP levels were not significantly improved by either drug. Acute energy failure in our brain slice model, intended to simulate oxygenated penumbral tissue, probably occurs primarily in neurons. The reason that dizocilpine best preserves high-energy phosphate levels might relate to its mechanism ofN-methyl-d-aspartate receptor blockade. Additional energy protection from dizocilpine might also arise from a partial blockade of voltage-dependent Na+channels, which is possible at the concentration used.