Marks Jeremy D, Boriboun Chan, Wang Janice
Department of Pediatrics, University of Chicago, Chicago, Illinois 60637, USA.
J Neurosci. 2005 Jul 13;25(28):6561-75. doi: 10.1523/JNEUROSCI.1450-05.2005.
Mitochondrial membrane potential (DeltaPsim)-dependent Ca2+ uptake plays a central role in neurodegeneration after NMDA receptor activation. NMDA-induced DeltaPsim dissipation increases during postnatal development, coincident with increasing vulnerability to NMDA. NMDA receptor activation also produces nitric oxide (NO), which can inhibit mitochondrial respiration, dissipating DeltaPsim. Because DeltaPsim dissipation reduces mitochondrial Ca2+ uptake, we hypothesized that NO mediates the NMDA-induced DeltaPsim dissipation in immature neurons, underlying their decreased vulnerability to excitotoxicity. Using hippocampal neurons cultured from 5- and 19-d-old rats, we measured NMDA-induced changes in [Ca2+]cytosol, DeltaPsim, NO, and [Ca2+]mito. In postnatal day 5 (P5) neurons, NMDA mildly dissipated DeltaPsim in a NO synthase (NOS)-dependent manner and increased NO. The NMDA-induced NO increase was abolished with carbonyl cyanide 4-(trifluoromethoxy)phenyl-hydrazone and regulated by [Ca2+]mito. Mitochondrial Ca2+ uptake inhibition prevented the NO increase, whereas inhibition of mitochondrial Ca2+ extrusion increased it. Consistent with this mitochondrial regulation, NOS and cytochrome oxidase immunoreactivity demonstrated mitochondrial localization of NOS. Furthermore, NOS blockade increased mitochondrial Ca2+ uptake during NMDA. Finally, at physiologic O2 tensions (3% O2), NMDA had little effect on survival of P5 neurons, but NOS blockade during NMDA markedly worsened survival, demonstrating marked neuroprotection by mitochondrial NO. In P19 neurons, NMDA dissipated DeltaPsim in an NO-insensitive manner. NMDA-induced NO production was not regulated by DeltaPsim, and NOS immunoreactivity was cytosolic, without mitochondrial localization. NOS blockade also protected P19 neurons from NMDA. These data demonstrate that mitochondrial NOS mediates much of the decreased vulnerability to NMDA in immature hippocampal neurons and that cytosolic NOS contributes to NMDA toxicity in mature neurons.
线粒体膜电位（ΔΨm）依赖性Ca2+摄取在NMDA受体激活后的神经退行性变中起核心作用。NMDA诱导的ΔΨm耗散在出生后发育过程中增加，这与对NMDA的易感性增加相吻合。NMDA受体激活还产生一氧化氮（NO），它可以抑制线粒体呼吸，使ΔΨm耗散。由于ΔΨm耗散会减少线粒体Ca2+摄取，我们推测NO介导了未成熟神经元中NMDA诱导的ΔΨm耗散，这是它们对兴奋性毒性易感性降低的基础。使用从5日龄和19日龄大鼠培养的海马神经元，我们测量了NMDA诱导的胞质Ca2+（[Ca2+]cytosol）、ΔΨm、NO和线粒体Ca2+（[Ca2+]mito）的变化。在出生后第5天（P5）的神经元中，NMDA以一种依赖于一氧化氮合酶（NOS）的方式轻度耗散ΔΨm并增加NO。用羰基氰化物4-（三氟甲氧基）苯基腙消除了NMDA诱导的NO增加，并且它受[Ca2+]mito调节。线粒体Ca2+摄取抑制阻止了NO增加，而线粒体Ca2+外排抑制则增加了NO。与这种线粒体调节一致，NOS和细胞色素氧化酶免疫反应性表明NOS定位于线粒体。此外，NOS阻断增加了NMDA作用期间的线粒体Ca2+摄取。最后，在生理氧张力（3% O2）下，NMDA对P5神经元的存活影响很小，但在NMDA作用期间进行NOS阻断会显著恶化存活情况，这表明线粒体NO具有显著的神经保护作用。在P19神经元中，NMDA以一种对NO不敏感的方式耗散ΔΨm。NMDA诱导的NO产生不受ΔΨm调节，并且NOS免疫反应性位于胞质中，没有线粒体定位。NOS阻断也保护P19神经元免受NMDA的损伤。这些数据表明，线粒体NOS介导了未成熟海马神经元对NMDA易感性降低的大部分原因，并且胞质NOS促成了成熟神经元中的NMDA毒性。
