Rameau Gerald A, Chiu Ling-Yu, Ziff Edward B
Department of Biochemistry, Howard Hughes Medical Institute, NYU School of Medicine, New York, NY 10016, USA.
Neurobiol Aging. 2003 Dec;24(8):1123-33. doi: 10.1016/j.neurobiolaging.2003.07.002.
Stimulation of NMDA receptors activates neuronal nitric oxide synthase (nNOS) and the production of nitric oxide (NO). Dephosphorylation of nNOS increases nNOS enzymatic activity. We have examined the regulation of nNOS phosphorylation in rat cortical neurons following NMDA receptor activation. We show that nNOS is constitutively phosphorylated and that NMDA receptor activation decreases the level of nNOS phosphorylation by a mechanism that is blocked specifically by NMDA receptor antagonists and inhibitors of the Ca2+-regulated phosphatases calcineurin and PP1/PP2A. Using quantitative digital microscopy, we show that NMDA receptor activation induces the accumulation of nitrotyrosine, a measure of nNOS activity, and TdT-mediated fluorescein-dUTP nick end labeling (TUNEL) positivity, a measure of cell death. A calcineurin inhibitor blocked the increase in both TUNEL and nitrotyrosine positivity. Notably, TUNEL was increased in those neurons that were most strongly positive for nitrotyrosine. We conclude that NMDA receptor activation induces death of neurons by a cell autonomous pathway involving nNOS dephosphorylation by a calcineurin-dependent mechanism.
N-甲基-D-天冬氨酸（NMDA）受体的激活会激活神经元型一氧化氮合酶（nNOS）并产生一氧化氮（NO）。nNOS的去磷酸化会增加nNOS的酶活性。我们研究了NMDA受体激活后大鼠皮质神经元中nNOS磷酸化的调节情况。我们发现nNOS是组成性磷酸化的，并且NMDA受体激活通过一种被NMDA受体拮抗剂以及Ca2+调节的磷酸酶钙调神经磷酸酶和蛋白磷酸酶1/蛋白磷酸酶2A（PP1/PP2A）抑制剂特异性阻断的机制降低了nNOS的磷酸化水平。使用定量数字显微镜，我们发现NMDA受体激活会诱导硝基酪氨酸的积累（一种nNOS活性的指标）以及TdT介导的荧光素-dUTP缺口末端标记（TUNEL）阳性（一种细胞死亡的指标）。一种钙调神经磷酸酶抑制剂阻断了TUNEL和硝基酪氨酸阳性的增加。值得注意的是，TUNEL在那些硝基酪氨酸阳性最强的神经元中增加。我们得出结论，NMDA受体激活通过一种涉及钙调神经磷酸酶依赖性机制使nNOS去磷酸化的细胞自主途径诱导神经元死亡。
