Survey of peroxynitrite scavengers and its application for pathophysiolgy

在本研究项目中，我们试图研究过氧亚硝酸盐特异性清除剂，并将其应用于研究，以澄清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γ-氨基丁酸（GABA）和乙酰胆碱等神经递质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作为确认其清除剂活性的工具。我们证实过氧亚硝酸盐能促进原代培养小鼠大脑皮层神经元中GABA和乙酰胆碱的释放。利用这个实验系统，我们研究了过氧亚硝酸盐诱发GABA释放的机制。过氧亚硝酸盐诱导的GABA释放被神经元膜去极化抑制剂所消除，提示过氧亚硝酸盐具有神经元膜去极化的活性。过氧亚硝酸盐也增加了Ca . More ^<2+>流入神经元。硝苯地平和ω-agatoxin VIA （ω-ATX）分别是L-型和P/ q型电压依赖性Ca^<2+>通道（VDCCs）的特异性抑制剂，它们都能显著抑制过氧亚硝酸盐释放GABA，并且同时存在的这些抑制剂完全消除了过氧亚硝酸盐诱导的内流，而ω- concontoxin GVIA （ω-CTX）是n型VDCCs的抑制剂，对GABA没有影响。综上所述，过氧亚硝酸盐诱导了L-型和P/ q型VDCCs打开后的GABA释放，而对n型VDCCs的功能没有影响。羟基自由基清除剂也促进过氧亚硝酸盐诱发的GABA释放，而这种由过氧亚硝酸盐降解过程中形成的羟基自由基的抑制作用是由于其对l型vdcc的抑制作用，表明羟基自由基改变了过氧亚硝酸盐诱导的神经递质释放。此外，过氧亚硝酸盐的活性诱导Ca^<2+>通过MnTBPA流入神经元，MnTBPA是一种已知的过氧亚硝酸盐清除率。目前，我们正试图利用上述实验系统来检测过氧亚硝酸盐清除剂的活性，以消除过氧亚硝酸盐的活性。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