Bickler P E, Fahlman C S, Ferriero D M
Departments of Anesthesia Neurology, University of California at San Francisco, San Francisco, California 94143-0542, USA.
J Neurochem. 2004 Feb;88(4):878-84. doi: 10.1046/j.1471-4159.2003.02203.x.
The effects of 30 s to 10 min hypoxia (PO2-10 mmHg) on glutamate receptor activity were studied in murine cortical neurons. Receptor activity was assessed as a rise in intracellular calcium concentration ([Ca2+]i) following a 10 s application of 1 mm glutamate or 100 micro mN-methy-d-aspartate (NMDA) in the presence of 0.1 mm Mg2+ and 10 micro m glycine. Change in [Ca2+]i elicited by glutamate increased 26% (n = 192, p < 0.001) and that to NMDA by 74% (n = 9, p < 0.01) during a 100-s period of hypoxia. After 10 min hypoxia, responses to glutamate were 62% smaller than those in normoxia, with increased basal intracellular [Ca2+]i predicting reduced receptor activity. When neurons were exposed to NMDA after 10 min of hypoxia, [Ca2+]i increases were 12% smaller than after 100 s hypoxia, but still 53% larger than in oxygenated neurons (n = 9, p = 0.01). Neurons expressed relatively similar amounts of NR2A, -B, -C, and -D subunits. The phosphorylation of NMDA NR1 subunits increased during hypoxia. Pre-treatment of neurons with a protein kinase C (PKC) inhibitor (chelerythrine, 10 micro m) prevented increases in N-methy-d-aspartate receptor (NMDAR) activity during hypoxia and reduced the phosphorylation of NR1 subunits. These results suggest that enhancement of glutamate receptor activity during the first minutes of hypoxia is mediated by phosphorylation of NMDARs by PKC and that other mechanisms, possibly involving intracellular calcium, limit glutamate receptor-mediated calcium influx during longer periods of hypoxia.
研究了30秒至10分钟低氧（PO₂ - 10 mmHg）对小鼠皮层神经元谷氨酸受体活性的影响。在存在0.1 mM Mg²⁺和10 μM甘氨酸的情况下，通过施加1 mM谷氨酸或100 μM N-甲基-D-天冬氨酸（NMDA）10秒后细胞内钙浓度（[Ca²⁺]i）的升高来评估受体活性。在100秒的低氧期间，谷氨酸引起的[Ca²⁺]i变化增加了26%（n = 192，p < 0.001），而NMDA引起的增加了74%（n = 9，p < 0.01）。低氧10分钟后，对谷氨酸的反应比常氧时小62%，基础细胞内[Ca²⁺]i升高预示着受体活性降低。当神经元在低氧10分钟后暴露于NMDA时，[Ca²⁺]i的增加比低氧100秒后小12%，但仍比充氧神经元大53%（n = 9，p = 0.01）。神经元表达相对相似量的NR2A、-B、-C和-D亚基。低氧期间NMDA NR1亚基的磷酸化增加。用蛋白激酶C（PKC）抑制剂（白屈菜红碱，10 μM）预处理神经元可防止低氧期间N-甲基-D-天冬氨酸受体（NMDAR）活性增加，并减少NR1亚基的磷酸化。这些结果表明，低氧最初几分钟内谷氨酸受体活性的增强是由PKC对NMDAR的磷酸化介导的，而其他机制，可能涉及细胞内钙，在更长时间的低氧期间限制了谷氨酸受体介导的钙内流。
