Survey of peroxynitrite scavengers and its application for pathophysiolgy

在本研究项目中，我们试图调查过氧亚硝酸盐的特异性清除剂，并将其应用于研究过氧亚硝酸盐在神经细胞损伤中的病理生理作用。在调查过氧亚硝酸盐清除剂之前，我们已经研究了过氧亚硝酸盐在神经递质γ-氨基丁酸和乙酰胆碱释放中的作用及其机制，因为我们试图利用过氧亚硝酸盐诱导神经递质释放的潜力来确定其清除剂的活性。我们证实了过氧亚硝酸盐促进原代培养小鼠大脑皮层神经元释放氨基丁酸和乙酰胆碱。利用这个实验系统，我们研究了过氧亚硝酸盐诱导的GABA释放的机制。过氧亚硝酸盐诱导的GABA释放可被神经元膜去极化的抑制剂所阻断，提示过氧亚硝酸盐具有去极化神经元膜的作用。过氧亚硝酸根还可增加Ca-…更多的[2+]涌入神经元。硝苯地平和ω-agatoxin分别是L型和P/Q型电压依赖性钙通道(VDCC)的特异性阻断剂，均能显著抑制过氧亚硝酸盐引起的ω释放，并可完全阻断过氧亚硝酸盐引起的内流，而N型VDCC的阻断剂ω-芋螺毒素GVIA(ω-CTX)对此无明显影响。这些结果表明，过氧亚硝酸根通过开放L和P/Q型VDCC而诱导γ-氨基丁酸的释放，而不改变N型VDCC的功能。羟基自由基清除剂也促进了过氧亚硝酸根诱导的GABABA的释放，而过亚硝酸根在降解过程中形成的羟基自由基的这种抑制作用是由于它抑制了L式的VDCC，说明羟基自由基修饰了过氧亚硝酸根诱导的神经递质的表观释放。此外，已知的过氧亚硝酸根清除剂MnTBPA可诱导过亚硝酸根离子内流到神经元内。目前，我们正在尝试使用上述实验系统来调查过亚硝酸根的清除者，以检查候选人取消过亚硝酸根活性的活动。较少

In this research project, we have attempted to survey scavengers specific for peroxynitrite and to apply them to investigation for clarification of pathophysiological role of peroxynitrite in neuronal cell injury. Prior to the survey of peroxynitrite scavengers, we have investigated the roles of peroxynitrite in release of neurotransmitters such as γ-aminobutyric acid (GABA) and acetylcholine and its mechanisms, because we attempts to use the potential of peroxynitrite to induce neurotransmitter release as a tool to confirm the activity of its scavengers.We confirmed that peroxynitrite enhanced the release of GABA and acetylcholine from mouse cerebral cortical neurons in primary culture. Using this experimental system we have examined mechanisms for peroxynitrite-evoked GABA release. Peroxynitrite-induced GABA release was abolished by inhibitors of neuronal membrane depolarization, suggesting the activity of peroxynitrite to depolarize neuronal membrane. Peroxynitrite also increased Ca … More ^<2+> influx into neurons. Each of nifedipine and ω-agatoxin VIA (ω-ATX), inhibitors specific for L- and P/Q-type voltage-dependent Ca^<2+> channels (VDCCs) respectively, inhibited significantly GABA release by peroxynitrite and the concomitant presence of these inhibitors completely abolished the influx induced by peroxynitrite, whereas ω-conotoxin GVIA (ω-CTX), an inhibitor for N-type VDCCs affected no changes. From these results it is concluded that peroxynitrite induced GABA release consequent to opening L- and P/Q-type VDCCs and did not modify the function of N-type VDCCs.Hydroxyl radical scavengers also facilitated peroxynitrite-evoked GABA release and this inhibitory action of hydroxyl radical formed from peroxynitrite during its degradation is due to its inhibition of L-type VDCCs, indicating hydroxyl radical modifies the apparent peroxynitrite-induced neurotransmitter release. In addition, the activity of peroxynitrite to induce Ca^<2+> influx into the neurons by MnTBPA, a known scavenger for peroxynitrite. In present, we are trying to survey scavengers for peroxynitrite using the experimental system described above for checking activities of candidates to abolish peroxynitrite activity. Less