Yamauchi Tomofusa, Kashii Satoshi, Yasuyoshi Hiroki, Zhang Shen, Honda Yoshihito, Akaike Akinori
Department of Ophthalmology and Visual Sciences, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Invest Ophthalmol Vis Sci. 2003 Jun;44(6):2750-6. doi: 10.1167/iovs.02-0815.
It has been shown that bradykinin (BK) protects retinal neurons against glutamate excitotoxicity, but it was not clear how BK inhibits glutamate excitotoxicity. The purpose of this study was to investigate the effect of opening the mitochondrial adenosine triphosphate (ATP)-sensitive potassium (Mit K (ATP)) channel on glutamate excitotoxicity and the protective effect of BK using cultured retinal neurons.
Primary cultures were obtained from the retina of fetal rats (gestation days 17-19). Glutamate neurotoxicity was assessed by 10-minute exposure to 1 mM glutamate followed by 1-hour incubation in glutamate-free medium, using the trypan blue exclusion method. BK, diazoxide (the opener of the Mit K (ATP) channel), 5HD, and glibenclamide (blockers of the Mit K (ATP) channel) were applied simultaneously with glutamate. Mitochondrial membrane potential was measured as the ratio of 590:527 nm fluorescence of JC-1.
Cell viability was markedly reduced by 10-minute exposure to 1 mM glutamate followed by 1-hour incubation in glutamate-free medium, and glutamate induced mitochondrial depolarization of retinal neurons. BK and diazoxide protected retinal neurons against glutamate excitotoxicity and inhibited glutamate-induced mitochondrial depolarization. These actions of BK and diazoxide were inhibited by the coapplication of 5HD and glibenclamide. Furthermore, diazoxide inhibited the sodium nitroprusside (SNP, NO donor) toxicity, but did not inhibit the 3-morpholinosydnonimine (SIN-1, NO, and superoxide donor) toxicity.
These results suggest that BK and diazoxide protect retinal neurons against glutamate excitotoxicity by opening the Mit K (ATP) channel. It is suggested that opening of the Mit K (ATP) channel inhibited glutamate-induced generation of superoxide.
已表明缓激肽（BK）可保护视网膜神经元免受谷氨酸兴奋性毒性作用，但BK如何抑制谷氨酸兴奋性毒性尚不清楚。本研究的目的是利用培养的视网膜神经元，研究开放线粒体三磷酸腺苷（ATP）敏感性钾通道（Mit K（ATP）通道）对谷氨酸兴奋性毒性的影响以及BK的保护作用。
从胎鼠（妊娠第17 - 19天）的视网膜获得原代培养物。使用台盼蓝排斥法，通过10分钟暴露于1 mM谷氨酸，随后在无谷氨酸培养基中孵育1小时来评估谷氨酸神经毒性。BK、二氮嗪（Mit K（ATP）通道开放剂）、5HD和格列本脲（Mit K（ATP）通道阻滞剂）与谷氨酸同时应用。以JC - 1的590:527 nm荧光比值测量线粒体膜电位。
10分钟暴露于1 mM谷氨酸，随后在无谷氨酸培养基中孵育1小时，细胞活力显著降低，且谷氨酸诱导视网膜神经元线粒体去极化。BK和二氮嗪保护视网膜神经元免受谷氨酸兴奋性毒性作用，并抑制谷氨酸诱导的线粒体去极化。5HD和格列本脲共同应用可抑制BK和二氮嗪的这些作用。此外，二氮嗪抑制硝普钠（SNP，NO供体）毒性，但不抑制3 - 吗啉代辛二酮（SIN - 1，NO和超氧化物供体）毒性。
这些结果表明，BK和二氮嗪通过开放Mit K（ATP）通道保护视网膜神经元免受谷氨酸兴奋性毒性作用。提示Mit K（ATP）通道的开放抑制了谷氨酸诱导的超氧化物生成。
